EP0973394A1 - NUCLEIC ACID AND AMINO ACID SEQUENCES RELATING TO $i(HELICOBACTER PYLORI) AND VACCINE COMPOSITIONS THEREOF - Google Patents

NUCLEIC ACID AND AMINO ACID SEQUENCES RELATING TO $i(HELICOBACTER PYLORI) AND VACCINE COMPOSITIONS THEREOF

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Publication number
EP0973394A1
EP0973394A1 EP97913847A EP97913847A EP0973394A1 EP 0973394 A1 EP0973394 A1 EP 0973394A1 EP 97913847 A EP97913847 A EP 97913847A EP 97913847 A EP97913847 A EP 97913847A EP 0973394 A1 EP0973394 A1 EP 0973394A1
Authority
EP
European Patent Office
Prior art keywords
seq
pylori
polypeptide
nucleic acid
fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97913847A
Other languages
German (de)
French (fr)
Other versions
EP0973394A4 (en
Inventor
Douglas Smith
Richard A. Alm
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AstraZeneca AB
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Astra AB
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Publication date
Application filed by Astra AB filed Critical Astra AB
Publication of EP0973394A1 publication Critical patent/EP0973394A1/en
Publication of EP0973394A4 publication Critical patent/EP0973394A4/en
Withdrawn legal-status Critical Current

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86Viral vectors
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04Antibacterial agents
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide

Definitions

  • H. pylori is a gram-negative, S-shaped, microaerophilic bacterium that was discovered and cultured from a human gastric biopsy specimen. (Warren, J.R. and B. Marshall, (1983) Lancet ⁇ : 1273-1275; and Marshall et al., (1984) Microbios Lett. 25: 83-88). H. pylori has been strongly linked to chronic gastritis and duodenal ulcer disease. (Rathbone et. al.. (1986) Gut 27_: 635-641).
  • H. pylori colonizes the human gastric mucosa, establishing an infection that usually persists for decades. Infection by H. pylori is prevalent worldwide. Developed countries have infection rates over 50% of the adult population, while developing countries have infection rates reaching 90% of the adults over the age of 20. (Hopkins R. J. and J. G. Morris (1994) Am. J. Med. 97: 265-277).
  • urease an enzyme that may play a role in neutralizing gastric acid pH
  • Ferrero, R.L. and A. Lee 1991 ) Microb. Ecol Hlth. Dis. 4: 121-134; Labigne et al., (1991 ) J. Bacterial. 173: 1920-1931
  • the bacterial flagellar proteins responsible for motility across the mucous layer (Hazell et al., (1986) J. Inf. Dis.
  • This invention relates to novel genes, e.g., genes encoding polypeptides such as bacterial surface proteins, from the organism Helicobacter pylori (H. pylori), and other related genes, their products, and uses thereof.
  • the nucleic acids and peptides of the present invention have utility for diagnostic and therapeutics for H. pylori and other Helicobacter species. They can also be used to detect the presence of//, pylori and other Helicobacter species in a sample; and for use in screening compounds for the ability to interfere with the H. pylori life cycle or to inhibit H. pylori infection. More specifically, this invention features compositions of nucleic acids corresponding to entire coding sequences of H.
  • pylori proteins including surface or secreted proteins or parts thereof, nucleic acids capable of binding mRNA from H. pylori proteins to block protein translation, and methods for producing H. pylori proteins or parts thereof using peptide synthesis and recombinant DNA techniques.
  • This invention also features antibodies and nucleic acids useful as probes to detect H. pylori infection.
  • vaccine compositions and methods for the protection or treatment of infection by //. pylori are within the scope of this invention.
  • Figure 1 is a bar graph that depicts the antibody titer in serum of mice following immunization with specific H. pylori antigens.
  • Figure 2 is a bar graph that depicts the antibody titer in mucous of mice following immunization with specific H. pylori antigens.
  • Figure 3 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in HEPES buffer.
  • Figure 4 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in buffer containing DOC. - :> -
  • Figure 5 depicts the amino acid sequence alignment in a portion of the sequence of five H. pylori proteins (depicted in the single letter amino acid code; shown N- terminal to C-terminal, left to right).
  • Figure 6 depicts the amino acid sequence alignment in a portion of the sequence of four H. pylori proteins (depicted in the single letter amino acid code; shown N- terminal to C-terminal, left to right).
  • Figure 7 depicts the amino acid sequence alignment in a portion of the sequence of two H. pylori proteins (depicted in the single letter amino acid code; shown N- terminal to C-terminal, left to right).
  • Figure 8 depicts the amino acid sequence alignment in a portion of the sequence of two H. pylori proteins (depicted in the single letter amino acid code; shown N- terminal to C-terminal, left to right).
  • the invention features a recombinant or substantially pure preparation of //, pylori polypeptide of SEQ ID NO: 74.
  • the invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide of SEQ ID NO: 74, such nucleic acid is contained in SEQ ID NO: 1.
  • the H. pylori polypeptide sequences of the invention described herein are contained in the Sequence Listing, and the nucleic acids encoding H. pylori polypeptides of the invention are contained in the Sequence Listing.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 75, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 2.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 76, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 3.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 77, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 4.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 78, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 5.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 79, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 6.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 80, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 7.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 81, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 8.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 82, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 9.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 83, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 10.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 84, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 11.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 85, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 12.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 86, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 13.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 87, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 14.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 88, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 15.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 89, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 16.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 90, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 17.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 91 , such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 18.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 92, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 19.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 93, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 20.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 94, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 21.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 95, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 22.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 96, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 23.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 97, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 24.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 98, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 25.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 99, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 26.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 100, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 27.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 101, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 28.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 102, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 29.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 103, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 30.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 104, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 31.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 105, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 32.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 106, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 33.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 107, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 34.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 108, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 35.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 109, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 36.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 110, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 37.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 11, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 38.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 12, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 39.
  • the invention features a substantially pure nucleic acid encoding an H pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 13, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 40.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 14, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 41.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 115, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 42.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 16, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:43.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 117, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 44.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 18, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 45.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 19, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 46.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 120, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 47.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 121, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 48.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 122, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 49.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 123, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 50.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 124, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 51.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 125, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 52.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 126, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 53.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 127, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 54.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 128, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 55.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 129, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 56.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 130, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 57.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 131, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 58.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 132, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 59.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 133, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 60.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 134, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 61.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 135, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 62.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 136, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 63.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 137, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 64.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 138, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 65.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 139, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 66.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 140, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 67.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 141 , such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 68.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 142, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 69.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 143, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 70.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 144, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 71.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 145, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 72.
  • the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 146, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 73.
  • nucleic acid comprising a nucleotide sequence encoding an H. pylori cell envelope polypeptide or a fragment thereof.
  • nucleic acid is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 48, SEQ ID NO: 16, SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 7, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 1 1 , SEQ ID NO: 71, SEQ ID NO: 17, SEQ ID NO: 57, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 25, and SEQ ID NO: 48.
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H.
  • the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C- terminal tyrosine cluster or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 42, SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 1 1, and SEQ ID NO:71.
  • the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 45, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 7, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58.
  • nucleic acid comprising a nucleotide sequence encoding an H. pylori secreted polypeptide or a fragment thereof.
  • nucleic acid is selected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 32, SEQ ID NO: 51, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40.
  • SEQ ID NO: 41 SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68.
  • nucleic acid comprising a nucleotide sequence encoding an H. pylori cellular polypeptide or a fragment thereof.
  • nucleic acid is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 73.
  • H. pylori cell envelope polypeptide or a fragment thereof wherein the polypeptide is selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 98, SEQ ID NO: 121, SEQ ID NO: 89, SEQ ID NO: 83, SEQ ID NO: 1 18, SEQ ID NO: 108, SEQ ID NO: 1 10, SEQ ID NO: 80, SEQ ID NO: 1 12, SEQ ID NO: 128, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 74, SEQ ID NO: 1 15, SEQ ID NO: 87, SEQ ID NO: 1 16, SEQ ID NO: 84, SEQ ID NO: 144, SEQ ID NO: 90, SEQ ID NO: 130, SEQ ID NO: 78
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 98, and SEQ ID NO: 121.
  • the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 83, SEQ ID NO: 1 18, SEQ ID NO: 108, SEQ ID NO: 1 10, SEQ ID NO: 80, SEQ ID NO: 1 12, SEQ ID NO: 128, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131 , SEQ ID NO: 74.
  • the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C- terminal tyrosine cluster or a fragment thereof selected from the group consisting of SEQ ID NO: 74, SEQ ID NO: 1 15, SEQ ID NO: 87, SEQ ID NO: 1 16. SEQ ID NO: 84, and SEQ ID NO: 144.
  • the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof selected from the group consisting of SEQ ID NO: 89.
  • H. pylori secreted polypeptide or a fragment thereof wherein the polypeptide is selected from the group consisting of SEQ ID NO: 145, SEQ ID NO: 105, SEQ ID NO: 124, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 95, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 1 1 1, SEQ ID NO: 113, SEQ ID NO: 1 14, SEQ ID NO: 1 17, SEQ ID NO: 1 19, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 145, SEQ ID NO
  • H. pylori cellular polypeptide or a fragment thereof wherein the polypeptide is selected from the group consisting of SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 93, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 100.
  • the invention pertains to any individual H. pylori polypeptide member or nucleic acid encoding such a member from the above-identified groups of//. pylori polypeptides.
  • the invention features nucleic acids capable of binding mRNA of //, pylori. Such nucleic acid is capable of acting as antisense nucleic acid to control the translation of mRNA of H. pylori.
  • a further aspect features a nucleic acid which is capable of binding specifically to an H pylori nucleic acid. These nucleic acids are also referred to herein as complements and have utility as probes and as capture reagents.
  • the invention features an expression system comprising an open reading frame corresponding to H. pylori nucleic acid.
  • the nucleic acid further comprises a control sequence compatible with an intended host.
  • the expression system is useful for making polypeptides corresponding to H pylori nucleic acid.
  • the invention features a cell transformed with the expression system to produce H. pylori polypeptides.
  • the invention features a method of generating antibodies against H. pylori polypeptides which are capable of binding specifically to H pylori polypeptides.
  • Such antibodies have utility as reagents for immunoassays to evaluate the abundance and distribution of , antigens.
  • the invention features a method of generating vaccines for immunizing an individual against H pylori.
  • the vaccination method includes: immunizing a subject with at least one H pylori polypeptide according to the present invention, e.g., a surface or secreted polypeptide, or active portion thereof, and a pharmaceutically acceptable carrier.
  • H pylori polypeptide e.g., a surface or secreted polypeptide, or active portion thereof, and a pharmaceutically acceptable carrier.
  • Such vaccines have therapeutic and/or prophylactic utilities.
  • the invention provides a method for generating a vaccine comprising a modified immunogenic H. pylori polypeptide, e.g., a surface or secreted polypeptide, or active portion thereof, and a pharmacologically acceptable carrier.
  • the invention features a method of evaluating a compound, e.g. a polypeptide, e.g., a fragment of a host cell polypeptide, for the ability to bind an H. pylori polypeptide. The method includes: contacting the candidate compound with an H pylori polypeptide and determining if the compound binds or otherwise interacts with an H. pylori polypeptide. Compounds which bind H.
  • the invention features a method of evaluating a compound, e.g. a polypeptide, e.g., a fragment of a host cell polypeptide, for the ability to bind an H. pylori nucleic acid, e.g., DNA or RNA.
  • the method includes: contacting the candidate compound with an H pylori nucleic acid and determining if the compound binds or otherwise interacts with an H. pylori polypeptide.
  • Compounds which bind H pylori are candidates as activators or inhibitors of the bacterial life cycle. These assays can be performed in vitro or in vivo.
  • the invention features H. pylori polypeptides, preferably a substantially pure preparation of an H. pylori polypeptide, or a recombinant H. pylori polypeptide.
  • the polypeptide has biological activity; the polypeptide has an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical or homologous to an amino acid sequence of the invention contained in the Sequence Listing, preferably it has about 65% sequence identity with an amino acid sequence of the invention contained in the Sequence Listing, and most preferably it has about 92% to about 99% sequence identity with an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acid residues in length; the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino
  • the amino acid sequence which differs in sequence identity by about 7% to about 8% from the H. pylori amino acid sequences of the invention contained in the Sequence Listing is also encompassed by the invention.
  • the H pylori polypeptide is encoded by a nucleic acid of the invention contained in the Sequence Listing, or by a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a nucleic acid of the invention contained in the Sequence Listing.
  • the subject H. pylori polypeptide differs in amino acid sequence at 1 , 2, 3, 5, 10 or more residues from a sequence of the invention contained in the Sequence Listing. The differences, however, are such that the H. pylori polypeptide exhibits an H. pylori biological activity, e.g., the H. pylori polypeptide retains a biological activity of a naturally occurring H. pylori polypeptide.
  • the polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' or 3' to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.
  • the H. pylori polypeptide is a recombinant fusion protein having a first H. pylori polypeptide portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to
  • the second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain.
  • the fusion protein can be used in a two-hybrid assay.
  • Polypeptides of the invention include those which arise as a result of alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events.
  • the invention also encompasses an immunogenic component which includes at least one H. pylori polypeptide in an immunogenic preparation; the immunogenic component being capable of eliciting an immune response specific for the H. pylori polypeptide, e.g., a humoral response, an antibody response, or a cellular response.
  • the immunogenic component comprises at least one antigenic determinant from a polypeptide of the invention contained in the Sequence Listing.
  • the invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an H. pylori polypeptide.
  • the encoded polypeptide has biological activity; the encoded polypeptide has an amino acid sequence at least 60%), 70%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence of the invention contained in the Sequence
  • the encoded polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the Sequence Listing; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded polypeptide comprises at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids of the invention contained in the Sequence Listing.
  • the nucleic acid of the invention is that contained in the Sequence Listing; the nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or
  • the encoded H. pylori polypeptide differs (e.g., by amino acid substitution, addition or deletion of at least one amino acid residue) in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence of the invention contained in the Sequence Listing.
  • the differences are such that: the H. pylori encoded polypeptide exhibits a H. pylori biological activity, e.g., the encoded H. pylori enzyme retains a biological activity of a naturally occurring H. pylori.
  • the encoded polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' or 3' to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.
  • the subject H. pylori nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the H. pylori gene sequence, e.g., to render the H. pylori gene sequence suitable for expression in a recombinant host cell.
  • a transcriptional regulatory sequence e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence
  • operably linked to the H. pylori gene sequence e.g., to render the H. pylori gene sequence suitable for expression in a recombinant host cell.
  • the nucleic acid which encodes an H. pylori polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 8 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 12 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 20 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 40 consecutive nucleotides of the invention contained in the Sequence Listing.
  • the nucleic acid encodes a peptide which differs by at least one amino acid residue from the sequences of the invention contained in the Sequence Listing.
  • the nucleic acid differs by at least one nucleotide from a nucleotide sequence of the invention contained in the Sequence Listing which encodes amino acids of the invention contained in the Sequence Listing.
  • the invention encompasses: a vector including a nucleic acid which encodes an H. pylori polypeptide or an H pylori polypeptide variant as described herein; a host cell transfected with the vector; and a method of producing a recombinant H. pylori polypeptide or H. pylori polypeptide variant; including culturing the cell, e.g., in a cell culture medium, and isolating the H. pylori or H. pylori polypeptide variant, e.g., from the cell or from the cell culture medium.
  • the invention features, a purified recombinant nucleic acid having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a sequence of the invention contained in the Sequence Listing.
  • the invention also provides a probe or primer which includes a substantially purified oligonucleotide.
  • the oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 8 consecutive nucleotides of sense or antisense sequence of the invention contained in the Sequence Listing, or naturally occurring mutants thereof.
  • the probe or primer further includes a label group attached thereto.
  • the label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor.
  • the oligonucleotide is at least 8 and less than 10, 20, 30, 50, 100, or 150 nucleotides in length.
  • the invention also provides an isolated H. pylori polypeptide which is encoded by a nucleic acid which hybridizes under stringent hybridization conditions to a nucleic acid contained in the Sequence Listing.
  • the invention further provides nucleic acids, e.g., RNA or DNA, encoding a polypeptide of the invention.
  • nucleic acids e.g., RNA or DNA
  • the H. pylori strain from which genomic sequences have been sequenced, has been deposited in the American Type Culture Collection (ATCC # 55679; deposited by Genome Therapeutics Corporation, 100 Beaver Street, Waltham, MA 02154) as strain HP-J99.
  • allelic variations include allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid which encodes a polypeptide of the invention contained in the Sequence Listing (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6 and 6.4.1-6.4.10, hereby incorporated by reference); and, polypeptides specifically bound by antisera to H. pylori polypeptides, especially by antisera to an active site or binding domain of//. pylori polypeptide.
  • the invention also includes fragments, preferably biologically active fragments. These and other polypeptides are also referred to herein as H. pylori polypeptide analogs or variants.
  • H. pylori polypeptides characterized as shown in Table 1 below, including: H. pylori cell envelope proteins, H. pylori secreted proteins, and H. pylori cellular proteins. Members of these groups were identified by BLAST homology searches and by searches for secretion signal or transmembrane protein motifs. Polypeptides related by significant homology to the polypeptides of Table 1 are also considered to be classified in the manner of the homologs shown in Table 1. TABLE 1
  • nt represents nucleotide Seq. ID number and “aa” represents amino acid Seq. ID number]
  • purified polypeptide and “isolated polypeptide” and “a substantially pure preparation of a polypeptide” are used interchangeably herein and. as used herein, from other proteins, lipids, and nucleic acids with which it naturally occurs.
  • the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it.
  • the polypeptide constitutes at least 10, 20, 50 70, 80 or 95%> dry weight of the purified preparation.
  • the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 ⁇ g of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
  • purified polypeptide and isolated polypeptide and “a substantially pure preparation of a polypeptide,” as used herein, refer to both a polypeptide obtained from nature or produced by recombinant DNA techniques as described herein.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the H pylori protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of//, pylori protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of H. pylori protein having less than about 30%) (by dry weight) of non-H. pylori protein (also referred to herein as a "contaminating protein"), more preferably less than about 20%) of non-H. pylori protein, still more preferably less than about 10% of non-H. pylori protein, and most preferably less than about 5%> non-H. pylori protein.
  • H pylori protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%>, and most preferably less than about 5%> of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of H pylori protein in which the protein is separated from chemical precusors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of H pylori protein having less than about 30%> (by dry weight) of chemical precursors or non-H. pylori chemicals, more preferably less than about 20%) chemical precursors or non-H. pylori chemicals, still more preferably less than about 10%> chemical precursors or non-H. pylori chemicals, and most preferably less than about 5% chemical precursors or non-H pylori chemicals.
  • a purified preparation of cells refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10%> and more preferably 50%) of the subject cells.
  • a purified or isolated or a substantially pure nucleic acid is a nucleic acid which is one or both of the following: not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid with which it occurs in the organism from which the nucleic acid is derived.
  • the term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences.
  • Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional H. pylori DNA sequence.
  • a "contig” as used herein is a nucleic acid representing a continuous stretch of genomic sequence of an organism.
  • ORF an "open reading frame”, also referred to herein as ORF, is a region of nucleic acid which encodes a polypeptide. This region may represent a portion of a coding sequence or a total sequence and can be determined from a stop to stop codon or from a start to stop codon.
  • a "coding sequence” is a nucleic acid which is transcribed into messenger RNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the five prime terminus and a translation stop code at the three prime terminus.
  • a coding sequence can include but is not limited to messenger RNA, synthetic DNA, and recombinant nucleic acid sequences.
  • a "complement" of a nucleic acid as used herein referes to an anti-parallel or antisense sequence that participates in Watson-Crick base-pairing with the original sequence.
  • a “gene product” is a protein or structural RNA which is specifically encoded by a gene.
  • probe refers to a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest. Probes are often associated with or capable of associating with a label.
  • a label is a chemical moiety capable of detection. Typical labels comprise dyes, radioisotopes, luminescent and chemiluminescent moieties, fluorophores, enzymes, precipitating agents, amplification sequences, and the like.
  • a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest and immobilizes such molecule is referred herein as a "capture ligand".
  • Capture ligands are typically associated with or capable of associating with a support such as nitro-cellulose, glass, nylon membranes, beads, particles and the like.
  • the specificity of hybridization is dependent on conditions such as the base pair composition of the nucleotides, and the temperature and salt concentration of the reaction. These conditions are readily discernable to one of ordinary skill in the art using routine experimentation.
  • Homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60%) homologous.
  • the DNA sequences ATTGCC and TATGGC share 50%> homology.
  • a comparison is made when two sequences are aligned to give maximum homology.
  • Nucleic acids are hybridizable to each other when at least one strand of a nucleic acid can anneal to the other nucleic acid under defined stringency conditions. Stringency of hybridization is determined by: (a) the temperature at which hybridization and/or washing is performed; and (b) the ionic strength and polarity of the hybridization and washing solutions. Hybridization requires that the two nucleic acids contain complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated.
  • hybridization of two sequences at high stingency requires that the sequences be essentially completely homologous.
  • Conditions of intermediate stringency such as, for example, 2X SSC at 65 ° C
  • low stringency such as, for example 2X SSC at 55° C
  • IX SSC is 0.15 M NaCl, 0.015 M Na citrate.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
  • SSC sodium chloride/sodium citrate
  • peptides, proteins, and polypeptides are used interchangeably herein.
  • surface protein refers to all surface accessible proteins, e.g. inner and outer membrane proteins, proteins adhering to the cell wall, and secreted proteins.
  • a polypeptide has H pylori biological activity if it has one, two and preferably more of the following properties: (1) if when expressed in the course of an H pylori infection, it can promote, or mediate the attachment of H pylori to a cell; (2) it has an enzymatic activity, structural or regulatory function characteristic of an H pylori protein; (3) the gene which encodes it can rescue a lethal mutation in an H. pylori gene; (4) or it is immunogenic in a subject.
  • a polypeptide has biological activity if it is an antagonist, agonist, or super-agonist of a polypeptide having one of the above-listed properties.
  • a biologically active fragment or analog is one having an in vivo or in vitro activity which is characteristic of the H pylori polypeptides of the invention contained in the Sequence Listing, or of other naturally occurring H. pylori polypeptides, e.g., one or more of the biological activities described herein.
  • fragments which exist in vivo e.g., fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's. Fragments include those expressed in native or endogenous cells as well as those made in expression systems, e.g., in C ⁇ O cells. Because peptides such as H.
  • H pylori polypeptides often exhibit a range of physiological properties and because such properties may be attributable to different portions of the molecule, a useful H pylori fragment or H pylori analog is one which exhibits a biological activity in any biological assay for H pylori activity. Most preferably the fragment or analog possesses 10%>, preferably 40%>, more preferably 60%>, 70%), 80%) or 90%) or greater of the activity of H. pylori, in any in vivo or in vitro assay. Analogs can differ from naturally occurring H. pylori polypeptides in amino acid sequence or in ways that do not involve sequence, or both.
  • Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation.
  • Preferred analogs include H. pylori polypeptides (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not substantially diminish the biological activity of the H pylori polypeptide.
  • Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • Other conservative substitutions can be made in view of the table below.
  • analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., ⁇ or ⁇ amino acids; and cyclic analogs.
  • fragment as applied to an H pylori analog, will ordinarily be at least about 20 residues, more typically at least about 40 residues, preferably at least about 60 residues in length. Fragments of H pylori polypeptides can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of H pylori polypeptide can be assessed by methods known to those skilled in the art as described herein. Also included are H. pylori polypeptides containing residues that are not required for biological activity of the peptide or that result from alternative mRNA splicing or alternative protein processing events.
  • an "immunogenic component” as used herein is a moiety, such as an H pylori polypeptide, analog or fragment thereof, that is capable of eliciting a humoral and/or cellular immune response in a host animal alone or in combination with an adjuvant.
  • an "antigenic component” as used herein is a moiety, such as an H. pylori polypeptide, analog or fragment thereof, that is capable of binding to a specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex.
  • transgene means a nucleic acid (encoding, e.g., one or more polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the cell's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
  • transgenic cell refers to a cell containing a transgene.
  • a "transgenic animal” is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene.
  • the transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by a process of transformation of competent cells or by microinjection or by infection with a recombinant virus.
  • This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
  • the term "antibody” as used herein is intended to include fragments thereof which are specifically reactive with H pylori polypeptides.
  • the term "cell-specific promoter” means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue.
  • the term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
  • Misexpression refers to a non-wild type pattern of gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post- transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
  • host cells and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refers to cells which can become or have been used as recipients for a recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood by individuals skilled in the art that the progeny of a single parental cell may not necessarily be completely identical in genomic or total DNA compliment to the original parent, due to accident or deliberate mutation.
  • control sequence refers to a nucleic acid having a base sequence which is recognized by the host organism to effect the expression of encoded sequences to which they are ligated.
  • the nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include a promoter, ribosomal binding site, terminators, and in some cases operators; in eukaryotes, generally such control sequences include promoters, terminators and in some instances, enhancers.
  • the term control sequence is intended to include at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences.
  • operably linked refers to sequences joined or ligated to function in their intended manner.
  • a control sequence is operably linked to coding sequence by ligation in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence and host cell.
  • the metabolism of a substance means any aspect of the, expression, function, action, or regulation of the substance.
  • the metabolism of a substance includes modifications, e.g., covalent or non-covalent modifications of the substance.
  • the metabolism of a substance includes modifications, e.g., covalent or non- covalent modification, the substance induces in other substances.
  • the metabolism of a substance also includes changes in the distribution of the substance.
  • the metabolism of a substance includes changes the substance induces in the distribution of other substances.
  • sample refers to a biological sample, such as, for example, tissue or fluid isloated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment.
  • tissue or fluid isloated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment.
  • tissue or fluid isloated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment.
  • the practice of the invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Sambrook, Fritsch, and Maniatis,
  • This invention provides nucleotide sequences of the genome of H. pylori which thus comprises a DNA sequence library of H. pylori genomic DNA.
  • the detailed description that follows provides nucleotide sequences of H. pylori, and also describes how the sequences were obtained and how ORFs and protein-coding sequences were identified. Also described are methods of using the disclosed H. pylori sequences in methods including diagnostic and therapeutic applications.
  • the library can be used as a database for identification and comparison of medically important sequences in this and other strains of H. pylori. To determine the genomic sequence of H. pylori.
  • DNA was isolated from a strain of H pylori (ATCC # 55679; deposited by Genome Therapeutics Corporation, 100 Beaver Street, Waltham, MA 02154) and mechanically sheared by nebulization to a median size of 2 kb. Following size fractionation by gel electrophoresis, the fragments were blunt-ended, ligated to adapter oligonucleotides, and cloned into each of 20 different pMPX vectors (Rice et al., abstracts of Meeting of Genome Mapping and
  • DNA sequencing was achieved using multiplex sequencing procedures essentially as disclosed in Church et al., 1988, Science 240:185; U.S. Patents No. 4,942,124 and 5,149,625).
  • DNA was extracted from pooled cultures and subjected to chemical or enzymatic sequencing. Sequencing reactions were resolved by electrophoresis, and the products were transferred and covalently bound to nylon membranes. Finally, the membranes were sequentially hybridized with a series of labelled oligonucleotides complimentary to "tag" sequences present in the different shotgun cloning vectors. In this manner, a large number of sequences could be obtained from a single set of sequencing reactions. The cloning and sequencing procedures are described in more detail in the Exemplification.
  • Synthetic oligonucleotides are designed that are complementary to sequences at the end of each contig. These oligonucleotides may be hybridized to libaries of H. pylori genomic DNA in, for example, lambda phage vectors or plasmid vectors to identify clones that contain sequences corresponding to the junctional regions between individual contigs. Such clones are then used to isolate template DNA and the same oligonucleotides are used as primers in polymerase chain reaction (PCR) to amplify junctional fragments, the nucleotide sequence of which is then determined.
  • PCR polymerase chain reaction
  • ORFs open reading frames
  • ORFs open reading frames
  • These ORFs may not correspond to the ORF of a naturally-occurring H. pylori polypeptide.
  • These ORFs may contain start codons which indicate the initiation of protein synthesis of a naturally- occurring H. pylori polypeptide.
  • Such start codons within the ORFs provided herein can be identified by those of ordinary skill in the relevant art, and the resulting ORF and the encoded H. pylori polypeptide is within the scope of this invention.
  • a codon such as AUG or GUG encoding methionine or valine
  • the ORF modified to correspond to a naturally-occurring H pylori polypeptide.
  • the predicted coding regions were defined by evaluating the coding potential of such sequences with the program GENEMARKTM (Borodovsky and Mclninch, 1993, Comp. Chem. J :123).
  • the nucleic acids of this invention may be obtained directly from the DNA of the above referenced H. pylori strain by using the polymerase chain reaction (PCR). See “PCR, A Practical Approach” (McPherson, Quirke, and Taylor, eds., IRL Press, Oxford, UK, 1991) for details about the PCR. High fidelity PCR can be used to ensure a faithful DNA copy prior to expression. In addition, the authenticity of amplified products can be checked by conventional sequencing methods.
  • PCR polymerase chain reaction
  • Clones carrying the desired sequences described in this invention may also be obtained by screening the libraries by means of the PCR or by hybridization of synthetic oligonucleotide probes to filter lifts of the library colonies or plaques as known in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd edition, 1989, Cold Spring Harbor Press, NY).
  • nucleic acids encoding H. pylori polypeptides from a cDNA library in accordance with protocols herein described.
  • a cDNA encoding an H. pylori polypeptide can be obtained by isolating total mRNA from an appropriate strain. Double stranded cDNAs can then be prepared from the total mRNA. Subsequently, the cDNAs can be inserted into a suitable plasmid or viral (e.g., bacteriophage) vector using any one of a number of known techniques.
  • nucleic acids of the invention can be DNA or RNA.
  • Preferred nucleic acids of the invention are contained in the Sequence Listing.
  • the nucleic acids of the invention can also be chemically synthesized using standard techniques.
  • Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
  • nucleic acids isolated or synthesized in accordance with features of the present invention are useful, by way of example, without limitation, as probes, primers, capture ligands. antisense genes and for developing expression systems for the synthesis of proteins and peptides corresponding to such sequences.
  • the nucleic acid normally consists of all or part (approximately twenty or more nucleotides for specificity as well as the ability to form stable hybridization products) of the nucleic acids of the invention contained in the Sequence Listing. These uses are described in further detail below.
  • Probes A nucleic acid isolated or synthesized in accordance with the sequence of the invention contained in the Sequence Listing can be used as a probe to specifically detect H pylori. With the sequence information set forth in the present application, sequences of twenty or more nucleotides are identified which provide the desired inclusivity and exclusivity with respect to H pylori, and extraneous nucleic acids likely to be encountered during hybridization conditions. More preferably, the sequence will comprise at least twenty to thirty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules.
  • nucleic acids for use as probes, can be provided with a label to facilitate detection of a hybridization product.
  • nucleic acid selected in the manner described above with respect to probes can be readily associated with a support.
  • the manner in which nucleic acid is associated with supports is well known.
  • Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing have utility to separate H. pylori nucleic acid from the nucleic acid of each other and other organisms.
  • Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing can also have utility to separate other Helicobacter species from each other and from other organisms.
  • the sequence will comprise at least twenty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules. Sequences larger than 1000 nucleotides in length are difficult to synthesize but can be generated by recombinant DNA techniques.
  • Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility as primers for the amplification of H pylori nucleic acid. These nucleic acids may also have utility as primers for the amplification of nucleic acids in other Helicobacter species.
  • PCR polymerase chain reaction
  • nucleic acid sequences of > 10-15 nucleotides of the invention contained in the Sequence Listing have utility in conjunction with suitable enzymes and reagents to create copies of H. pylori nucleic acid. More preferably, the sequence will comprise twenty or more nucleotides to convey stability to the hybridization product formed between the primer and the intended target molecules. Binding conditions of primers greater than 100 nucleotides are more difficult to control to obtain specificity. High fidelity PCR can be used to ensure a faithful DNA copy prior to expression. In addition, amplified products can be checked by conventional sequencing methods.
  • the copies can be used in diagnostic assays to detect specific sequences, including genes from H. pylori and/or other Helicobacter species.
  • the copies can also be incorporated into cloning and expression vectors to generate polypeptides corresponding to the nucleic acid synthesized by PCR, as is described in greater detail herein.
  • Antisense Nucleic acid or nucleic acid-hybridizing derivatives isolated or synthesized in accordance with the sequences described herein have utility as antisense agents to prevent the expression of H. pylori genes. These sequences also have utility as antisense agents to prevent expression of genes of other Helicobacter species.
  • nucleic acid or derivatives corresponding to H. pylori nucleic acids is loaded into a suitable carrier such as a liposome or bacteriophage for introduction into bacterial cells.
  • a nucleic acid having twenty or more nucleotides is capable of binding to bacteria nucleic acid or bacteria messenger RNA.
  • the antisense nucleic acid is comprised of 20 or more nucleotides to provide necessary stability of a hybridization product of non-naturally occurring nucleic acid and bacterial nucleic acid and/or bacterial messenger RNA.
  • Nucleic acid having a sequence greater than 1000 nucleotides in length is difficult to synthesize but can be generated by recombinant DNA techniques.
  • H pylori Nucleic Acids Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility to generate polypeptides.
  • the nucleic acid of the invention exemplified in the Sequence Listing or fragments of said nucleic acid encoding active portions of H pylori polypeptides can be cloned into suitable vectors or used to isolate nucleic acid.
  • the isolated nucleic acid is combined with suitable DNA linkers and cloned into a suitable vector.
  • a gene product may be produced in large quantities in an expressing strain for use as an antigen, an industrial reagent, for structural studies, etc. This expression can be accomplished in a mutant strain which lacks the activity of the gene to be tested, or in a strain that does not produce the same gene product(s). This includes, but is not limited to other Helicobacter strains, or other bacterial strains such as E. coli, Norcardia, Corynebacterium, Campylobacter, and Streptomyces species.
  • the expression host will utilize the natural Helicobacter promoter whereas in others, it will be necessary to drive the gene with a promoter sequence derived from the expressing organism (e.g., an E. coli beta-galactosidase promoter for expression in E. coli).
  • a promoter sequence derived from the expressing organism e.g., an E. coli beta-galactosidase promoter for expression in E. coli.
  • telomere sequence data is cloned into an appropriate recombinant plasmid containing an origin of replication that functions in the host organism and an appropriate selectable marker. This can be accomplished by a number of procedures known to those skilled in the art. It is most preferably done by cutting the plasmid and the fragment to be cloned with the same restriction enzyme to produce compatible ends that can be ligated to join the two pieces together.
  • the recombinant plasmid is introduced into the host organism by, for example, electroporation and cells containing the recombinant plasmid are identified by selection for the marker on the plasmid. Expression of the desired gene product is detected using an assay specific for that gene product.
  • a suitable host cell for expression of a gene can be any procaryotic or eucaryotic cell.
  • an H pylori polypeptide can be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cell (C ⁇ O).
  • Other suitable host cells are known to those skilled in the art.
  • yeast S. cerivisae examples include pYepSecl (Baldari. et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and ⁇ erskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:1 13-123), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., (1983) Mol Cell Biol 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39).
  • COS cells Gluzman, Y., (1981) Cell 23: 175-182
  • pCDM 8 Aruffo, A. and Seed, B., (1987) Proc. Natl Acad. Sci. USA 84:8573-8577
  • C ⁇ O dhfr Chinese
  • Hamster Ovary cells are used with vectors such as pMT2PC (Kaufman et al. (1987), EMBO J. 6: 187- 195) for stable amplification/expression in mammalian cells.
  • Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation. DEAE-dextran-mediated transfection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.
  • Fusion vectors usually add a number of NH2 terminal amino acids to the expressed target gene. These NH2 terminal amino acids often are referred to as a reporter group. Such reporter groups usually serve two purposes: 1) to increase the solubility of the target recombinant protein; and 2) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the reporter group and the target recombinant protein to enable separation of the target recombinant protein from the reporter group subsequent to purification of the fusion protein.
  • Such enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly. MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • a preferred reporter group is poly(His), which may be fused to the amino or carboxy terminus of the protein and which renders the recombinant fusion protein easily purifiable by metal chelate chromatography.
  • Inducible non-fusion expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pETl Id (Studier et al.. Gene Expression Technology: Methods in Enzvmology 185. Academic Press, San Diego, California ( 1990) 60-89). While target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc, expression of target genes inserted into pETl Id relies on transcription from the T7 gnlO-lac 0 fusion promoter mediated by coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl under the transcriptional control of the lacUV 5 promoter.
  • T7 gnl coexpressed viral RNA polymerase
  • a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding an H. pylori polypeptide can be cultured under appropriate conditions to allow expression of the polypeptide to occur.
  • the polypeptide may be secreted and isolated from a mixture of cells and medium containing the peptide.
  • the polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
  • a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
  • Polypeptides of the invention can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such polypeptides. Additionally, in many situations, polypeptides can be produced by chemical cleavage of a native protein (e.g., tryptic digestion) and the cleavage products can then be purified by standard techniques.
  • membrane bound proteins these can be isolated from a host cell by contacting a membrane-associated protein fraction with a detergent forming a solubilized complex, where the membrane-associated protein is no longer entirely embedded in the membrane fraction and is solubilized at least to an extent which allows it to be chromatographically isolated from the membrane fraction.
  • a detergent suitable for solubilizing these complexes For example, one property considered is the ability of the detergent to solubilize the H. pylori protein within the membrane fraction at minimal denaturation of the membrane- associated protein allowing for the activity or functionality of the membrane-associated protein to return upon reconstitution of the protein.
  • CMC critical micelle concentration
  • a third property considered when selecting a detergent is the hydrophobicity of the detergent.
  • membrane-associated proteins are very hydrophobic and therefore detergents which are also hydrophobic, e.g., the triton series, would be useful for solubilizing the hydrophobic proteins.
  • Another property important to a detergent can be the capability of the detergent to remove the H pylori protein with minimal protein-protein interaction facilitating further purification.
  • a fifth property of the detergent which should be considered is the charge of the detergent.
  • detergent should be an uncharged detergent.
  • Chromatographic techniques which can be used in the final purification step are known in the art and include hydrophobic interaction, lectin affinity, ion exchange, dye affinity and immunoaffinity.
  • One strategy to maximize recombinant H pylori peptide expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzvmology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy would be to alter the nucleic acid encoding an H pylori peptide to be inserted into an expression vector so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada et al., (1992) Nuc. Acids Res. 20:21 1 1-2118).
  • Such alteration of nucleic acids of the invention can be carried out by standard DNA synthesis techniques.
  • the nucleic acids of the invention can also be chemically synthesized using standard techniques.
  • Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See, e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
  • This invention encompasses isolated H pylori polypeptides encoded by the disclosed H. pylori genomic sequences, including the polypeptides of the invention contained in the Sequence Listing. Polypeptides of the invention are preferably at least 5 amino acid residues in length. Using the DNA sequence information provided herein, the amino acid sequences of the polypeptides encompassed by the invention can be deduced using methods well-known in the art. It will be understood that the sequence of an entire nucleic acid encoding an H pylori polypeptide can be isolated and identified based on an ORF that encodes only a fragment of the cognate protein-coding region.
  • polypeptides of the invention can be isolated from wild-type or mutant H pylori cells or from heterologous organisms or cells (including, but not limited to, bacteria, yeast, insect, plant and mammalian cells) into which an H. pylori nucleic acid has been introduced and expressed.
  • the polypeptides can be part of recombinant fusion proteins.
  • H pylori polypeptides of the invention can be chemically synthesized using commercially automated procedures such as those referenced herein.
  • the disclosed H. pylori genome sequence includes segments that direct the synthesis of ribonucleic acids and polypeptides, as well as origins of replication, promoters, other types of regulatory sequences, and intergenic nucleic acids.
  • the invention encompasses nucleic acids encoding immunogenic components of vaccines and targets for agents effective against H pylori. Identification of said immunogenic components involved in the determination of the function of the disclosed sequences can be achieved using a variety of approaches. Non-limiting examples of these approaches are described briefly below.
  • H. pylori nucleic acid and polypeptide sequences Computer-assisted comparison of the disclosed H. pylori sequences with previously reported sequences present in publicly available databases is useful for identifying functional H. pylori nucleic acid and polypeptide sequences.
  • protein-coding sequences may be compared as a whole, and that a high degree of sequence homology between two proteins (such as, for example, >80-90%>) at the amino acid level indicates that the two proteins also possess some degree of functional homology, such as, for example, among enzymes involved in metabolism, DNA synthesis, or cell wall synthesis, and proteins involved in transport, cell division, etc.
  • H pylori proteins identified as containing putative signal sequences and/or transmembrane domains are useful as immunogenic components of vaccines.
  • H pylori genes Nucleic acids that encode proteins essential for growth or viability of H pylori are preferred drug targets. H pylori genes can be tested for their biological relevance to the organism by examining the effect of deleting and/or disrupting the genes, i.e., by so-called gene "knockout", using techniques known to those skilled in the relevant art. In this manner, essential genes may be identified. Strain-specific sequences: Because of the evolutionary relationship between different H pylori strains, it is believed that the presently disclosed H pylori sequences are useful for identifying, and/or discriminating between, previously known and new H pylori strains. It is believed that other H.
  • pylori strains will exhibit at least 70% sequence homology with the presently disclosed sequence.
  • Systematic and routine analyses of DNA sequences derived from samples containing H pylori strains, and comparison with the present sequence allows for the identification of sequences that can be used to discriminate between strains, as well as those that are common to all H pylori strains.
  • the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that discriminate between different strains of H pylori.
  • Strain-specific components can also be identified functionally by their ability to elicit or react with antibodies that selectively recognize one or more H pylori strains.
  • the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that are common to all H. pylori strains but are not found in other bacterial species.
  • the selection of candidate protein antigens for vaccine development can be derived from the nucleic acids encoding H. pylori polypeptides.
  • the ORF's can be analyzed for homology to other known exported or membrane proteins and analyzed using the discriminant analysis described by Klein, et al. (Klein, P., Kanehsia, M., and DeLisi, C. (1985) Biochimica et Biophysica Acta 815, 468-476) for predicting exported and membrane proteins.
  • Homology searches can be performed using the BLAST algorithm contained in the Wisconsin Sequence Analysis Package (Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1 ) to compare each predicted ORF amino acid sequence with all sequences found in the current GenBank, SWISS-PROT and PIR databases.
  • BLAST searches for local alignments between the ORF and the databank sequences and reports a probability score which indicates the probability of finding this sequence by chance in the database.
  • ORF's with significant homology e.g. probabilities lower than lxlO" 6 that the homology is only due to random chance
  • membrane or exported proteins represent protein antigens for vaccine development. Possible functions can be provided to H. pylori genes based on sequence homology to genes cloned in other organisms.
  • ORF amino acid sequence and compares it to information derived from the properties of known membrane and exported proteins. This comparison predicts which proteins will be exported, membrane associated or cytoplasmic. ORF amino acid sequences identified as exported or membrane associated by this algorithm are likely protein antigens for vaccine development.
  • outer membrane proteins are likely to represent the best antigens to provide a protective immune response against H. pylori.
  • algorithms that can be used to aid in prediction of these outer membrane proteins include the presence of an amphipathic beta-sheet region at their C-terminus. This region which has been detected in a large number of outer membrane proteins in Gram negative bacteria is often characterized by hydrophobic residues (Phe or Tyr) clustered at alternating positions from the C-terminus (e.g., see Figure 5, block F; Figure 7, block E). Importantly, these sequences have not been detected at the C-termini of periplasmic proteins, thus allowing preliminary distinction between these classes of proteins based on primary sequence data. This phenomenon has been reported previously by Struyve et al. (J. Mol Biol. 218: 141-148. 1991).
  • FIG. 5 Also illustrated in Figure 5 are additional amino acid sequence motifs found in many outer membrane proteins of H. pylori.
  • the amino acid sequence alignment in Figure 5 depicts portions of the sequence of five H. pylori proteins (depicted in the single letter amino acid code) labeled with their amino acid Sequence ID Numbers and shown N-terminal to C-terminal, left to right.
  • Five or six distinct blocks (labeled A through E or F) of similar amino acid residues are found including the distinctive hydrophobic residues (Phe or Tyr; F or Y according to the single letter code for amino acid residues) frequently found at positions near the C-terminus of outer membrane proteins.
  • the presence of several shared motifs clearly establishes the similarity between members of this group of proteins.
  • V Not-T (not-U) ⁇ A or C or G)
  • amino acid translations of this invention account for the ambiguity in the nucleic acid sequence by translating the ambiguous codon as the letter "X". In all cases, the permissible amino acid residues at a position are clear from an examination of the nucleic acid sequence based on the standard genetic code.
  • H pylori Nucleic Acids and Polypeptides
  • one skilled in the art can alter the disclosed structure (of H pylori genes), e.g., by producing fragments or analogs, and test the newly produced structures for activity. Examples of techniques known to those skilled in the relevant art which allow the production and testing of fragments and analogs are discussed below.
  • These, or analogous methods can be used to make and screen libraries of polypeptides, e.g., libraries of random peptides or libraries of fragments or analogs of cellular proteins for the ability to bind H pylori polypeptides. Such screens are useful for the identification of inhibitors of H. pylori.
  • Fragments of a protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis.
  • Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide.
  • Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNA's which encode an array of fragments.
  • DNA's which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination of the above-discussed methods.
  • Fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • peptides of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.
  • Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are elsewhere herein). (A) PCR Mutagenesis
  • PCR mutagenesis reduced Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA (Leung et al., 1989, Technique 1:11- 15).
  • the DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase. e.g., by using a dGTP/dATP ratio of five and adding Mn2+ to the PCR reaction.
  • the pool of amplified DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries.
  • Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242).
  • This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complimentary DNA strand.
  • the mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements.
  • a library of homologs can also be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem.
  • Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein.
  • the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.
  • Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244: 1081-1085, 1989).
  • a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine).
  • Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.
  • Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et al., (DNA 2: 183, 1983). Briefly, the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein.
  • a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA.
  • oligonucleotides of at least 25 nucleotides in length are used.
  • An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single- stranded DNA template molecule.
  • the oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765[1978]).
  • the starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated.
  • the codon(s) in the protein subunit DNA to be mutated are identified.
  • a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques.
  • This double-stranded oligonucleotide is referred to as the cassette.
  • This cassette is designed to have 3' and 5' ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.
  • This plasmid now contains the mutated desired protein subunit DNA sequence.
  • Combinatorial mutagenesis can also be used to generate mutants (Ladner et al., WO 88/06630).
  • the amino acid sequences for a group of homologs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to create a degenerate set of combinatorial sequences.
  • the variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences.
  • Other Modifications of H pylori Nucleic Acids and Polypeptides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences.
  • H. pylori polypeptide it is possible to modify the structure of an H. pylori polypeptide for such purposes as increasing solubility, enhancing stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo).
  • a modified H. pylori protein or peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition as described herein.
  • H pylori peptide can also be modified by substitution of cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid residues to minimize dimerization via disulfide linkages.
  • amino acid side chains of fragments of the protein of the invention can be chemically modified. Another modification is cyclization of the peptide.
  • an H. pylori polypeptide can be modified to incorporate one or more polymorphisms in the amino acid sequence of the protein resulting from any natural allelic variation. Additionally, D-amino acids, non- natural amino acids, or non-amino acid analogs can be substituted or added to produce a modified protein within the scope of this invention. Furthermore, an H pylori polypeptide can be modified using polyethylene glycol (PEG) according to the method of A. Sehon and co-workers (Wie et al., supra) to produce a protein conjugated with PEG. In addition, PEG can be added during chemical synthesis of the protein.
  • PEG polyethylene glycol
  • H pylori proteins include reduction/alky lation (Tarr, Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton NJ 155-194 (1986)); acylation (Tarr, supra); chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology, WH Freeman, San Francisco, CA (1980), U.S. Patent 4,939,239; or mild formalin treatment (Marsh, (1971) Int. Arch, of Allergy andAppl Immunol. , 4J_: 199 - 215).
  • an amino acid fusion moiety to the peptide backbone.
  • hexa-histidine can be added to the protein for purification by immobilized metal ion affinity chromatography (Hochuli, E. et al., (1988) Bio/Technology, 6: 1321 - 1325).
  • specific endoprotease cleavage sites can be introduced between the sequences of the fusion moiety and the peptide.
  • canonical protease sensitive sites can be engineered between regions, each comprising at least one epitope via recombinant or synthetic methods.
  • charged amino acid pairs such as KK or RR
  • the resulting peptide can be rendered sensitive to cleavage by cathepsin and/or other trypsin-like enzymes which would generate portions of the protein containing one or more epitopes.
  • such charged amino acid residues can result in an increase in the solubility of the peptide.
  • Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, e.g., in this case, binding to H pylori polypeptide or an interacting protein, facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • detection of a desired activity e.g., in this case, binding to H pylori polypeptide or an interacting protein.
  • Two hybrid assays such as the system described above (as with the other screening methods described herein), can be used to identify polypeptides, e.g., fragments or analogs of a naturally-occurring H. pylori polypeptide, e.g., of cellular proteins, or of randomly generated polypeptides which bind to an H. pylori protein.
  • the H. pylori domain is used as the bait protein and the library of variants are expressed as fish fusion proteins.
  • a two hybrid assay (as with the other screening methods described herein), can be used to find polypeptides which bind a H. pylori polypeptide.
  • the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay".
  • the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371 ; and Goward et al. (1992) TIBS 18:136-140).
  • a detectably labeled ligand can be used to score for potentially functional peptide homologs.
  • Fluorescently labeled ligands e.g., receptors, can be used to detect homologs which retain ligand- binding activity.
  • the use of fluorescently labeled ligands allows cells to be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, to be separated by a fluorescence-activated cell sorter.
  • a gene library can be expressed as a fusion protein on the surface of a viral particle.
  • foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
  • coli filamentous phages M13, fd., and fl are most often used in phage display libraries. Either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle.
  • Foreign epitopes can be expressed at the NH2- terminal end of pill and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267: 16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et al. ( ⁇ 99 ⁇ ) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).
  • E. coli the outer membrane protein, LamB
  • LamB the outer membrane protein
  • Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands. e.g., to antibodies, and can elicit an immune response when the cells are administered to animals.
  • Other cell surface proteins e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392), PhoE (Agterberg, et al.
  • Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al. (1989) Appl Environ. Microbiol. 55, 984-993). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment.
  • Another large surface structure used for peptide display is the bacterial motive organ, the flagellum.
  • Fusion of peptides to the subunit protein flagellin offers a dense array of many peptide copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083).
  • Surface proteins of other bacterial species have also served as peptide fusion partners. Examples include the Staphylococcus protein A and the outer membrane IgA protease of Neisseria (Hansson et al. (1992) J. Bacteriol 174, 4239-4245 and Klauser et al. (1990) EMBO J. 9, 1991 - 1999).
  • the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface.
  • An alternative scheme uses the DNA-binding protein Lad to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89: 1865-1869).
  • This system uses a plasmid containing the Lad gene with an oligonucleotide cloning site at its 3 '-end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced. This fusion retains the natural ability of Lad to bind to a short DNA sequence known as LacO operator (LacO). By installing two copies of LacO on the expression plasmid, the Lacl-peptide fusion binds tightly to the plasmid that encoded it.
  • LacO LacO operator
  • the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis.
  • the cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides.
  • the associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands.
  • the peptides are attached to the C-terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini.
  • Both of the filamentous phage coat proteins, pill and pVIII are anchored to the phage through their C-termini, and the guest peptides are placed into the outward-extending N-terminal domains.
  • the phage- displayed peptides are presented right at the amino terminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6378-6382)
  • a second difference is the set of biological biases affecting the population of peptides actually present in the libraries.
  • the Lad fusion molecules are confined to the cytoplasm of the host cells.
  • the phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles.
  • the peptides in the Lad and phage libraries may differ significantly as a result of their exposure to different proteolytic activities.
  • the phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to inco ⁇ oration into phage.
  • RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening.
  • the polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector. This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification.
  • polysome-derived peptides on phage By expressing the polysome-derived peptides on phage, one can either continue the affinity selection procedure in this format or assay the peptides on individual clones for binding activity in a phage ⁇ LISA, or for binding specificity in a completion phage ⁇ LISA (Barret, et al. (1992) Anal. Biochem 204,357-364). To identify the sequences of the active peptides one sequences the DNA produced by the phagemid host.
  • the high through-put assays described above can be followed by secondary screens in order to identify further biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists.
  • the type of a secondary screen used will depend on the desired activity that needs to be tested.
  • an assay can be developed in which the ability to inhibit an interaction between a protein of interest and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated though one of the primary screens described above.
  • the invention also provides for reduction of the protein binding domains of the subject H pylori polypeptides to generate mimetics, e.g. peptide or non-peptide agents.
  • mimetics e.g. peptide or non-peptide agents.
  • the peptide mimetics are able to disrupt binding of a polypeptide to its counter ligand, e.g., in the case of an H. pylori polypeptide binding to a naturally occurring ligand.
  • the critical residues of a subject H pylori polypeptide which are involved in molecular recognition of a polypeptide can be determined and used to generate H /r ⁇ / ⁇ r/-derived peptidomimetics which competitively or noncompetitively inhibit binding of the H pylori polypeptide with an interacting polypeptide (see, for example, European patent applications EP-412,762A and EP-B31,080A).
  • scanning mutagenesis can be used to map the amino acid residues of a particular H pylori polypeptide involved in binding an interacting polypeptide
  • peptidomimetic compounds e.g. diazepine or isoquinoline derivatives
  • non- hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R.
  • This invention also features vaccine compositions or formulations (used interchangeably herein) for protection against infection by H pylori or for treatment of H. pylori infection.
  • treatment of H pylori infection refers to therapeutic treatment of an existing or established H. pylori infection.
  • protection against H. pylori infection or “prophylactic treatment” refer to the use of H pylori vaccine formulation for reducing the risk of or preventing an infection in a subject at risk for H pylori infection.
  • the vaccine compositions contain one or more immunogenic components, such as a surface protein, from H pylori, or portion thereof, and a pharmaceutically acceptable carrier.
  • the vaccine formulations of the invention contain at least one or combination of H pylori polypeptides or fragments thereof, from same or different H pylori antigens.
  • Nucleic acids and H. pylori polypeptides for use in the vaccine formulations of the invention include the nucleic acids and polypeptides set forth in the Sequence Listing, preferably those H pylori nucleic acids that encode surface proteins and surface proteins or fragments thereof.
  • a preferred nucleic acid and H. pylori polypeptide for use in a vaccine composition of the invention is selected from the group of nucleic acids which encode cell envelope proteins and H pylori cell envelope proteins as set forth in Table 1.
  • any nucleic acid encoding an immunogenic H. pylori protein and H. pylori polypetide, or portion thereof can be used in the present invention. These vaccines have therapeutic and/or prophylactic utilities.
  • One aspect of the invention provides a vaccine composition for protection against infection by H. pylori which contains at least one immunogenic fragment of an H. pylori protein and a pharmaceutically acceptable carrier.
  • Preferred fragments include peptides of at least about 10 amino acid residues in length, preferably about 10-20 amino acid residues in length, and more preferably about 12-16 amino acid residues in length.
  • Immunogenic components of the invention can be obtained, for example, by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding the full-length H pylori protein.
  • fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • immunogenic components are identified by the ability of the peptide to stimulate T cells.
  • Peptides which stimulate T cells as determined by, for example, T cell proliferation or cytokine secretion are defined herein as comprising at least one T cell epitope.
  • T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to the protein allergen which is responsible for the clinical symptoms of allergy. These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate HLA molecule on the surface of an antigen presenting cell, thereby stimulating the T cell subpopulation with the relevant T cell receptor for the epitope.
  • a T cell epitope is the basic element, or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acids essential to receptor recognition (e.g., approximately 6 or 7 amino acid residues). Amino acid sequences which mimic those of the T cell epitopes are within the scope of this invention.
  • immunogenic components of the invention are identified through genomic vaccination.
  • the basic protocol is based on the idea that expression libraries consisting of all or parts of a pathogen genome, e.g., an H. pylori genome, can confer protection when used to genetically immunize a host.
  • This expression library immunization (ELI) is analogous to expression cloning and involves reducing a genomic expression library of a pathogen, e.g., H. pylori, into plasmids that can act as genetic vaccines.
  • the plasmids can also be designed to encode genetic adjuvants which can dramatically stimulate the humoral response. These genetic adjuvants can be introduced at remote sites and act as well extracelluraly as intracellularly.
  • An expression library of pathogen DNA is used to immunize a host thereby producing the effects of antigen presentation of a live vaccine without the risk.
  • random fragments from the H. pylori genome or from cosmid or plasmid clones, as well as PCR products from genes identified by genomic sequencing can be used to immunize a host.
  • ELI is a technique that allows for production of a non-infectious multipartite vaccine, even when little is known about pathogen's biology, because ELI uses the immune system to screen candidate genes. Once isolated, these genes can be used as genetic vaccines or for development of recombinant protein vaccines. Thus, ELI allows for production of vaccines in a systematic, largely mechanized fashion.
  • Screening immunogenic components can be accomplished using one or more of several different assays.
  • peptide T cell stimulatory activity is assayed by contacting a peptide known or suspected of being immunogenic with an antigen presenting cell which presents appropriate MHC molecules in a T cell culture.
  • Presentation of an immunogenic H. pylori peptide in association with appropriate MHC molecules to T cells in conjunction with the necessary costimulation has the effect of transmitting a signal to the T cell that induces the production of increased levels of cytokines, particularly of interleukin-2 and interleukin-4.
  • the culture supernatant can be obtained and assayed for interleukin-2 or other known cytokines.
  • any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc. Natl. Acad. Sci USA, 86: 1333 (1989) the pertinent portions of which are inco ⁇ orated herein by reference.
  • a kit for an assay for the production of interferon is also available from Genzyme Co ⁇ oration (Cambridge, MA).
  • a common assay for T cell proliferation entails measuring tritiated thymidine inco ⁇ oration.
  • the proliferation of T cells can be measured in vitro by determining the amount of ⁇ H-labeled thymidine inco ⁇ orated into the replicating DNA of cultured cells. Therefore, the rate of DNA synthesis and, in turn, the rate of cell division can be quantified.
  • Vaccine compositions or formulations of the invention containing one or more immunogenic components preferably include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the H. pylori nucleic acid or polypeptide.
  • auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the H. pylori nucleic acid or polypeptide.
  • the polypeptide is preferably coadministered with a suitable adjuvant and/or a delivery system described herein.
  • the therapeutically effective amount of DNA or protein of this invention will depend, inter alia, upon the administration schedule, the unit dose of an H pylori nucleic acid or polypeptide administered, whether the protein or nucleic acid is administered in combination with other therapeutic agents, the immune status and health of the patient, and the therapeutic activity of the particular protein or nucleic acid.
  • Vaccine formulations are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Methods for intramuscular immunization are described by Wolff et al. (1990) Science 247: 1465-1468 and by Sedegah et al. (1994) Immunology 9j 9866-9870. Other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral immunization is preferred over parenteral methods for inducing protection against infection by H pylori. Czinn et. al. (1993) Vaccine V ⁇ _: 637-642. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • the vaccine formulation includes, as a pharmaceutically acceptable carrier, an adjuvant.
  • suitable adjuvants for use in the vaccine formulations of the invention include, but are not limited, to aluminum hydroxide; N-acetyl-muramyl—L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine (CGP 1 1637, referred to as nor-MDP); N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'-dipalmitoyl-sn-glycero-3- hydroxyphos-phoryloxy)-ethylamine (CGP 19835 A, referred to a MTP-PE); RIBI, which contains three components from bacteria; monophosphoryl lipid A; trehalose dimycoloate; cell wall skeleton (MPL +
  • Non-toxic derivatives of cholera toxin including its B subunit, and/or conjugates or genetically engineered fusions of the H pylori polypeptide with cholera toxin or its B subunit, procholeragenoid, fungal polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters, labile toxin of E. coli. non-H. pylori bacterial lysates, block polymers or saponins.
  • the vaccine formulation includes, as a pharmaceutically acceptable carrier, a delivery system.
  • Suitable delivery systems for use in the vaccine formulations of the invention include biodegradable microcapsules or immuno- stimulating complexes (ISCOMs), cochleates, or liposomes, genetically engineered attenuated live vectors such as viruses or bacteria, and recombinant (chimeric) virus-like particles, e.g., bluetongue.
  • the vaccine formulation includes both a delivery system and an adjuvant.
  • Delivery systems in humans may include enteric release capsules protecting the antigen from the acidic environment of the stomach, and including H pylori polypeptide in an insoluble form as fusion proteins.
  • Suitable carriers for the vaccines of the invention are enteric coated capsules and polylactide-glycolide microspheres.
  • Suitable diluents are 0.2 N Na ⁇ CO3 and/or saline.
  • Vaccines of the invention can be administered as a primary prophylactic agent in adults or in children, as a secondary prevention, after successful eradication of H. pylori in an infected host, or as a therapeutic agent in the aim to induce an immune response in a susceptible host to prevent infection by H. pylon.
  • the vaccines of the invention are administered in amounts readily determined by persons of ordinary skill in the art.
  • a suitable dosage will be in the range of 10 ⁇ g to 10 g, preferably 10 ⁇ g to 100 mg, for example 50 ⁇ g to 50 mg.
  • a suitable dosage for adults will also be in the range of 5 ⁇ g to 500 mg. Similar dosage ranges will be applicable for children.
  • the amount of adjuvant employed will depend on the type of adjuvant used.
  • the mucosal adjuvant is cholera toxin
  • it is suitably used in an amount of 5 ⁇ g to 50 ⁇ g, for example 10 ⁇ g to 35 ⁇ g.
  • the amount used will depend on the amount employed in the matrix of the microcapsule to achieve the desired dosage. The determination of this amount is within the skill of a person of ordinary skill in the art.
  • the optimal dose may be more or less depending upon the patient's body weight, disease, the route of administration, and other factors.
  • appropriate dosage levels can be obtained based on results with known oral vaccines such as, for example, a vaccine based on an E. coli lysate (6 mg dose daily up to total of 540 mg) and with an enterotoxigenic E. coli purified antigen (4 doses of 1 mg) (Schulman et al., J. Urol 150:917-921 (1993)); Boedecker et al., American Gastroenterological Assoc. 999:A-222 (1993)).
  • the number of doses will depend upon the disease, the formulation, and efficacy data from clinical trials. Without intending any limitation as to the course of treatment, the treatment can be administered over 3 to 8 doses for a primary immunization schedule over 1 month (Boedeker, American Gastroenterological Assoc. 888 :A-222 (1993)).
  • a vaccine composition of the invention can be based on a killed whole E. coli preparation with an immunogenic fragment of an H. pylori protein of the invention expressed on its surface or it can be based on an E. coli lysate, wherein the killed E. coli acts as a carrier or an adjuvant. It will be apparent to those skilled in the art that some of the vaccine compositions of the invention are useful only for preventing H pylori infection, some are useful only for treating H. pylori infection, and some are useful for both preventing and treating H pylori infection. In a preferred embodiment, the vaccine composition of the invention provides protection against H.
  • H. pylori infection by stimulating humoral and/or cell-mediated immunity against H pylori. It should be understood that amelioration of any of the symptoms of H. pylori infection is a desirable clinical goal, including a lessening of the dosage of medication used to treat H. ⁇ y/or ' -caused disease, or an increase in the production of antibodies in the serum or mucous of patients.
  • the invention also includes antibodies specifically reactive with the subject H pylori polypeptide.
  • Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • An immunogenic portion of the subject H. pylori polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • the subject antibodies are immunospecific for antigenic determinants of the H. pylori polypeptides of the invention, e.g. antigenic determinants of a polypeptide of the invention contained in the Sequence Listing, or a closely related human or non-human mammalian homolog (e.g., 90% homologous, more preferably at least 95%> homologous).
  • the anti-H. pylori antibodies do not substantially cross react (i.e., react specifically) with a protein which is for example, less than 80%> percent homologous to a sequence of the invention contained in the Sequence Listing.
  • the antibody has a binding affinity for a non-homologous protein which is less than 10 percent, more preferably less than 5 percent, and even more preferably less than 1 percent, of the binding affinity for a protein of the invention contained in the Sequence Listing. In a most preferred embodiment, there is no crossreactivity between bacterial and mammalian antigens.
  • antibody as used herein is intended to include fragments thereof which are also specifically reactive with H pylori polypeptides.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.
  • the antibody of the invention is further intended to include bispecific and chimeric molecules having an anti-H. pylori portion.
  • Both monoclonal and polyclonal antibodies (Ab) directed against H pylori polypeptides or H pylori polypeptide variants, and antibody fragments such as Fab' and F(ab')2, can be used to block the action of H. pylori polypeptide and allow the study of the role of a particular H. pylori polypeptide of the invention in aberrant or unwanted intracellular signaling, as well as the normal cellular function of the H. pylori and by microinjection of anti-H. pylori polypeptide antibodies of the present invention.
  • Antibodies which specifically bind H pylori epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of H pylori antigens.
  • Anti H pylori polypeptide antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate H. pylori levels in tissue or bodily fluid as part of a clinical testing procedure.
  • the ability to monitor H. pylori polypeptide levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder.
  • pylori polypeptide can be measured in cells found in bodily fluid, such as in urine samples or can be measured in tissue, such as produced by gastric biopsy. Diagnostic assays using anti-H. pylori antibodies can include, for example, immunoassays designed to aid in early diagnosis of H. pylori infections. The present invention can also be used as a method of detecting antibodies contained in samples from individuals infected by this bacterium using specific H. pylori antigens.
  • anti-H. pylori polypeptide antibodies of the invention is in the immunological screening of cDNA libraries constructed in expression vectors such as ⁇ gtl 1, ⁇ gtl 8-23, ⁇ ZAP, and ⁇ ORF8.
  • Messenger libraries of this type having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.
  • ⁇ gtl 1 will produce fusion proteins whose amino termini consist of ⁇ -galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.
  • Antigenic epitopes of a subject H. pylori polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-H.
  • H pylori polypeptide antibodies Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence of H pylori gene homologs can be detected and cloned from other species, and alternate isoforms (including splicing variants) can be detected and cloned.
  • Kits Containing Nucleic Acids, Polypeptides or Antibodies of the Invention can be combined with other reagents and articles to form kits.
  • Kits for diagnostic pu ⁇ oses typically comprise the nucleic acid, polypeptides or antibodies in vials or other suitable vessels.
  • Kits typically comprise other reagents for performing hybridization reactions, polymerase chain reactions (PCR), or for reconstitution of lyophilized components, such as aqueous media, salts, buffers, and the like.
  • Kits may also comprise reagents for sample processing such as detergents, chaotropic salts and the like.
  • Kits may also comprise immobilization means such as particles, supports, wells, dipsticks and the like.
  • Kits may also comprise labeling means such as dyes, developing reagents, radioisotopes, fluorescent agents, luminescent or chemiluminescent agents, enzymes, intercalating agents and the like. With the nucleic acid and amino acid sequence information provided herein, individuals skilled in art can readily assemble kits to serve their particular pu ⁇ ose. Kits further can include instructions for use.
  • labeling means such as dyes, developing reagents, radioisotopes, fluorescent agents, luminescent or chemiluminescent agents, enzymes, intercalating agents and the like.
  • H. pylori Polypeptides By making available purified and recombinant H. pylori polypeptides, the present invention provides assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function, in this case, of the subject H pylori polypeptides, or of their role in intracellular signaling. Such inhibitors or potentiators may be useful as new therapeutic agents to combat H pylori infections in humans. A variety of assay formats will suffice and, in light of the present inventions, will be comprehended by the skilled artisan.
  • the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or change in enzymatic properties of the molecular target. Accordingly, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified H pylori polypeptide.
  • Screening assays can be constructed in vitro with a purified H pylori polypeptide or fragment thereof, such as an H. pylori polypeptide having enzymatic activity, such that the activity of the polypeptide produces a detectable reaction product.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay can also be performed to provide a baseline for comparison. Suitable products include those with distinctive abso ⁇ tion, fluorescence, or chemi-luminescence properties, for example, because detection may be easily automated.
  • a variety of synthetic or naturally occurring compounds can be tested in the assay to identify those which inhibit or potentiate the activity of the H pylori polypeptide. Some of these active compounds may directly, or with chemical alterations to promote membrane permeability or solubility, also inhibit or potentiate the same activity (e.g., enzymatic activity) in whole, live H pylori cells.
  • H pylori chromosomal DNA was isolated according to a basic DNA protocol outlined in Schleif R.F. and Wensink P.C, Practical Methods in Molecular Biology, p.98, Springer- Verlag, NY., 1981, with minor modifications. Briefly, cells were pelleted, resuspended in TE (10 mM Tris, 1 mM EDTA, p ⁇ 7.6) and GES lysis buffer (5.1 M guanidium thiocyanate, 0.1 M EDTA, p ⁇ 8.0, 0.5%) N-laurylsarcosine) was added. Suspension was chilled and ammonium acetate (N ⁇ 4 AC) was added to final concentration of 2.0 M. DNA was extracted, first with chloroform, then with phenol- chloroform, and reextracted with chloroform. DNA was precipitated with isopropanol, washed twice with 70% EtOH, dried and resuspended in TE.
  • the purified DNA fragments were then blunt-ended using T4 DNA polymerase.
  • the healed DNA was then ligated to unique BstXI-linker adapters in 100-1000 fold molar excess.
  • These linkers are complimentary to the BstXI-cut pMPX vectors, while the overhang is not self-complimentary. Therefore, the linkers will not concatemerize nor will the cut- vector religate itself easily.
  • the linker-adopted inserts were separated from the uninco ⁇ orated linkers on a 1%> agarose gel and purified using GeneClean. The linker-adopted inserts were then ligated to each of the 20 pMPX vectors to construct a series of "shotgun" subclone libraries.
  • the vectors contain an out-of- frame lacZ gene at the cloning site which becomes in-frame in the event that an adapter-dimer is cloned, allowing these to be avoided by their blue-color.
  • each of the 20 vectors was then transformed into DH5 competent cells (Gibco/BRL, DH5 ⁇ transformation protocol).
  • the libraries were assessed by plating onto antibiotic plates containing ampicillin, methicillin and IPTG/Xgal. The plates were incubated overnight at 37°C. Successful transformants were then used for plating of clones and pooling into the multiplex pools. The clones were picked and pooled into 40 ml growth medium cultures. The cultures were grown overnight at 37°C.
  • DNA was purified using the Qiagen Midi-prep kits and Tip- 100 columns (Qiagen, Inc.). In this manner, 100 ⁇ g of DNA was obtained per pool. Fifteen 96-well plates of DNA were generated to obtain a 5-10 fold sequence redundancy assuming 250-300 base average read-lengths. These purified DNA samples were then sequenced using the multiplex DNA sequencing based on chemical degradation methods (Church G.M. and Kieffer-Higgins S., Science 240:185-188, 1988) or by Sequithrem (Epicenter Technologies) dideoxy sequencing protocols. The sequencing reactions were electrophoresed and transferred onto nylon membranes by direct transfer electrophoresis from 40 cm gels (Richterich P. and Church G.M., Methods in Enzymology 218:187-222, 1993) or by electroblotting
  • each gel produced a large number of films, each containing new sequencing information. Whenever a new blot was processed, it was initially probed for an internal standard sequence added to each of the pools.
  • Digital images of the films were generated using a laser-scanning densitometer (Molecular Dynamics, Sunnyvale, CA).
  • the digitized images were processed on computer workstations (VaxStation 4000's) using the program REPLICATM (Church et al., Automated DNA Sequencing and Analysis (J.C. Venter, ed.), Academic Press, 1994).
  • Image processing included lane straightening, contrast adjustment to smooth out intensity differences, and resolution enhancement by iterative gaussian deconvolution.
  • the sequences were then automatically picked in REPLICATM and displayed for interactive proofreading before being stored in a project database. The proofreading was accomplished by a quick visual scan of the film image followed by mouse clicks on the bands of the displayed image to modify the base calls.
  • sequence errors could be detected and corrected because multiple sequence reads covering the same portion of the genomic DNA provide adequate sequence redundancy for editing.
  • Each sequence automatically received an identification number (corresponding to microtiter plate, probe information, and lane set number). This number serves as a permanent identifier of the sequence so it is always possible to identify the original of any particular sequence without recourse to a specialized database.
  • GelAssemble developed by the Genetics Computer Group (GCG) (Devereux et al., Nucleic Acid Res. 12:387-95, 1984) that interacts with REPLICATM. This provided for an integrated editor that allows multiple sequence gel images to be instantaneously called up from the REPLICATM database and displayed to allow rapid scanning of contigs and proofreading of gel traces where discrepancies occurred between different sequence reads in the assembly.
  • GCG Genetics Computer Group
  • the pET System Novagen
  • a DNA sequence encoding a peptide tag, the ⁇ is-Tag was fused to the 3 ' end of DNA sequences of interest in order to facilitate purification of the recombinant protein products.
  • the 3' end was selected for fusion in order to avoid alteration of any 5' terminal signal sequence.
  • ppiB a gene cloned for use as a control in the expression studies.
  • the sequence for H pylori ppiB contains a DNA sequence encoding a ⁇ is-Tag fused to the 5' end of the full length gene, because the protein product of this gene does not contain a signal sequence and is expressed as a cytosolic protein.
  • Sequences chosen (from the list of the DNA sequences of the invention) for cloning from the J99 strain of H. pylori were prepared for amplification cloning by polymerase chain reaction (PCR).
  • Synthetic oligonucleotide primers (Table 3) specific for the 5' and 3' ends of open reading frames (ORFs) were designed and purchased (GibcoBRL Life Technologies, Gaithersburg, MD, USA). All forward primers (specific for the 5' end of the sequence) were designed to include an Ncol cloning site at the extreme 5' terminus, except for ⁇ pSeq. 4821082 where Ndel was used.
  • H pylori sequence 4821082 where the initiator methionine is immediately followed by the remainder of the native H pylori DNA sequence.
  • All reverse primers included a EcoRI site at the extreme 5' terminus to permit cloning of each H pylori sequence into the reading frame of the pET-28b.
  • the pET-28b vector provides sequence encoding an additional 20 carboxy-terminal amino acids (only 19 amino acids in ⁇ pSeq.
  • Oligonucleotide primers used for PCR amplification of H pylori DNA sequences Oligonucleotide primers used for PCR amplification of H pylori DNA sequences
  • Genomic DNA prepared from the J99 strain of H. pylori (ATCC #55679; deposited by Genome Therapeutics Co ⁇ oration, 100 Beaver Street, Waltham, MA 02154) was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
  • genomic DNA 50 nanograms was introduced into a reaction vial containing 2 mM MgCl2, 1 micromolar synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H pylori ORF, 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP. dCTP, dTTP and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems. Inc., Branchburg, NJ, USA) in a final volume of 100 microliters.
  • the following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
  • each sample of amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). All amplified DNA samples were subjected to digestion with the restriction endonucleases, Ncol and EcoRI (New England BioLabs, Beverly, MA, USA), or in the case of HpSeq. 4821082 (SEQ ID NO: 1309), with Ndd and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
  • DNA samples were then subjected to electrophoresis on 1.0 %> NuSeive (FMC BioProducts, Rockland, ME USA) agarose gels. DNA was visualized by exposure to ethidium bromide and long wave uv irradiation. DNA contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, C A, USA).
  • the pET-28b vector was prepared for cloning by digestion with Ncol and EcoRI, or in the case of H pylori protein 4821082 with Ndel and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
  • cloning ppiB the pET-28a vector, which encodes a ⁇ is-Tag that can be fused to the 5' end of an inserted gene, was used and the cloning site prepared for cloning with the ppiB gene by digestion with Bam ⁇ I and Xhol restriction endonucleases.
  • DNA inserts were cloned (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) into the previously digested pET-28b expression vector, except for the amplified insert for ppiB, which was cloned into the pET-28a expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) as described below.
  • Competent bacteria E coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994).
  • the pET vector can be propagated in any E. coli K-12 strain e.g. ⁇ MS174, HB101, JM109, DH5, etc. for the pu ⁇ ose of cloning or plasmid preparation.
  • Hosts for expression include E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lad gene, the lacUV5 promoter and the gene for T7 RNA polymerase.
  • T7 RNA polymerase is induced by addition of isopropyl-B-D-thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying a T7 promoter and a gene of interest.
  • Strains used include: BL21(DE3) (Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60-89).
  • H. pylori sequences 50 nanograms of plasmid DNA isolated as described above was used to transform competent BL21(DE3) bacteria as described above (provided by Novagen as part of the pET expression system kit).
  • the lacZ gene (beta-galactosidase) was expressed in the pET- System as described for the H. pylori recombinant constructions.
  • Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 micrograms/ml kanamycin sulfate.
  • bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 micrograms/ml) to an optical density at 600 nM of 0.5 to 1.0 O.D. units, at which point, 1 millimolar IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions.
  • the concentrations of purified protein preparations were quantified spectrophotometrically using absorbance coefficients calculated from amino acid content (Perkins, S.J. 1986 Eur. J. Biochem. 157, 169-180). Protein concentrations were also measured by the method of Bradford, M.M. (1976) Anal. Biochem. 72, 248-254, and Lowry, O.H., Rosebrough, N., Farr, A.L. & Randall, R.J. (1951) J. Biol. Chem. 193, pages 265-275, using bovine serum albumin as a standard.
  • SDS-polyacrylamide gels (12%> or 4.0 to 25 %> acrylamide gradient gels) were purchased from BioRad (Hercules, CA, USA), and stained with Coomassie blue.
  • Molecular weight markers included rabbit skeletal muscle myosin (200 kDa), E. coli (- galactosidase (1 16 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
  • NTA Ni ⁇ " - nitrilotriacetate-agarose
  • the column was washed with 250 ml (50 bed volumes) of lysis buffer containing 10 %> glycerol, 0.1 %> Brij 35, and was eluted with sequential steps of lysis buffer containing 10 %> glycerol, 0.05 %> Brij 35, 1 mM PMSF, and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD28O nm ' anc peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole.
  • Fractions containing the recombinant proteins from the Ni2 + -NTA-agarose columns were pooled and then concentrated to approximately 5 ml by centrifugal filtration (Centriprep-10, Amicon, MA), and loaded directly onto a 180-ml column (1.6 X 91 cm) of Sephacryl S-100 HR gel filtration medium equilibrated in Buffer A (10 mM Hepes, pH 7.5. 150 mM NaCl, 0.1 mM EGTA) and run in Buffer A at 18 ml/h. Fractions containing the recombinant protein were identified by absorbance at 280 nm and analyzed by SDS-PAGE. Fractions were pooled and concentrated by centrifugal filtration.
  • buffer B (10 mM MOPS, pH 6.5, 0.1 mM EGTA) containing 50 mM NaCl.
  • the column was washed with 10 bed volumes of buffer B containing 50 mM NaCl, and developed with a 50-ml linear gradient of increasing NaCl (50 to 500 mM).
  • Recombinant protein 71 16626 eluted as a sha ⁇ peak at 300 mM NaCl.
  • the pellets were washed with lysis buffer containing 10 %> glycerol, 10 mM EDTA, 1% Triton X-100, 1 mM PMSF and 0.1% -mercaptoethanol, followed by several washes with lysis buffer containing 1 M urea, 1 mM PMSF and 0.1 % 2- mercaptoethanol.
  • the resulting white pellet was composed primarily of inclusion bodies, free of unbroken cells and membranous materials..
  • the column was washed with 250 ml (50 bed volumes) of lysis buffer containing 8 M urea, 1.0 mM PMSF and 0.1 % 2-mercaptoethanol, and developed with sequential steps of lysis buffer containing 8M urea, 1 mM PMSF, 0.1 %> 2- mercaptoethanol and 20, 100, 200, and 500 mM imidazole in succession.
  • Fractions were monitored by absorbance at OD28O nrn - an ⁇ ⁇ P ea ⁇ fractions were analyzed by SDS- PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole.
  • the pellet containing the inclusion bodies was solubilized in buffer B containing
  • H. pylori To investigate the immunomodulatory effect of H. pylori proteins, a mouse/H pylori model was used. This model mimics the human H. pylori infection in many respects. The focus is on the effect of oral immunization in H. pylori infected animals in order to test the concept of therapeutic oral immuno therapy.
  • mice Female SPF BALB/c mice were purchased from Bomholt Breeding center
  • H pylori H pylori
  • strain 244 originally isolated from an ulcer patient
  • This strain has earlier proven to be a good colonizer of the mouse stomach.
  • the bacteria were grown overnight in Brucella broth supplemented with 10 %> fetal calf serum, at 37°C in a microaerophilic atmosphere (10% CO 2 , 5%>O ).
  • the animals were given an oral dose of omeprazole (400 ⁇ mol/kg) and 3-5 h after this an oral inoculation of H. pylori in broth (approximately 10 cfu/animal). Positive take of the infection was checked in some animals 2-3 weeks after the inoculation.
  • Recombinant H pylori antigens were chosen based on their association with externally exposed H. pylori cell membrane. These antigens were selected from the following groups: (1.) Outer Membrane Proteins; (2.) Periplastic/Secreted proteins; (3.) Outer Surface proteins; and (4.) Inner Membrane proteins. All recombinant proteins were constructed with a hexa- ⁇ IS tag for purification reasons and the non-Helicobacter pylori control protein (b-galactosidase from E. coli; LacZ), was constructed in the same way.
  • the antigens are listed in Table 5 below.
  • mice in each group were immunized 4 times over a 34 day period (day 1, 15, 25 and 35).
  • Purified antigens in solution or suspension were given at a dose of 100 mg/mouse.
  • CT Cholera toxin
  • Omeprazole (400 mmol/kg) was given orally to the animals 3-5 h prior to immunization as a way of protecting the antigens from acid degradation.
  • Infected control animals received HEPES buffer + CT or DOC buffer + CT. Animals were sacrificed 2-4 weeks after final immunization. A general outline of the study is shown in Table 6 below.
  • mice were all infected with H. pylori strain Ah244 at day 30
  • Protein 26054702 100 ⁇ g + CT 10 ⁇ gg BBaallbb//cc 0.3 ml 0, 14, 24, 34 5. Protein 26380318, 100 ⁇ g + CT 10 ⁇ g Balb/c 0.3 ml 0, 14, 24, 34
  • Mucosal infection The mice were sacrificed by CO 2 and cervical dislocation. The abdomen was opened and the stomach removed. After cutting the stomach along the greater curvature, it was rinsed in saline. The mucosa from the antrum and co ⁇ us of an area of 25mm was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth and plated onto Blood Skirrow selective plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted. The identity of H pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining.
  • the urease test was performed essentially as follows.
  • the reagent, Urea Agar Base Concentrate, was purchased from DIFCO Laboratories, Detroit, MI (Catalog # 0284-61-3).
  • Urea agar base concentrate was diluted 1 : 10 with water. 1 ml of if the diluted concentrate was mixed with 100-200 ml of actively growing H. pylori cells. Color change to magenta indicated that cells were urease positive.
  • the catalase test was performed essentially as follows.
  • a solution of the regent (1% w/v in water) was prepared.
  • H. pylori cells were swabbed onto Whatman filter paper and overlaid with the 1%> solution. Color change to dark blue indicated that the cells were catalase positive.
  • Serum antibodies From all mice serum was prepared from blood drawn by heart puncture. Serum antibodies were identified by regular ELISA techniques, where the specific antigens of Helicobacter pylori were plated.
  • Mucosal antibodies Gentle scrapings of a defined part of the co ⁇ us and of 4 cm of duodenum were performed in 50%> of the mice in order to detect the presence of antibodies in the mucous.
  • the antibody titers were determined by regular ELISA technique as for serum antibodies.
  • Antibodies in mucus In the mucus scrapings, specific antibodies against all antigens tested were seen. By far the strongest response was seen with Protein 30100332, followed by Protein 14640637, and Protein 26380318 (see Figure 2).
  • H pylori associated proteins included in this study, when used as oral immunogens in conjunction with the oral adjuvant CT, resulted in stimulation of an immune response as measured by specific serum and mucosal antibodies.
  • a majority of the proteins led to a reduction, and in some cases complete clearance of the colonization of H pylori in this animal model.
  • the reduction or clearance was due to heterologous protection rather than homologous protection (the polypeptides were based on the H. pylori J99 strain sequence and used in the therapeutic immunization studies against a different (AH244) challenge strain, indicating the vaccine potential against a wide variety of H. pylori strains.
  • H. pylori Four genes were cloned and sequenced from several strains of H. pylori to compare the DNA and deduced amino acid sequences. This information was used to determine the sequence variation between the H. pylori strain, J99, and other H. pylori strains isolated from human patients.
  • H. pylori strains (as listed in Table 9) were grown in BLBB (1% Tryptone, 1% Peptamin 0.1% Glucose, 0.2% Yeast Extract 0.5% Sodium Chloride, 5% Fetal Bovine Serum) to an OD 600 of 0.2. Cells were centrifuged in a Sorvall RC-3B at 3500 x g at 4°C for 15 minutes and the pellet resuspended in 0.95 mis of 10 mM Tris- HCl, 0.1 mM EDTA (TE).
  • BLBB 1% Tryptone, 1% Peptamin 0.1% Glucose, 0.2% Yeast Extract 0.5% Sodium Chloride, 5% Fetal Bovine Serum
  • Lysozyme was added to a final concentration of 1 mg/ml along with, SDS to 1% and RNAse A + TI to 0.5mg/ml and 5 units/ml respectively, and incubated at 37°C for one hour. Proteinase K was then added to a final concentration of 0.4mg/ml and the sample was incubated at 55 C for more than one hour. NaCl was added to the sample to a concentration of 0.65 M, mixed carefully, and 0.15 ml of 10% CTAB in 0.7M NaCL (final is 1% CTAB/70mM NaCL) was added followed by incubation at 65°C for 20 minutes.
  • Genomic DNA prepared from twelve strains of Helicobacter pylori was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994).
  • genomic DNA (10 nanograms) was introduced into a reaction vial containing 2 mM MgCl 2 , 1 micromolar synthetic oligonucleotide primers (forward and reverse primers, see Table 7) complementary to and flanking a defined H.
  • pylori ORF 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP, dCTP, dTTP and 0.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 20 microliters in duplicate reactions.
  • each pair of samples were combined and used directly for cloning into the pCR cloning vector as described below.
  • Competent bacteria E coli strain TOPI OF' or E. coli strain INVaF' were transformed with recombinant pCR expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). Briefly, 2 microliters of 0.5 micromolar BM ⁇ was added to each vial of 50 microliters of competent cells.
  • Oligonucleotide primers used for sequencing of H. pylori DNA sequences Oligonucleotide primers used for sequencing of H. pylori DNA sequences.
  • the data demonstrate that there is variation in the DNA sequence ranging from as little as 0.12 % difference (Protein 346, J99 strain) to approximately 7% change (Protein 26054702, strain A ⁇ 5).
  • the deduced protein sequences show either no variation ( Protein 346, strains AH 18 and AH24) or up to as much as 7.66% amino acid changes (Protein 26054702, Strain AH5).
  • Therapeutic targets are chosen from genes whose protein products appear to play key roles in essential cell pathways such as cell envelope synthesis, DNA synthesis, transcription, translation, regulation and colonization/virulence.
  • H. pylori Gene Sequences The sequences of the genes or ORFs (open reading frames) selected as knock-out targets are identified from the H. pylori genomic sequence and used to design primers to specifically amplify the genes/ORFs. All synthetic oligonucleotide primers are designed with the aid of the OLIGO program (National Biosciences, Inc., Madison, MN 55447, USA), and can be purchased from Gibco/BRL Life Technologies (Gaithersburg, MD, USA). If the ORF is smaller than 800 to 1000 base pairs, flanking primers are chosen outside of the open reading frame.
  • Genomic DNA prepared from the Helicobacter pylori ⁇ pJ99 strain (ATCC 55679; deposited by Genome Therapeutics Co ⁇ oration, 100 Beaver Street, Waltham, MA 02154) is used as the source of template DNA for amplification of the ORFs by PCR (polymerase chain reaction) (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al.. editors, 1994).
  • PCR polymerase chain reaction
  • PCR products are cloned into the pT7Blue T-Vector (catalog#69820-l, Novagen, Inc., Madison, WI, USA) using the TA cloning strategy (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994).
  • the ligation of the PCR product into the vector is accomplished by mixing a 6 fold molar excess of the PCR product, 10 ng of pT7Blue-T vector (Novagen), 1 microliter of T4 DNA Ligase Buffer (New England Biolabs, Beverly, MA, USA), and 200 units of T4 DNA Ligase (New England Biolabs) into a final reaction volume of 10 microliters. Ligation is allowed to proceed for 16 hours at 16°C.
  • Ligation products are electroporated (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al.. editors, 1994) into electroporation- competent XL-1 Blue or DH5-a E.coli cells (Clontech Lab., Inc. Palo Alto, CA, USA).
  • 1 microliter of ligation reaction is mixed with 40 microliters of electrocompetent cells and subjected to a high voltage pulse (25 microFarads, 2.5 kV, 200 ohms) after which the samples are incubated in 0.45 ml SOC medium (0.5%) yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 and 20 mM glucose) at 37°C with shaking for 1 hour.
  • a high voltage pulse 25 microFarads, 2.5 kV, 200 ohms
  • these pT7Blue plasmid DNAs are used as templates for PCR amplification of the cloned inserts, using the same forward and reverse primers used for the initial amplification of the J99 Hpylori sequence. Recognition of the primers and a PCR product of the correct size as visualized on a 2% TAE, ethidium bromide stained agarose gel are confirmation that the correct inserts had been cloned. Two to six such verified clones are obtained for each knock-out target, and frozen at -70°C for storage.
  • plasmid DNA from these verified clones are pooled, and used in subsequent cloning steps.
  • the sequences of the genes/ORFs are again used to design a second pair of primers which flank the region of H. pylori DNA to be either interrupted or deleted (up to 250 basepairs) within the ORFs but are oriented away from each other.
  • the pool of circular plasmid DNAs of the previously isolated clones are used as templates for this round of PCR. Since the orientation of amplification of this pair of deletion primers is away from each other, the portion of the ORF between the primers is not included in the resultant PCR product.
  • the PCR product is a linear piece of DNA with H.
  • a Kanamycin-resistance cassette (Labigne-Roussel et al, 1988 J. Bacteriology 170, 1704- 1708) is ligated to this PCR product by the TA cloning method used previously (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994).
  • the Kanamycin cassette containing a Campylobacter kanamycin resistance gene is obtained by carrying out an EcoRI digestion of the recombinant plasmid pCTB8:te? (Cover et al.,1994, J. Biological Chemistry 269, pp. 10566-10573).
  • the proper fragment (1.4 kb) is isolated on a 1% TAE gel, and isolated using the QIAquick gel extraction kit (Qiagen, Gaithersburg, MD, USA).
  • the fragment is end repaired using the Klenow fill-in protocol, which involved mixing 4ug of the DNA fragment, 1 microliter of dATP,dGTP, dCTP, dTTP at 0.5 mM, 2 microliter of Klenow Buffer (New England Biolabs) and 5 units of Klenow DNA Polymerase I Large (Klenow) Fragment (New England Biolabs) into a 20 microliter reaction, incubating at 30°C for 15 min, and inactivating the enzyme by heating to 75°C for 10 minutes.
  • This blunt-ended Kanamycin cassette is then purified through a Qiaquick column (Qiagen, Gaithersburg, MD, USA) to eliminate nucleotides.
  • the "T" overhang is then generated by mixing 5 micrograms of the blunt-ended kanamycin cassette, 10 mM Tris pH 8.3, 50 mM KCl, 2 mM MgCl 2 , 5 units of DNA Polymerase (Amplitaq, Roche Molecular
  • the "Kan-T" cassette is purified using a QIAquick column (Qiagen, Gaithersburg, MD, USA).
  • the PCR product of the deletion primers (F2 and R2) is ligated to the Kan-T cassette by mixing 10 to 25 ng of deletion primer PCR product, 50 - 75 ng Kan-T cassette DNA, 1 microliter lOx T4 DNA Ligase reaction mixture, 0.5 microliter T4 DNA Ligase (New England Biolabs, Beverly, MA, USA) in a 10 microliter reaction and incubating for 16 hours at 16°C.
  • the ligation products are transformed into XL-1 Blue or DH5-a E.coli cells by electroporation as described previously. After recovery in SOC, cells are plated onto LB plates containing 100 microgram/ml Ampicillin and grown overnight at 37°C. These plates are then replica plated onto plates containing 25 microgram ml Kanamycin and allowed to grow overnight. Resultant colonies have both the Ampicillin resistance gene present in the pT7Blue vector, and the newly introduced Kanamycin resistance gene. Colonies are picked into LB containing 25 microgram/ml Kanamycin and plasmid DNA is isolated from the cultured cells using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD. USA).
  • Several tests by PCR amplification are conducted on these plasmids to verify that the Kanamycin is inserted in the H. pylori gene/ORF, and to determine the orientation of the insertion of the Kanamycin-resistance gene relative to the H. pylori gene/ORF.
  • the plasmid DNAs are used as templates for PCR amplification with the set of primers originally used to clone the H pylori gene/ORFs.
  • the correct PCR product is the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette.
  • the orientation of the Kanamycin resistance gene with respect to the knock-out gene/ORF is determined and both orientations are eventually used in H pylori transformations (see below).
  • primers are designed from the ends of the kanamycin resistance gene ("Kan-1 " 5'-ATCTTACCTATCACCTCAAAT-3' (SEQ ID NO:207)), and "Kan-2" 5'-AGACAGCAACATCTTTGTGAA-3' (SEQ ID NO:208)).
  • the orientation of the Kanamycin cassette relative to the H. pylori sequence is determined. Positive clones are classified as either in the "A" orientation (the same direction of transcription is present for both the H. pylori gene and the Kanamycin resistance gene), or in the "B" orientation (the direction of transcription for the H. pylori gene is opposite to that of the Kanamycin resistance gene). Clones which share the same orientation (A or B) are pooled for subsequent experiments and independently transformed into H pylori.
  • H. pylori cells Two strains of H pylori are used for transformation: ATCC 55679, the clinical isolate which provided the DNA from which the H. pylori sequence database is obtained, and A ⁇ 244, an isolate which had been passaged in, and has the ability to colonize the mouse stomach.
  • Cells for transformation are grown at 37°C, 10%> CO 2 , 100%) humidity, either on Sheep-Blood agar plates or in Brucella Broth liquid. Cells are grown to exponential phase, and examined microscopically to determine that the cells are "healthy" (actively moving cells) and not contaminated.
  • cells are harvested by scraping cells from the plate with a sterile loop, suspended in 1 ml of Brucella Broth, spun down (1 minute, top speed in eppendorf microfuge) and resuspended in 200 microliters Brucella Broth. If grown in Brucella Broth liquid, cells are centrifuged (15 minutes at 3000 rpm in a Beckman TJ6 centrifuge) and the cell pellet resuspended in 200 microliters of Brucella broth. An aliquot of cells is taken to determine the optical density at 600 nm, in order to calculate the concentration of cells.
  • Cells are then spread onto that plate using a swab wetted in Brucella broth, and grown for 20 hours at 37°C, 6%o CO 2 .
  • Cells are then transferred to a Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin, and allowed to grow for 3 to 5 days at 37°C, 6% CO 2 , 100%) humidity. If colonies appear, they are picked and regrown as patches on a fresh Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin.
  • the template for PCR (DNA from the colony) is obtained by a rapid boiling DNA preparation method as follows. An aliquot of the colony (stab of the colony with a toothpick) is introduced into 100 microliters of 1% Triton X-100, 20 mM Tris, pH 8.5, and boiled for 6 minutes. An equal volume of phenol : chloroform (1 : 1) is added and vortexed. The mixture is microfuged for 5 minutes and the supernatant is used as DNA template for PCR with combinations of the following primers to verify homologous recombination at the proper chromosomal location.
  • TEST 1 PCR with cloning primers originally used to amplify the gene/ORF.
  • a positive result of homologous recombination at the correct chromosomal location should show a single PCR product whose size is expected to be the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette.
  • a PCR product of just the size of the gene/ORF is proof that the gene had not been knocked out and that the transformant is not the result of homologous recombination at the correct chromosome location.
  • TEST 2 PCR with F3 (primer designed from sequences upstream of the gene/ORF and not present on the plasmid), and either primer Kan-1 or Kan-2 (primers designed from the ends of the kanamycin resistance gene), depending on whether the plasmid DNA used was of "A" or "B” orientation. Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the location of F3 to the insertion site of kanamycin resistance gene). No PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out. TEST 3.
  • PCR with R3 primer designed from sequences downstream of the gene/ORF and not present on the plasmid
  • primer Kan-1 or Kan-2 depending on whether the plasmid DNA used was of "A" or "B” orientation.
  • Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the insertion site of kanamycin resistance gene to the downstream location of R3).
  • no PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out.
  • Transformants showing positive results for all three tests above indicate that the gene is not essential for survival in vitro.
  • a negative result in any of the three above tests for each transformant indicates that the gene had not been disrupted, and that the gene is essential for survival in vitro.
  • the plasmid DNA is further analyzed by PCR on DNA from transformant populations prior to plating for colony formation. This will verify that the plasmid can enter the cells and undergo homologous recombination at the correct site. Briefly, plasmid DNA is incubated according to the transformation protocol described above. DNA is extracted from the H.
  • TEST 2 and TEST 3 Positive results in TEST 2 and TEST 3 would verify that the plasmid DNA could enter the cells and undergo homologous recombination at the correct chromosomal location. If TEST
  • H. pylori target gene and its protein product e.g., an H. pylori enzyme
  • the assay is essentially as described by Fisher (Fischer, G., et.al. (1984) Biomed. Biochim. Ada 43:1101-1111).
  • the assay measures the cis-trans isomerization of the Ala-Pro bond in the test peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma # S- 7388, lot # 84H5805).
  • the assay is coupled with ⁇ -chymotrypsin, where the ability of the protease to cleave the test peptide occurs only when the Ala-Pro bond is in trans.
  • test peptide The conversion of the test peptide to the trans isomer in the assay is followed at 390 nm on a Beckman Model DU-650 spectophotometer. The data are collected every second with an average scanning of time of 0.5 second. Assays are carried out in 35 mM Hepes, pH 8.0, in a final volume of 400 ul, with 10 ⁇ M ⁇ -chymotrypsin (type 1-5 from bovine Pancreas, Sigma # C-7762, lot 23H7020) and 10 nM PPIase. To initiate the reaction. 10 ⁇ l of the substrate ( 2 mM N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in DMSO) is added to 390 ⁇ l of reaction mixture at room temperature.
  • substrate 2 mM N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in DMSO
  • a 50 ml culture of Helicobacter pylori (strain J99) in Brucella broth is harvested at mid-log phase (OD oo nm ⁇ 1 ) an d resuspended in lysis buffer with the following protease inhibitors: 1 mM PMSF, and 10 ⁇ g/ml of each of aprotinin, leupeptin, pepstatine, TLCK, TPCK, and soybean trypsin inhibitor.
  • the suspension is subjected to 3 cycles of freeze-thaw (15 minutes at -70 C, then 30 minutes at room temperature), followed by sonication (three 20 second bursts).
  • the lysate is centrifuged (12,000 g x 30 minutes) and the supernatant is assayed for enzymatic activity as described above.
  • H. pylori enzymes can be expressed at high levels and in an active form in E. coli. Such high yields of purified proteins provide for the design of various high throughput drug screening assays.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • ORGANISM Helicobacter pylori
  • FEATURE FEATURE
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • ORGANISM Helicobacter pylori
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Helicobacter pylori
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Helicobacter pylori
  • FEATURE FEATURE
  • CTAGGTGCTA CTGCTCCCTT AATGGCAAAG CCTTTATTAA GCGATGAAGA CTTATTGAAA 120
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Helicobacter pylori
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Helicobacter pylori
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Helicobacter pylori
  • TTCTTGCCTT ATAATTTAAA TAATGTTAAG CTTAGTTTTA CAGACGCTCA AGGCAATGTG 360 ATCGATCTAG GCGTGATAGA GACTATCCCC AAACACTCTA AGATTGTTTT GCCCGGAGAG 420 GCATTTGATA GTCTAAAAAT TGACCCCTAT ACTTTATTTC TTCCAAAAAT TGAAGCCACT 480
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • GATTTAGGTA AACAAGTTTA TGCACCTAAT AAAATCCAGT TGGATATGGT CTCTTGGGGT 420 GTGGGGAGCG ATTTGTTAGC TGATATTATT GATAAAGACA ACGCTTCTTT TGGTATTTTT 480
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ORGANISM Helicobacter pylori
  • GACTACGCTC ATGCCAATTC TATTAAGCTT AAAAACCCTA ACTATAATAG CGAAGCGGCG 300 CAAGTGGCTA GTCAAATTCT TGGGAAACAA GAAATCAATC GTTTAACAAA CATTGCCGAT 360
  • ORGANISM Helicobacter pylori
  • GAAAGCGCTA CAACGCAAAT AAACGCCAAT AAGCAAGAAG CAATAAATAA CATCACGCAA 720
  • ORGANISM Helicobacter pylori
  • CTCATCAATC AAAACGCCTT GCCGATCAAC TACGCTAACT TGGGGAGTCA AACAAACTAC 1020
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • ORGANISM Helicobacter pylori

Abstract

Recombinant or substantially pure preparations of H. pylori polypeptides are described. The nucleic acids encoding the polypeptides also are described. The H. pylori polypeptides are useful for diagnostics and vaccine compositions.

Description

NUCLEIC ACID AND AMINO ACID SEQUENCES RELATING TO HELICOBACTER PYLORI AND VACCINE COMPOSITIONS THEREOF
Background of the Invention Helicobacter pylori is a gram-negative, S-shaped, microaerophilic bacterium that was discovered and cultured from a human gastric biopsy specimen. (Warren, J.R. and B. Marshall, (1983) Lancet \ : 1273-1275; and Marshall et al., (1984) Microbios Lett. 25: 83-88). H. pylori has been strongly linked to chronic gastritis and duodenal ulcer disease. (Rathbone et. al.. (1986) Gut 27_: 635-641). Moreover, evidence is accumulating for an etiologic role of /, pylori in nonulcer dyspepsia, gastric ulcer disease, and gastric adenocarcinoma. (Blaser M. J., (1993) Trends Microbiol J_: 255- 260). Transmission of the bacteria occurs via the oral route, and the risk of infection increases with age. (Taylor, D.N. and M. J. Blaser, (1991) Epidemiol. Rev 3: 42-50). H. pylori colonizes the human gastric mucosa, establishing an infection that usually persists for decades. Infection by H. pylori is prevalent worldwide. Developed countries have infection rates over 50% of the adult population, while developing countries have infection rates reaching 90% of the adults over the age of 20. (Hopkins R. J. and J. G. Morris (1994) Am. J. Med. 97: 265-277).
The bacterial factors necessary for colonization of the gastric environment, and for virulence of this pathogen, are poorly understood. Examples of the putative virulence factors include the following: urease, an enzyme that may play a role in neutralizing gastric acid pH (Eaton et al., ( 1991 ) Infect. Immunol. 59: 2470-2475; Ferrero, R.L. and A. Lee ( 1991 ) Microb. Ecol Hlth. Dis. 4: 121-134; Labigne et al., (1991 ) J. Bacterial. 173: 1920-1931); the bacterial flagellar proteins responsible for motility across the mucous layer. (Hazell et al., (1986) J. Inf. Dis. 153: 658-663; Leying et al., (1992) Mol Microbiol. 6: 2863-2874; and Haas et al., (1993) Mol Microbiol 8: 753-760); Vac A, a bacterial toxin that induces the formation of intracellular vacuoles in epithelial cells (Schmitt, W. and R. Haas, (1994) Molecular Microbiol. 12(2): 307-319); and several gastric tissue-specific adhesins. (Boren et al., (1993) Science 262: 1892- 1895; Evans et al., (1993) J. Bacteriol L75: 674-683; and Falk et al., (1993) Proc. Natl. Acad. Sci. USA 90: 2035-203).
Numerous therapeutic agents are currently available that eradicate H. pylori infections in vitro. (Huesca et. al., (1993) Zbl Bakt. 280: 244-252; Hopkins, R. J. and J. G. Morris, supra). However, many of these treatments are suboptimally effective in vivo because of bacterial resistance, altered drug distribution, patient non-compliance or poor drug availabilty. (Hopkins, R. J. and j. G. Morris, supra). Treatment with antibiotics combined with bismuth are part of the standard regime used to treat H. pylori infection. (Malfertheiner. P. and J. E. Dominguez-Munoz (1993) Clinical Therapeutics 5 Supp. B: 37-48). Recently, combinations of a proton pump inhibitors and a single antibiotic have been shown to ameliorate duodenal ulcer disease. (Malfertheiner, P. and J. E. Dominguez-Munoz supra). However, methods employing antibiotic agents can have the problem of the emergence of bacterial strains which are resistant to these agents.
(Hopkins, R. J. and J. G. Morris, supra). These limitations demonstrate that new more effective methods are needed to combat H. pylori infections in vivo. In particular, the design of new vaccines that may prevent infection by this bacterium is highly desirable.
Summary of the Invention
This invention relates to novel genes, e.g., genes encoding polypeptides such as bacterial surface proteins, from the organism Helicobacter pylori (H. pylori), and other related genes, their products, and uses thereof. The nucleic acids and peptides of the present invention have utility for diagnostic and therapeutics for H. pylori and other Helicobacter species. They can also be used to detect the presence of//, pylori and other Helicobacter species in a sample; and for use in screening compounds for the ability to interfere with the H. pylori life cycle or to inhibit H. pylori infection. More specifically, this invention features compositions of nucleic acids corresponding to entire coding sequences of H. pylori proteins, including surface or secreted proteins or parts thereof, nucleic acids capable of binding mRNA from H. pylori proteins to block protein translation, and methods for producing H. pylori proteins or parts thereof using peptide synthesis and recombinant DNA techniques. This invention also features antibodies and nucleic acids useful as probes to detect H. pylori infection. In addition, vaccine compositions and methods for the protection or treatment of infection by //. pylori are within the scope of this invention.
Detailed Description of the Drawings
Figure 1 is a bar graph that depicts the antibody titer in serum of mice following immunization with specific H. pylori antigens. Figure 2 is a bar graph that depicts the antibody titer in mucous of mice following immunization with specific H. pylori antigens.
Figure 3 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in HEPES buffer.
Figure 4 is a bar graph that depicts therapeutic immunization of H. pylori infected mice with specific antigens dissolved in buffer containing DOC. - :> -
Figure 5 depicts the amino acid sequence alignment in a portion of the sequence of five H. pylori proteins (depicted in the single letter amino acid code; shown N- terminal to C-terminal, left to right).
Figure 6 depicts the amino acid sequence alignment in a portion of the sequence of four H. pylori proteins (depicted in the single letter amino acid code; shown N- terminal to C-terminal, left to right).
Figure 7 depicts the amino acid sequence alignment in a portion of the sequence of two H. pylori proteins (depicted in the single letter amino acid code; shown N- terminal to C-terminal, left to right). Figure 8 depicts the amino acid sequence alignment in a portion of the sequence of two H. pylori proteins (depicted in the single letter amino acid code; shown N- terminal to C-terminal, left to right).
Detailed Description of the Invention In one aspect, the invention features a recombinant or substantially pure preparation of //, pylori polypeptide of SEQ ID NO: 74. The invention also includes substantially pure nucleic acid encoding an H. pylori polypeptide of SEQ ID NO: 74, such nucleic acid is contained in SEQ ID NO: 1. The H. pylori polypeptide sequences of the invention described herein are contained in the Sequence Listing, and the nucleic acids encoding H. pylori polypeptides of the invention are contained in the Sequence Listing.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 75, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 2. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 76, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 3.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 77, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 4.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 78, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 5.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 79, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 6. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 80, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 7.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 81, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 8.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 82, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 9. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 83, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 10.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 84, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 11.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 85, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 12.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 86, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 13.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 87, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 14. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 88, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 15.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 89, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 16.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 90, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 17.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 91 , such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 18. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 92, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 19.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 93, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 20.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 94, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 21. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 95, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 22.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 96, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 23.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 97, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 24.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 98, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 25.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 99, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 26. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 100, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 27.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 101, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 28.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 102, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 29.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 103, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 30. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 104, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 31.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 105, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 32.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 106, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 33. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 107, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 34.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 108, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 35.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 109, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 36.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 110, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 37.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 11, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 38. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 12, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 39.
In another aspect, the invention features a substantially pure nucleic acid encoding an H pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 13, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 40.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 14, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 41.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 115, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 42. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 16, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO:43.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 117, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 44.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 18, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 45. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 1 19, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 46.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 120, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 47.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 121, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 48.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 122, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 49.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 123, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 50. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 124, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 51.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 125, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 52.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 126, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 53.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 127, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 54. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 128, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 55.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 129, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 56.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 130, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 57. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 131, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 58.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 132, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 59.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 133, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 60.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 134, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 61.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 135, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 62. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 136, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 63.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 137, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 64.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 138, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 65.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 139, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 66. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 140, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 67.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 141 , such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 68.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 142, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 69. In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 143, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 70.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 144, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 71.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 145, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 72.
In another aspect, the invention features a substantially pure nucleic acid encoding an H. pylori polypeptide having an amino acid sequence of SEQ ID NO: 146, such as a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 73.
Particularly perferred is an isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori cell envelope polypeptide or a fragment thereof. Such nucleic acid is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 48, SEQ ID NO: 16, SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 7, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 1 1 , SEQ ID NO: 71, SEQ ID NO: 17, SEQ ID NO: 57, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 21.
In another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 25, and SEQ ID NO: 48. In another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 7, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 1 1, and SEQ ID NO: 71.
In another embodiment, the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C- terminal tyrosine cluster or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 42, SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 1 1, and SEQ ID NO:71.
In yet another embodiment, the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof encoded by the nucleic acid selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 45, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 7, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58.
Particularly preferred is an isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori secreted polypeptide or a fragment thereof. Such nucleic acid is selected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 32, SEQ ID NO: 51, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40. SEQ ID NO: 41 , SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68.
Particularly preferred is an isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori cellular polypeptide or a fragment thereof. Such nucleic acid is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 73.
Particularly preferred is a purified or isolated H. pylori cell envelope polypeptide or a fragment thereof, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 98, SEQ ID NO: 121, SEQ ID NO: 89, SEQ ID NO: 83, SEQ ID NO: 1 18, SEQ ID NO: 108, SEQ ID NO: 1 10, SEQ ID NO: 80, SEQ ID NO: 1 12, SEQ ID NO: 128, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 74, SEQ ID NO: 1 15, SEQ ID NO: 87, SEQ ID NO: 1 16, SEQ ID NO: 84, SEQ ID NO: 144, SEQ ID NO: 90, SEQ ID NO: 130, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 81, and SEQ ID NO: 94. In another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 98, and SEQ ID NO: 121.
In another embodiment, the H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 83, SEQ ID NO: 1 18, SEQ ID NO: 108, SEQ ID NO: 1 10, SEQ ID NO: 80, SEQ ID NO: 1 12, SEQ ID NO: 128, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 101 , SEQ ID NO: 103, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131 , SEQ ID NO: 74. SEQ ID NO: 115, SEQ ID NO: 87, SEQ ID NO: 1 16, SEQ ID NO: 84, SEQ ID NO: 144, SEQ ID NO: 90, and SEQ ID NO: 130.
In another embodiment, the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C- terminal tyrosine cluster or a fragment thereof selected from the group consisting of SEQ ID NO: 74, SEQ ID NO: 1 15, SEQ ID NO: 87, SEQ ID NO: 1 16. SEQ ID NO: 84, and SEQ ID NO: 144.
In another embodiment, the H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof selected from the group consisting of SEQ ID NO: 89. SEQ ID NO: 118, SEQ ID NO: 108, SEQ ID NO: 1 10, SEQ ID NO: 80, SEQ ID NO: 1 12, SEQ ID NO: 128, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131.
Particularly preferred is a purified or isolated H. pylori secreted polypeptide or a fragment thereof, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 145, SEQ ID NO: 105, SEQ ID NO: 124, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 95, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 1 1 1, SEQ ID NO: 113, SEQ ID NO: 1 14, SEQ ID NO: 1 17, SEQ ID NO: 1 19, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141. Particularly preferred is a purified or isolated H. pylori cellular polypeptide or a fragment thereof, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 93, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 100. SEQ ID NO: 120, SEQ ID NO: 123, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 146.
In another aspect, the invention pertains to any individual H. pylori polypeptide member or nucleic acid encoding such a member from the above-identified groups of//. pylori polypeptides.
In another aspect, the invention features nucleic acids capable of binding mRNA of //, pylori. Such nucleic acid is capable of acting as antisense nucleic acid to control the translation of mRNA of H. pylori. A further aspect features a nucleic acid which is capable of binding specifically to an H pylori nucleic acid. These nucleic acids are also referred to herein as complements and have utility as probes and as capture reagents.
In another aspect, the invention features an expression system comprising an open reading frame corresponding to H. pylori nucleic acid. The nucleic acid further comprises a control sequence compatible with an intended host. The expression system is useful for making polypeptides corresponding to H pylori nucleic acid. In another aspect, the invention features a cell transformed with the expression system to produce H. pylori polypeptides.
In another aspect, the invention features a method of generating antibodies against H. pylori polypeptides which are capable of binding specifically to H pylori polypeptides. Such antibodies have utility as reagents for immunoassays to evaluate the abundance and distribution of , antigens.
In another aspect, the invention features a method of generating vaccines for immunizing an individual against H pylori. The vaccination method includes: immunizing a subject with at least one H pylori polypeptide according to the present invention, e.g., a surface or secreted polypeptide, or active portion thereof, and a pharmaceutically acceptable carrier. Such vaccines have therapeutic and/or prophylactic utilities.
In another aspect, the invention provides a method for generating a vaccine comprising a modified immunogenic H. pylori polypeptide, e.g., a surface or secreted polypeptide, or active portion thereof, and a pharmacologically acceptable carrier. In another aspect, the invention features a method of evaluating a compound, e.g. a polypeptide, e.g., a fragment of a host cell polypeptide, for the ability to bind an H. pylori polypeptide. The method includes: contacting the candidate compound with an H pylori polypeptide and determining if the compound binds or otherwise interacts with an H. pylori polypeptide. Compounds which bind H. pylori are candidates as activators or inhibitors of the bacterial life cycle. These assays can be performed in vitro or in vivo. In another aspect, the invention features a method of evaluating a compound, e.g. a polypeptide, e.g., a fragment of a host cell polypeptide, for the ability to bind an H. pylori nucleic acid, e.g., DNA or RNA. The method includes: contacting the candidate compound with an H pylori nucleic acid and determining if the compound binds or otherwise interacts with an H. pylori polypeptide. Compounds which bind H pylori are candidates as activators or inhibitors of the bacterial life cycle. These assays can be performed in vitro or in vivo.
The invention features H. pylori polypeptides, preferably a substantially pure preparation of an H. pylori polypeptide, or a recombinant H. pylori polypeptide. In preferred embodiments: the polypeptide has biological activity; the polypeptide has an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical or homologous to an amino acid sequence of the invention contained in the Sequence Listing, preferably it has about 65% sequence identity with an amino acid sequence of the invention contained in the Sequence Listing, and most preferably it has about 92% to about 99% sequence identity with an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the Sequence Listing; the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acid residues in length; the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acid residues of the invention contained in the Sequence Listing. In yet another preferred embodiment, the amino acid sequence which differs in sequence identity by about 7% to about 8% from the H. pylori amino acid sequences of the invention contained in the Sequence Listing is also encompassed by the invention. In preferred embodiments: the H pylori polypeptide is encoded by a nucleic acid of the invention contained in the Sequence Listing, or by a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a nucleic acid of the invention contained in the Sequence Listing.
In a preferred embodiment, the subject H. pylori polypeptide differs in amino acid sequence at 1 , 2, 3, 5, 10 or more residues from a sequence of the invention contained in the Sequence Listing. The differences, however, are such that the H. pylori polypeptide exhibits an H. pylori biological activity, e.g., the H. pylori polypeptide retains a biological activity of a naturally occurring H. pylori polypeptide.
In preferred embodiments, the polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' or 3' to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.
In yet other preferred embodiments, the H. pylori polypeptide is a recombinant fusion protein having a first H. pylori polypeptide portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to
H. pylori. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
Polypeptides of the invention include those which arise as a result of alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events.
The invention also encompasses an immunogenic component which includes at least one H. pylori polypeptide in an immunogenic preparation; the immunogenic component being capable of eliciting an immune response specific for the H. pylori polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogenic component comprises at least one antigenic determinant from a polypeptide of the invention contained in the Sequence Listing.
In another aspect, the invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an H. pylori polypeptide. In preferred embodiments: the encoded polypeptide has biological activity; the encoded polypeptide has an amino acid sequence at least 60%), 70%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence of the invention contained in the Sequence
Listing; the encoded polypeptide has an amino acid sequence essentially the same as an amino acid sequence of the invention contained in the Sequence Listing; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded polypeptide comprises at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids of the invention contained in the Sequence Listing.
In preferred embodiments: the nucleic acid of the invention is that contained in the Sequence Listing; the nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or
99%o homologous with a nucleic acid sequence of the invention contained in the
Sequence Listing.
In a preferred embodiment, the encoded H. pylori polypeptide differs (e.g., by amino acid substitution, addition or deletion of at least one amino acid residue) in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence of the invention contained in the Sequence Listing. The differences, however, are such that: the H. pylori encoded polypeptide exhibits a H. pylori biological activity, e.g., the encoded H. pylori enzyme retains a biological activity of a naturally occurring H. pylori.
In preferred embodiments, the encoded polypeptide includes all or a fragment of an amino acid sequence of the invention contained in the Sequence Listing; fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' or 3' to the genomic DNA which encodes a sequence of the invention contained in the Sequence Listing.
In preferred embodiments, the subject H. pylori nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the H. pylori gene sequence, e.g., to render the H. pylori gene sequence suitable for expression in a recombinant host cell.
In yet a further preferred embodiment, the nucleic acid which encodes an H. pylori polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 8 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 12 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 20 consecutive nucleotides of the invention contained in the Sequence Listing; more preferably to at least 40 consecutive nucleotides of the invention contained in the Sequence Listing.
In a preferred embodiment, the nucleic acid encodes a peptide which differs by at least one amino acid residue from the sequences of the invention contained in the Sequence Listing.
In a preferred embodiment, the nucleic acid differs by at least one nucleotide from a nucleotide sequence of the invention contained in the Sequence Listing which encodes amino acids of the invention contained in the Sequence Listing. In another aspect, the invention encompasses: a vector including a nucleic acid which encodes an H. pylori polypeptide or an H pylori polypeptide variant as described herein; a host cell transfected with the vector; and a method of producing a recombinant H. pylori polypeptide or H. pylori polypeptide variant; including culturing the cell, e.g., in a cell culture medium, and isolating the H. pylori or H. pylori polypeptide variant, e.g., from the cell or from the cell culture medium.
In another aspect, the invention features, a purified recombinant nucleic acid having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a sequence of the invention contained in the Sequence Listing.
The invention also provides a probe or primer which includes a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 8 consecutive nucleotides of sense or antisense sequence of the invention contained in the Sequence Listing, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 8 and less than 10, 20, 30, 50, 100, or 150 nucleotides in length.
The invention also provides an isolated H. pylori polypeptide which is encoded by a nucleic acid which hybridizes under stringent hybridization conditions to a nucleic acid contained in the Sequence Listing.
The invention further provides nucleic acids, e.g., RNA or DNA, encoding a polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
The H. pylori strain, from which genomic sequences have been sequenced, has been deposited in the American Type Culture Collection (ATCC # 55679; deposited by Genome Therapeutics Corporation, 100 Beaver Street, Waltham, MA 02154) as strain HP-J99.
Included in the invention are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid which encodes a polypeptide of the invention contained in the Sequence Listing (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3.1 - 6.3.6 and 6.4.1-6.4.10, hereby incorporated by reference); and, polypeptides specifically bound by antisera to H. pylori polypeptides, especially by antisera to an active site or binding domain of//. pylori polypeptide. The invention also includes fragments, preferably biologically active fragments. These and other polypeptides are also referred to herein as H. pylori polypeptide analogs or variants.
Putative functions have been determined for several of the H. pylori polypeptides of the invention, as shown in Table 1.
Accordingly, uses of the claimed H. pylori polypeptides based on these identified functions, as well as other functions as described herein, are also within the scope of the invention.
In addition, the present invention encompasses H. pylori polypeptides characterized as shown in Table 1 below, including: H. pylori cell envelope proteins, H. pylori secreted proteins, and H. pylori cellular proteins. Members of these groups were identified by BLAST homology searches and by searches for secretion signal or transmembrane protein motifs. Polypeptides related by significant homology to the polypeptides of Table 1 are also considered to be classified in the manner of the homologs shown in Table 1. TABLE 1
[In Table 1, "nt" represents nucleotide Seq. ID number and "aa" represents amino acid Seq. ID number]
Definitions
The terms "purified polypeptide" and "isolated polypeptide" and "a substantially pure preparation of a polypeptide" are used interchangeably herein and. as used herein, from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. Preferably, the polypeptide constitutes at least 10, 20, 50 70, 80 or 95%> dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 μg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide. Furthermore, the terms "purified polypeptide" and "isolated polypeptide" and "a substantially pure preparation of a polypeptide," as used herein, refer to both a polypeptide obtained from nature or produced by recombinant DNA techniques as described herein. For example, an "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the H pylori protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of//, pylori protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of H. pylori protein having less than about 30%) (by dry weight) of non-H. pylori protein (also referred to herein as a "contaminating protein"), more preferably less than about 20%) of non-H. pylori protein, still more preferably less than about 10% of non-H. pylori protein, and most preferably less than about 5%> non-H. pylori protein. When the H pylori protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%>, and most preferably less than about 5%> of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals" includes preparations of H pylori protein in which the protein is separated from chemical precusors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of H pylori protein having less than about 30%> (by dry weight) of chemical precursors or non-H. pylori chemicals, more preferably less than about 20%) chemical precursors or non-H. pylori chemicals, still more preferably less than about 10%> chemical precursors or non-H. pylori chemicals, and most preferably less than about 5% chemical precursors or non-H pylori chemicals. A purified preparation of cells refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10%> and more preferably 50%) of the subject cells.
A purified or isolated or a substantially pure nucleic acid, e.g., a substantially pure DNA, (are terms used interchangeably herein) is a nucleic acid which is one or both of the following: not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA also includes a recombinant DNA which is part of a hybrid gene encoding additional H. pylori DNA sequence.
A "contig" as used herein is a nucleic acid representing a continuous stretch of genomic sequence of an organism.
An "open reading frame", also referred to herein as ORF, is a region of nucleic acid which encodes a polypeptide. This region may represent a portion of a coding sequence or a total sequence and can be determined from a stop to stop codon or from a start to stop codon.
As used herein, a "coding sequence" is a nucleic acid which is transcribed into messenger RNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the five prime terminus and a translation stop code at the three prime terminus. A coding sequence can include but is not limited to messenger RNA, synthetic DNA, and recombinant nucleic acid sequences.
A "complement" of a nucleic acid as used herein referes to an anti-parallel or antisense sequence that participates in Watson-Crick base-pairing with the original sequence.
A "gene product" is a protein or structural RNA which is specifically encoded by a gene.
As used herein, the term "probe" refers to a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest. Probes are often associated with or capable of associating with a label. A label is a chemical moiety capable of detection. Typical labels comprise dyes, radioisotopes, luminescent and chemiluminescent moieties, fluorophores, enzymes, precipitating agents, amplification sequences, and the like. Similarly, a nucleic acid, peptide or other chemical entity which specifically binds to a molecule of interest and immobilizes such molecule is referred herein as a "capture ligand". Capture ligands are typically associated with or capable of associating with a support such as nitro-cellulose, glass, nylon membranes, beads, particles and the like. The specificity of hybridization is dependent on conditions such as the base pair composition of the nucleotides, and the temperature and salt concentration of the reaction. These conditions are readily discernable to one of ordinary skill in the art using routine experimentation.
Homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60%) homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50%> homology. Generally, a comparison is made when two sequences are aligned to give maximum homology. Nucleic acids are hybridizable to each other when at least one strand of a nucleic acid can anneal to the other nucleic acid under defined stringency conditions. Stringency of hybridization is determined by: (a) the temperature at which hybridization and/or washing is performed; and (b) the ionic strength and polarity of the hybridization and washing solutions. Hybridization requires that the two nucleic acids contain complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated. Typically, hybridization of two sequences at high stingency (such as, for example, in a solution of 0.5X SSC, at 65° C) requires that the sequences be essentially completely homologous. Conditions of intermediate stringency (such as, for example, 2X SSC at 65 ° C) and low stringency (such as, for example 2X SSC at 55° C), require correspondingly less overall complementarity between the hybridizing sequences. (IX SSC is 0.15 M NaCl, 0.015 M Na citrate). A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C. The terms peptides, proteins, and polypeptides are used interchangeably herein. As used herein, the term "surface protein" refers to all surface accessible proteins, e.g. inner and outer membrane proteins, proteins adhering to the cell wall, and secreted proteins.
A polypeptide has H pylori biological activity if it has one, two and preferably more of the following properties: (1) if when expressed in the course of an H pylori infection, it can promote, or mediate the attachment of H pylori to a cell; (2) it has an enzymatic activity, structural or regulatory function characteristic of an H pylori protein; (3) the gene which encodes it can rescue a lethal mutation in an H. pylori gene; (4) or it is immunogenic in a subject. A polypeptide has biological activity if it is an antagonist, agonist, or super-agonist of a polypeptide having one of the above-listed properties.
A biologically active fragment or analog is one having an in vivo or in vitro activity which is characteristic of the H pylori polypeptides of the invention contained in the Sequence Listing, or of other naturally occurring H. pylori polypeptides, e.g., one or more of the biological activities described herein. Especially preferred are fragments which exist in vivo, e.g., fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's. Fragments include those expressed in native or endogenous cells as well as those made in expression systems, e.g., in CΗO cells. Because peptides such as H. pylori polypeptides often exhibit a range of physiological properties and because such properties may be attributable to different portions of the molecule, a useful H pylori fragment or H pylori analog is one which exhibits a biological activity in any biological assay for H pylori activity. Most preferably the fragment or analog possesses 10%>, preferably 40%>, more preferably 60%>, 70%), 80%) or 90%) or greater of the activity of H. pylori, in any in vivo or in vitro assay. Analogs can differ from naturally occurring H. pylori polypeptides in amino acid sequence or in ways that do not involve sequence, or both. Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation. Preferred analogs include H. pylori polypeptides (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not substantially diminish the biological activity of the H pylori polypeptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be made in view of the table below. TABLE 2
CONSERVATIVE AMINO ACID REPLACEMENTS
Other analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids; and cyclic analogs.
As used herein, the term "fragment", as applied to an H pylori analog, will ordinarily be at least about 20 residues, more typically at least about 40 residues, preferably at least about 60 residues in length. Fragments of H pylori polypeptides can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of H pylori polypeptide can be assessed by methods known to those skilled in the art as described herein. Also included are H. pylori polypeptides containing residues that are not required for biological activity of the peptide or that result from alternative mRNA splicing or alternative protein processing events.
An "immunogenic component" as used herein is a moiety, such as an H pylori polypeptide, analog or fragment thereof, that is capable of eliciting a humoral and/or cellular immune response in a host animal alone or in combination with an adjuvant.
An "antigenic component" as used herein is a moiety, such as an H. pylori polypeptide, analog or fragment thereof, that is capable of binding to a specific antibody with sufficiently high affinity to form a detectable antigen-antibody complex.
As used herein, the term "transgene" means a nucleic acid (encoding, e.g., one or more polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the cell's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
As used herein, the term "transgenic cell" refers to a cell containing a transgene.
As used herein, a "transgenic animal" is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by a process of transformation of competent cells or by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
The term "antibody" as used herein is intended to include fragments thereof which are specifically reactive with H pylori polypeptides. As used herein, the term "cell-specific promoter" means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue. The term also covers so-called "leaky" promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
Misexpression, as used herein, refers to a non-wild type pattern of gene expression. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post- transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
As used herein, "host cells" and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refers to cells which can become or have been used as recipients for a recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood by individuals skilled in the art that the progeny of a single parental cell may not necessarily be completely identical in genomic or total DNA compliment to the original parent, due to accident or deliberate mutation.
As used herein, the term "control sequence" refers to a nucleic acid having a base sequence which is recognized by the host organism to effect the expression of encoded sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include a promoter, ribosomal binding site, terminators, and in some cases operators; in eukaryotes, generally such control sequences include promoters, terminators and in some instances, enhancers. The term control sequence is intended to include at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences.
As used herein, the term "operably linked" refers to sequences joined or ligated to function in their intended manner. For example, a control sequence is operably linked to coding sequence by ligation in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence and host cell.
The metabolism of a substance, as used herein, means any aspect of the, expression, function, action, or regulation of the substance. The metabolism of a substance includes modifications, e.g., covalent or non-covalent modifications of the substance. The metabolism of a substance includes modifications, e.g., covalent or non- covalent modification, the substance induces in other substances. The metabolism of a substance also includes changes in the distribution of the substance. The metabolism of a substance includes changes the substance induces in the distribution of other substances.
A "sample" as used herein refers to a biological sample, such as, for example, tissue or fluid isloated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment. The practice of the invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning; Laboratory Manual 2nd ed. (1989); DNA Cloning, Volumes I and II (D.N Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed, 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); the series, Methods in Enzymoloqy (Academic Press, Inc.), particularly Vol. 154 and Vol. 155 (Wu and Grossman, eds.) and PCR-A Practical Approach (McPherson, Quirke, and Taylor, eds., 1991).
I. Isolation of Nucleic Acids of H. pylori and Uses Therefor
H. pylori Genomic Sequence
This invention provides nucleotide sequences of the genome of H. pylori which thus comprises a DNA sequence library of H. pylori genomic DNA. The detailed description that follows provides nucleotide sequences of H. pylori, and also describes how the sequences were obtained and how ORFs and protein-coding sequences were identified. Also described are methods of using the disclosed H. pylori sequences in methods including diagnostic and therapeutic applications. Furthermore, the library can be used as a database for identification and comparison of medically important sequences in this and other strains of H. pylori. To determine the genomic sequence of H. pylori. DNA was isolated from a strain of H pylori (ATCC # 55679; deposited by Genome Therapeutics Corporation, 100 Beaver Street, Waltham, MA 02154) and mechanically sheared by nebulization to a median size of 2 kb. Following size fractionation by gel electrophoresis, the fragments were blunt-ended, ligated to adapter oligonucleotides, and cloned into each of 20 different pMPX vectors (Rice et al., abstracts of Meeting of Genome Mapping and
Sequencing, Cold Spring Harbor, NY, 5/1 1-5/15, 1994, p. 225) to construct a series of "shotgun" subclone libraries.
DNA sequencing was achieved using multiplex sequencing procedures essentially as disclosed in Church et al., 1988, Science 240:185; U.S. Patents No. 4,942,124 and 5,149,625). DNA was extracted from pooled cultures and subjected to chemical or enzymatic sequencing. Sequencing reactions were resolved by electrophoresis, and the products were transferred and covalently bound to nylon membranes. Finally, the membranes were sequentially hybridized with a series of labelled oligonucleotides complimentary to "tag" sequences present in the different shotgun cloning vectors. In this manner, a large number of sequences could be obtained from a single set of sequencing reactions. The cloning and sequencing procedures are described in more detail in the Exemplification.
Individual sequence reads obtained in this manner were assembled using the FALCON™ program (Church et al, 1994, Automated DNA Sequencing and Analysis, J.C. Venter, ed., Academic Press) and PHRAP (P. Green, Abstracts of DOE Human Genome Program Contractor-Grantee Workshop V, Jan. 1996, p.157). The average contig length was about 3-4 kb.
A variety of approaches are used to order the contigs so as to obtain a continuous sequence representing the entire H. pylori genome. Synthetic oligonucleotides are designed that are complementary to sequences at the end of each contig. These oligonucleotides may be hybridized to libaries of H. pylori genomic DNA in, for example, lambda phage vectors or plasmid vectors to identify clones that contain sequences corresponding to the junctional regions between individual contigs. Such clones are then used to isolate template DNA and the same oligonucleotides are used as primers in polymerase chain reaction (PCR) to amplify junctional fragments, the nucleotide sequence of which is then determined. The H. pylori sequences were analyzed for the presence of open reading frames (ORFs) comprising at least 180 nucleotides. As a result of the analysis of ORFs based on stop-to-stop codon reads, it should be understood that these ORFs may not correspond to the ORF of a naturally-occurring H. pylori polypeptide. These ORFs may contain start codons which indicate the initiation of protein synthesis of a naturally- occurring H. pylori polypeptide. Such start codons within the ORFs provided herein can be identified by those of ordinary skill in the relevant art, and the resulting ORF and the encoded H. pylori polypeptide is within the scope of this invention. For example, within the ORFs a codon such as AUG or GUG (encoding methionine or valine) which is part of the initiation signal for protein synthesis can be identified and the ORF modified to correspond to a naturally-occurring H pylori polypeptide. The predicted coding regions were defined by evaluating the coding potential of such sequences with the program GENEMARK™ (Borodovsky and Mclninch, 1993, Comp. Chem. J :123).
Other H pylori Nucleic Acids
The nucleic acids of this invention may be obtained directly from the DNA of the above referenced H. pylori strain by using the polymerase chain reaction (PCR). See "PCR, A Practical Approach" (McPherson, Quirke, and Taylor, eds., IRL Press, Oxford, UK, 1991) for details about the PCR. High fidelity PCR can be used to ensure a faithful DNA copy prior to expression. In addition, the authenticity of amplified products can be checked by conventional sequencing methods. Clones carrying the desired sequences described in this invention may also be obtained by screening the libraries by means of the PCR or by hybridization of synthetic oligonucleotide probes to filter lifts of the library colonies or plaques as known in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd edition, 1989, Cold Spring Harbor Press, NY).
It is also possible to obtain nucleic acids encoding H. pylori polypeptides from a cDNA library in accordance with protocols herein described. A cDNA encoding an H. pylori polypeptide can be obtained by isolating total mRNA from an appropriate strain. Double stranded cDNAs can then be prepared from the total mRNA. Subsequently, the cDNAs can be inserted into a suitable plasmid or viral (e.g., bacteriophage) vector using any one of a number of known techniques. Genes encoding H. pylori polypeptides can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention. The nucleic acids of the invention can be DNA or RNA. Preferred nucleic acids of the invention are contained in the Sequence Listing.
The nucleic acids of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein). Nucleic acids isolated or synthesized in accordance with features of the present invention are useful, by way of example, without limitation, as probes, primers, capture ligands. antisense genes and for developing expression systems for the synthesis of proteins and peptides corresponding to such sequences. As probes, primers, capture ligands and antisense agents, the nucleic acid normally consists of all or part (approximately twenty or more nucleotides for specificity as well as the ability to form stable hybridization products) of the nucleic acids of the invention contained in the Sequence Listing. These uses are described in further detail below. Probes A nucleic acid isolated or synthesized in accordance with the sequence of the invention contained in the Sequence Listing can be used as a probe to specifically detect H pylori. With the sequence information set forth in the present application, sequences of twenty or more nucleotides are identified which provide the desired inclusivity and exclusivity with respect to H pylori, and extraneous nucleic acids likely to be encountered during hybridization conditions. More preferably, the sequence will comprise at least twenty to thirty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules.
Sequences larger than 1000 nucleotides in length are difficult to synthesize but can be generated by recombinant DNA techniques. Individuals skilled in the art will readily recognize that the nucleic acids, for use as probes, can be provided with a label to facilitate detection of a hybridization product.
Nucleic acid isolated and synthesized in accordance with the sequence of the invention contained in the Sequence Listing can also be useful as probes to detect homologous regions (especially homologous genes) of other Helicobacter species using appropriate stringency hybridization conditions as described herein. Capture Ligand
For use as a capture ligand, the nucleic acid selected in the manner described above with respect to probes, can be readily associated with a support. The manner in which nucleic acid is associated with supports is well known. Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing have utility to separate H. pylori nucleic acid from the nucleic acid of each other and other organisms. Nucleic acid having twenty or more nucleotides in a sequence of the invention contained in the Sequence Listing can also have utility to separate other Helicobacter species from each other and from other organisms. Preferably, the sequence will comprise at least twenty nucleotides to convey stability to the hybridization product formed between the probe and the intended target molecules. Sequences larger than 1000 nucleotides in length are difficult to synthesize but can be generated by recombinant DNA techniques.
Primers
Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility as primers for the amplification of H pylori nucleic acid. These nucleic acids may also have utility as primers for the amplification of nucleic acids in other Helicobacter species. With respect to polymerase chain reaction (PCR) techniques, nucleic acid sequences of > 10-15 nucleotides of the invention contained in the Sequence Listing have utility in conjunction with suitable enzymes and reagents to create copies of H. pylori nucleic acid. More preferably, the sequence will comprise twenty or more nucleotides to convey stability to the hybridization product formed between the primer and the intended target molecules. Binding conditions of primers greater than 100 nucleotides are more difficult to control to obtain specificity. High fidelity PCR can be used to ensure a faithful DNA copy prior to expression. In addition, amplified products can be checked by conventional sequencing methods.
The copies can be used in diagnostic assays to detect specific sequences, including genes from H. pylori and/or other Helicobacter species. The copies can also be incorporated into cloning and expression vectors to generate polypeptides corresponding to the nucleic acid synthesized by PCR, as is described in greater detail herein.
Antisense Nucleic acid or nucleic acid-hybridizing derivatives isolated or synthesized in accordance with the sequences described herein have utility as antisense agents to prevent the expression of H. pylori genes. These sequences also have utility as antisense agents to prevent expression of genes of other Helicobacter species.
In one embodiment, nucleic acid or derivatives corresponding to H. pylori nucleic acids is loaded into a suitable carrier such as a liposome or bacteriophage for introduction into bacterial cells. For example, a nucleic acid having twenty or more nucleotides is capable of binding to bacteria nucleic acid or bacteria messenger RNA. Preferably, the antisense nucleic acid is comprised of 20 or more nucleotides to provide necessary stability of a hybridization product of non-naturally occurring nucleic acid and bacterial nucleic acid and/or bacterial messenger RNA. Nucleic acid having a sequence greater than 1000 nucleotides in length is difficult to synthesize but can be generated by recombinant DNA techniques. Methods for loading antisense nucleic acid in liposomes is known in the art as exemplified by U.S. Patent 4,241 ,046 issued December 23, 1980 to Papahadjopoulos et al.
II. Expression of H pylori Nucleic Acids Nucleic acid isolated or synthesized in accordance with the sequences described herein have utility to generate polypeptides. The nucleic acid of the invention exemplified in the Sequence Listing or fragments of said nucleic acid encoding active portions of H pylori polypeptides can be cloned into suitable vectors or used to isolate nucleic acid. The isolated nucleic acid is combined with suitable DNA linkers and cloned into a suitable vector.
The function of a specific gene or operon can be ascertained by expression in a bacterial strain under conditions where the activity of the gene product(s) specified by the gene or operon in question can be specifically measured. Alternatively, a gene product may be produced in large quantities in an expressing strain for use as an antigen, an industrial reagent, for structural studies, etc. This expression can be accomplished in a mutant strain which lacks the activity of the gene to be tested, or in a strain that does not produce the same gene product(s). This includes, but is not limited to other Helicobacter strains, or other bacterial strains such as E. coli, Norcardia, Corynebacterium, Campylobacter, and Streptomyces species. In some cases the expression host will utilize the natural Helicobacter promoter whereas in others, it will be necessary to drive the gene with a promoter sequence derived from the expressing organism (e.g., an E. coli beta-galactosidase promoter for expression in E. coli).
To express a gene product using the natural H pylori promoter, a procedure such as the following can be used. A restriction fragment containing the gene of interest, together with its associated natural promoter element and regulatory sequences
(identified using the DNA sequence data) is cloned into an appropriate recombinant plasmid containing an origin of replication that functions in the host organism and an appropriate selectable marker. This can be accomplished by a number of procedures known to those skilled in the art. It is most preferably done by cutting the plasmid and the fragment to be cloned with the same restriction enzyme to produce compatible ends that can be ligated to join the two pieces together. The recombinant plasmid is introduced into the host organism by, for example, electroporation and cells containing the recombinant plasmid are identified by selection for the marker on the plasmid. Expression of the desired gene product is detected using an assay specific for that gene product.
In the case of a gene that requires a different promoter, the body of the gene (coding sequence) is specifically excised and cloned into an appropriate expression plasmid. This subcloning can be done by several methods, but is most easily accomplished by PCR amplification of a specific fragment and ligation into an expression plasmid after treating the PCR product with a restriction enzyme or exonuclease to create suitable ends for cloning. A suitable host cell for expression of a gene can be any procaryotic or eucaryotic cell. For example, an H pylori polypeptide can be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cell (CΗO). Other suitable host cells are known to those skilled in the art.
Expression in eucaryotic cells such as mammalian, yeast, or insect cells can lead to partial or complete glycosylation and/or formation of relevant inter- or intra-chain disulfide bonds of a recombinant peptide product. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari. et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Ηerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:1 13-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series (Smith et al., (1983) Mol Cell Biol 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39). Generally, COS cells (Gluzman, Y., (1981) Cell 23: 175-182) are used in conjunction with such vectors as pCDM 8 (Aruffo, A. and Seed, B., (1987) Proc. Natl Acad. Sci. USA 84:8573-8577) for transient amplification/expression in mammalian cells, while CΗO (dhfr Chinese
Hamster Ovary) cells are used with vectors such as pMT2PC (Kaufman et al. (1987), EMBO J. 6: 187- 195) for stable amplification/expression in mammalian cells. Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation. DEAE-dextran-mediated transfection, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.
Expression in procaryotes is most often carried out in E. coli with either fusion or non-fusion inducible expression vectors. Fusion vectors usually add a number of NH2 terminal amino acids to the expressed target gene. These NH2 terminal amino acids often are referred to as a reporter group. Such reporter groups usually serve two purposes: 1) to increase the solubility of the target recombinant protein; and 2) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the reporter group and the target recombinant protein to enable separation of the target recombinant protein from the reporter group subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly. MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein. A preferred reporter group is poly(His), which may be fused to the amino or carboxy terminus of the protein and which renders the recombinant fusion protein easily purifiable by metal chelate chromatography.
Inducible non-fusion expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pETl Id (Studier et al.. Gene Expression Technology: Methods in Enzvmology 185. Academic Press, San Diego, California ( 1990) 60-89). While target gene expression relies on host RNA polymerase transcription from the hybrid trp-lac fusion promoter in pTrc, expression of target genes inserted into pETl Id relies on transcription from the T7 gnlO-lac 0 fusion promoter mediated by coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gnl under the transcriptional control of the lacUV 5 promoter.
For example, a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding an H. pylori polypeptide can be cultured under appropriate conditions to allow expression of the polypeptide to occur. The polypeptide may be secreted and isolated from a mixture of cells and medium containing the peptide. Alternatively, the polypeptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. Polypeptides of the invention can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such polypeptides. Additionally, in many situations, polypeptides can be produced by chemical cleavage of a native protein (e.g., tryptic digestion) and the cleavage products can then be purified by standard techniques.
In the case of membrane bound proteins, these can be isolated from a host cell by contacting a membrane-associated protein fraction with a detergent forming a solubilized complex, where the membrane-associated protein is no longer entirely embedded in the membrane fraction and is solubilized at least to an extent which allows it to be chromatographically isolated from the membrane fraction. Several different criteria are used for choosing a detergent suitable for solubilizing these complexes. For example, one property considered is the ability of the detergent to solubilize the H. pylori protein within the membrane fraction at minimal denaturation of the membrane- associated protein allowing for the activity or functionality of the membrane-associated protein to return upon reconstitution of the protein. Another property considered when selecting the detergent is the critical micelle concentration (CMC) of the detergent in that the detergent of choice preferably has a high CMC value allowing for ease of removal after reconstitution. A third property considered when selecting a detergent is the hydrophobicity of the detergent. Typically, membrane-associated proteins are very hydrophobic and therefore detergents which are also hydrophobic, e.g., the triton series, would be useful for solubilizing the hydrophobic proteins. Another property important to a detergent can be the capability of the detergent to remove the H pylori protein with minimal protein-protein interaction facilitating further purification. A fifth property of the detergent which should be considered is the charge of the detergent. For example, if it is desired to use ion exchange resins in the purification process then preferably detergent should be an uncharged detergent. Chromatographic techniques which can be used in the final purification step are known in the art and include hydrophobic interaction, lectin affinity, ion exchange, dye affinity and immunoaffinity.
One strategy to maximize recombinant H pylori peptide expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzvmology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy would be to alter the nucleic acid encoding an H pylori peptide to be inserted into an expression vector so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada et al., (1992) Nuc. Acids Res. 20:21 1 1-2118). Such alteration of nucleic acids of the invention can be carried out by standard DNA synthesis techniques.
The nucleic acids of the invention can also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See, e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
III. H pylori Polypeptides
This invention encompasses isolated H pylori polypeptides encoded by the disclosed H. pylori genomic sequences, including the polypeptides of the invention contained in the Sequence Listing. Polypeptides of the invention are preferably at least 5 amino acid residues in length. Using the DNA sequence information provided herein, the amino acid sequences of the polypeptides encompassed by the invention can be deduced using methods well-known in the art. It will be understood that the sequence of an entire nucleic acid encoding an H pylori polypeptide can be isolated and identified based on an ORF that encodes only a fragment of the cognate protein-coding region. This can be acheived, for example, by using the isolated nucleic acid encoding the ORF, or fragments thereof, to prime a polymerase chain reaction with genomic H. pylori DNA as template; this is followed by sequencing the amplified product.
The polypeptides of the invention can be isolated from wild-type or mutant H pylori cells or from heterologous organisms or cells (including, but not limited to, bacteria, yeast, insect, plant and mammalian cells) into which an H. pylori nucleic acid has been introduced and expressed. In addition, the polypeptides can be part of recombinant fusion proteins.
H pylori polypeptides of the invention can be chemically synthesized using commercially automated procedures such as those referenced herein.
IV. Identification of Nucleic Acids Encoding Vaccine Components and Targets for
Agents Effective Against H. pylori
The disclosed H. pylori genome sequence includes segments that direct the synthesis of ribonucleic acids and polypeptides, as well as origins of replication, promoters, other types of regulatory sequences, and intergenic nucleic acids. The invention encompasses nucleic acids encoding immunogenic components of vaccines and targets for agents effective against H pylori. Identification of said immunogenic components involved in the determination of the function of the disclosed sequences can be achieved using a variety of approaches. Non-limiting examples of these approaches are described briefly below.
Homology to known sequences: Computer-assisted comparison of the disclosed H. pylori sequences with previously reported sequences present in publicly available databases is useful for identifying functional H. pylori nucleic acid and polypeptide sequences. It will be understood that protein-coding sequences, for example, may be compared as a whole, and that a high degree of sequence homology between two proteins (such as, for example, >80-90%>) at the amino acid level indicates that the two proteins also possess some degree of functional homology, such as, for example, among enzymes involved in metabolism, DNA synthesis, or cell wall synthesis, and proteins involved in transport, cell division, etc. In addition, many structural features of particular protein classes have been identified and correlate with specific consensus sequences, such as, for example, binding domains for nucleotides, DNA, metal ions, and other small molecules; sites for covalent modifications such as phosphorylation, acylation, and the like; sites of proteimprotein interactions, etc. These consensus sequences may be quite short and thus may represent only a fraction of the entire protein-coding sequence. Identification of such a feature in an H pylori sequence is therefore useful in determining the function of the encoded protein and identifying useful targets of antibacterial drugs.
Of particular relevance to the present invention are structural features that are common to secretory, transmembrane, and surface proteins, including secretion signal peptides and hydrophobic transmembrane domains. H pylori proteins identified as containing putative signal sequences and/or transmembrane domains are useful as immunogenic components of vaccines.
Identification of essential genes: Nucleic acids that encode proteins essential for growth or viability of H pylori are preferred drug targets. H pylori genes can be tested for their biological relevance to the organism by examining the effect of deleting and/or disrupting the genes, i.e., by so-called gene "knockout", using techniques known to those skilled in the relevant art. In this manner, essential genes may be identified. Strain-specific sequences: Because of the evolutionary relationship between different H pylori strains, it is believed that the presently disclosed H pylori sequences are useful for identifying, and/or discriminating between, previously known and new H pylori strains. It is believed that other H. pylori strains will exhibit at least 70% sequence homology with the presently disclosed sequence. Systematic and routine analyses of DNA sequences derived from samples containing H pylori strains, and comparison with the present sequence allows for the identification of sequences that can be used to discriminate between strains, as well as those that are common to all H pylori strains. In one embodiment, the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that discriminate between different strains of H pylori. Strain-specific components can also be identified functionally by their ability to elicit or react with antibodies that selectively recognize one or more H pylori strains.
In another embodiment, the invention provides nucleic acids, including probes, and peptide and polypeptide sequences that are common to all H. pylori strains but are not found in other bacterial species.
Specific Example: Determination Of Candidate Protein Antigens For Antibody And Vaccine Development
The selection of candidate protein antigens for vaccine development can be derived from the nucleic acids encoding H. pylori polypeptides. First, the ORF's can be analyzed for homology to other known exported or membrane proteins and analyzed using the discriminant analysis described by Klein, et al. (Klein, P., Kanehsia, M., and DeLisi, C. (1985) Biochimica et Biophysica Acta 815, 468-476) for predicting exported and membrane proteins.
Homology searches can be performed using the BLAST algorithm contained in the Wisconsin Sequence Analysis Package (Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1 ) to compare each predicted ORF amino acid sequence with all sequences found in the current GenBank, SWISS-PROT and PIR databases. BLAST searches for local alignments between the ORF and the databank sequences and reports a probability score which indicates the probability of finding this sequence by chance in the database. ORF's with significant homology (e.g. probabilities lower than lxlO"6 that the homology is only due to random chance) to membrane or exported proteins represent protein antigens for vaccine development. Possible functions can be provided to H. pylori genes based on sequence homology to genes cloned in other organisms.
Discriminant analysis (Klein, et al. supra) can be used to examine the ORF amino acid sequences. This algorithm uses the intrinsic information contained in the
ORF amino acid sequence and compares it to information derived from the properties of known membrane and exported proteins. This comparison predicts which proteins will be exported, membrane associated or cytoplasmic. ORF amino acid sequences identified as exported or membrane associated by this algorithm are likely protein antigens for vaccine development.
Surface exposed outer membrane proteins are likely to represent the best antigens to provide a protective immune response against H. pylori. Among the algorithms that can be used to aid in prediction of these outer membrane proteins include the presence of an amphipathic beta-sheet region at their C-terminus. This region which has been detected in a large number of outer membrane proteins in Gram negative bacteria is often characterized by hydrophobic residues (Phe or Tyr) clustered at alternating positions from the C-terminus (e.g., see Figure 5, block F; Figure 7, block E). Importantly, these sequences have not been detected at the C-termini of periplasmic proteins, thus allowing preliminary distinction between these classes of proteins based on primary sequence data. This phenomenon has been reported previously by Struyve et al. (J. Mol Biol. 218: 141-148. 1991).
Also illustrated in Figure 5 are additional amino acid sequence motifs found in many outer membrane proteins of H. pylori. The amino acid sequence alignment in Figure 5 depicts portions of the sequence of five H. pylori proteins (depicted in the single letter amino acid code) labeled with their amino acid Sequence ID Numbers and shown N-terminal to C-terminal, left to right. Five or six distinct blocks (labeled A through E or F) of similar amino acid residues are found including the distinctive hydrophobic residues (Phe or Tyr; F or Y according to the single letter code for amino acid residues) frequently found at positions near the C-terminus of outer membrane proteins. The presence of several shared motifs clearly establishes the similarity between members of this group of proteins.
Additional amino acid alignments for four outer membrane proteins isolated from H. pylori are depicted in Figure 6.
Outer membrane proteins isolated from H. pylori frequently share additional motifs as depicted for two proteins in Figure 7 which also share the C-terminal hydrophobic residues, and as depicted for two proteins in Figure 8 which do not share the C-terminal hydrophobic residue motif but share a different C-terminal motif.
One skilled in the art would know that these shared sequence motifs are highly significant and establish a similarity among this group of proteins.
Infrequently it is not possible to distinguish between multiple possible nucleotides at a given position in the nucleic acid sequence. In those cases the ambiguities are denoted by an extended alphabet as follows:
These are the official IUPAC-IUB single-letter base codes
Code Base Description
G Guanine
A Adenine
T Thymine
C Cytosine
R Purine A or G)
Y Pyrimidine C or T or U)
M Amino [A or C)
K Ketone [G or T)
S Strong interaction C or G) w Weak interaction ^A or T)
Η Not-G [A or C or T)
B Not-A [C or G or T)
V Not-T (not-U) ^A or C or G)
D Not-C A or G or T)
N Any A or C or G or T)
The amino acid translations of this invention account for the ambiguity in the nucleic acid sequence by translating the ambiguous codon as the letter "X". In all cases, the permissible amino acid residues at a position are clear from an examination of the nucleic acid sequence based on the standard genetic code.
V. Production of Fragments and Analogs of H pylori Nucleic Acids and Polypeptides Based on the discovery of the H pylori gene products of the invention provided in the Sequence Lsiting, one skilled in the art can alter the disclosed structure (of H pylori genes), e.g., by producing fragments or analogs, and test the newly produced structures for activity. Examples of techniques known to those skilled in the relevant art which allow the production and testing of fragments and analogs are discussed below. These, or analogous methods can be used to make and screen libraries of polypeptides, e.g., libraries of random peptides or libraries of fragments or analogs of cellular proteins for the ability to bind H pylori polypeptides. Such screens are useful for the identification of inhibitors of H. pylori.
Generation of Fragments
Fragments of a protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNA's which encode an array of fragments. DNA's which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination of the above-discussed methods. Fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, peptides of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.
Alteration of Nucleic Acids and Polypeptides: Random Methods
Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are elsewhere herein). (A) PCR Mutagenesis
In PCR mutagenesis, reduced Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA (Leung et al., 1989, Technique 1:11- 15). The DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase. e.g., by using a dGTP/dATP ratio of five and adding Mn2+ to the PCR reaction. The pool of amplified DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries.
(B) Saturation Mutagenesis
Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242). This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complimentary DNA strand. The mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements.
(C) Degenerate Oligonucleotides
A library of homologs can also be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429- 2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378- 6382; as well as U.S. Patents Nos. 5,223,409, 5,198,346, and 5,096,815). Alteration of Nucleic Acids and Polypeptides: Methods for Directed Mutagenesis
Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.
(A) Alanine Scanning Mutagenesis
Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244: 1081-1085, 1989). In alanine scanning, a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine). Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.
(B) Oligonucleotide-Mediated Mutagenesis Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et al., (DNA 2: 183, 1983). Briefly, the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA. Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single- stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765[1978]).
(C) Cassette Mutagenesis
Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al. (Gene, 34:315 [1985]). The starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated. The codon(s) in the protein subunit DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the desired protein subunit DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3' and 5' ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated desired protein subunit DNA sequence.
(D) Combinatorial Mutagenesis
Combinatorial mutagenesis can also be used to generate mutants (Ladner et al., WO 88/06630). In this method, the amino acid sequences for a group of homologs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to create a degenerate set of combinatorial sequences. The variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences. Other Modifications of H pylori Nucleic Acids and Polypeptides
It is possible to modify the structure of an H. pylori polypeptide for such purposes as increasing solubility, enhancing stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo). A modified H. pylori protein or peptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition as described herein.
An H pylori peptide can also be modified by substitution of cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid residues to minimize dimerization via disulfide linkages. In addition, amino acid side chains of fragments of the protein of the invention can be chemically modified. Another modification is cyclization of the peptide.
In order to enhance stability and/or reactivity, an H. pylori polypeptide can be modified to incorporate one or more polymorphisms in the amino acid sequence of the protein resulting from any natural allelic variation. Additionally, D-amino acids, non- natural amino acids, or non-amino acid analogs can be substituted or added to produce a modified protein within the scope of this invention. Furthermore, an H pylori polypeptide can be modified using polyethylene glycol (PEG) according to the method of A. Sehon and co-workers (Wie et al., supra) to produce a protein conjugated with PEG. In addition, PEG can be added during chemical synthesis of the protein. Other modifications of H pylori proteins include reduction/alky lation (Tarr, Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton NJ 155-194 (1986)); acylation (Tarr, supra); chemical coupling to an appropriate carrier (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology, WH Freeman, San Francisco, CA (1980), U.S. Patent 4,939,239; or mild formalin treatment (Marsh, (1971) Int. Arch, of Allergy andAppl Immunol. , 4J_: 199 - 215).
To facilitate purification and potentially increase solubility of an H. pylori protein or peptide, it is possible to add an amino acid fusion moiety to the peptide backbone. For example, hexa-histidine can be added to the protein for purification by immobilized metal ion affinity chromatography (Hochuli, E. et al., (1988) Bio/Technology, 6: 1321 - 1325). In addition, to facilitate isolation of peptides free of irrelevant sequences, specific endoprotease cleavage sites can be introduced between the sequences of the fusion moiety and the peptide.
To potentially aid proper antigen processing of epitopes within an H. pylori polypeptide, canonical protease sensitive sites can be engineered between regions, each comprising at least one epitope via recombinant or synthetic methods. For example, charged amino acid pairs, such as KK or RR, can be introduced between regions within a protein or fragment during recombinant construction thereof. The resulting peptide can be rendered sensitive to cleavage by cathepsin and/or other trypsin-like enzymes which would generate portions of the protein containing one or more epitopes. In addition, such charged amino acid residues can result in an increase in the solubility of the peptide.
Primary Methods for Screening Polypeptides and Analogs
Various techniques are known in the art for screening generated mutant gene products. Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, e.g., in this case, binding to H pylori polypeptide or an interacting protein, facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the techniques described below is amenable to high through-put analysis for screening large numbers of sequences created, e.g., by random mutagenesis techniques.
(A) Two Hybrid Systems
Two hybrid assays such as the system described above (as with the other screening methods described herein), can be used to identify polypeptides, e.g., fragments or analogs of a naturally-occurring H. pylori polypeptide, e.g., of cellular proteins, or of randomly generated polypeptides which bind to an H. pylori protein. (The H. pylori domain is used as the bait protein and the library of variants are expressed as fish fusion proteins.) In an analogous fashion, a two hybrid assay (as with the other screening methods described herein), can be used to find polypeptides which bind a H. pylori polypeptide.
(B) Display Libraries
In one approach to screening assays, the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay". For example, the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371 ; and Goward et al. (1992) TIBS 18:136-140). In a similar fashion, a detectably labeled ligand can be used to score for potentially functional peptide homologs. Fluorescently labeled ligands, e.g., receptors, can be used to detect homologs which retain ligand- binding activity. The use of fluorescently labeled ligands, allows cells to be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, to be separated by a fluorescence-activated cell sorter.
A gene library can be expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at concentrations well over 10^ phage per milliliter, a large number of phage can be screened at one time. Second, since each infectious phage displays a gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages M13, fd., and fl are most often used in phage display libraries. Either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle. Foreign epitopes can be expressed at the NH2- terminal end of pill and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267: 16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et al. (\99\) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).
A common approach uses the maltose receptor of E. coli (the outer membrane protein, LamB) as a peptide fusion partner (Charbit et al. (1986) EMBO 5, 3029-3037). Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands. e.g., to antibodies, and can elicit an immune response when the cells are administered to animals. Other cell surface proteins, e.g., OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392), PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchs et al. (1991) Bio/Tech 9, 1369-1372), as well as large bacterial surface structures have served as vehicles for peptide display. Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al. (1989) Appl Environ. Microbiol. 55, 984-993). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment. Another large surface structure used for peptide display is the bacterial motive organ, the flagellum. Fusion of peptides to the subunit protein flagellin offers a dense array of many peptide copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083). Surface proteins of other bacterial species have also served as peptide fusion partners. Examples include the Staphylococcus protein A and the outer membrane IgA protease of Neisseria (Hansson et al. (1992) J. Bacteriol 174, 4239-4245 and Klauser et al. (1990) EMBO J. 9, 1991 - 1999).
In the filamentous phage systems and the LamB system described above, the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface.
Capturing the peptide captures the particle and the DNA within. An alternative scheme uses the DNA-binding protein Lad to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89: 1865-1869). This system uses a plasmid containing the Lad gene with an oligonucleotide cloning site at its 3 '-end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced. This fusion retains the natural ability of Lad to bind to a short DNA sequence known as LacO operator (LacO). By installing two copies of LacO on the expression plasmid, the Lacl-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides. The associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands. As a demonstration of the practical utility of the method, a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dynorphin B. A cohort of peptides was recovered, all related by a consensus sequence corresponding to a six-residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl Acad. Sci. U.S.A. 89-1869) This scheme, sometimes referred to as peptides-on-plasmids, differs in two important ways from the phage display methods. First, the peptides are attached to the C-terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini. Both of the filamentous phage coat proteins, pill and pVIII, are anchored to the phage through their C-termini, and the guest peptides are placed into the outward-extending N-terminal domains. In some designs, the phage- displayed peptides are presented right at the amino terminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6378-6382) A second difference is the set of biological biases affecting the population of peptides actually present in the libraries. The Lad fusion molecules are confined to the cytoplasm of the host cells. The phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles. The peptides in the Lad and phage libraries may differ significantly as a result of their exposure to different proteolytic activities. The phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to incoφoration into phage. Certain peptides exert a deleterious effect on these processes and are underrepresented in the libraries (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251). These particular biases are not a factor in the Lad display system.
The number of small peptides available in recombinant random libraries is enormous. Libraries of 10' -10° independent clones are routinely prepared. Libraries as large as 10^ recombinants have been created, but this size approaches the practical limit for clone libraries. This limitation in library size occurs at the step of transforming the DNA containing randomized segments into the host bacterial cells. To circumvent this limitation, an in vitro system based on the display of nascent peptides in polysome complexes has recently been developed. This display library method has the potential of producing libraries 3-6 orders of magnitude larger than the currently available phage/phagemid or plasmid libraries. Furthermore, the construction of the libraries, expression of the peptides, and screening, is done in an entirely cell-free format. In one application of this method (Gallop et al. (1994) J. Med. Chem. 37(9):1233-1251), a molecular DNA library encoding 10^ decapeptides was constructed and the library expressed in an E. coli S30 in vitro coupled transcription/translation system. Conditions were chosen to stall the ribosomes on the mRNA, causing the accumulation of a substantial proportion of the RNA in polysomes and yielding complexes containing nascent peptides still linked to their encoding RNA. The polysomes are sufficiently robust to be affinity purified on immobilized receptors in much the same way as the more conventional recombinant peptide display libraries are screened. RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening. The polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector. This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification. By expressing the polysome-derived peptides on phage, one can either continue the affinity selection procedure in this format or assay the peptides on individual clones for binding activity in a phage ΕLISA, or for binding specificity in a completion phage ΕLISA (Barret, et al. (1992) Anal. Biochem 204,357-364). To identify the sequences of the active peptides one sequences the DNA produced by the phagemid host.
Secondary Screening of Polypeptides and Analogs The high through-put assays described above can be followed by secondary screens in order to identify further biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists. The type of a secondary screen used will depend on the desired activity that needs to be tested. For example, an assay can be developed in which the ability to inhibit an interaction between a protein of interest and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated though one of the primary screens described above.
Therefore, methods for generating fragments and analogs and testing them for activity are known in the art. Once the core sequence of interest is identified, it is routine for one skilled in the art to obtain analogs and fragments.
Peptide Mimetics of H pylori Polypeptides
The invention also provides for reduction of the protein binding domains of the subject H pylori polypeptides to generate mimetics, e.g. peptide or non-peptide agents. The peptide mimetics are able to disrupt binding of a polypeptide to its counter ligand, e.g., in the case of an H. pylori polypeptide binding to a naturally occurring ligand. The critical residues of a subject H pylori polypeptide which are involved in molecular recognition of a polypeptide can be determined and used to generate H /rμ/ør/-derived peptidomimetics which competitively or noncompetitively inhibit binding of the H pylori polypeptide with an interacting polypeptide (see, for example, European patent applications EP-412,762A and EP-B31,080A).
For example, scanning mutagenesis can be used to map the amino acid residues of a particular H pylori polypeptide involved in binding an interacting polypeptide, peptidomimetic compounds (e.g. diazepine or isoquinoline derivatives) can be generated which mimic those residues in binding to an interacting polypeptide, and which therefore can inhibit binding of an H. pylori polypeptide to an interacting polypeptide and thereby interfere with the function of H pylori polypeptide. For instance, non- hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden,
Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto- methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (\986) J Chem Soc Perkin Trans 1 : 1231), and β-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res
Commun\26:4\9; and Dann et al. (1986) Biochem Biophys Res Commun 134:71).
VI. Vaccine Formulations for H pylori Nucleic Acids and Polypeptides
This invention also features vaccine compositions or formulations (used interchangeably herein) for protection against infection by H pylori or for treatment of H. pylori infection. As used herein, the term "treatment of H pylori infection" refers to therapeutic treatment of an existing or established H. pylori infection. The terms "protection against H. pylori infection" or "prophylactic treatment" refer to the use of H pylori vaccine formulation for reducing the risk of or preventing an infection in a subject at risk for H pylori infection. In one embodiment, the vaccine compositions contain one or more immunogenic components, such as a surface protein, from H pylori, or portion thereof, and a pharmaceutically acceptable carrier. For example, in one embodiment, the vaccine formulations of the invention contain at least one or combination of H pylori polypeptides or fragments thereof, from same or different H pylori antigens. Nucleic acids and H. pylori polypeptides for use in the vaccine formulations of the invention include the nucleic acids and polypeptides set forth in the Sequence Listing, preferably those H pylori nucleic acids that encode surface proteins and surface proteins or fragments thereof. For example, a preferred nucleic acid and H. pylori polypeptide for use in a vaccine composition of the invention is selected from the group of nucleic acids which encode cell envelope proteins and H pylori cell envelope proteins as set forth in Table 1. However, any nucleic acid encoding an immunogenic H. pylori protein and H. pylori polypetide, or portion thereof, can be used in the present invention. These vaccines have therapeutic and/or prophylactic utilities.
One aspect of the invention provides a vaccine composition for protection against infection by H. pylori which contains at least one immunogenic fragment of an H. pylori protein and a pharmaceutically acceptable carrier. Preferred fragments include peptides of at least about 10 amino acid residues in length, preferably about 10-20 amino acid residues in length, and more preferably about 12-16 amino acid residues in length. Immunogenic components of the invention can be obtained, for example, by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding the full-length H pylori protein. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
In one embodiment, immunogenic components are identified by the ability of the peptide to stimulate T cells. Peptides which stimulate T cells, as determined by, for example, T cell proliferation or cytokine secretion are defined herein as comprising at least one T cell epitope. T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to the protein allergen which is responsible for the clinical symptoms of allergy. These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate HLA molecule on the surface of an antigen presenting cell, thereby stimulating the T cell subpopulation with the relevant T cell receptor for the epitope. These events lead to T cell proliferation, lymphokine secretion, local inflammatory reactions, recruitment of additional immune cells to the site of antigen T cell interaction, and activation of the B cell cascade, leading to the production of antibodies. A T cell epitope is the basic element, or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acids essential to receptor recognition (e.g., approximately 6 or 7 amino acid residues). Amino acid sequences which mimic those of the T cell epitopes are within the scope of this invention.
In another embodiment, immunogenic components of the invention are identified through genomic vaccination. The basic protocol is based on the idea that expression libraries consisting of all or parts of a pathogen genome, e.g., an H. pylori genome, can confer protection when used to genetically immunize a host. This expression library immunization (ELI) is analogous to expression cloning and involves reducing a genomic expression library of a pathogen, e.g., H. pylori, into plasmids that can act as genetic vaccines. The plasmids can also be designed to encode genetic adjuvants which can dramatically stimulate the humoral response. These genetic adjuvants can be introduced at remote sites and act as well extracelluraly as intracellularly.
This is a new approach to vaccine production that has many of the advantages of live/attenuated pathogens but no risk of infection. An expression library of pathogen DNA is used to immunize a host thereby producing the effects of antigen presentation of a live vaccine without the risk. For example, in the present invention, random fragments from the H. pylori genome or from cosmid or plasmid clones, as well as PCR products from genes identified by genomic sequencing, can be used to immunize a host. The feasibility of this approach has been demonstrated with Mycoplasma pulmonis (Barry et al., Nature 377:632-635, 1995), where even partial expression libraries of Mycoplasma pulmonis, a natural pathogen in rodents, provided protection against challenge from the pathogen. ELI is a technique that allows for production of a non-infectious multipartite vaccine, even when little is known about pathogen's biology, because ELI uses the immune system to screen candidate genes. Once isolated, these genes can be used as genetic vaccines or for development of recombinant protein vaccines. Thus, ELI allows for production of vaccines in a systematic, largely mechanized fashion.
Screening immunogenic components can be accomplished using one or more of several different assays. For example, in vitro, peptide T cell stimulatory activity is assayed by contacting a peptide known or suspected of being immunogenic with an antigen presenting cell which presents appropriate MHC molecules in a T cell culture. Presentation of an immunogenic H. pylori peptide in association with appropriate MHC molecules to T cells in conjunction with the necessary costimulation has the effect of transmitting a signal to the T cell that induces the production of increased levels of cytokines, particularly of interleukin-2 and interleukin-4. The culture supernatant can be obtained and assayed for interleukin-2 or other known cytokines. For example, any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc. Natl. Acad. Sci USA, 86: 1333 (1989) the pertinent portions of which are incoφorated herein by reference. A kit for an assay for the production of interferon is also available from Genzyme Coφoration (Cambridge, MA).
Alternatively, a common assay for T cell proliferation entails measuring tritiated thymidine incoφoration. The proliferation of T cells can be measured in vitro by determining the amount of ^H-labeled thymidine incoφorated into the replicating DNA of cultured cells. Therefore, the rate of DNA synthesis and, in turn, the rate of cell division can be quantified.
Vaccine compositions or formulations of the invention containing one or more immunogenic components (e.g., H. pylori polypeptide or fragment thereof or nucleic acid encoding an H. pylori polypeptide or fragment thereof) preferably include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents, and the like, compatible with pharmaceutical administration. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the H. pylori nucleic acid or polypeptide. For vaccine formulations of the invention containing H. pylori polypeptides, the polypeptide is preferably coadministered with a suitable adjuvant and/or a delivery system described herein. It will be apparent to those of skill in the art that the therapeutically effective amount of DNA or protein of this invention will depend, inter alia, upon the administration schedule, the unit dose of an H pylori nucleic acid or polypeptide administered, whether the protein or nucleic acid is administered in combination with other therapeutic agents, the immune status and health of the patient, and the therapeutic activity of the particular protein or nucleic acid.
Vaccine formulations are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Methods for intramuscular immunization are described by Wolff et al. (1990) Science 247: 1465-1468 and by Sedegah et al. (1994) Immunology 9j 9866-9870. Other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Oral immunization is preferred over parenteral methods for inducing protection against infection by H pylori. Czinn et. al. (1993) Vaccine V\_: 637-642. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
In one embodiment, the vaccine formulation includes, as a pharmaceutically acceptable carrier, an adjuvant. Examples of the suitable adjuvants for use in the vaccine formulations of the invention include, but are not limited, to aluminum hydroxide; N-acetyl-muramyl—L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine (CGP 1 1637, referred to as nor-MDP); N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'-dipalmitoyl-sn-glycero-3- hydroxyphos-phoryloxy)-ethylamine (CGP 19835 A, referred to a MTP-PE); RIBI, which contains three components from bacteria; monophosphoryl lipid A; trehalose dimycoloate; cell wall skeleton (MPL + TDM + CWS) in a 2% squalene/Tween 80 emulsion; and cholera toxin. Others which may be used are non-toxic derivatives of cholera toxin, including its B subunit, and/or conjugates or genetically engineered fusions of the H pylori polypeptide with cholera toxin or its B subunit, procholeragenoid, fungal polysaccharides, including schizophyllan, muramyl dipeptide, muramyl dipeptide derivatives, phorbol esters, labile toxin of E. coli. non-H. pylori bacterial lysates, block polymers or saponins.
In another embodiment, the vaccine formulation includes, as a pharmaceutically acceptable carrier, a delivery system. Suitable delivery systems for use in the vaccine formulations of the invention include biodegradable microcapsules or immuno- stimulating complexes (ISCOMs), cochleates, or liposomes, genetically engineered attenuated live vectors such as viruses or bacteria, and recombinant (chimeric) virus-like particles, e.g., bluetongue. In another embodiment of the invention, the vaccine formulation includes both a delivery system and an adjuvant.
Delivery systems in humans may include enteric release capsules protecting the antigen from the acidic environment of the stomach, and including H pylori polypeptide in an insoluble form as fusion proteins. Suitable carriers for the vaccines of the invention are enteric coated capsules and polylactide-glycolide microspheres. Suitable diluents are 0.2 N NaΗCO3 and/or saline.
Vaccines of the invention can be administered as a primary prophylactic agent in adults or in children, as a secondary prevention, after successful eradication of H. pylori in an infected host, or as a therapeutic agent in the aim to induce an immune response in a susceptible host to prevent infection by H. pylon. The vaccines of the invention are administered in amounts readily determined by persons of ordinary skill in the art. Thus, for adults a suitable dosage will be in the range of 10 μg to 10 g, preferably 10 μg to 100 mg, for example 50 μg to 50 mg. A suitable dosage for adults will also be in the range of 5 μg to 500 mg. Similar dosage ranges will be applicable for children.
The amount of adjuvant employed will depend on the type of adjuvant used. For example, when the mucosal adjuvant is cholera toxin, it is suitably used in an amount of 5 μg to 50 μg, for example 10 μg to 35 μg. When used in the form of microcapsules, the amount used will depend on the amount employed in the matrix of the microcapsule to achieve the desired dosage. The determination of this amount is within the skill of a person of ordinary skill in the art.
Those skilled in the art will recognize that the optimal dose may be more or less depending upon the patient's body weight, disease, the route of administration, and other factors. Those skilled in the art will also recognize that appropriate dosage levels can be obtained based on results with known oral vaccines such as, for example, a vaccine based on an E. coli lysate (6 mg dose daily up to total of 540 mg) and with an enterotoxigenic E. coli purified antigen (4 doses of 1 mg) (Schulman et al., J. Urol 150:917-921 (1993)); Boedecker et al., American Gastroenterological Assoc. 999:A-222 (1993)). The number of doses will depend upon the disease, the formulation, and efficacy data from clinical trials. Without intending any limitation as to the course of treatment, the treatment can be administered over 3 to 8 doses for a primary immunization schedule over 1 month (Boedeker, American Gastroenterological Assoc. 888 :A-222 (1993)).
In a preferred embodiment, a vaccine composition of the invention can be based on a killed whole E. coli preparation with an immunogenic fragment of an H. pylori protein of the invention expressed on its surface or it can be based on an E. coli lysate, wherein the killed E. coli acts as a carrier or an adjuvant. It will be apparent to those skilled in the art that some of the vaccine compositions of the invention are useful only for preventing H pylori infection, some are useful only for treating H. pylori infection, and some are useful for both preventing and treating H pylori infection. In a preferred embodiment, the vaccine composition of the invention provides protection against H. pylori infection by stimulating humoral and/or cell-mediated immunity against H pylori. It should be understood that amelioration of any of the symptoms of H. pylori infection is a desirable clinical goal, including a lessening of the dosage of medication used to treat H. τy/or'-caused disease, or an increase in the production of antibodies in the serum or mucous of patients.
VII. Antibodies Reactive With H. pylori Polypeptides
The invention also includes antibodies specifically reactive with the subject H pylori polypeptide. Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. An immunogenic portion of the subject H. pylori polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
In a preferred embodiment, the subject antibodies are immunospecific for antigenic determinants of the H. pylori polypeptides of the invention, e.g. antigenic determinants of a polypeptide of the invention contained in the Sequence Listing, or a closely related human or non-human mammalian homolog (e.g., 90% homologous, more preferably at least 95%> homologous). In yet a further preferred embodiment of the invention, the anti-H. pylori antibodies do not substantially cross react (i.e., react specifically) with a protein which is for example, less than 80%> percent homologous to a sequence of the invention contained in the Sequence Listing. By "not substantially cross react", it is meant that the antibody has a binding affinity for a non-homologous protein which is less than 10 percent, more preferably less than 5 percent, and even more preferably less than 1 percent, of the binding affinity for a protein of the invention contained in the Sequence Listing. In a most preferred embodiment, there is no crossreactivity between bacterial and mammalian antigens.
The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with H pylori polypeptides. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. The antibody of the invention is further intended to include bispecific and chimeric molecules having an anti-H. pylori portion.
Both monoclonal and polyclonal antibodies (Ab) directed against H pylori polypeptides or H pylori polypeptide variants, and antibody fragments such as Fab' and F(ab')2, can be used to block the action of H. pylori polypeptide and allow the study of the role of a particular H. pylori polypeptide of the invention in aberrant or unwanted intracellular signaling, as well as the normal cellular function of the H. pylori and by microinjection of anti-H. pylori polypeptide antibodies of the present invention. Antibodies which specifically bind H pylori epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of H pylori antigens. Anti H pylori polypeptide antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate H. pylori levels in tissue or bodily fluid as part of a clinical testing procedure. Likewise, the ability to monitor H. pylori polypeptide levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder. The level of an H. pylori polypeptide can be measured in cells found in bodily fluid, such as in urine samples or can be measured in tissue, such as produced by gastric biopsy. Diagnostic assays using anti-H. pylori antibodies can include, for example, immunoassays designed to aid in early diagnosis of H. pylori infections. The present invention can also be used as a method of detecting antibodies contained in samples from individuals infected by this bacterium using specific H. pylori antigens.
Another application of anti-H. pylori polypeptide antibodies of the invention is in the immunological screening of cDNA libraries constructed in expression vectors such as λgtl 1, λgtl 8-23, λZAP, and λORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, λgtl 1 will produce fusion proteins whose amino termini consist of β-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of a subject H. pylori polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-H. pylori polypeptide antibodies. Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence of H pylori gene homologs can be detected and cloned from other species, and alternate isoforms (including splicing variants) can be detected and cloned.
VIII. Kits Containing Nucleic Acids, Polypeptides or Antibodies of the Invention The nucleic acid, polypeptides and antibodies of the invention can be combined with other reagents and articles to form kits. Kits for diagnostic puφoses typically comprise the nucleic acid, polypeptides or antibodies in vials or other suitable vessels. Kits typically comprise other reagents for performing hybridization reactions, polymerase chain reactions (PCR), or for reconstitution of lyophilized components, such as aqueous media, salts, buffers, and the like. Kits may also comprise reagents for sample processing such as detergents, chaotropic salts and the like. Kits may also comprise immobilization means such as particles, supports, wells, dipsticks and the like. Kits may also comprise labeling means such as dyes, developing reagents, radioisotopes, fluorescent agents, luminescent or chemiluminescent agents, enzymes, intercalating agents and the like. With the nucleic acid and amino acid sequence information provided herein, individuals skilled in art can readily assemble kits to serve their particular puφose. Kits further can include instructions for use.
IX. Drug Screening Assays Using H. pylori Polypeptides By making available purified and recombinant H. pylori polypeptides, the present invention provides assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function, in this case, of the subject H pylori polypeptides, or of their role in intracellular signaling. Such inhibitors or potentiators may be useful as new therapeutic agents to combat H pylori infections in humans. A variety of assay formats will suffice and, in light of the present inventions, will be comprehended by the skilled artisan.
In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or change in enzymatic properties of the molecular target. Accordingly, in an exemplary screening assay of the present invention, the compound of interest is contacted with an isolated and purified H pylori polypeptide.
Screening assays can be constructed in vitro with a purified H pylori polypeptide or fragment thereof, such as an H. pylori polypeptide having enzymatic activity, such that the activity of the polypeptide produces a detectable reaction product. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. Suitable products include those with distinctive absoφtion, fluorescence, or chemi-luminescence properties, for example, because detection may be easily automated. A variety of synthetic or naturally occurring compounds can be tested in the assay to identify those which inhibit or potentiate the activity of the H pylori polypeptide. Some of these active compounds may directly, or with chemical alterations to promote membrane permeability or solubility, also inhibit or potentiate the same activity (e.g., enzymatic activity) in whole, live H pylori cells.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references and published patent applications cited throughout this application are hereby incoφorated by reference.
EXEMPLIFICATION
I. Cloning and Sequencing of H. pylori DNA
H pylori chromosomal DNA was isolated according to a basic DNA protocol outlined in Schleif R.F. and Wensink P.C, Practical Methods in Molecular Biology, p.98, Springer- Verlag, NY., 1981, with minor modifications. Briefly, cells were pelleted, resuspended in TE (10 mM Tris, 1 mM EDTA, pΗ 7.6) and GES lysis buffer (5.1 M guanidium thiocyanate, 0.1 M EDTA, pΗ 8.0, 0.5%) N-laurylsarcosine) was added. Suspension was chilled and ammonium acetate (NΗ4AC) was added to final concentration of 2.0 M. DNA was extracted, first with chloroform, then with phenol- chloroform, and reextracted with chloroform. DNA was precipitated with isopropanol, washed twice with 70% EtOH, dried and resuspended in TE.
Following isolation whole genomic H. pylori DNA was nebulized (Bodenteich et al., Automated DNA Sequencing and Analysis (J.C. Venter, ed.), Academic Press, 1994) to a median size of 2000 bp. After nebulization, the DNA was concentrated and separated on a standard 1%> agarose gel. Several fractions, corresponding to approximate sizes 900-1300 bp. 1300-1700 bp, 1700-2200 bp, 2200-2700 bp, were excised from the gel and purified by the GeneClean procedure (Biol 01, Inc.).
The purified DNA fragments were then blunt-ended using T4 DNA polymerase. The healed DNA was then ligated to unique BstXI-linker adapters in 100-1000 fold molar excess. These linkers are complimentary to the BstXI-cut pMPX vectors, while the overhang is not self-complimentary. Therefore, the linkers will not concatemerize nor will the cut- vector religate itself easily. The linker-adopted inserts were separated from the unincoφorated linkers on a 1%> agarose gel and purified using GeneClean. The linker-adopted inserts were then ligated to each of the 20 pMPX vectors to construct a series of "shotgun" subclone libraries. The vectors contain an out-of- frame lacZ gene at the cloning site which becomes in-frame in the event that an adapter-dimer is cloned, allowing these to be avoided by their blue-color.
All subsequent steps were based on the multiplex DNA sequencing protocols outlined in Church G.M. and Kieffer-Higgins S., Science 240:185-188, 1988. Only major modifications to the protocols are highlighted. Briefly, each of the 20 vectors was then transformed into DH5 competent cells (Gibco/BRL, DH5α transformation protocol). The libraries were assessed by plating onto antibiotic plates containing ampicillin, methicillin and IPTG/Xgal. The plates were incubated overnight at 37°C. Successful transformants were then used for plating of clones and pooling into the multiplex pools. The clones were picked and pooled into 40 ml growth medium cultures. The cultures were grown overnight at 37°C. DNA was purified using the Qiagen Midi-prep kits and Tip- 100 columns (Qiagen, Inc.). In this manner, 100 μg of DNA was obtained per pool. Fifteen 96-well plates of DNA were generated to obtain a 5-10 fold sequence redundancy assuming 250-300 base average read-lengths. These purified DNA samples were then sequenced using the multiplex DNA sequencing based on chemical degradation methods (Church G.M. and Kieffer-Higgins S., Science 240:185-188, 1988) or by Sequithrem (Epicenter Technologies) dideoxy sequencing protocols. The sequencing reactions were electrophoresed and transferred onto nylon membranes by direct transfer electrophoresis from 40 cm gels (Richterich P. and Church G.M., Methods in Enzymology 218:187-222, 1993) or by electroblotting
(Church, supra). 24 samples were run per gel. 45 successful membranes were produced by chemical sequencing and 8 were produced by dideoxy sequencing. The DNA was covalently bound to the membranes by exposure to ultraviolet light, and hybridized with labeled oligonucleotides complimentary to tag sequences on the vectors (Church, supra). The membranes were washed to rinse off non-specifically bound probe, and exposed to X-ray film to visualize individual sequence ladders. After autoradiography, the hybridized probe was removed by incubation at 65° C, and the hybridization cycle repeated with another tag sequence until the membrane had been probed 38 times for chemical sequencing membranes and 10 times for the dideoxy sequencing membranes. Thus, each gel produced a large number of films, each containing new sequencing information. Whenever a new blot was processed, it was initially probed for an internal standard sequence added to each of the pools.
Digital images of the films were generated using a laser-scanning densitometer (Molecular Dynamics, Sunnyvale, CA). The digitized images were processed on computer workstations (VaxStation 4000's) using the program REPLICA™ (Church et al., Automated DNA Sequencing and Analysis (J.C. Venter, ed.), Academic Press, 1994). Image processing included lane straightening, contrast adjustment to smooth out intensity differences, and resolution enhancement by iterative gaussian deconvolution. The sequences were then automatically picked in REPLICA™ and displayed for interactive proofreading before being stored in a project database. The proofreading was accomplished by a quick visual scan of the film image followed by mouse clicks on the bands of the displayed image to modify the base calls. Many of the sequence errors could be detected and corrected because multiple sequence reads covering the same portion of the genomic DNA provide adequate sequence redundancy for editing. Each sequence automatically received an identification number (corresponding to microtiter plate, probe information, and lane set number). This number serves as a permanent identifier of the sequence so it is always possible to identify the original of any particular sequence without recourse to a specialized database.
Routine assembly of H. pylori sequences was done using the program FALCON (Church, Church et al., Automated DNA Sequenicng and Analysis (J.C. Venter, ed.), Academic Press, 1994). This program has proven to be fast and reliable for most sequences. The assembled contigs were displayed using a modified version of
GelAssemble, developed by the Genetics Computer Group (GCG) (Devereux et al., Nucleic Acid Res. 12:387-95, 1984) that interacts with REPLICA™. This provided for an integrated editor that allows multiple sequence gel images to be instantaneously called up from the REPLICA™ database and displayed to allow rapid scanning of contigs and proofreading of gel traces where discrepancies occurred between different sequence reads in the assembly.
II. Identification, cloning and expression of recombinant H. pylori DNA sequences
To facilitate the cloning, expression and purification of membrane and secreted proteins from H. pylori a powerful gene expression system, the pET System (Novagen), for cloning and expression of recombinant proteins in E. coli, was selected. Also, a DNA sequence encoding a peptide tag, the Ηis-Tag, was fused to the 3 ' end of DNA sequences of interest in order to facilitate purification of the recombinant protein products. The 3' end was selected for fusion in order to avoid alteration of any 5' terminal signal sequence. The exception to the above was ppiB, a gene cloned for use as a control in the expression studies. In this study, the sequence for H pylori ppiB contains a DNA sequence encoding a Ηis-Tag fused to the 5' end of the full length gene, because the protein product of this gene does not contain a signal sequence and is expressed as a cytosolic protein.
PCR Amplification and cloning of DNA sequences containing ORF's for membrane and secreted proteins from the J99 Strain of Helicobacter pylori.
Sequences chosen (from the list of the DNA sequences of the invention) for cloning from the J99 strain of H. pylori were prepared for amplification cloning by polymerase chain reaction (PCR). Synthetic oligonucleotide primers (Table 3) specific for the 5' and 3' ends of open reading frames (ORFs) were designed and purchased (GibcoBRL Life Technologies, Gaithersburg, MD, USA). All forward primers (specific for the 5' end of the sequence) were designed to include an Ncol cloning site at the extreme 5' terminus, except for ΗpSeq. 4821082 where Ndel was used. These primers were designed to permit initiation of protein translation at a methionine residue followed by a valine residue and the coding sequence for the remainder of the native H. pylori DNA sequence. An exception is H pylori sequence 4821082 where the initiator methionine is immediately followed by the remainder of the native H pylori DNA sequence. All reverse primers (specific for the 3' end of any H pylori ORF) included a EcoRI site at the extreme 5' terminus to permit cloning of each H pylori sequence into the reading frame of the pET-28b. The pET-28b vector provides sequence encoding an additional 20 carboxy-terminal amino acids (only 19 amino acids in ΗpSeq. 26380318 and ΗpSeq.14640637) including six histidine residues (at the extreme C-terminus), which comprise the Ηis-Tag. An exception to the above, as noted earlier, is the vector construction for the ppiB gene. A synthetic oligonucleotide primer specific for the 5' end of ppiB gene encoded a BamΗI site at its extreme 5' terminus and the primer for the 3' end of the ppiB gene encoded a Xhol site at its extreme 5' terminus. TABLE 3
Oligonucleotide primers used for PCR amplification of H pylori DNA sequences
Genomic DNA prepared from the J99 strain of H. pylori (ATCC #55679; deposited by Genome Therapeutics Coφoration, 100 Beaver Street, Waltham, MA 02154) was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). To amplify a DNA sequence containing an H pylori ORF, genomic DNA (50 nanograms) was introduced into a reaction vial containing 2 mM MgCl2, 1 micromolar synthetic oligonucleotide primers (forward and reverse primers) complementary to and flanking a defined H pylori ORF, 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP. dCTP, dTTP and 2.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems. Inc., Branchburg, NJ, USA) in a final volume of 100 microliters. The following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler:
Protein 26054702, Protein 7116626, Protein 29479681, Protein 30100332, and
Protein 4821082;
Denaturation at 94°C for 2 min, .
2 cycles at 94°C for 15 sec, 30°C for 15 sec and 72°C for 1.5 min 23 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes.
Protein 16225006;
Denaturation at 94°C for 2 min. 25 cycles at 95°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min Reaction was concluded at 72°C for 6 minutes.
Protein 4721061;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 36°C for 15 sec and 72°C for 1.5 min 23 cycles at 94°C for 15 sec, 60°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes.
Protein 26380318;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 38°C for 15 sec and 72°C for 1.5 min 23 cycles at 94°C for 15 sec, 62°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes.
Protein 14640637;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 33°C for 15 sec and 72°C for 1.5 min 30 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes.
Conditions for amplification of H. pylori ppiB;
Denaturation at 94°C for 2 min.
2 cycles at 94°C for 15 sec, 32°C for 15 sec and 72°C for 1.5 min 25 cycles at 94°C for 15 sec, 56°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes
Upon completion of thermal cycling reactions, each sample of amplified DNA was washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). All amplified DNA samples were subjected to digestion with the restriction endonucleases, Ncol and EcoRI (New England BioLabs, Beverly, MA, USA), or in the case of HpSeq. 4821082 (SEQ ID NO: 1309), with Ndd and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). DNA samples were then subjected to electrophoresis on 1.0 %> NuSeive (FMC BioProducts, Rockland, ME USA) agarose gels. DNA was visualized by exposure to ethidium bromide and long wave uv irradiation. DNA contained in slices isolated from the agarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, C A, USA).
Cloning ofH pylori DNA sequences into the pET-28b prokaryotic expression vector.
The pET-28b vector was prepared for cloning by digestion with Ncol and EcoRI, or in the case of H pylori protein 4821082 with Ndel and EcoRI (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). In the case of cloning ppiB, the pET-28a vector, which encodes a Ηis-Tag that can be fused to the 5' end of an inserted gene, was used and the cloning site prepared for cloning with the ppiB gene by digestion with BamΗI and Xhol restriction endonucleases.
Following digestion, DNA inserts were cloned (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) into the previously digested pET-28b expression vector, except for the amplified insert for ppiB, which was cloned into the pET-28a expression vector. Products of the ligation reaction were then used to transform the BL21 strain of E. coli (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994) as described below.
Transformation of competent bacteria with recombinant plasmids
Competent bacteria, E coli strain BL21 or E. coli strain BL21(DE3), were transformed with recombinant pET expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). Briefly, 1 microliter of ligation reaction was mixed with 50 microliters of electrocompetent cells and subjected to a high voltage pulse, after which, samples were incubated in 0.45 milliliters SOC medium (0.5%> yeast extract, 2.0 % tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgC12, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate for growth overnight. Transformed colonies of BL21 were then picked and analyzed to evaluate cloned inserts as described below.
Identification of recombinant pET expression plasmids carrying H. pylori sequences
Individual BL21 clones transformed with recombinant pET-28b-Η. pylori ORFs were analyzed by PCR amplification of the cloned inserts using the same forward and reverse primers, specific for each H. pylori sequence, that were used in the original PCR amplification cloning reactions. Successful amplification verified the integration of the H pylori sequences in the expression vector (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., eds., 1994). Isolation and Preparation of plasmid DNA from BL21 transformants
Individual clones of recombinant pET-28b vectors carrying properly cloned H. pylori ORFs were picked and incubated in 5 mis of LB broth plus 25 microgram/ml kanamycin sulfate overnight. The following day plasmid DNA was isolated and purified using the Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA, USA). Expression of recombinant H. pylori sequences in E. coli
The pET vector can be propagated in any E. coli K-12 strain e.g. ΗMS174, HB101, JM109, DH5, etc. for the puφose of cloning or plasmid preparation. Hosts for expression include E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that carries the lad gene, the lacUV5 promoter and the gene for T7 RNA polymerase. T7 RNA polymerase is induced by addition of isopropyl-B-D-thiogalactoside (IPTG), and the T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying a T7 promoter and a gene of interest. Strains used include: BL21(DE3) (Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60-89).
To express recombinant H. pylori sequences, 50 nanograms of plasmid DNA isolated as described above was used to transform competent BL21(DE3) bacteria as described above (provided by Novagen as part of the pET expression system kit). The lacZ gene (beta-galactosidase) was expressed in the pET- System as described for the H. pylori recombinant constructions. Transformed cells were cultured in SOC medium for 1 hour, and the culture was then plated on LB plates containing 25 micrograms/ml kanamycin sulfate. The following day, bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 micrograms/ml) to an optical density at 600 nM of 0.5 to 1.0 O.D. units, at which point, 1 millimolar IPTG was added to the culture for 3 hours to induce gene expression of the H. pylori recombinant DNA constructions.
After induction of gene expression with IPTG, bacteria were pelleted by centrifugation in a Sorvall RC-3B centrifuge at 3500 x g for 15 minutes at 4°C. Pellets were resuspended in 50 milliliters of cold 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). Cells were then centrifuged at 2000 x g for 20 min at 4°C. Wet pellets were weighed and frozen at -80°C until ready for protein purification.
HI. Purification of recombinant proteins from E. coli Analytical Methods
The concentrations of purified protein preparations were quantified spectrophotometrically using absorbance coefficients calculated from amino acid content (Perkins, S.J. 1986 Eur. J. Biochem. 157, 169-180). Protein concentrations were also measured by the method of Bradford, M.M. (1976) Anal. Biochem. 72, 248-254, and Lowry, O.H., Rosebrough, N., Farr, A.L. & Randall, R.J. (1951) J. Biol. Chem. 193, pages 265-275, using bovine serum albumin as a standard.
SDS-polyacrylamide gels (12%> or 4.0 to 25 %> acrylamide gradient gels) were purchased from BioRad (Hercules, CA, USA), and stained with Coomassie blue. Molecular weight markers included rabbit skeletal muscle myosin (200 kDa), E. coli (- galactosidase (1 16 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
/. Purification of soluble proteins
All steps were carried out at 4°C. Frozen cells were thawed, resuspended in 5 volumes of lysis buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 5 mM imidazole with 10%> glycerol, 0.1 %> 2-mercaptoethanol, 200 μg/ ml lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 ug/ml each of leupeptin, aprotinin, pepstatin, L-l-chloro-3-[4- tosylamido]-7-amino-2-heptanone (TLCK), L-l-chloro-3-[4-tosylamido]-4-phenyl-2- butanone (TPCK), and soybean trypsin inhibitor, and ruptured by several passages through a small volume microfluidizer (Model M-l 10S, Microfluidics International Coφoration, Newton, MA). The resultant homogenate was made 0.1 %> Brij 35, and centrifuged at 100,000 x g for 1 hour to yield a clear supernatant (crude extract).
Following filtration through a 0.8 μm Supor filter (Gelman Sciences, FRG) the crude extract was loaded directly onto a Ni^ "- nitrilotriacetate-agarose (NTA) with a 5 milliliter bed volume (Hochuli, E., Dbeli, H., and Schacheer, A. (1987) J. Chromatography 41 1, 177-184) pre-equilibrated in lysis buffer containing 10 %> glycerol, 0.1 % Brij 35 and 1 mM PMSF. The column was washed with 250 ml (50 bed volumes) of lysis buffer containing 10 %> glycerol, 0.1 %> Brij 35, and was eluted with sequential steps of lysis buffer containing 10 %> glycerol, 0.05 %> Brij 35, 1 mM PMSF, and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD28O nm' anc peak fractions were analyzed by SDS-PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole.
Recombinant protein 14640637 and proteins, beta-galactosidase (lacZ) andpeptidyl- prolyl cis-trans isomerase (ppiB)
Fractions containing the recombinant proteins from the Ni2+-NTA-agarose columns were pooled and then concentrated to approximately 5 ml by centrifugal filtration (Centriprep-10, Amicon, MA), and loaded directly onto a 180-ml column (1.6 X 91 cm) of Sephacryl S-100 HR gel filtration medium equilibrated in Buffer A (10 mM Hepes, pH 7.5. 150 mM NaCl, 0.1 mM EGTA) and run in Buffer A at 18 ml/h. Fractions containing the recombinant protein were identified by absorbance at 280 nm and analyzed by SDS-PAGE. Fractions were pooled and concentrated by centrifugal filtration.
Recombinant protein 7116626
Fractions containing the recombinant protein from the Ni^+ -NTA-agarose column were pooled and dialyzed overnight against 1 liter of dialysis buffer (10 mM MOPS, pH 6.5, 50 mM NaCl, 0.1 mM EGTA, 0.02% Brij 35 and 1 mM PMSF). In the morning, a fine white precipitate was removed by centrifugation and the resulting supernatant was loaded onto an 8 ml (8 x 75 mm) MonoS high performance liquid chromatography column (Pharmacia Biotechnology, Inc.. Piscataway. NJ, USA) equilibrated in buffer B (10 mM MOPS, pH 6.5, 0.1 mM EGTA) containing 50 mM NaCl. The column was washed with 10 bed volumes of buffer B containing 50 mM NaCl, and developed with a 50-ml linear gradient of increasing NaCl (50 to 500 mM). Recombinant protein 71 16626 eluted as a shaφ peak at 300 mM NaCl.
2. Purification of insoluble proteins from inclusion bodies
The following steps were carried out at 4°C. Cell pellets were resuspended in lysis buffer with 10%o glycerol 200 μg/ ml lysozyme, 5 mM EDTA, ImM PMSF and 0.1 % -mercaptoethanol. After passage through the cell disrupter, the resulting homogenate was made 0.2 %> deoxycholate. stirred 10 minutes, then centrifuged at 20,000 x g, for 30 min. The pellets were washed with lysis buffer containing 10 %> glycerol, 10 mM EDTA, 1% Triton X-100, 1 mM PMSF and 0.1% -mercaptoethanol, followed by several washes with lysis buffer containing 1 M urea, 1 mM PMSF and 0.1 % 2- mercaptoethanol. The resulting white pellet was composed primarily of inclusion bodies, free of unbroken cells and membranous materials..
Recombinant proteins 26054702, 16225006, 30100332, 4721061
The following steps were carried out at room temperature. Purified inclusion bodies were dissolved in 20 ml 8.0 M urea in lysis buffer with 1 mM PMSF and 0.1 % 2-mercaptoethanol, and incubated at room temperature for 1 hour. Materials that did not dissolve were removed by centrifugation. The clear supernatant was filtered, then loaded onto a Ni^+ -NTA agarose column pre-equilibrated in 8.0 M urea in Lysis Buffer. The column was washed with 250 ml (50 bed volumes) of lysis buffer containing 8 M urea, 1.0 mM PMSF and 0.1 % 2-mercaptoethanol, and developed with sequential steps of lysis buffer containing 8M urea, 1 mM PMSF, 0.1 %> 2- mercaptoethanol and 20, 100, 200, and 500 mM imidazole in succession. Fractions were monitored by absorbance at OD28O nrn- an<^ Pea^ fractions were analyzed by SDS- PAGE. Fractions containing the recombinant protein eluted at 100 mM imidazole.
Recombinant proteins 29479681, 26380318
The pellet containing the inclusion bodies was solubilized in buffer B containing
8 M urea, 1 mM PMSF and 0.1 % 2-mercaptoethanol, and incubated for 1 hour at room temperature. Insoluble materials were removed by centrifugation at 20,000 x g for 30 min, and the cleared supernatant was loaded onto a 15 ml ( 1.6 x 7.5 cm ) SP-Sepharose column pre-equilibrated in buffer B, 6 M urea, 1 mM PMSF, 0.1 % 2-mercaptoethanol.
After washing the column with 10 bed volumes, the column was developed with a linear gradient from 0 to 500 mM NaCl. Dialysis and concentration of protein samples
Urea was removed slowly from the protein samples by dialysis against Tris- buffered saline (TBS; 10 mM Tris pH 8.0, 150 mM NaCl) containing 0.5 % deoxycholate (DOC) with sequential reduction in urea concentration as follows; 6M,
4M, 3M, 2M, IM, 0.5 M and finally TBS without any urea. Each dialysis step was conducted for a minimum of 4 hours at room temperature.
After dialysis, samples were concentrated by pressure filtration using Amicon stirred-cells. Protein concentrations were measured using the methods of Perkins (1986
Eur. J. Biochem. 157, 169-180), Bradford ((1976) Anal. Biochem. 72, 248-254) and
Lowry ((1951) J. Biol. Chem. 193, pages 265-275). The recombinant proteins purified by the methods described above are summarized in Table 4 below.
TABLE 4
Outer Membrane Proteins
Periplasmic/Secreted Protein
Other Surface Proteins
Inner Membrane Proteins
IV. Analysis of H pylori proteins as Vaccine candidates
To investigate the immunomodulatory effect of H. pylori proteins, a mouse/H pylori model was used. This model mimics the human H. pylori infection in many respects. The focus is on the effect of oral immunization in H. pylori infected animals in order to test the concept of therapeutic oral immuno therapy.
Animals
Female SPF BALB/c mice were purchased from Bomholt Breeding center
(Denmark). They were kept in ordinary makrolon cages with free supply of water and food. The animals were 4-6 weeks old at arrival.
Infection
After a minimum of one week of acclimatization, the animals were infected with a type 2 strain (VacA negative) of H pylori (strain 244, originally isolated from an ulcer patient). In our hands, this strain has earlier proven to be a good colonizer of the mouse stomach. The bacteria were grown overnight in Brucella broth supplemented with 10 %> fetal calf serum, at 37°C in a microaerophilic atmosphere (10% CO2, 5%>O ). The animals were given an oral dose of omeprazole (400 μmol/kg) and 3-5 h after this an oral inoculation of H. pylori in broth (approximately 10 cfu/animal). Positive take of the infection was checked in some animals 2-3 weeks after the inoculation.
Antigens
Recombinant H pylori antigens were chosen based on their association with externally exposed H. pylori cell membrane. These antigens were selected from the following groups: (1.) Outer Membrane Proteins; (2.) Periplastic/Secreted proteins; (3.) Outer Surface proteins; and (4.) Inner Membrane proteins. All recombinant proteins were constructed with a hexa-ΗIS tag for purification reasons and the non-Helicobacter pylori control protein (b-galactosidase from E. coli; LacZ), was constructed in the same way.
All antigens were given in a soluble form, i.e. dissolved in either a ΗΕPΕS buffer or in a buffer containing 0.5%> Deoxycholate (DOC).
The antigens are listed in Table 5 below.
Table 5
Helicobacter pylori proteins
Outer membrane Proteins
Protein 7116626 Protein 4721061 Protein 16225006 Protein 29479681 Protein 14640637
Periplasmic/Secreted Proteins
Protein 30100332
Other cell envelope proteins
Protein 4821082
Flagella-associated proteins
Protein 26380318
Control proteins b-galactosidase (LacZ)
Immunizations
Ten animals in each group were immunized 4 times over a 34 day period (day 1, 15, 25 and 35). Purified antigens in solution or suspension were given at a dose of 100 mg/mouse. As an adjuvant, the animals were also given 10 μg/mouse of Cholera toxin (CT) with each immunization. Omeprazole (400 mmol/kg) was given orally to the animals 3-5 h prior to immunization as a way of protecting the antigens from acid degradation. Infected control animals received HEPES buffer + CT or DOC buffer + CT. Animals were sacrificed 2-4 weeks after final immunization. A general outline of the study is shown in Table 6 below.
Table 6
Study outline, therapeutic immunization:
Mice were all infected with H. pylori strain Ah244 at day 30
Substance Mouse strain Dose/mouse Dates for n=10 dosinε
1. Controls, PBS Balb/c 0.3 ml 0, 14, 24, 34
2. Cholera toxin, 10 μg Balb/c 0.3 ml 0, 14, 24, 34
3. Protein 16225006, 100 μg + CT 10 μμgg BBaallbb//cc 0.3 ml 0, 14, 24, 34
4. Protein 26054702, 100 μg + CT 10 μμgg BBaallbb//cc 0.3 ml 0, 14, 24, 34 5. Protein 26380318, 100 μg + CT 10 μg Balb/c 0.3 ml 0, 14, 24, 34
6. Protein 29479681, 100 μg + CT 10 μg Balb/c 0.3 ml 0, 14, 24, 34
7. Protein 30100332, 100 μg + CT 10 μgBalb/c 0.3 ml 0, 14, 24, 34
8. Protein 4721061 , lOO μg + CT lO μg Balb/c 0.3 ml 0, 14, 24, 34
9. Protein 4821082, 100 μg + CT 10 μg Balb/c 0.3 ml 0, 14, 24, 34
10. Protein 71 16626, 100 μg + CT 10 μg Balb/c 0.3 ml 0, 14, 24, 34
1 1. Protein 14640637, 100 μg + CT 10 μg Balb/c 0.3 ml 0, 14, 24, 34
Analysis of infection
Mucosal infection: The mice were sacrificed by CO2 and cervical dislocation. The abdomen was opened and the stomach removed. After cutting the stomach along the greater curvature, it was rinsed in saline. The mucosa from the antrum and coφus of an area of 25mm was scraped separately with a surgical scalpel. The mucosa scraping was suspended in Brucella broth and plated onto Blood Skirrow selective plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies was counted. The identity of H pylori was ascertained by urease and catalase test and by direct microscopy or Gram staining.
The urease test was performed essentially as follows. The reagent, Urea Agar Base Concentrate, was purchased from DIFCO Laboratories, Detroit, MI (Catalog # 0284-61-3). Urea agar base concentrate was diluted 1 : 10 with water. 1 ml of if the diluted concentrate was mixed with 100-200 ml of actively growing H. pylori cells. Color change to magenta indicated that cells were urease positive.
The catalase test was performed essentially as follows. The reagent, N,N,N',N'- Tetramethyl-p-Phenylenediamine, was purchased from Sigma, St. Louis, MO (Catalog # T3134). A solution of the regent (1% w/v in water) was prepared. H. pylori cells were swabbed onto Whatman filter paper and overlaid with the 1%> solution. Color change to dark blue indicated that the cells were catalase positive.
Serum antibodies: From all mice serum was prepared from blood drawn by heart puncture. Serum antibodies were identified by regular ELISA techniques, where the specific antigens of Helicobacter pylori were plated.
Mucosal antibodies: Gentle scrapings of a defined part of the coφus and of 4 cm of duodenum were performed in 50%> of the mice in order to detect the presence of antibodies in the mucous. The antibody titers were determined by regular ELISA technique as for serum antibodies.
Statistical analysis: Wilcoxon-Mann- Whitney sign rank test was used for determination of significant effects of the antigens on Helicobacter pylori colonization. P<0.05 was considered significant. Because the antrum is the major colonization site for Helicobacter most emphasis was put upon changes in the antral colonization.
Results
Antibodies in sera: All antigens tested given together with CT gave rise to a measurable specific titer in serum. The highest responses were seen with Protein
7116626, Protein 4721061, Protein 26380318, Protein 14640637 and Protein 4821082 (see Figure 1).
Antibodies in mucus: In the mucus scrapings, specific antibodies against all antigens tested were seen. By far the strongest response was seen with Protein 30100332, followed by Protein 14640637, and Protein 26380318 (see Figure 2).
Therapeutic immunization effects:
All control animals (BALB/c mice) were well colonized with H. pylori (strain AΗ244) in both antrum and coφus of the stomach. Of the antigens tested 3 proteins (Protein 4721061, Protein 4821082, and Protein 14640637) gave a good and significant reduction and/or eradication of the H. pylori infection. The degree of colonization of the antrum was lower following immunization with Protein 7116626 and Protein 26380318 compared to control. The effect of Proteins 16225006, 29479681, and 30100332 did not differ from control. The control protein lacZ, i.e. the non-H pylori protein, had no eradication effect and in fact had higher Helicobacter colonization compared to the ΗEPES + CT control. All data are shown in Figures 3 and 4 for proteins dissolved in ΗEPES and DOC respectively. Data is shown as geometric mean values. n=8-10 Wilcoxon-Mann- Whitney sign rank test * = p<0.05; x 10 = number of mice showing eradication of H pylori over the total number of mice examined.
The data presented indicate that all of the H pylori associated proteins included in this study, when used as oral immunogens in conjunction with the oral adjuvant CT, resulted in stimulation of an immune response as measured by specific serum and mucosal antibodies. A majority of the proteins led to a reduction, and in some cases complete clearance of the colonization of H pylori in this animal model. It should be noted that the reduction or clearance was due to heterologous protection rather than homologous protection (the polypeptides were based on the H. pylori J99 strain sequence and used in the therapeutic immunization studies against a different (AH244) challenge strain, indicating the vaccine potential against a wide variety of H. pylori strains.
The highest colonization in the antrum was seen in animals treated with the non- Helicobacter protein LacZ, indicating that the effects seen with the Helicobacter pylori antigens were specific.
Taken together these data strongly support the use of these H. pylori proteins in a pharmaceutical formulation for the use in humans to treat and/or prevent H. pylori infections.
V. Sequence Variance Analysis of genes in Helicobacter pylori strains
Four genes were cloned and sequenced from several strains of H. pylori to compare the DNA and deduced amino acid sequences. This information was used to determine the sequence variation between the H. pylori strain, J99, and other H. pylori strains isolated from human patients.
Preparation of Chromosomal DNA.
Cultures of H. pylori strains (as listed in Table 9) were grown in BLBB (1% Tryptone, 1% Peptamin 0.1% Glucose, 0.2% Yeast Extract 0.5% Sodium Chloride, 5% Fetal Bovine Serum) to an OD600 of 0.2. Cells were centrifuged in a Sorvall RC-3B at 3500 x g at 4°C for 15 minutes and the pellet resuspended in 0.95 mis of 10 mM Tris- HCl, 0.1 mM EDTA (TE). Lysozyme was added to a final concentration of 1 mg/ml along with, SDS to 1% and RNAse A + TI to 0.5mg/ml and 5 units/ml respectively, and incubated at 37°C for one hour. Proteinase K was then added to a final concentration of 0.4mg/ml and the sample was incubated at 55 C for more than one hour. NaCl was added to the sample to a concentration of 0.65 M, mixed carefully, and 0.15 ml of 10% CTAB in 0.7M NaCL (final is 1% CTAB/70mM NaCL) was added followed by incubation at 65°C for 20 minutes. At this point, the samples were extracted with chloroformύsoamyl alcohol, extracted with phenol, and extracted again with chloroformύsoamyl alcohol. DNA was precipitated with either EtOH (1.5 x volumes) or isopropanol (0.6 x volumes) at -70°C for lOminutes, washed in 70%) EtOH and resuspended in TE.
PCR Amplification and cloning. Genomic DNA prepared from twelve strains of Helicobacter pylori was used as the source of template DNA for PCR amplification reactions (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). To amplify a DNA sequence containing an H pylori ORF, genomic DNA (10 nanograms) was introduced into a reaction vial containing 2 mM MgCl2, 1 micromolar synthetic oligonucleotide primers (forward and reverse primers, see Table 7) complementary to and flanking a defined H. pylori ORF, 0.2 mM of each deoxynucleotide triphosphate; dATP, dGTP, dCTP, dTTP and 0.5 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 20 microliters in duplicate reactions.
Table 7 Oligonucleotide primers used for PCR amplification of H pylori DNA sequences.
The following thermal cycling conditions were used to obtain amplified DNA products for each ORF using a Perkin Elmer Cetus/ GeneAmp PCR System 9600 thermal cycler: Protein 7116626 and Protein 346; Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 30°C for 15 sec and 72°C for 1.5 min 23 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min Reactions were concluded at 72°C for 6 minutes.
Protein 26054702 for strains AH5, 5155, 7958, AH24,and J99; Denaturation at 94°C for 2 min, 2 cycles at 94°C for 15 sec, 30°C for 15 sec and 72°C for 1.5 min 25 cycles at 94°C for 15 sec, 55°C for 15 sec and 72°C for 1.5 min Reaction was concluded at 72°C for 6 minutes.
Protein 26054702 and Protein 294796813 for strains AH4, AH15, AH61, 5294, 5640, AH 18, and Hp244 ;
Denaturation at 94°C for 2 min,
2 cycles at 94°C for 15 sec, 30°C for 20 sec and 72°C for 2 min
25 cycles at 94°C for 15 sec, 55°C for 20 sec and 72°C for 2 min
Reactions were concluded at 72°C for 8 minutes.
Upon completion of thermal cycling reactions, each pair of samples were combined and used directly for cloning into the pCR cloning vector as described below.
Cloning ofH. pylori DNA sequences into the pCR TA cloning vector. All amplified inserts were cloned into the pCR 2.1 vector by the method described in the Original TA cloning kit (Invitrogen, San Diego, CA). Products of the ligation reaction were then used to transform the TOPI OF' (INVaF' in the case of H. pylori sequence 350) strain of E. coli as described below.
Transformation of competent bacteria with recombinant plasmids
Competent bacteria, E coli strain TOPI OF' or E. coli strain INVaF' were transformed with recombinant pCR expression plasmids carrying the cloned H. pylori sequences according to standard methods (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). Briefly, 2 microliters of 0.5 micromolar BMΕ was added to each vial of 50 microliters of competent cells.
Subsequently, 2 microliters of ligation reaction was mixed with the competent cells and incubated on ice for 30 minutes. The cells and ligation mixture were then subjected to a "heat shock" at 42°C for 30 seconds, and were subsequently placed on ice for an additional 2 minutes, after which, samples were incubated in 0.45 milliliters SOC medium (0.5%) yeast extract, 2.0 % tryptone. 10 mM NaCl, 2.5 mM KCl, 10 mM MgC12, 10 mM MgSO4 and 20, mM glucose) at 37°C with shaking for 1 hour. Samples were then spread on LB agar plates containing 25 microgram/ml kanamycin sulfate or 100 micrograms/ml ampicillan for growth overnight. Transformed colonies of TOPI OF' or INVaF' were then picked and analyzed to evaluate cloned inserts as described below. Identification of recombinant PCR plasmids carrying H. pylori sequences
Individual TOPI OF' or INVaF' clones transformed with recombinant pCR- Hpylori ORFs were analyzed by PCR amplification of the cloned inserts using the same forward and reverse primers, specific for each H. pylori sequence, that were used in the original PCR amplification cloning reactions. Successful amplification verified the integration of the H. pylori sequences in the cloning vector (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). Individual clones of recombinant pCR vectors carrying properly cloned H. pylori
ORFs were picked for sequence analysis. Sequence analysis was performed on ABI
Sequencers using standard protocols (Perkin Elmer) using vector-specific primers (as found in PCRII or pCR2.1, Invitrogen, San Diego, CA) and sequencing primers specific to the ORF as listed in Table 8 below.
Table 8
Oligonucleotide primers used for sequencing of H. pylori DNA sequences.
Results
To establish the PCR error rate in these experiments, five individual clones of Protein 26054702, prepared from five separate PCR reaction mixtures from H. pylori strain J99, were sequenced over a total length of 897 nucleotides for a cumulative total of 4485 bases of DNA sequence. DNA sequence for the five clones was compared to a DNA sequence obtained previously by a different method, i.e., random shotgun cloning and sequencing. The PCR error rate for the experiments described herein was determined to be 2 base changes out of 4485 bases, which is equivalent to an estimated error rate of less than or equal to 0.04%.
DNA sequence analysis was performed on four different open reading frames identified as genes and amplified by PCR methods from a dozen different strains of the bacterium Helicobacter pylori. The deduced amino acid sequences of three of the four open reading frames that were selected for this study showed statistically significant BLAST homology to defined proteins present in other bacterial species. Those ORFs included: Protein 26054702, homologous to the val A & B genes encoding an ABC transporter in F. novicida; Protein 71 16626, homologous to lipoprotein e (P4) present in the outer membrane of Η. influenzae; Protein 29479681 , homologous to fecA, an outer membrane receptor in iron (III) dicitrate transport in E. coli. Protein 346 was identified as an unknown open reading frame, because it showed low homology with sequences in the public databases.
To assess the extent of conservation or variance in the ORFs across various strains of H pylori, changes in DNA sequence and the deduced protein sequence were compared to the DNA and deduced protein sequences found in the J99 strain of H pylori (see Table 9 below). Results are presented as percent identity to the J99 strain of H. pylori sequenced by random shotgun cloning. To control for any variations in the J99 sequence each of the four open reading frames were cloned and sequenced again from the J99 bacterial strain and that sequence information was compared to the sequence information that had been collected from inserts cloned by random shotgun sequencing of the J99 strain. The data demonstrate that there is variation in the DNA sequence ranging from as little as 0.12 % difference (Protein 346, J99 strain) to approximately 7% change (Protein 26054702, strain AΗ5). The deduced protein sequences show either no variation ( Protein 346, strains AH 18 and AH24) or up to as much as 7.66% amino acid changes (Protein 26054702, Strain AH5).
Table 9
Multiple Strain DNA Sequence analysis of H. pylori Vaccine Candidates
J99 Protein #: 26054702 2054702 71166267116626 29479681 29479681 346 346
Length ofRegion
Sequenced: 248 a.a. 746 nt. 232 a.a. 96 nt. 182a.a. 548 nt. 273 a.a. 819 nt.
Strain Tested
AA Nuc. AA Nuc. AA Nuc. AA Nuc. identity identity identity identity identity identity identity identity
J99 100.00% 100.00% 100.00% 100.00% 100.00% 100.00% 99.63% 99.88%
AH244 95.16% 95.04% n.d. n.d. 99.09% 96.71% 98.90% 96.45%
AH4 95.97% 95.98% 97.84% 95.83% n.d. n.d. 97.80% 95.73%
AH5 92.34% 93.03% 98.28% 96.12% 98.91% 96.90% 98.53% 95.73%
AH 15 95.16% 94.91% 97.41 % 95.98% 99.82% 97.99% 99.63% 96.09%
AH61 n.d. n.d. 97.84% 95.98% 99.27% 97.44% n.d. n.d.
5155 n.d. n.d. n.d. n.d. 99.45% 97.08% 98.53% 95.60%
5294 94.35% 94.37% 98.28% 95.40% 99.64% 97.26% 97.07% 95.48%
7958 94.35% 94.10% 97.84% 95.40% n.d. n.d. 99.63% 96.46%
5640 95.16% 94.37% 97.41 % 95.69% 99.09% 97.63% 98.53% 95.48%
AH 18 n.d. n.d. 98.71% 95.69% 99.64% 97.44% 100.00% 95.97%
AH24 94.75% 95.04% 97.84% 95.40% 99.27% 96.71% 100.00% 96.46%
n.d.= not done. VI. Experimental Knock-Out Protocol for the Determination of Essential H pylori Genes as Potential Therapeutic Targets
Therapeutic targets are chosen from genes whose protein products appear to play key roles in essential cell pathways such as cell envelope synthesis, DNA synthesis, transcription, translation, regulation and colonization/virulence.
The protocol for the deletion of portions of H. pylori genes/ORFs and the insertional mutagenesis of a kanamycin-resistance cassette in order to identify genes which are essential to the cell is modified from previously published methods (Labigne- Roussel et al., 1988, J. Bacteriology 170, pp. 1704-1708; Cover et al.,1994, J. Biological Chemistry 269, pp. 10566-10573; Reyrat et al., 1995, Proc. Natl. Acad. Sci. 92, pp 8768-8772). The result is a gene "knock-out."
Identification and Cloning ofH. pylori Gene Sequences The sequences of the genes or ORFs (open reading frames) selected as knock-out targets are identified from the H. pylori genomic sequence and used to design primers to specifically amplify the genes/ORFs. All synthetic oligonucleotide primers are designed with the aid of the OLIGO program (National Biosciences, Inc., Plymouth, MN 55447, USA), and can be purchased from Gibco/BRL Life Technologies (Gaithersburg, MD, USA). If the ORF is smaller than 800 to 1000 base pairs, flanking primers are chosen outside of the open reading frame.
Genomic DNA prepared from the Helicobacter pylori ΗpJ99 strain (ATCC 55679; deposited by Genome Therapeutics Coφoration, 100 Beaver Street, Waltham, MA 02154) is used as the source of template DNA for amplification of the ORFs by PCR (polymerase chain reaction) (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al.. editors, 1994). For the preparation of genomic DNA from H. pylori, see Example I. PCR amplification is carried out by introducing 10 nanograms of genomic HpJ99 DNA into a reaction vial containing 10 mM Tris pH 8.3, 50 mM KCl, 2 mM MgCl2, 2 microMolar synthetic oligonucleotide primers (forward=Fl and reverse=Rl), 0.2 mM of each deoxynucleotide triphosphate
(dATP,dGTP, dCTP, dTTP), and 1.25 units of heat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc.. Branchburg, NJ, USA) in a final volume of 40 microliters. The PCR is carried out with Perkin Elmer Cetus/GeneAmp PCR System 9600 thermal cyclers. Upon completion of thermal cycling reactions, each sample of amplified DNA is visualized on a 2%> TAE agarose gel stained with Ethidium Bromide (Current Protocols in Molecular Biology, John Wiley and Sons, Inc.. F. Ausubel et al., editors, 1994) to determine that a single product of the expected size had resulted from the reaction. Amplified DNA is then washed and purified using the Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA).
PCR products are cloned into the pT7Blue T-Vector (catalog#69820-l, Novagen, Inc., Madison, WI, USA) using the TA cloning strategy (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). The ligation of the PCR product into the vector is accomplished by mixing a 6 fold molar excess of the PCR product, 10 ng of pT7Blue-T vector (Novagen), 1 microliter of T4 DNA Ligase Buffer (New England Biolabs, Beverly, MA, USA), and 200 units of T4 DNA Ligase (New England Biolabs) into a final reaction volume of 10 microliters. Ligation is allowed to proceed for 16 hours at 16°C.
Ligation products are electroporated (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al.. editors, 1994) into electroporation- competent XL-1 Blue or DH5-a E.coli cells (Clontech Lab., Inc. Palo Alto, CA, USA). Briefly, 1 microliter of ligation reaction is mixed with 40 microliters of electrocompetent cells and subjected to a high voltage pulse (25 microFarads, 2.5 kV, 200 ohms) after which the samples are incubated in 0.45 ml SOC medium (0.5%) yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose) at 37°C with shaking for 1 hour. Samples are then spread onto LB (10 g/1 bacto tryptone, 5 g/1 bacto yeast extract, 10 g/1 sodium chloride) plates containing 100 microgram/ml of Ampicillin, 0.3% X-gal, and 100 microgram/ml IPTG. These plates are incubated overnight at 37°C. Ampicillin-resistant colonies with white color are selected, grown in 5 ml of liquid LB containing 100 microgram/ml of Ampicillin, and plasmid DNA is isolated using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD, USA).
To verify that the correct Hpylori DNA inserts had been cloned, these pT7Blue plasmid DNAs are used as templates for PCR amplification of the cloned inserts, using the same forward and reverse primers used for the initial amplification of the J99 Hpylori sequence. Recognition of the primers and a PCR product of the correct size as visualized on a 2% TAE, ethidium bromide stained agarose gel are confirmation that the correct inserts had been cloned. Two to six such verified clones are obtained for each knock-out target, and frozen at -70°C for storage. To minimize errors due to PCR, plasmid DNA from these verified clones are pooled, and used in subsequent cloning steps. The sequences of the genes/ORFs are again used to design a second pair of primers which flank the region of H. pylori DNA to be either interrupted or deleted (up to 250 basepairs) within the ORFs but are oriented away from each other. The pool of circular plasmid DNAs of the previously isolated clones are used as templates for this round of PCR. Since the orientation of amplification of this pair of deletion primers is away from each other, the portion of the ORF between the primers is not included in the resultant PCR product. The PCR product is a linear piece of DNA with H. pylori DNA at each end and the pT7Blue vector backbone between them which, in essence, resultes in the deletion of a portion of the ORFs. The PCR product is visualized on a 1% TAE, ethidium bromide stained agarose gel to confirm that only a single product of the correct size has been amplified.
A Kanamycin-resistance cassette (Labigne-Roussel et al, 1988 J. Bacteriology 170, 1704- 1708) is ligated to this PCR product by the TA cloning method used previously (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., F. Ausubel et al., editors, 1994). The Kanamycin cassette containing a Campylobacter kanamycin resistance gene is obtained by carrying out an EcoRI digestion of the recombinant plasmid pCTB8:te? (Cover et al.,1994, J. Biological Chemistry 269, pp. 10566-10573). The proper fragment (1.4 kb) is isolated on a 1% TAE gel, and isolated using the QIAquick gel extraction kit (Qiagen, Gaithersburg, MD, USA). The fragment is end repaired using the Klenow fill-in protocol, which involved mixing 4ug of the DNA fragment, 1 microliter of dATP,dGTP, dCTP, dTTP at 0.5 mM, 2 microliter of Klenow Buffer (New England Biolabs) and 5 units of Klenow DNA Polymerase I Large (Klenow) Fragment (New England Biolabs) into a 20 microliter reaction, incubating at 30°C for 15 min, and inactivating the enzyme by heating to 75°C for 10 minutes. This blunt-ended Kanamycin cassette is then purified through a Qiaquick column (Qiagen, Gaithersburg, MD, USA) to eliminate nucleotides. The "T" overhang is then generated by mixing 5 micrograms of the blunt-ended kanamycin cassette, 10 mM Tris pH 8.3, 50 mM KCl, 2 mM MgCl2, 5 units of DNA Polymerase (Amplitaq, Roche Molecular
Systems, Inc., Branchburg, NJ, USA), 20 microliters of 5 mM dTTP, in a 100 microliter reaction and incubating the reaction for 2 hours at 37°C. The "Kan-T" cassette is purified using a QIAquick column (Qiagen, Gaithersburg, MD, USA). The PCR product of the deletion primers (F2 and R2) is ligated to the Kan-T cassette by mixing 10 to 25 ng of deletion primer PCR product, 50 - 75 ng Kan-T cassette DNA, 1 microliter lOx T4 DNA Ligase reaction mixture, 0.5 microliter T4 DNA Ligase (New England Biolabs, Beverly, MA, USA) in a 10 microliter reaction and incubating for 16 hours at 16°C.
The ligation products are transformed into XL-1 Blue or DH5-a E.coli cells by electroporation as described previously. After recovery in SOC, cells are plated onto LB plates containing 100 microgram/ml Ampicillin and grown overnight at 37°C. These plates are then replica plated onto plates containing 25 microgram ml Kanamycin and allowed to grow overnight. Resultant colonies have both the Ampicillin resistance gene present in the pT7Blue vector, and the newly introduced Kanamycin resistance gene. Colonies are picked into LB containing 25 microgram/ml Kanamycin and plasmid DNA is isolated from the cultured cells using the Qiagen miniprep protocol (Qiagen, Gaithersburg, MD. USA).
Several tests by PCR amplification are conducted on these plasmids to verify that the Kanamycin is inserted in the H. pylori gene/ORF, and to determine the orientation of the insertion of the Kanamycin-resistance gene relative to the H. pylori gene/ORF. To verify that the Kanamycin cassette is inserted into the H pylori sequence, the plasmid DNAs are used as templates for PCR amplification with the set of primers originally used to clone the H pylori gene/ORFs. The correct PCR product is the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette. To avoid potential polar effects of the kanamycin resistance cassette on H pylori gene expression, the orientation of the Kanamycin resistance gene with respect to the knock-out gene/ORF is determined and both orientations are eventually used in H pylori transformations (see below). To determine the orientation of insertion of the kanamycin resistance gene, primers are designed from the ends of the kanamycin resistance gene ("Kan-1 " 5'-ATCTTACCTATCACCTCAAAT-3' (SEQ ID NO:207)), and "Kan-2" 5'-AGACAGCAACATCTTTGTGAA-3' (SEQ ID NO:208)). By using each of the cloning primers in conjunction with each of the Kan primers (4 combinations of primers), the orientation of the Kanamycin cassette relative to the H. pylori sequence is determined. Positive clones are classified as either in the "A" orientation (the same direction of transcription is present for both the H. pylori gene and the Kanamycin resistance gene), or in the "B" orientation (the direction of transcription for the H. pylori gene is opposite to that of the Kanamycin resistance gene). Clones which share the same orientation (A or B) are pooled for subsequent experiments and independently transformed into H pylori.
Transformation of Plasmid DNA into H. pylori cells Two strains of H pylori are used for transformation: ATCC 55679, the clinical isolate which provided the DNA from which the H. pylori sequence database is obtained, and AΗ244, an isolate which had been passaged in, and has the ability to colonize the mouse stomach. Cells for transformation are grown at 37°C, 10%> CO2, 100%) humidity, either on Sheep-Blood agar plates or in Brucella Broth liquid. Cells are grown to exponential phase, and examined microscopically to determine that the cells are "healthy" (actively moving cells) and not contaminated. If grown on plates, cells are harvested by scraping cells from the plate with a sterile loop, suspended in 1 ml of Brucella Broth, spun down (1 minute, top speed in eppendorf microfuge) and resuspended in 200 microliters Brucella Broth. If grown in Brucella Broth liquid, cells are centrifuged (15 minutes at 3000 rpm in a Beckman TJ6 centrifuge) and the cell pellet resuspended in 200 microliters of Brucella broth. An aliquot of cells is taken to determine the optical density at 600 nm, in order to calculate the concentration of cells. An aliquot (1 to 5 OD60o units/25 microliter) of the resuspended cells is placed onto a prewarmed Sheep-Blood agar plate, and the plate is further incubated at 37°C, 6%> CO2, 100%) humidity for 4 hours. After this incubation, 10 microliters of plasmid DNA (100 micrograms per microliter) is spotted onto these cells. A positive control (plasmid DNA with the ribonuclease H gene disrupted by kanamycin resistance gene) and a negative control (no plasmid DNA) are done in parallel. The plates are returned to 37°C, 6% CO2 for an additional 4 hours of incubation. Cells are then spread onto that plate using a swab wetted in Brucella broth, and grown for 20 hours at 37°C, 6%o CO2. Cells are then transferred to a Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin, and allowed to grow for 3 to 5 days at 37°C, 6% CO2, 100%) humidity. If colonies appear, they are picked and regrown as patches on a fresh Sheep-Blood agar plate containing 25 micrograms/ml Kanamycin.
Three sets of PCR tests are done to verify that the colonies of transformants have arisen from homologous recombination at the proper chromosomal location. The template for PCR (DNA from the colony) is obtained by a rapid boiling DNA preparation method as follows. An aliquot of the colony (stab of the colony with a toothpick) is introduced into 100 microliters of 1% Triton X-100, 20 mM Tris, pH 8.5, and boiled for 6 minutes. An equal volume of phenol : chloroform (1 : 1) is added and vortexed. The mixture is microfuged for 5 minutes and the supernatant is used as DNA template for PCR with combinations of the following primers to verify homologous recombination at the proper chromosomal location.
TEST 1. PCR with cloning primers originally used to amplify the gene/ORF. A positive result of homologous recombination at the correct chromosomal location should show a single PCR product whose size is expected to be the size of the deleted gene/ORF but increased in size by the addition of a 1.4 kilobase Kanamycin cassette. A PCR product of just the size of the gene/ORF is proof that the gene had not been knocked out and that the transformant is not the result of homologous recombination at the correct chromosome location.
TEST 2. PCR with F3 (primer designed from sequences upstream of the gene/ORF and not present on the plasmid), and either primer Kan-1 or Kan-2 (primers designed from the ends of the kanamycin resistance gene), depending on whether the plasmid DNA used was of "A" or "B" orientation. Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the location of F3 to the insertion site of kanamycin resistance gene). No PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out. TEST 3. PCR with R3 (primer designed from sequences downstream of the gene/ORF and not present on the plasmid) and either primer Kan-1 or Kan-2, depending on whether the plasmid DNA used was of "A" or "B" orientation. Homologous recombination at the correct chromosomal location will result in a single PCR product of the expected size (i.e., from the insertion site of kanamycin resistance gene to the downstream location of R3). Again, no PCR product or PCR product(s) of incorrect size(s) will prove that the plasmid had not integrated at the correct site and that the gene had not been knocked out.
Transformants showing positive results for all three tests above indicate that the gene is not essential for survival in vitro. A negative result in any of the three above tests for each transformant indicates that the gene had not been disrupted, and that the gene is essential for survival in vitro. In the event that no colonies result from two independent transformations while the positive control with the disrupted ribonuclease H plasmid DNA produces transformants, the plasmid DNA is further analyzed by PCR on DNA from transformant populations prior to plating for colony formation. This will verify that the plasmid can enter the cells and undergo homologous recombination at the correct site. Briefly, plasmid DNA is incubated according to the transformation protocol described above. DNA is extracted from the H. pylori cells immediately after incubation with the plasmid DNAs and the DNA is used as template for the above TEST 2 and TEST 3. Positive results in TEST 2 and TEST 3 would verify that the plasmid DNA could enter the cells and undergo homologous recombination at the correct chromosomal location. If TEST
2 and TEST 3 are positive, then failure to obtain viable transformants indicates that the gene is essential, and cells suffering a disruption in that gene are incapable of colony formation
VII. High-throughput drug screen assay
Cloning, expression and protein purification
Cloning, transformation, expression and purification of the H. pylori target gene and its protein product,e.g., an H. pylori enzyme, to be used in a high-throughput drug screen assay, is carried out essentially as described in Examples II and III above.
Development and application of a screening assay for a particular H. pylori gene product, peptidyl-propyl cis-trans isomerase. is described below as a specific example. Enzymatic Assay
The assay is essentially as described by Fisher (Fischer, G., et.al. (1984) Biomed. Biochim. Ada 43:1101-1111). The assay measures the cis-trans isomerization of the Ala-Pro bond in the test peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma # S- 7388, lot # 84H5805). The assay is coupled with α-chymotrypsin, where the ability of the protease to cleave the test peptide occurs only when the Ala-Pro bond is in trans. The conversion of the test peptide to the trans isomer in the assay is followed at 390 nm on a Beckman Model DU-650 spectophotometer. The data are collected every second with an average scanning of time of 0.5 second. Assays are carried out in 35 mM Hepes, pH 8.0, in a final volume of 400 ul, with 10 μM α-chymotrypsin (type 1-5 from bovine Pancreas, Sigma # C-7762, lot 23H7020) and 10 nM PPIase. To initiate the reaction. 10 μl of the substrate ( 2 mM N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in DMSO) is added to 390 μl of reaction mixture at room temperature.
Enzymatic assay in crude bacterial extract.
A 50 ml culture of Helicobacter pylori (strain J99) in Brucella broth is harvested at mid-log phase (OD oo nm ~ 1 ) and resuspended in lysis buffer with the following protease inhibitors: 1 mM PMSF, and 10 μg/ml of each of aprotinin, leupeptin, pepstatine, TLCK, TPCK, and soybean trypsin inhibitor. The suspension is subjected to 3 cycles of freeze-thaw (15 minutes at -70 C, then 30 minutes at room temperature), followed by sonication (three 20 second bursts). The lysate is centrifuged (12,000 g x 30 minutes) and the supernatant is assayed for enzymatic activity as described above.
Many H. pylori enzymes can be expressed at high levels and in an active form in E. coli. Such high yields of purified proteins provide for the design of various high throughput drug screening assays.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims. SEQUENCE LISTING
1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Astra Aktiebolag
(B) STREET: S-151 85
(C) CITY: Sodertalje (D) STATE:
(E) COUNTRY: Sweden
(F) POSTAL CODE (ZIP)
(ii) TITLE OF INVENTION: NUCLEIC ACID AND AMINO ACID SEQUENCES RELATING TO HELICOBACTER PYLORI AND
VACCINE COMPOSITIONS THEREOF
(iii) NUMBER OF SEQUENCES: 208
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE:
(B) COMPUTER:
(C) OPERATING SYSTEM: (D) SOFTWARE:
(v) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER
(B) FILING DATE:
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:US 08/739,150
(B) FILING DATE: 28-OCT-1996 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/759,739
(B) FILING DATE: 06-DEC-1996
(viii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/891,928
(B) FILING DATE: 14 -JULY- 1997
(ix) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: LAHIVE & COCKFIELD (B) STREET: 28 State Street
(C) CITY: Boston
(D) STATE: Massachusetts
(E) COUNTRY: USA
(F) ZIP: 02109-1875
(x) ATTORNEY/AGENT INFORMATION:
(A) NAME : Mandragouras , Amy E .
(B) REGISTRATION NUMBER: 36,207
(C) REFERENCE/DOCKET NUMBER: GTN-001CP10PC (xi) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617)227-7400
(B) TELEFAX: (617)742-4214 (2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 561 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...561
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
ATGATTAAAA GAATTGCTTG TATTTTAAGC TTGAGCGCGA GTTTAGCGTT AGCTGGCGAA 60
GTGAATGGGT TTTTCATGGG TGCGGGTTAT CAACAAGGTC GTTATGGCCC TTATAACAGC 120 AATTACTCTG ATTGGCGTCA TGGCAATGAC CTTTATGGTT TGAATTTCAA ATTAGGTTTT 180
GTAGGCTTTG CCAATAAATG GTTTGGGGCT AGGGTGTATG GCTTTTTAGA TTGGTTTAAC 240
ACTTCAGGGA CTGAACACAC CAAAACCAAT TTGCTCACCT ATGGCGGCGG TGGCGATTTG 300
ATTGTCAATC TCATTCCTTT GGATAAATTC GCTCTAGGTC TCATTGGTGG CGTTCAATTA 360
GCCGGAAACA CTTGGATGTT CCCTTATGAT GTCAATCAAA CCAGATTCCA GTTCTTATGG 420 AATTTAGGCG GAAGAATGCG TGTTGGGGAT CGCAGTGCGT TTGAAGCGGG CGTGAAATTC 480
CCTATGGTTA ATCAGGGTAG CAAAGATGTA GGGCTTATCC GCTACTATTC TTGGTATGTG 540
GATTATGTCT TCACTTTCTA G 561
(2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 351 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori ( ix) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION 1...351 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
TTGATGCGCA TTATCATAAG GTTACTTTCA TTTAAAATGA ACGCTTTTTT AAAACTCGCG 60
CTCGCTTCTT TGATGGGGGG GCTTTGGTAT GCTTTCAATG GCGAAGGCTC TGAGATTGTC 120
GCTATAGGGA TTTTTGTGTT GATCTTGTTT GTTTTTTTTA TCCGCCCTGT GAGTTTCCAA 180 GACCCAGAAA AACGAGAAGA ATACATAGAA CGGCTTAAAA AAAACCATGA GAGGAAAATG 240
ATCTTACAAG ACAAGCAAAA AGAAGAGCAA ATGCGCCTCT ATCAAGCCAA AAAAGAGCGA 300
GAGAGCAGGC AAAAACAAGA CCTTAAAGAA CAAATGAAAA AATACTCATA A 351
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1038 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1038 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
ATGGTTAAAC ACTATCTTTT CATGGCGGTT TCGCAGGTCT TTTTCTCCTT CTTTTTAGTG 60
CTGTTTTTTA TCTCTTCCAT TGTGTTATTA ATCAGTATTG CAAGCGTAAC GCTCGTGATT 120
AAAGTGAGCT TTTTGGATCT GGTGCAACTC TTTTTGTATT CCTTGCCAGG AACCATTTTT 180 TTTATTTTGC CGATCACTTT TTTTGCGGCT TGCGCTTTGG GGCTTTCAAG GCTTAGCTAT 240
GACCATGAAT TGTTAGTGTT TTTCTCTTTA GGGGTTTCGC CTAAAAAAAT GACTAAAGCG 300
TTTGTGCCTT TAAGTTTGTT AGTGAGCGCG ATTTTATTAG CGTTTTCGCT CATCTTAATC 360
CCCACTTCTA AGAGCGCTTA TTACGGGTTT TTGCGTCAAA AAAAAGACAA GATTGACATT 420
AACATCAGAG CGGGTGAATT CGGGCAAAAA TTAGGCGATT GGCTCGTGTA TGTGGATAAG 480 ACTGAAAACA ATTCCTATGA TAATTTGGTG CTTTTTTCTA ATAAAAGTCT CTCTCAAGAA 540
AGCTTTATTT TGGCTCAAAA AGGCAATATC AACAATCAAA ACGGCGTGTT TGAATTGAAT 600
TTGTATAACG GGCATGCGTA TTTCACTCAA GGCGATAAAA TGCGTAAGGT TGATTTTGAA 660
GAATTGCATT TGCGCAACAA GCTCAAGTCT TTCAATTCTA ATGATGCGGC TTATTTGCAA 720
GGCACGGATT ATTTGGGTTA TTGGAAAAAA GCCTTTGGTA AAAACGCTAA TAAAAATCAA 780 AAACGCCGTT TTTCTCAAGC GATCTTAGTT TCCTTGTTCC CTTTAGCGAG CGTGTTTTTA 840
ATCCCCTTAT TTGGCATCGC CAACCCGCGA TTCAAAACGA ATTGGAGTTA TTTCTATGTC 900
CTTGGAGCGG TTGGGGTTTA TTTTTTAATG GTGCATGTGA TTTCTACGGA TTTGTTTTTG 960
ATGACCTTTT TCTTCCCCTT TATTTGGGCG TTTATTTCTT ATTTATTGTT TAGAAAATTC 1020
ATTTTAAAGC GTTATTAA 1038 (2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 831 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...831
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
ATGAAGAAAA AAGCAAAAGT CTTTTGGTGT TGTTTTAAAA TGATTCGTTG GTTGTATTTG 60 GCGGTCTTTT TTTTGTTGAG CGTATCAGAC GCTAAAGAAA TCGCTATGCA ACGATTTGAC 120
AAACAAAACC ATAAGATTTT TGAAATCCTT GCGGATAAAG TGAGCGCCAA AGACAATGTG 180
ATAACCGCCT CAGGGAATGC GATCCTATTG AATTATGACG TGTATATTCT AGCGGATAAG 240
GTGCGTTATG ACACCAAGAC TAAAGAAGCG TTATTAGAAG GCAATATTAA GGTTTATAGG 300
GGCGAGGGCT TGCTCGTTAA AACCGATTAT GTGAAATTGA GTTTGAACGA AAAATATGAG 360 ATCATTTTCC CCTTTTATGT CCAAGACAGC GTGAGCGGGA TTTGGGTGAG CGCGGATATT 420
GCTAGCGGGA AGGATCAAAA ATATAAGATT AAAAACATGA GCGCTTCAGG GTGCAGCATT 480
GACAACCCCA TTTGGCATGT CAATGCGACT TCAGGCTCAT TTAACATGCA AAAATCGCAT 540
TTGTCAATGT GGAATCCTAA GATTTATGTC GGCGATATTC CTGTATTGTA TTTGCCCTAT 600
ATTTTCATGT CCACGAGCAA TAAAAGAACT ACCGGGTTTT TATACCCTGA GTTTGGCACT 660 TCCAACTTAG ACGGCTTTAT TTATTTGCAA CCCTTTTATT TAGCCCCCAA AAACTCATGG 720
GATATGACCT TTACCCCACA AATCCGTTAC AAAAGGGGTT TTGGCTTGAA TTTTGAAGCG 780
CGCTACATCA ACTCTAAGAC GCAGGTTTTT ATTCAATGCG CGCTATTTTA G 831
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 675 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori ( ix) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION 1...675 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
ATGATTAGAT TAAAAGGTTT GAATAAAACT TTAAAAACAA GCTTATTAGC TGGGGTTTTA 60
CTAGGTGCTA CTGCTCCCTT AATGGCAAAG CCTTTATTAA GCGATGAAGA CTTATTGAAA 120
CGAGTAAAAC TACAC ATAT CAAAGAAGAT ACGCTGACTA GCTGTAATGC TAAGGTGGAC 180 GGCTCTCAAT ACTTGAATAG TGGTTGGAAT TTATCTAAAG AATTTCCGCA AGAATATAGA 240
GAAAAGATTT TTGAATGCGT AGAAGAAGAA AAACATAAAC AAGCCCTTAA TTTAATCAAT 300
AAAGAAGACA CTAAAGATAA AGAAGAACTT GCAAAAAAAA TCAAAGAAAT TAAAGAAAAA 360
GCTAAAGTTT TAAGGCAAAA ATTTATGGCT TTTGAAATGA AAGAACACTC TAAAGAATTC 420
CCAAATAAAA AGCAACTTCA AACCATGCTT GAGAACGCTT TTGATAATGG AGCTGAAAGT 480 TTTATTGATG ATTGGCACGA ACGCTTTGGG GGTATAAGTA GAGAGAATAC TTATAAAGCA 540
CTTGGCATTA AAGAATATAG TGATGAAGGA AAGATATTGC CTTTGGCGAA AGAAGTTATA 600
TTAGACAATA TAAAAAAGAT TTTGAAGAAA GCACTTATGA TACTAGACAA CCCTTATCTG 660
CTATGGCTAG TATGA 675 (2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1290 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1290
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
ATGCCATACG CCTTAAGAAA AAGATTTTTC AAACGCCTTT TATTGTTTTT TTTAATTGTT 60
TGTATGATAA ATTTGCATGC CAAAAGCTAT CTGTTTTCTC CTTTGCCCCC AGCGCACCAG 120 CAAATCATTA AGACAGAGCC TTGCTCTTTG GAGTGCTTGA AAGACTTGAT GCTGCAAAAT 180
CAAATCTTTT CTTTTGTATC CCAATACGAT GATAACAACC AAGATGAGAG CCTTAAAACT 240
TATTACAAGG ACATCTTAAA CAAACTCAAC CCCGTATTCA TCGCTTCTCA AACTCCAGCT 300
AAAGAAAGCT ATGAGCCTAA GATTGAATTA GCGATTTTAC TGCCTAAAAA GGTGGTGGGC 360
CGTTATGCGA TTTTAGTGAT GAACACCCTT TTAGCGTATT TGAACACCAG AAACAACGAT 420 TTCAATATCC AAGTCTTTGA CAGCGATGAA GAAAGCCCTG AAAAATTAGA AGAAACCTAT 480
AAAGAAATTG AAAAAGAAAA ATTCCCTTTT ATCATCGCTT TATTGACTAA AGAGGGCGTG 540
GAAAATTTGC TCCAAAATAC GACTATCAAT ACCCCTACTT ATGTGCCTAC GGTGAATAAA 600
ACGCAATTAG AAAATCATAC CGAGCTTTCT TTAAGCGAGC GCTTGTATTT TGGGGGGATT 660
GATTATAAAG AGCAATTAGG CATGCTCGCA ACTTTCATTA GCCCTAATTC GCCCGTGATT 720 GAATACGATG ATGATGGCCT GATAGGTGAA CGCTTGAGGC AAATCACGGA GTCTTTAAAC 780 GTTGAAGTCA AACACCAAGA AAACATTTCT TACAAACAAG CGACCAGTTT TTCTAAAAAT 840
TTTAGAAAAC ATGATGCGTT TTTTAAAAAT TCTACCTTAA TTTTGAACAC CCCTACCACT 900
AAAAGCGGTC TGATCCTTTC TCAAATAGGG CTTTTAGAGT ATAAGCCTCT TAAAATCCTT 960
TCCACACAAA TCAATTTCAA CCCCTCTTTA CTCTTGCTCA CCCAGCCTAA AGACAGGAAA 1020
AATTTATTCA TTGTCAATGC CTTGCAAAAC AGCGATGAAA CGCTGATAGA ATACGCTTCC 1080
TTATTAGAGA GCGATTTAAG GCATGATTGG GTGAATTATT CCAGCGCGAT AGGGCTAGAG 1140
ATGTTTTTAA ACACGCTAGA TCCGCATTTT AAAAAGTCTT TTCAAGAGAG TTTGGAAGAC 1200
AATCAAGTCC GTTACCACAA TCAAATTTAT CAGGCTTTAG GGTATTCTTT TGAGCCGATA 1260
AAAAACGAAA GCGAAACAAA AAAAGAATAA 1290
(2) INFORMATION FOR SEQ ID NO : 7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1368 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...1368
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
GTGTTAAAAT TTCAAAAATT ACCCTTATTG TTTGTTTCCA TTCTTTATAA TCAAAGCCCT 60 TTATTGGCTT TTGATTATAA GTTTAGTGGG GTAGCGGAAT CTGTTTCTAA AGTGGGGTTT 120
AACCATTCCA AACTCAATTC CAAAGAAGGG ATTTTCCCTA CAGCCACCTT TGTAACCGCC 180
ACGATCAAGC TTCAAGTGGA TTCCAATCTG CTCCCTAAAA ACATTGAAAA ACACAGCTTA 240
AAAATAGGCG TTGGGGGGAT TTTAGGAGCG CTCGCTTACG ATTCCACCAA AACGCTCATA 300
GACCAAGCCA CGCATCAAAT CTATGGCTCA GAACTTTTTT ACCTCATAGG GCGTTGGTGG 360 GGGTTTTTAG GCAACGCTCC TTGGAAAGAC TCCCTCATAG AATCTGACGC TCACACCCGT 420
AATTATGTGC TGTATAATTC CTATCTGTTT TATTCTTATG GCGATAAATT CCACCTAAAA 480
TTAGGGCGTT ATCTCTCTAA CATGGATTTT ATGAGTTCCT ACACACAGGG TTTTGAACTG 540
GATTATAAAA TCAATTCTAA AATAGCGTTA AAATGGTTTA GCTCTTTTGG GAGGGCGTTG 600
GCTTTTGGGC AATGGATACG GGATTGGTAT GCCCCTATTG TAACTGAAGA TGGCAGAAAA 660 GAAGTTTATG ATGGCATCCA TGCCGCGCAA CTCTATTTTT CTAGCAAGCA TGTTCAAGTC 720
ATGCCTTTTG CTTATTTTTC GCCTAAGATT TACGGAGCGC CCGGTGTTAA AATCCATATT 780
GATAGCAACC CGAAATTCAA AGGCTTAGGG TTAAGGGCTC AAACCACTAT TAATGTGATT 840
TTCCCTGTTT ATGCTAAAGA TTTATACGAT GTGTATTGGC GTAACTCTAA GATTGGCGAG 900
TGGGGCGCAT CGCTTTTGAT CCACCAACGC TTTGACTACA ACGAATTTAA CTTTGGCTTT 960 GGTTATTACC AAAATTTTGG CAACGCTAAC GCAAGGATTG GCTGGTATGG TAACCCCATC 1020
CCTTTTAATT ATAGAAATAA CAGCGTTTAT GGTGGGGTCT TCAGTAACGC TATTACCGCA 1080
GACGCCGTTT CTGGGTATGT CTTTGGTGGG GGGGTGTATA GAGGGTTTTT ATGGGGTATT 1140
TTAGGCAGAT ACACTTATGC CACTAGAGCG AGCGAAAGAT CCATCAACTT GAACTTGGGC 1200
TATAAATGGG GTTCTTTTGC TAGAGTTGAT GTGAATTTAG AATACTATGT GGTCAGCATG 1260 CACAACGGCT ATAGATTAGA CTATCTCACC GGCCCTTTCA ACAAAGCCTT TAAGGCTGAC 1320 GCACAAGATA GGAGTAACCT TATGGTTAGC ATGAAATTCT TTTTTTAA 1368
(2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 849 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...849
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
ATGGGGTGTT CGTTTATCTT TAAAAAAGTT AGGGTTTATT CTAAAATGTT GGTTGCTTTG 60
GGGCTTTCAA GCGTGTTGAT CGGTTGCGCG ATGAATCCAA GCGCTGAGAC AAAAAAACCA 120
AATGACGCCA AAAACCAACA ACCAGTTCAA ACTCATGAAA GAATGACAAC AAGTTCTGAA 180
CATGTTACGC CACTAGATTT TAATTACCCG GTGCATATTG TTCAAGCCCC ACAAAACCAT 240 CATGTTGTAG GTATTTTAAT GCCACGCATT CAAGTGAGCG ATAATCTAAA ACCCTATATT 300
GATAAGTTTC AAGACGCTTT AATTAATCAA ATCCAAACTA TTTTTGAAAA AAGAGGCTAT 360
CAAGTGTTGC GTTTTCAAGA TGAAAAAGCT TTGAATGTGC AAGATAAGAA AAAGATTTTT 420
TCCGTTTTGG ATTTGAAAGG GTGGGTAGGA ATCTTAGAAG ATTTGAAAAT GAATTTAAAA 480
GATCCCAATA GTCCCAATTT AGACACGCTA GTGGATCAAA GCTCAGGCTC TGTATGGTTT 540 AATTTTTATG AACCAGAAAG CAATCGTGTC GTCCATGATT TTGCTGTAGA AGTAGGAACT 600
TTTCAGGCAA TAACATACAC ATACACCTCT ACTAATAACG CTTCAGGAGG GTTTAATTCT 660
TCAAAAAGCG TTATCCATGA AAATTTGGAT AAGAATAGAG AAGACGCGAT ACACAAGATT 720
TTAAACAGAA TGTATGCGGT TGTCATGAAA AAAGCTGTAA CAGAACTTAC AAAAGAAAAT 780
ATCGCCAAAT ACAGAGACGC TATTGATAGA ATGAAAGGCT TTAAAAGTTC TATGCCTCAA 840 AAAAAGTAG 849
(2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 843 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...843
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 : ATGAAACTGA GAGCAAGTGT TTTAATCGGT GTGGCAATTC TGTGCTTAAT TTTAAGTGCG 60
TGCAGTAACT ATGCGAAAAA AGTGGTGAAA CAAAAGAACC ATGTTTATAC GCCTGTGTAT 120
AATGAACTGA TAGAGAAGTA TAGTGAGATC CCCTTAAATG ACAAACTCAA AGACACACCA 180
TTCATGGTGC AAGTGAAGTT GCCAAATTAC AAGGACTATT TGTTGGATAA TAAACAAGTT 240
GTACTAACTT TCAAACTTGT TCACCATTCT AAAAAGATTA CGCTCATAGG CGATGCCAAT 300 AAGATCCTCC AATACAAGAA TTACTTCCAA GCTAACGGGG CAAGATCTGA CATTGATTTT 360
TACTTGCAAC CCACTTTGAA TCAAAAGGGT GTGGTGATGA TAGCGAGTAA CTACAATGAT 420
AATCCCAACA ACAAAGAAAA ACCACAGACC TTTGATGTGT TGCAAGGAAG TCAGCCAATG 480
CTAGGAGCTA ACACAAAAAA CTTGCATGGC TATGATGTGA GTGGAGCAAA CAACAAGCAA 540
GTGATCAATG AAGTGGCAAG AGAAAAAGCT CAGCTAGAAA AAATCAATCA GTATTACAAG 600 ACTCTCTTGC AAGACAAGGA ACAAGAATAT ACCACTAGGA AAAATAACCA ACGAGAAATT 660
TTAGAAACAT TGAGTAATCG TGCAGGTTAT CAAATGAGGC AGAATGTGAT TAGTTCTGAG 720
ATTTTTAAGA ATGGCAACTT GAACATGCAA GCCAAAGAAG AAGAAGTTAG GGAGAAGCTA 780
CAAGAAGAAA GAGAGAATGA ATACTTGCGC AATCAAATCA GAAGTTTGCT CAGTGGTAAG 840
TGA 843
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1179 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...1179
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
ATGAGAAAAC TATTCATCCC ACTTTTATTA TTCAGCGCTT TAGAAGCGAA CGAGAAAAAC 60 GGCTTTTTCA TAGAAGCCGG CTTTGAAACT GGGCTATTAG AAGGCACACA AACGCAAGAA 120
AAAAGACACA CCACCACAAA AAACACTTAC GCAACTTACA ATTATTTACC CACAGACACG 180
ATTTTAAAAA GAGCGGCTAA TTTATTCACC AATGCCGAAG CGATTTCAAA ATTAAAATTC 240
TCATCTTTAT CCCCTGTTAG AGTGTTGTAT ATGTATAATG GTCAATTAAC TATAGAAAAC 300
TTCTTGCCTT ATAATTTAAA TAATGTTAAG CTTAGTTTTA CAGACGCTCA AGGCAATGTG 360 ATCGATCTAG GCGTGATAGA GACTATCCCC AAACACTCTA AGATTGTTTT GCCCGGAGAG 420 GCATTTGATA GTCTAAAAAT TGACCCCTAT ACTTTATTTC TTCCAAAAAT TGAAGCCACT 480
AGCACTTCTA TTTCTGACGC TAACACGCAG AGGGTGTTTG AAACGCTCAA TAAGATTAAG 540
ACAAATTTGG TCGTAAATTA TAGGAATGAA AACAAATTTA AAGATCACGA AAATCATTGG 600
GAAGCCTTTA CCCCACAAAC CGCAGAAGAA TTCACTAATT TAATGTTGAA CATGATCGCT 660 GTTTTAGACT CCCAATCTTG GGGCGATGCG ATCTTAAACG CTCCTTTTGA GTTCACTAAC 720
AGCCCAACAG ATTGCGATAA TGATCCTTCA AAATGCGTAA ATCCTGGGAC AAACGGGCTT 780
GTCAATTCTA AAGTCGATCA AAAATATGTG TTAAACAAAC AAGACATTGT CAATAAATTT 840
AAAAACAAAG CGGATCTTGA TGTAATTGTT TTAAAGGATT CAGGGGTTGT AGGGCTTGGG 900
AGTGATATTA CCCCTAGCAA CAATGATGAT GGCAAGCATT ATGGCCAGTT AGGGGTAGTA 960 GCTTCTGCTT TAGATCCTAA AAAACTCTTT GGCGATAACC TTAAGACTAT CAATTTAGAG 1020
GATTTAAGAA CCATCTTGCA TGAATTCAGC CACACTAAAG GCTATGGGCA TAACGGGAAT 1080
ATGACCTATC AAAGAGTGCC GGTAACGAAA GATGGTCAAG TGGAAAAGGA TAGTAATGGC 1140
AAGCCAAAAG ATTCTGATGG CCTCCCCTAT AATGTGTGT 1179 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 813 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...813
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
ATGAAAAAGT TTGTAGCTTT AGGGCTTCTA TCCGCGGTTT TAAGCTCTTC GTTGTTAGCC 60
GAAGGTGATG GTGTTTATAT AGGGACTAAT TATCAGCTTG GACAAGCCCG TTTGAATAGC 120 AATATTTATA ATACAGGGGA TTGCACAGGG AGTGTTGTAG GTTGCCCCCC AGGTCTTACC 180
GCTAATAAGC ATAATCCAGG AGGCACCAAT ATCAATTGGC ACTCCAAATA CGCTAATGGG 240
GCTTTGAATG GTTTTGGGTT GAATGTGGGT TATAAGAAAT TCTTCCAATT CAAGTCGCTA 300
GATATGACAA GCAAGTGGTT TGGTTTTAGA GTGTATGGGC TTTTTGATTA CGGGCATGCC 360
GATTTAGGTA AACAAGTTTA TGCACCTAAT AAAATCCAGT TGGATATGGT CTCTTGGGGT 420 GTGGGGAGCG ATTTGTTAGC TGATATTATT GATAAAGACA ACGCTTCTTT TGGTATTTTT 480
GGTGGGGTCG CTATCGGCGG TAACACTTGG AAAAGCTCTG CAGCAAACTA TTGGAAAGAG 540
CAAATCATTG AAGCCAAAGG TCCTGATGTT TGTACCCCTA CTTATTGTAA CCCTAATGCC 600
CCTTATAGCA CCAACACTTC AACCGTCGCT TTTCAAGTGT GGTTGAATTT TGGGGTGAGA 660
GCCAATATCT ACAAGCATAA TGGCGTGGAA TTTGGCGTGA GAGTGCCGCT ACTCATCAAT 720 AAATTTTTGA GCGCGGGTCC TAACGCTACT AACCTTTATT ACCATTTGAA ACGGGATTAT 780
TCGCTTTATT TGGGGTATAA CTACACTTTT TAA 813
(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 423 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...423
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
ATGCATCCTA TAATGTTTGC CTATATCGCT AACGCGCTCG CTCAAGCTAG AAAGATCAAC 60
GGAACACTTT GCATGGCGTT TCAAAAAATA TCTCAAGTCA AAGAATTAGG CATTGATAAA 120
GCAAAGAGTT TGATAGGCAA CCTTTCTCAA GTGATTATCT ACCCCACAAA AGATACTGAT 180
GAATTAATAG AATGTGGCGT CCCATTAAGC GATAGTGAAA TCAATTTCTT ACACAACACG 240 GACATGAGAG CCAGACAAGT GCTAGTAAAA AATATCGTTA CAAACGCTTC AGCTTTTATT 300
GAAATTGATT TAAAAAAGAT TTGCAAGAAC TACTTTATAT TCTTGATAGC AATGCTGGTA 360
ATAGAAAAAT CCTCAATGAT CTTAAAAAAG CAAACCAAGA AACTTATAAG GAAGAGTATT 420
TAA 423 (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 771 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...771
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
ATGTTGGGGA GCGTCAAAAA AGCGGTTTTT AGGGTTTTGT GTTTGGGGGC GTTGTGTTTA 60
TGCGGGGGGT TAATGGCAGA GCAAGATCCT AAAGAGCTTA TATTTTCAGG TATAACTATT 120 TACACGGATA AAAATTTCAC TAGAGCTAAG AAATATTTTG AAAAAGCTTG CAAATCAAAC 180 GATGCTGATG GCTGTGCAAT CTTAAGAGAG GTTTATTCTA GTGGTAAAGC CATAGCGAGA 240
GAAAACGCAA GAGAGAGCAT TGAAAAAGCT CTTGAACACA CCGCTACTGC TAAAGTTTGT 300
AAATTAAACG ATGCTGAAAA ATGCAAGGAC TTAGCAGAGT TTTATTTTAA TGTAAACGAT 360
CTTAAAAATG CTTTAGAATA TTACTCTAAA TCTTGTAAGT TAAATAATGT TGAAGGGTGT 420 ATGCTGTCAG CAACTTTTTA TAACGATATG ATAAAGGGTT TGAAAAAAGA TAAAAAAGAT 480
CTAGAATATT ATTCTAAAGC TTGCGAGTTA AATAACGGTG GAGGGTGTTC TAAATTAGGA 540
GGGGATTATT TTTTTGGTGA AGGCGTAACA AAAGATTTCA AAAAAGCTTT TGAATATTCT 600
GCCAAAGCTT GTGAGTTGAA CGATGCTAAA GGGTGTTACG CTCTAGCAGC GTTTTATAAT 660
GAGGGTAAAG GCGTGGCAAA GGATGAAAAG CAAACGACAG AAAACCTTGA AAAGAGTTGC 720 AAGCTAGGAT TAAAAGAAGC ATGCGATATT CTCAAAGAAC AAAAACAATA A 771
(2) INFORMATION FOR SEQ ID NO : 14 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 729 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...729
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ATGAAAAAAT TTTTTTCTCA ATCTTTGTTA GCTCTTATTA TCTCTATGAA TGCGGTATCT 60
GGCATGGATG GTAATGGCGT TTTTTTAGGG GCGGGTTATT TGCAAGGACA GGCGCAAATG 120
CATGCGGATA TTAATTCTCA AAAACAAGCC ACCAACGCTA CGATCAAAGG CTTTGACGCG 180
CTCTTGGGGT ATCAATTTTT CTTTGAAAAA CACTTTGGCT TACGCCTTTA TGGGTTTTTT 240
GACTACGCTC ATGCCAATTC TATTAAGCTT AAAAACCCTA ACTATAATAG CGAAGCGGCG 300 CAAGTGGCTA GTCAAATTCT TGGGAAACAA GAAATCAATC GTTTAACAAA CATTGCCGAT 360
CCCAGAACTT TTGAGCCGAA CATGCTCACT TATGGGGGGG CTATGGACGT GATGGTTAAT 420
GTCATCAATA ACGGCATCAT GAGTTTGGGG GCTTTTGGCG GGATACAATT GGCCGGCAAT 480
TCATGGCTTA TGGCGACACC GAGCTTTGAG GGCATTTTAG TGGAACAAGC CCTTGTGAGC 540
AAGAAAGCCA CTTCTTTCCA ATTTTTATTC AATGTGGGGG CTCGCTTAAG GATCTTAAAA 600 CATTCTAGCA TTGAAGCGGG CGTGAAATTC CCCATGCTAA AGAAAAACCC CTACATCACT 660
GCAAAAAATT TGGATATAGG GTTTAGGCGC GTGTATTCGT GGTATGTGAA TTACGTGTTC 720
ACTTTCTAG 729
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...804 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
ATGAACTACC CTAATCTACC TAACAGCGCT TTAGAGATAA GCGAACAGCC AGAAGTGAAA 60
GAAATCACTA ACGAGCTTTT AAAGCAATTA CAAAACGCTT TAAGGAGCAA CGCGCATTTT 120
AGCGAGCAAG TGGAATTAAG CCTTAAATGC ATCGTTAGGA TTTTAGAAGT GCTTTTGAGT 180 TTGGATTTTT TTAAGAATGC GAATGAGATT GATAGCAGTT TAAGAAATTC CATTGAGTGG 240
CTGACTAACG CCGGCGAGAG CTTGAAATTA AAAATGAAAG AATACGAGCG CTTTTTTAGC 300
GAGTTTAATA CGAGCATGCA TGCCAACGAG CAGGAAGTAA CCAATACCTT AAACGCTAAC 360
GCCGAGAACA TTAAAAGCGA AATTAAAAAG CTAGAAAATC AATTGATAGA AACCACGACA 420
AGACTTTTAA CGAGCTATCA AATCTTTTTA AACCAAGCCA GAGATAACGC TAACAACCAA 480 ATCACAAAAA ACAAAACCCA AAGCCTTGAA GCGATTACAC AAGCTAAAAA CAACGCTAAT 540
AATGAAATAA GCAACAATCA AACGCAAGCG ATAACTAATA TCACCGAAGC GAAAACGAAC 600
GCTAATAATG AAATAAGCAA CAATCAAACG CAAGCGATAA CTAACATTAA CGAAGCCAAA 660
GAAAGCGCTA CAACGCAAAT AAACGCCAAT AAGCAAGAAG CAATAAATAA CATCACGCAA 720
GAAAAAACCC AAGCCACAAG CGAGATCACC GAAGCGAAAA AGACCGATCA TTATCAAAAC 780 ATTGATTTTT TTGAGTTTGA AT A 804
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1632 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...1632
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GTGATAGAGA CCATCCCCAA ACACTCTAAG ATTGTTTTAC CCGGGGAGGC GTTTGATAGT 60 TTAAAAGAGG CGTTTGATAA AATTGACCCC TATACTTTCT TTTTTCCAAA ATTTGAAGCC 120
ACTAGCACTT CTATTTCTGA TACTAACACG CAGAGGGTGT TTGAAACGCT CAATAACATT 180
AAAACAAATC TTATAATGAA ATATAGTAAT GAAAATCCAA ACAATTTCAA CACTTGTCCT 240
TACAATAATA ATGGTAATAC AAAAAATGAT TGTTGGCAAA ATTTCACCCC ACAAACCGCA 300 GAAGAATTCA CCAATTTAAT GTTGAACATG ATCGCTGTCT TAGACTCCCA ATCTTGGGGC 360
GATGCGATCT TAAACGCTCC TTTTGAATTC ACTAACAGCT CAACAGATTG CGATAGCGAT 420
CCTTCAAAAT GCGTAAATCC CGGAGTAAAT GGGCGTGTTG ATACTAAAGT CGATCAACAA 480
TATATACTCA ACAAACAAGG TATTATTAAT AATTTTAGAA AAAAAATAGA AATTGATGCG 540
GTTGTTTTAA AAAATTCAGG GGTTGTAGGG TTAGCCAATG GATATGGCAA TGATGGTGAA 600 TATGGCACAT TAGGGGTAGA AGCCTATGCT TTAGATCCTA AAAAACTCTT TGGCAACGAC 660
CTTAAGACTA TCAATTTAGA AGATTTAAGA ACCATCTTGC ATGAATTCAG CCACACTAAA 720
GGCTATGGGC ATAACGGGAA TATGACCTAT CAAAGAGTGC CGGTAACGAA AGATGGTCAA 780
GTGGAAAAGG ATAGTAATGG CAAGCCAAAA GATTCTGATG GCCTCCCCTA TAATGTGTGT 840
TCGCTTTATG GGGGATCCAA TCAGCCCGCT TTCCCTAGCA ACTACCCTAA TTCCATCTAT 900 CACAATTGTG CGGATGTCCC GGCTGGCTTT TTAGGGGTAA CAGCAGCGGT TTGGCAGCAG 960
CTCATCAATC AAAACGCCTT GCCGATCAAC TACGCTAACT TGGGGAGTCA AACAAACTAC 1020
AACCTAAACG CTAGTTTAAA CACGCAAGAT TTAGCCAATT CCATGCTCAG CACCATCCAA 1080
AAAACCTTTG TAACTTCTAG CGTTACCAAC CACCATTTTT CAAACGCATC GCAAAGTTTT 1140
AGAAGCCCTA TTTTAGGGGT TAACGCTAAA ATAGGCTATC AAAACTACTT TAATGATTTC 1200 ATAGGGTTGG CTTATTATGG CATCATCAAA TACAATTACG CTAAAGCTGT TAATCAAAAA 1260
GTCCAGCAAT TGAGCTATGG TGGGGGGATA GATTTGTTAT TGGATTTCAT CACCACTTAC 1320
TCCAATAAAA ATAGCCCTAC AGGCATTCAA ACCAAAAGGA ATTTTTCTTC ATCTTTTGGT 1380
ATCTTTGGGG GGTTAAGGGG CTTGTATAAC AGCTATTATG TGTTGAACAA AGTCAAAGGA 1440
AGCGGCAATT TAGATGTGGC TACCGGGTTG AACTACCGCT ATAAGCATTC TAAATATTCT 1500 GTAGGGATTA GCATCCCTTT AATCCAAAGA AAAGCTAGCG TCGTTTCTAG CGGTGGCGAT 1560
TATACGAACT CTTTTGTTTT CAATGAAGGG GCTAGCCACT TTAAGGTGTT TTTCAATTAC 1620
GGTGGGTGTT TT 1632
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1071 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1071 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
TTGATGAAAA GCATTTTGCT CTTTATGATT TTTGTAGTTT GTCAGTTAGA AGGCAAAAAA 60
TTTTCACAAG ATAATTTTAA GGTGGATTAT AACTACTATT TGCGCAAACA GGATTTGCAC 120
ATCATTAAAA CGCAAAACGA TTTGTCCAAT GCCTGGTATC TCCCTCCACA AAAAGCCCCC 180 AAAGAACATT CTTGGGTGGA TTTTGCTAAA AAATATTTAA ACATGATGGA TTATCTAGGC 240 ACTTATTTTT TGCCTTTTTA TCATAGTTTC ACCCCCATTT TTCAATGGTA CCACCCTAAT 300
ATCAACCCCT ACCAACGCAA TGAGTTTAAG TTCCAAATCA GTTTTAGAGT GCCTGTATTT 360
AGGCATATTC TTTGGACTAA AGGCACGCTT TATCTGGCTT ATACCCAAAC TAACTGGTTT 420
CAAATTTATA ATGACCCTCA ATCCGCCCCC ATGCGAATGA TCAATTTCAT GCCTGAACTC 480 ATCTATGTTT ATCCTATTAA TTTTAAACCT TTTGGGGGTA AAATAGGGAA TTTTTCTGAA 540
ATTTGGATAG GTTGGCAGCA CATTTCTAAT GGTGTGGGGG GTGCGCAATG TTACCAGCCT 600
TTTAATAAAG AAGGTAATCC TGAAAACCAG TTTCCAGGAC AACCTGTAAT CGTTAAAGAT 660
TATAACGGGC AAAAAGATGT GCGCTGGGGG GGGTGTCKTT CGGTGARCSC GGGCAACSCC 720
CTGTGTTTCG TTTTGGTGTG GGAAAAGGGA GGCCTAAAAA TCATGGTCGC TTATTGGCCC 780 TATGTCCCTT ATGATCAATC CAACCCTCAA TTGATTGATT ACATGGGGTA TGGTAACGCT 840
AAAATTGATT ACAGGAGAGG GCGCCACCAT TTTGAATTGC AACTTTATGA TATTTTCACG 900
CAATACTGGC GTTATGATCG CTGGCATGGA GCTTTCCGCT TAGGCTATAC CTACCGCATT 960
AACCCTTTTG TGGGGATTTA TGCGCAGTGG TTTAACGGCT ATGGCGATGG CTTGTATGAA 1020
TACGATGTTT TTTCCAATCG TATAGGGGTA GGAATACGCT TGAACCCTTA A 1071
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2028 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...2028
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
TTGTCTAAAG GTTTGAGTAT CGGTAATAAA ATCATATTGT GCGTGGCGTT GATTGTGATC 60 GTGTGCGTGA GCATTTTAGG GGTGTCCTTA AACAGCAGGG TGAAAGAGAT TTTAAAAGAA 120
AGCGCTCTGC ATTCAATGCA AGATAGTTTG CATTTCAAGG TTAAGGAAGT GCAAAGTGTT 180
TTGGAAAACA CTTATACGAG CATGGGCATT GTCAAAGAAA TGCTCCCTGA AGACACCAAA 240
AGAGAAATCA AAATCCAGTT GTTAAAAAAC TTCATTTTAG CCAATTCGCA TGTCGCTGGG 300
GTGAGCATGT TTTTTAAAGA CAGAGAGGAT TTGAGATTGA CGCTTTTACG AGATAACGAT 360 ACGATCAAGT TGATGGAAAA CCCGTCATTA GGGAGTAACC CTTTAGCGCA AAAAGCGATG 420
AAAAATAAAG AAATTTCTAA AAGCTTGCCT TATTACAGGA AAATGCCTAA CGGGGCGGAA 480
GTTTATGGCG TGGATATTCT TTTACCACTA TTCAAGGAAA ACACGCAAGA AGTGGTGGGG 540
GTTCTGATGA TTTTCTTTTC CATTGACAGC TTCAGTAATG AAATCACTAA AAACAGGAGC 600
GATTTATTTT TAATTGGCGT TAAAGGTAAA GTGCTTTTGA GCGCGAATAA AAGCTTGCAA 660 GACAAATCCA TCACCGAAAT TTATAAAAGC GTGCCTAAAG CCACTAATGA AGTGATGGCT 720
ATTTTAGAAA ATGGCTCTAA AGCGACTTTA GAATACTTGG ATCCCTTTAG CCATAAGGAG 780
AATTTTTTAG CCGTTGAAAC CTTTAAAATG CTAGGCAAAA CAGAAAGTAA AGACAATCTT 840
AATTGGATGA TCGCTTTGAT CATTGAAAAA GACAAGGTCT ATGAGCAAGT GGGATCGGTG 900
CGTTTTGTGG TGGTTGCAGC GAGTGCTATC ATGGTGTTAG CCTTAATCAT AGCGATCACT 960 CTTTTAATGC GAGCGATCGT GAGCAATCGT TTGGAAGTCG TTTCTAGCAC CTTGTCTCAT 1020 TTCTTTAAAT TATTGAACAA TCAAGCCCAT TCTAGCGACA TTAAATTGGT TGAAGCGCGA 1080
TCTAATGACG AATTAGGGCG CATGCAAACA GCGATCAATA AAAATATCTT GCAAACCCAA 1140
AAAACCATGC AAGAAGACAG GCAAGCCGTC CAAGACACCA TTAAAGTGGT TTCAGACGTG 1200
AAAGCGGGGA ATTTTGCGGT GCGCATCACG GCTGAACCCG CAAGCCCTGA TTTGAAAGAA 1260 TTGAGAGACG CGCTAAATGG GATCATGGAT TATTTGCAAG AAAGCGTAGG GACTCACATG 1320
CCAAGCATTT TCAAAATCTT TGAAAGCTAT TCTGGCTTGG ATTTTAGAGG GCGGATCCAA 1380
AACGCTTCGG GTAGGGTGGA ATTGGTTACT AACGCTTTAG GGCAAGAAAT CCAAAAAATG 1440
CTAGAAACTT CGTCTAATTT TGCCAAAGAT CTAGCGAACG ATAGCGCGAA TTTAAAAGAA 1500
TGCGTGCAAA ATTTAGAAAA GGCTTCAAAC TCCCAACACA AAAGCCTGAT GGAAACTTCC 1560 AAAACGATAG AAAATATCAC CACTTCCATT CAAGGCGTGA GCTCTCAAAG TGAAGCCATG 1620
ATTGAACAAG GGAAAGACAT TAAAAGCATT GTAGAAATCA TTAGAGATAT TGCCGATCAA 1680
ACGAATCTAT TAGCCCTAAA CGCTGCTATT GAAGCCGCAC GAGCCGGCGA GCATGGCAGA 1740
GGCTTTGCGG TGGTGGCTGA TGAGGTGAGG AAGCTCGCTG AAAGGACGCA AAAATCCCTC 1800
AGTGAGATTG AAGCCAATAT TAATATTCTC GTTCAAAGCA TTTCAGACAC GAGCGAAAGC 1860 ATTAAAAACC AGGTTAAAGA AGTAGAAGAG ATCAACGCTT CTATTGAAGC CTTAAGATCG 1920
GTTACTGAGG GCAATCTAAA AATCGCTAGC GATTCTTTAG AAATCAGTCA AGAAATTGAC 1980
AAAGTCTCTA ACGATATTTT AGAAGATGTG AATAAAAAGC AGTTTTAA 2028
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 816 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...816 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
ATGAACATAT TCAAGCGTAT TATTTGCGTA ACCGCTATTG TTTTAGGTTT TTTTAACCTT 60
TTAGACGCCA AACACCACAA AGAAAAAAAA GAAGACCACA AAATCACTCG TGAGCTTAAA 120
GTGGGCGCTA ACCCTGTGCC GCATGCGCAA ATCTTGCAAT CAGTTGTGGA TGATTTGAAA 180 GAGAAAGGGA TCAAATTAGT GATCGTGTCT TTTACGGATT ATGTGTTGCC TAATTTAGCG 240
CTCAATGACG GCTCTTTAGA CGCGAATTAC TTCCAGCACC GCCCTTATTT GGATCGGTTT 300
AATTTGGACA GAAAAATGCA CCTTGTTGGT TTGGCCAATA TCCATGTGGA GCCTTTAAGA 360
TTTTATTCTC AAAAAATCAC AGACATTAAA AACCTTAAAA AAGGCTCAGT GATTGCTGTG 420
CCAAATGATC CGGCCAATCA AGGCAGGGCG TTGATTTTAC TCCATAAACA AGGCCTTATC 480 GCTCTCAAAG ACCCAAGCAA TCTATACGCT ACGGAGTTTG ATATTGTCAA AAATCCTTAC 540
AACATCAAAA TCAAACCCCT AGAAGCTGCG TTATTGCCTA AGGTTTTAGG GGATGTGGAT 600
GGGGCTATCA TAACAGGGAA TTATGCCTTG CAAGCAAAAC TCACCGGAGC CTTATTTTCA 660
GAAGATAAGG ACTCGCCTTA TGCTAATCTT GTAGCCTCTC GTGAGGATAA TGCGCAAGAT 720
GAAGCGATAA AAGCGTTGAT TGAAGCCTTA CAGAGCGAAA AGACCAGGAA ATTCATTTTG 780 GATACCTATA AGGGGGCGAT TATCCCGGCT TTTTAA 816 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 486 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...486
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: ATGTTTTTTA AAACTTATCA AAAATTACTG GGCGCGAGCT GTTTGGCGCT GTATTTAGTG 60
GGCTGTGGGA ATGGTGGTGG CGGTGAATCG CCGGTTGAGA TGATTGCAAA TAGCGAGGGT 120
ACGTTTCAAA TCGACTCCAA AGCAGATAGC ATTACTATTC AAGGCGTGAA GCTTAATAGA 180
GGTAATTGTG CTGTCAATTT TGTTCCAGTA AGTGAGACGT TTCAAATGGG TGTTTTAAGT 240
CAAGTTACTC CAATCTCTAT ACAGGATTTT AAAGATATGG CAAGCACTTA TAAGATATTT 300 GATCAAAAGA AAGGGTTGGC AAACATAGCA AATAAAATTT CTCAATTAGA GCAAAAGGGT 360
GTGATGATGG AACCTCAAAC CCTTAATTTT GGAGAAAGTT TAAAAGGCAT TTCTCAAGGG 420
TGCAATATTA TAGAGGCAGA AATACAAACC GACAAAGGCG CTTGGACTTT TAACTTTGAT 480
AAATAA 486 (2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1014 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1014 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
ATGATTAGAT TAAAAGGTTT GAATAAAACT TTAAAAACAA GCTTATTAGC TGGGGTTTTA 60
CTAGGTGCTA CTGCTCCCTT AATGGCAAAG CCTTTATTAA GCGATGAAGA CTTATTGAAA 120 CGAGTAAAAC TACACAATAT CAAAGAAGAT ACGCTGACTA GCTGTAATGC TAAGGTGGAC 180
GGCTCTCAAT ACTTGAATAG TGGTTGGAAT TTATCTAAAG AATTTCCGCA AGAATATAGA 240
GAAAAGATTT TTGAATGCGT AGAAGAAGAA AAACATAAAC AAGCCCTTAA TTTAATCAAT 300
AAAGAAGACA CTGAAGATAA AGAAGAACTT GCAAAAAAAA TCAAAGAAAT TAAAGAAAAA 360
GCTAAAGTTT TAAGGCAAAA ATTTATGGCT TTTGAAATGA AAGAACACTC TAAAGAATTC 420 CCAAATAAAA AGCAACTTCA AACCATGCTT GAGAACGCTT TTGATAATGG AGCTGAAAGT 480
TTTATTGATG ATTGGCACGA ACGCTTTGGG GGTATAAGTA GAGAGAATAC TTATAAAGCA 540
CTTGGCATTA AAGAATATAG TGATGAAGGA AAGATATTAG CCTTTGGCGA AAGAAGTTAT 600
ATTAGACAAT ATAAAAAAGA TTTTGAAGAA AGCACTTATG ATACTAGACA AACCTTATCT 660
GCTATGGCTA ATATGAGTGG CGAAAACGAT TATAAAATTA CTTGGTTAAA ACCCAAATAT 720 CAGCTCCATA GTTCAAATAA TATTAAACCC TTAATGTCAA ACACAGAGTT GTTAAATATG 780
ATAGAGCTAA CCAATATCAA AAAAGAATAT GTTATGGGCT GTAATATGGA AATAGATGGT 840
TCTAAATATC CCATTCATAA AGATTGGGGA TTTTTTGGTA AGGCAAAAGT CCCAGAAACT 900
TGGAGAAATA AGATTTGGGA ATGTATTAAG AATAAAGTAA AGTCCTATGA CAACACTACC 960
GCTGAAATAG GAATAGTTTG GAAAAAAAAT ACTTATTCTA TCTCTCATCA CTAA 1014
(2) INFORMATION FOR SEQ ID NO: 22;
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1251 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...1251
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
ATGAAAAAAT TAGTTTTTAG CATGCTTTTA TGTTGTAAAA GCGTGTTTGC AGAGGGGGAA 60 ACTCCTTTGA TTGTCAATGA CCCAGAAACC CATGTAAGTC AAGCCACTAT CATAGGCAAA 120
ATGGTAGATA GTATCAAAAG ATACGAAGAG ATTATTTCTA AGGCTCAAGC TCAAGTCAAT 180
CAGTTACAAA AAGTCAATAA CATGATAAAT ACGACTAATT CTTTGATTAG TAGTAGTGCT 240
ATCACTTTAG CCAATCCTAT GCAAGTTTTA CAAAACGCTC AGTATCAAAT AGAGAGCATT 300
AGATACAACT ATGAGAATTT AAAGCAAAGC ATAGAAAATT GGAACGCACA AAATTTGTTA 360 AGAAACAAAT ACTTACAGCA ACAATGCCCT TGGCTTAATG TCAATGCTCT TACTAACAAT 420
AAGATTGTCA ATCTTAAAGA TCTCAATAAC CTAATCACCA AAAATGGCGA ACAAACCCAA 480
ACCGCAAGAG ATGTGCAAAA TCTCATTCAG TCCATTAGTG GCAGTGGCTA TGGAAACATG 540
CAATCACTTG CTGGGGAATT GAGTGGTAGA GCGTGGGGGG AAATGTTGTG TAAAATGGTA 600
AACGATAGTA ATTATGAAAG CGAGCAAGCT CTTTTAGCAA CAGGCAATAA CCCAGAAGAG 660 CAAAAACGAA GATTTTTGCT TAGAGTAAAG AAAAAGGTTA ATGATAATAA GCAGTTAAAA 720 GATAAACTTG ACCCATTTCT AAAAAGACTT GATGTCCTAC AAACTGAGTT TGGTGTAACT 780
GACCCTACAG CTAACCATAA TAAGCAAGGG ATACATTATT GCACAGAAAA TAAAGAGACA 840
GGTAAATGCG ACCCTATTAA AAATGTATTT AGGACAACTC GCTTAGATAA CGAATTAGAA 900
CAAGAAATCC AAACGCTCAC ACTTGATTTA ATCAAAGCCT CCAATAAAGA CGCTCAAAGC 960
CAAGCCTACG CAAATTTCAA TCAAAGGATT AAATTACTTA CTCTAAAATA TTTAAAAGAA 1020
ATTACCAATC AAATGCTCTT TTTAAATCAA ACAATGGCAA TGCAAAGCGA GATTATGACA 1080
GATGATTATT TTAGGCAAAA TAATGATGGC TTTGGGGAAA AAGAAAACCA TATAGACAAA 1140
CAATTAACGC AAAAAAGAAT AAACGAAAGA GAAAGAGCTA GAATATACTT TCAAAACCCT 1200
AATGTTAAAT TTGACCAATT TGGCTTTCCC ATTTTTAGTA TATGGGATTA A 1251
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1131 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...1131
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
GTGAATAAGT GGATTAAAGG GGCGGTTGTT TTTGTAGGGG GTTTTGCAAC GATTACAACC 60 TTTTCTTTAA TCTACCACCA AAAGCCAAAA GCCCCCCTAA ATAACCAGCC TAGCCTTTTG 120
AATGACGATG AGGTGAAATA CCCCTTACAA GACTACACTT TCACTCAAAA CCCACAGCCA 180
ACTAACACGG AAAGCTCCAA AGACGCTACC ATCAAAGCCT TACAAGAACA GCTCAAAGCC 240
GCTTTAAAAG CCCTAAACTC CAAAGAAATG AATTATTCCA AAGAAGAGAC TTTTACTAGC 300
CCTCCCATGG ATCCAAAAAC AACCCCCCCT AAAAAAGACT TTTCTCCAAA ACAATTAGAT 360 TTACTGGCCT CTCGCATCAC CCCTTTCAAG CAAAGCCCTA AAAATTACGA AGAAAACCTG 420
ATTTTCCCTG TGGATAACCC TAATGGCATT GATAGTTTCA CTAACCTTAA AGAAAAAGAC 480
ATCGCCACTA ATGAAAACAA GCTTTTACGC ACCATTACAG CTGACAAAAT GATACCCGCT 540
TTTTTGATTA CGCCCATTTC TAGCCAGATC GCTGGTAAAG TGATTGCGCA AGTGGAGAGC 600
GATATTTTTG CAAGCATGGG CAAAGCCGTC TTAATCCCCA AAGGCTCTAA AGTCATAGGC 660 TATTACAGCA ACAATAACAA AATGGGCGAA TACCGCTTGG ATATTGTATG GAGTCGAATC 720
ATCACTCCCC ATGGCATTAA TATCATGCTC ACTAACGCTA AAGGGGCGGA CATTAAAGGC 780
TATAACGGCT TAGTGGGGGA ATTGATTGAA AGGAATTTCC AACGCTATGG CGTGCCGTTA 840
CTGCTTTCTA CGCTCACTAA CGGCCTATTG ATTGGGATCA CTTCGGCTTT AAACAACAGA 900
GGCAATAAAG AAGAGGTGAC TAATTTCTTT GGGGATTATC TTTTATTGCA ATTGATGAGG 960 CAAAGCGGCA TGGGGATCAA TCAAGTGGTC AATCAAATTT TAAGAGACAA GAGCAAGATC 1020
GCCCCCATTG TGGTGATTAG AGAGGGGAGT AGGGTCTTCA TTTCGCCCAA TACTGACATC 1080
TTCTTCCCTA TACCCAGAGA GAATGAAGTC ATCGCTGAGT TTTTGAAGTG A 1131
(2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2751 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...2751 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
GTGGATTTGA GGATCCAATC TAAAGAAGTC AGTCATAATT TAAAGGAATT ATCAAAAACG 60
CTAATCAGCT ATCCTTTTGA AAAACATGTA GAAGCTTTAG GGGAACAATG CAGTAACTTC 120
GTTTCTATTC CCATTAACAA TGACGACTAT TCAAATATTT GCACTTTTGT GAGTGATTTT 180 ATAAATCTTA TAGCTTCTTA CAATTTATTA GAATCATTTT TAGATTTTTA TAAAGATAAA 240
TTAAAATTGA GCGAGCTTGT AACTGAATAT GCCAACGTAA CCAATAATCT GCTTTTCAAA 300
AAATTAATCA AACATTTAAG CGGCAACAAT CAATTGGTTA AAAATTTTTA TCAGTGTATA 360
AGAGAAATTA TAAAATACAA CGCCCCTAAT AAAGAATACA AACCCAATCA ATTTTTTATA 420
ATAGGGAAAG GCAAACAAAA ACAATTAGCA AAAATTTATT CTCATTTAAA AGAACTTAGT 480 GCAAGTGAAA TTAAACCACA AGATATGGAA GACATCTTAA AAAAGCTAGA GGAATTAGAT 540
AAAATTTTTA AAACTACCGA CTTTACAAAA TTCACACCAA AAACTGAAAT TAAGGATATT 600
ATTAAAGAAA TAGACGAAAA ATACCCTATC AATGAAAATT TTAAACGGCA ATTTAATGAG 660
TTTGAATCAA ATATTGAAAA ACATGATGAA ATAAAAAAGG ATTTTGAGCG AAACAAAGAG 720
TCGCTGATCC GAGAAATTGA AAATCACTGC AAAAATGAAT GCAATAGCGA AGAAGAGCCG 780 GAGTATAAGA TTAATGATCT GCTCAAAAAT ATCCAACAAA TATGCAAAAA TTATATAGAA 840
AGTCATGCCG TTAATGATGT GTCTAAAGAT ATTAAATCCA TGATGTGTCA GTTTTATTTG 900
AAACAGATAG ATTTATTAGT CAATTCAGAA ATTGTGCGAT ACAGATACAG CAATCTTTTT 960
GAACCAATAC AAAGATCTTT ATGGGAGAGT ATAAAAATTT TAGATAATGA AAGTGGCATT 1020
TATTTGTTCC CTAAAAATAT TGGTGAAATC AAGGATAAAT TTGAAGCAAA CAAGGAAAAA 1080 TTCAAACAAA GCAAAAATGT TTCTGAGTTC GCAGAATATT GCCGAGAGTG TAACCCCTAT 1140
ACAGCGTTTA ACTTTCATCT AAATATAAAT AATGGTTTAT CTCATCAATT TGAAAAATTC 1200
GTGCCAATCA TGAAAGAATA CAAAGAGCCA AAAATCACAG ATAATGACCT TGAAGCCATA 1260
TCAACCAAAG AGACTGGTCT TGCTAGCCAA TTATCTGGGC ACTGGTTTTT TCAGCTTTCG 1320
TTATTTAATA AAACAAACTT TAATCCTAAT AAAATTTGGA TTCCTTTAGA GTTCAATAAA 1380 AGATCAAAAA TAAAGTTTGA TAAAGATTTA GAAATCTATT TTGATAGTCA TGAATCGTTC 1440
AATATCTCTA AAAAATACTT GCAAGAAATA GATCAAGAAT CACTAAAAAA GATCAAACAA 1500
TCAAAAGATT TTTTTTCAAT TCAAAAAATA GAGAGTAAGC ATGATAATAA CGATATACTG 1560
CAACTTGAAT TTTTTGAGAA TGATACAAGT TTTCTTTTTG CTAAAGGAAG TTTTGCAGAA 1620
ATTTTAGAAT ACAACATGCA ATTAAAAATA GATTCTTTAA TTACAAAAGA ATTTAATAAG 1680 CTTTTAGCGA TCGTTCAAGA TAGTCCCCAA GATAGTTACC AATTAAAAAT TCGTGTCCGA 1740
CATAACAATA AGCTTCCTAG AGAGAAATAT ACGGAACATG AAATAAAACT TGAAGTTTAT 1800
GATTGCAGAA AATCCCACGA TCACAATGAG CCAATCATCT TAAGCCAGCA AAGCACCGGC 1860
TTCCAATGGG CGTTTAATTT CATGTTTGGC TTTCTTTATA ATGTGGGATC ACATTTTAGT 1920
TTTAACCATA ATATTATCTA TGTCATGGAC GAGCCAGCCA CTCATTTGAG CGTGCCAGCC 1980 AGAAAGGAGT TTAGGAAATT TTTAAAAGAA TACGCTCATA AAAATCATGT TACTTTTGTT 2040 TTAGCCACCC ATGACCCCTT TTTAGTGGAT ACGGATCATT TAGATGAAAT AAGGATTGTG 2100
GAAAAGGAAA CAGAAGGCTC TGTAATTAAG AATCACTTTA ACTATCCCCT AAATAATGCA 2160
AGCAAAGACT CCGACGCTTT GGACAAAATC AAACGCTCTT TAGGAGTGGG CCAGCATGTT 2220
TTTCATAACC CCCAAAAACA CCGAATCATT TTTGTAGAAG GCATCACGGA TTATTGTTAT 2280
TTGAGCGCTT TTAAATTGTA TTTGCGTTAC AAAGAATACA AGGACAACCC CATTCCTTTC 2340
ACTTTCTTAC CCATTTCAGG GCTTAAAAAC GATTCAAACG ATATGAAAGA AACCATTGAA 2400
AAACTTTGCG AGTTAGACAA TCACCCTATT GTTTTGACAG ACGATGACAG AAAATGCGTT 2460
TTTAACCAAC AAGCAACGAG CGAACGATTT AAAAGAGCTA ATGAAGAAAT GCATGATCCC 2520
ATCACCATCC TACAACTCTC AGACTGCGAT AGGCATTTCA AACAAATTGA AGATTGTTTC 2580
AGCGCAAACG ATAGAAACAA ATACGCTAAA AATAAGCAAA TGGAATTGAG CATGGCTTTT 2640
AAAACAAGGC TTTTGTATGG CGGAGAAGAT GCGATAGAAA AACAAACAAA AAGAAATTTT 2700
TTAAAATTAT TCAAATGGAT TGCATGGGCT ACAAACTTGA TCAAAAACTA A 2751
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 531 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...531 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
ATGACTGCAA TGATGCGTTA TTTTCACATC TATGCGACCA CTTTTTTCTT CCCTTTGGCG 60
CTTCTTTTTG CGGTTAGTGG GCTTTCATTG CTCTTTAAAG CGCGCCAAGA CACTGGCGCT 120
AAGATCAAAG AATGGGTTTT AGAAAAATCC TTAAAAAAAG AAGAACGATT GGACTTTTTA 180 AAAGGCTTTA TAAAAGAAAA CCATATCGCT ATGCCTAAAA AGATAGAGCC TAGAGAGTAT 240
AGGGGAGCGT TAGTCATTGG CACGCCTTTG TATGAAATCA ACCTTGAAAC TAAAGGCACT 300
CAAACGAAAA TCAAGACCAT TGAAAGGGGC TTTTTAGGCG CGCTCATCAT GCTGCATAAG 360
GCTAAGGTGG GCATCGTGTT TCAGGCGCTT TTAGGGATTT TTTGCGTGTT TTTATTGTTG 420
TTTTACTTGA GCGCGTTTTT AATGGTGGCT TTTAAAGACA CTAAACGCAT GTTTATAAGC 480 GTTTTAATAG GGAGCGTGGT GTTCTTTGGA GCGATCTATT GGTCTTTGTA G 531
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 669 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...669
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: ATGTTTAAAA ACGCTTTAAA TATACAAGAT TTTTCATTTA AAAATCATAC TAGTACAGCC 60
ATTATTGGCA CAAATGGTGC TGGAAAATCA ACGCTTATCA ACACTATTCT AGGCATTAGA 120
TCAGACTATA ATTTTAAAGC ACAAAACAAT AATATTCCAT ACCACGACAA TGTTATACCA 180
CAACGCAAGC AATTGGGAGT TGTCTCTAAC CTATTCAACT ACCCACCTGG ATTAAACGCA 240
AACGACCTTT TTAAATTCTA TCAATTTTTT CACAAAAACT GCACTCTAGA TTTGTTTGAA 300 AAAAATCTTT TAAATAAAAC CTACGAACAC CTAAGCGACG GACAAAAACA GCGCTTAAAA 360
ATTGACTTAG CTCTTAGCCA TCACCCACAA TTAGTTATTA TGGATGAACC AGAAACCAGT 420
TTAGAGCAAA ACGCTCTTAT AAGACTATCA AATCTCATAA GCTTGCGCAA CACCCAACAA 480
CTTACAAGTA TCATCGCCAC TCATGATCCT ATTGTCTTAG ATAGTTGCGA ATGGGTATTG 540
CTCCTTAAGA ATGGCAACAT TGCTCAATAC AAACCTTTAA ATTCTATATT AAAATCTGTA 600 GCTAAAACTT TTAACTTTAA AGAAAAACCA ACCACAAAAG ACTTATTAGC GTTACTAAAG 660
GATATTTAA 669
(2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1221 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1221
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
ATGTATGCGG CTCATCCTAT TAAACCCATA AAAGCCCCTA AACTCAAATC TCAATTTTTA 60
AGGCGTGTGT TTGTGGGCGC GTCCATTAGG CGCTGGAATG ACCAAGCATG CCCTTTGGAA 120
TTTGTGGAAT TAGACAAGCA AGCCCATAAA GCGATGATTG CGTATCTGCT CGCTAAAGAT 180
TTAAAAGATA GGGGTAAAGA TTTAGATTTA GATCTTTTAA TCAAATATTT TTGCTTTGAG 240 TTTTTGGAGC GCTTGGTTTT AACCGATATT AAACCCCCTA TTTTTTACGC CCTCCAACAA 300 ACGCATAGTA AAGAGTTAGC TTCCTATGTT GCGCAAAGTT TGCAAGATGA AATCAGTGCG 360
TATTTTTCTT TAGAGGAACT CAAAGAGTAT TTAAGCCACA GGCCTCAAAT TTTAGAAACT 420
CAAATTTTAG AGAGCGCGCA TTTTTATGCG TCTAAGTGGG AGTTTGATAT TATCTATCAT 480
TTTAACCCCA ACATGTATGG CGTGAAAGAG ATTAAAGATA AAATTGACAA GCAACTCCAC 540 AATAACGATC ATTTGTTTGA AGGGCTTTTT GGGGAAAAAG AAGATTTGAA AAAATTGGTG 600
AGCATGTTTG GGCAGTTGCG TTTCCAAAAG CGCTGGAGCC AAACCCCAAG AGTGCCACAA 660
ACCAGTGTTC TAGGGCATAC TTTATGCGTG GCGATTATGG GGTATTTATT GAGTTTTGAC 720
TTGAAAGCTT GTAAAAGCAT GCGGATCAAT CATTTTTTGG GCGGGCTTTT CCATGATTTA 780
CCCGAAATTT TAACCCGAGA CATTATCACG CCCATCAAAC AAAGCGTTGC AGGGCTTGAT 840 CATTGCATTA AAGAGATTGA AAAAAAGGAA ATGCAAAACA AAGTCTATTC CTTTGTGTCT 900
TTGGGCGTTC AAGAAGATTT GAAATATTTC ACCGAAAACG AGTTTAAAAA CCGCTACAAA 960
GACAAGTCTC ATCAAATCGT TTTCACTAAA GACGCTGAAG AATTATTCAC GCTTTATAAT 1020
AGCGATGAAT ATCTTGGGGT TTGCGGGGAG CTTTTGAAGG TGTGCGATCA TTTGAGCGCG 1080
TTTTTAGAAG CCCAAATCTC TCTTTCTCAT GGCATTTCTA GCTACGATTT AATCCAAGGA 1140 GCTAAAAACC TTTTAGAATT GCGATCCCAA ACGGAACTGC TTGATTTGGA TTTAGGGAAA 1200
TTGTTTAGAG ATTTTAAGTA A 1221
(2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1008 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1008
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
GTGTTGTGGG TGCTATATTT TTTAACCAGT TTATTTATTT GCTCTTTGAT TGTTTTGTGG 60
TCTAAAAAAT CCATGCTCTT TGTGGATAAC GCTAATAAAA TCCAAGGCTT CCATCATGCA 120
AGAACCCCAC GAGCCGGGGG GCTTGGGATC TTTCTTTCTT TTGCGTTGGC TTGTTATCTT 180
GAACCTTTTG AGATGCCTTT TAAGGGGCCT TTTGTTTTCT TAGGGCTATC GCTAGTGTTT 240 TTGAGCGGTT TTTTAGAAGA CATTAACCTT TCATTAAGCC CCAAAATACG CCTTATTTTG 300
CAAGCTGTAG GGGTCGTTTG CATCATTTCA TCAACGCCTT TAGTGGTGAG CGATTTTTCG 360
CCCCTTTTTA GCTTGCCTTA TTTCATCGCT TTTTTATTCG CTATTTTTAT GCTGGTGGGT 420
ATCAGTAACG CTATTAATAT CATTGACGGG TTTAACGGGC TTGCATCTGG GATTTGCGCG 480
ATCGCGCTTT TAGTCATTCA TTATATAGAC CCTAGCAGTT TGTCTTGTTT GCTCGCTTAC 540 ATGGTGCTTG GGTTTATGGT GTTAAATTTC CCTTCAGGAA AGATTTTTTT AGGCGATGGG 600
GGGGCGTATT TTTTGGGTTT GGTGTGCGGG ATTTCTCTCT TGCATTTGAG TTTGGAGCAA 660
AAAATCAGCG TGTTTTTTGG GCTCAATTTA ATGCTTTATC CGGTCATAGA GGTGCTTTTT 720
AGTATCCTTA GGCGCAAAAT AAAACGCCAG AAAGCCACCA TGCCGGATAA TTTGCATTTG 780
CACACCCTTT TATTTAAATT CTTGCAACAA CGCTCTTTCA ATTACCCTAA CCCTTTATGC 840 GCGTTTATCC TTATTCTATG CAACCTGCCT TTTATTTTAA TAAGCGTTTT GTTTCGCTTG 900 GACGCTTATG CGCTCATTGT GATTAGCCTA GTCTTTATCG CATGCTATTT AATAGGCTAT 960 GCTTATTTGA ATAGGCAAGT TTGCGCTTTA GAAAAGCGGG CGTTTTAA 1008
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 291 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...291 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
ATGAAAAAGG TTATTGTGGC TTTAGGCGTT TTGGCGTTCG CAAATGTTTT AATGGCAACC 60
GATGTTAAGG CTCTTGTAAA AGGTTGTGCC GCTTGCCATG GGGTTAAGTT TGAAAAGAAA 120
GCTTTAGGTA AAAGCAAAAT CGTTAACATG ATGAGCGAAA AAGAGATTGA AGAGGATCTT 180 ATGGCTTTTA AAAGCGGTGC CAACAAGAAT CCTGTCATGA CCGCGCAAGC TAAAAAATTA 240
AGCGATGAAG ACATCAAAGC TTTAGCCAAA TACATCCCCA CTCTCAAATA A 291
(2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 471 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...471
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: - I l l -
ATGCGAGATT TCAATAACAT TCAAATCACA CGCTTAAAAG TGCGTCAAAA TGCCGTTTTT 60
GAAAAACTGG ATCTGGAGTT TAAAGATGGC TTGAGCGCGA TTAGTGGGGC TAGTGGGGTG 120
GGGAAAAGCG TCCTTATTGC GAGCCTTTTA GGGGCGTTTG GGCTTAAAGA GAGCAACGCT 180
TCAAACATTG AAGTGGAATT GATCGCGCCT TTTTTAGACA CGGAAGAATA CGGCATTTTT 240 AGAGAAGATG AGCATGAACC CTTAGTTATT AGCGTGATTA AAAAAGAAAA AACACGCTAT 300
TTTTTAAACC AAACAAGCCT ATCTAAAAAC ACGCTCAAAG CGTTATTAAA GGGGCTTATT 360
AAACGCTTAT CTAACGACAG ATTCAGCCAG AATGAACTCA ACGATATTTT AATGCTCTCC 420
TTATTAGATG GCTATATCCA AAATAAAAAT AGGCGTTTAG CCCCCTTTTA G 471 (2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 357 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...357
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
GTGATGCTAA TGGCAATTTT TACCCCTTAT ATTCTTATTT TGAAAATGAT GAAAAAGTCT 60
ATGAGTTTAT TCGCCAATAT GGGGTTGGAG CAAATTTTTT GCAACAGAGA CATTAAAGAT 120 TTAAATGATT TTGTTTTTGG TATAGAAGTG GGGCTTGATA GCAATGCGAG AAAAAATCGT 180
AGCAGAAAGG CTATGGAAAA TCATCTTATC GGTCTTTTTG TCCAAGCTCA ATTAAATTTT 240
AAAGAACAAG TAGATATTAG AGAATTTGAG GATTTACGCC AGGCTTTTGG AAATGATACT 300
AAAAAATTTG ATTTTGTTAT TTTTAGCAAA GAGAAAACTT ATTTTCATAG AAGCTAA 357 (2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1068 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori ( ix) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1068
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
ATGAATATCA AAATTTTAAA AATATTAGTT GGAGGGTTAT TTTTTTTGAG CTTGAACGCC 60
CATTTATGGG GGAAACAAGA CAATAGCTTT TTAGGGATTG GTGAAAGAGC CTATAAAAGC 120 GGGAATTATT CTAAAGCGGC GTCTTATTTT AAAAAAGCAT GCAACGATGG GGTGAGTGAA 180
GGCTGCACGC AATTAGGAAT CATTTATGAA AACGGGCAAG GCACTAGAAT AGATTATAAA 240
AAAGCCCTAG AATATTATAA AACCGCATGC CAGGCTGATG ATAGGGAAGG GTGTTTTGGC 300
TTAGGGGGGC TTTATGATGA GGGTTTAGGC ACGGCTCAAA ATTATCAAGA AGCCATTGAC 360
GCTTACGCTA AGGCATGCGT TTTAAAACAC CCTGAGAGTT GCTACAATTT AGGCATCATT 420 TATGATAGAA AAATCAAAGG CAATGCCGCT CAAGCGGTTA CTTACTATCA AAAAAGCTGT 480
AATTTTGATA TGGCTAAGGG GTGTTATATT TTAGGCACTG CCTATGAAAA AGGCTTTTTA 540
GAAGTCAAAC AGAGCAACCA TAAAGCCGTT ATCTATTATT TGAAAGCGTG CCGATTGAAT 600
GAGGGGCAGG CTTGCCGAGC GTTAGGGAGT TTGTTTGAAA ATGGCGATGC AGGGCTTGAT 660
GAAGATTTTG AAGTGGCGTT TGATTATTTG CAAAAAGCTT GCGCTTTAAA CAATTCTGGT 720 GGTTGCGCGA GTTTAGGCTC TATGTATATG TTGGGCAGGT ATGTTAAAAA AGACCCCCAA 780
AAGGCTTTTA ACTATTTCAA GCAAGCATGC GATATGGGGA GCGCGGTGAG TTGCTCTAGG 840
ATGGGCTTTA TGTATTCGCA AGGGGACACT GTTTCAAAAG ACTTGAGGAA AGCCCTTGAT 900
AATTATGAAA GAGGTTGCGA TATGGGCGAT GAAGTGGGTT GCTTCGCTCT AGCGGGCATG 960
TATTACAACA TGAAAGATAA AGAAAACGCC ATAATGATTT ATGACAAGGG CTGTAAATTG 1020 GGCATGAAAC AGGCATGCGA AAATCTCACC AAACTCAGGG GGTATTAG 1068
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 582 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...582
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: ATGAAAGAAA AAAACTTTTG GCCTTTAGGA ATCATGAGCG TGCTTATTTT TGGGCTTGGG 60
ATCGTGGTGT TTTTAGTGGT GTTTGCCCTA AAAAATTCGC CTAAAAATGA TTTAGTGTAT 120
TTCAAGGGTC ATAACGAAGT GGATTTAAAC TTTAACGCCA TGCTTAAAAC TTATGAAAAC 180
TTTAAATCCA ATTATCGTTT TTCAGTGGGT TTAAAGCCTC TTACCGAAAG CCCTAAAACC 240
CCCATTTTGC CCTATTTTTC TAAAGGCACG CATGGGGATA AAAAAATCCA AGAAAACCTT 300 TTAAACAACG CTTTGATTTT AGAAAAGTCC AACACGCTTT ATGCACAATT GCAACCGCTC 360 AAACCCGCTT TAGATTCGCC AAATATTCAA GTGTATTTAG CGTTCTATCC CAGCCAATCC 420
CAGCCCAGAT TATTAGGAAC GCTTGATTGT AAAAACGCAT GCGAACCTTT AAAATTTGAT 480
TTGTTAGAGG GCGATAAAGT GGGGCGCTAT AAGATCCTTT TTAAATTTGT TTTTAAAAAT 540
AAAGAAGAAT TGATTTTGGA GCAACTGGCT TTTTTTAAGT AG 582
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 870 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...870
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
TTGGGTATCA ATATGTGTTC TAAAAAAATA AGAAATCTCA TTTTATGCTT TGGTTTTATT 60 TTAAGCTTGT GCGCTGAAGA AAATATCACC AAAGAAAACA TGACTGAAAC GAACACGACT 120
GAAGAAAACA CCCCTAAAGA CGCTCCCATT CTTTTGGAAG AAAAACGCGC CCAAACTCTA 180
GAGCTTAAAG AAGAAAATGA AGTGGCAAAA AAGATTGATG AAAAAAGCCT GCTTGAAGAA 240
ATCCATAAGA AAAAACGCCA GCTTTACATG CTCAAAGGGG AATTGCATGA AAAGAATGAA 300
TCCATCTTAT TCCAACAAAT GGCTAAAAAT AAGAGCGGCT TTTTTATAGG CGTGATCCTT 360 GGCGATATAG GGATTAACGC TAATCCTTAT GAGAAGTTTG AACTTTTAAG CAATATTCAA 420
GCTTCTCCCT TGCTGTATGG TTTAAGGAGC GGGTATCAAA AGTATTTCGC TAACGGGATT 480
AGCGCCTTAC GCTTTTATGG GGAATATTTA GGGGGGGCGA TGAAAGGGTT TAAAAGCGAT 540
TCTTTAGCTT CTTATCAAAC CGCAAGCTTG AATATTGATC TGTTGATGGA TAAGCCTATT 600
GACAAAGAAA AAAGGTTTGC GTTAGGGATA TTTGGAGGCG TTGGAGTGGG GTGGAATGGG 660 ATGTATCAAA ATTTAAAAGA GATTAGAGGG TATTCACAGC CTAACGCCTT TGGGTTGGTG 720
TTAAATTTAG GGGTGAGCAT GACGCTCAAC CTCAAACACC GCTTTGAATT AGCCCTAAAA 780
ATGCCTCCCT TAAAAGAAAC TTCGCAAACC TTTTTATATT ATTTTAAAAG CACTAATATT 840
TATTATATTA GTTACAACTA TTTATTGTAA 870 (2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2007 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO ( iv) ANTI - SENSE : NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...2007
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
ATGAGAAAAC TATTCATCCC ACTTTTATTA TTCAGCGCTT TAGAAGCGAA CGAGAAAAAC 60
GGCTTTTTCA TAGAAGCCGG CTTTGAAACT GGGCTATTAG AAGGCACACA AACGCAAGAA 120 AAAAGACACA CCACCACAAA AAACACTTAC GCAACTTACA ATTATTTACC CACAGACACG 180
ATTTTAAAAA GAGCGGCTAA TTTATTCACC AATGCCGAAG CGATTTCAAA ATTAAAATTC 240
TCATCTTTAT CCCCTGTTAG AGTGTTGTAT ATGTATAATG GTCAATTAAC TATAGAAAAC 300
TTCTTGCCTT ATAATTTAAA TAATGTTAAG CTTAGTTTTA CAGACGCTCA AGGCAACACG 360
ATTGATCTAG GCGTGATAGA GACCATCCCC AAACACTCTA AGATTGTTTT ACCCGGGGAG 420 GCGTTTGATA GTTTAAAAGA GGCGTTTGAT AAAATTGACC CCTATACTTT ATTTCTTCCA 480
AAATTTGAAG CCACTAGCAC TTCTATTTCT GATACTAACA CGCAGAGGGT GTTTGAAACG 540
CTCAATAACA TTAAAACAAA TCTTATAATG AAATATAGTA ATGAAAATCC AAACAATTTC 600
AACACTTGTC CTTACAATAA TAATGGTAAT ACAAAAAATG ATTGTTGGCA AAATTTCACC 660
CCACAAACCG CAGAAGAATT CACCAATTTA ATGTTGAACA TGATCGCTGT CTTAGACTCC 720 CAATCTTGGG GCGATGCGAT CTTAAACGCT CCTTTTGAAT TCACTAACAG CTCAACAGAT 780
TGCGATAGCG ATCCTTCAAA ATGCGTAAAT CCCGGAGTAA ATGGGCGTGT TGATACTAAA 840
GTCGATCAAC AATATATACT CAACAAACAA GGTATTATTA ATAATTTTAG AAAAAAAATA 900
GAAATTGATG CGGTTGTTTT AAAAAATTCA GGGGTTGTAG GGTTAGCCAA TGGATATGGC 960
AATGATGGTG AATATGGCAC ATTAGGGGTA GAAGCCTATG CTTTAGATCC TAAAAAACTC 1020 TTTGGCAACG ACCTTAAGAC TATCAATTTA GAAGATTTAA GAACCATCTT GCATGAATTC 1080
AGCCACACTA AAGGCTATGG GCATAACGGG AATATGACCT ATCAAAGAGT GCCGGTAACG 1140
AAAGATGGTC AAGTGGAAAA GGATAGTAAT GGCAAGCCAA AAGATTCTGA TGGCCTCCCC 1200
TATAATGTGT GTTCGCTTTA TGGGGGATCC AATCAGCCCG CTTTCCCTAG CAACTACCCT 1260
AATTCCATCT ATCACAATTG TGCGGATGTC CCGGCTGGCT TTTTAGGGGT AACAGCAGCG 1320 GTTTGGCAGC AGCTCATCAA TCAAAACGCC TTGCCGATCA ACTACGCTAA CTTGGGGAGT 1380
CAAACAAACT ACAACCTAAA CGCTAGTTTA AACACGCAAG ATTTAGCCAA TTCCATGCTC 1440
AGCACCATCC AAAAAACCTT TGTAACTTCT AGCGTTACCA ACCACCATTT TTCAAACGCA 1500
TCGCAAAGTT TTAGAAGCCC TATTTTAGGG GTTAACGCTA AAATAGGCTA TCAAAACTAC 1560
TTTAATGATT TCATAGGGTT GGCTTATTAT GGCATCATCA AATACAATTA CGCTAAAGCT 1620 GTTAATCAAA AAGTCCAGCA ATTGAGCTAT GGTGGGGGGA TAGATTTGTT ATTGGATTTC 1680
ATCACCACTT ACTCCAATAA AAATAGCCCT ACAGGCATTC AAACCAAAAG GAATTTTTCT 1740
TCATCTTTTG GTATCTTTGG GGGGTTAAGG GGCTTGTATA ACAGCTATTA TGTGTTGAAC 1800
AAAGTCAAAG GAAGCGGCAA TTTAGATGTG GCTACCGGGT TGAACTACCG CTATAAGCAT 1860
TCTAAATATT CTGTAGGGAT TAGCATCCCT TTAATCCAAA GAAAAGCTAG CGTCGTTTCT 1920 AGCGGTGGCG ATTATACGAA CTCTTTTGTT TTCAATGAAG GGGCTAGCCA CTTTAAGGTG 1980
TTTTTCAATT ACGGGTGGGT GTTTTAG 2007
(2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 192 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...192
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
ATGAATACAG AAATTTTAAC CATCATGTTA GTTGTCTCCG TGCTTATGGG ATTGGTAGGC 60
TTAATAGCGT TTTTATGGGG GGTTAAAAGC GGTCAGTTTG ACGATGAAAA ACGCATGCTT 120
GAAAGCGTGT TGTATGACAG CGCGAGCGAC TTGAACGAAG CGATTTTACA AGAAAAACGC 180
CAAAAGAATT AA 192
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1221 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...1221
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
ATGGTATTTT TTCATAAGAA AATTATTTTA AATTTTATCT ATTCTTTAAT GGTTGCTTTT 60 TTATTCCATT TATCCTATGG GGTTCTTTTA AAAGCCGATG GAATGGCTAA AAAGCAAACT 120
CTTTTAGTGG GTGAAAGGCT TGTGTGGGAT AAGCTCACGC TGTTAGGGTT TTTAGAAAAA 180
AACCATATCC CCCAAAAACT CTACTACAAT TTGAGCTCTC AAGATAAAGA ATTGAGTGCT 240
GAAATCCAAA GCAATGTTAC CTACTACACT TTAAGAGATG CAAATAACAC GCTCATTCAA 300
GCCCTTATCC CTATTAGCCA GGATTTGCAA ATCCATATTT ACAAAAAAGG AGAGGATTAT 360 TTTTTAGACT TTATCCCCAT TGTTTTCACT CGTAAAGAAA GAACCCTCCT TCTTTCCTTA 420
CAAACTTCGC CCTATCAAGA TATTGTCAAA GCCACCAATG ACCCCCTTTT AGCCAACCAA 480
TTGATGAACG CGTATAAAAA AAGCGTGCCT TTTAAACGCC TAGTGAAAAA CGATAAAATC 540
GCTATCGTTT ATACAAGGGA TTATCGTGTG GGGCAAGCGT TTGGCCAGCC GACCATCAAA 600
ATGGCGATGG TTAGCTCTCG TTTGCACCAA TACTATCTTT TTTCCCATTC AAACGGGCGT 660 TATTACGATT CAAAAGCGCA AGAAGTGGCA GGGTTTTTAC TAGAAACCCC GGTGAAATAC 720 ACCCGCATTT CTTCGCCTTT TTCGTATGGG AGGTTCCATC CTGTTTTAAA AGTTAAACGG 780
CCTCATTACG GCGTGGATTA TGCGGCTAAA CATGGCAGTT TGATCCATTC TGCTTCAGAC 840
GGCCGTGTGG GTTTTATAGG GGTTAAGGCG GGTTATGGGA AGGTGGTTGA AATCCATTTG 900
AATGAATTGC GCTTGGTGTA TGCTCACATG AGCGCGTTCG CTAACGGATT AAAAAAAGGC 960
TCGTTCGTTA AAAAAGGGCA AATCATAGGA AGAGTGGGAA GCACGGGTTT AAGCACCGGG 1020
CCGCATTTGC ATTTTGGCGT GTATAAAAAC TCCCGCCCCA TTAATCCTTT AGGCTATATC 1080
CGCACCGCTA AAAGCAAGCT GCATGGCAAA CAAAGAGAGG TTTTTTTAGA AAAAGCTCAG 1140
TATTCTAAGC AAAAATTAGA AGAACTTTTT AAAACCCATT CTTTTGAAAA AAATTCATTT 1200
TATCTTTTAG AGGGTTTTTA A 1221
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 891 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...891
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
TTGTTTTTAG TCAAAAAAAT AGGCGTGGTA ATAATGATTT TAGTCTGCTT TTTAGCTTGC 60 TCGCAAGAGA GCTTTATCAA AATGCAAAAA AAAGCCCAAG AGCAAGAAAA TGACGGCTCT 120
AAACGCCCCA GCTATGTGGA TTCGGATTAT GAAGTCTTTA GCGAAACGAT TTTTTTACAA 180
AACATGGTGT ATCAGCCTAT AGAGGAAAGA AACGCTTTTT TCCAACTGAC TAAAGATG.AA 240
GACAATTCTT TTAACCCTGA AAATTCCGTG ATTTTACTGA ATGAGCCAAG CGATAATAGT 300
GAAAAAAACC TACTCTCATA CCCAAACGAT CCCAATAACA ATGAAGACAA CGCTAATAAT 360 AGTCAAAAAA ATCCGTTCCT TTACAAGCCC AAAAGAAAAA CAAAAAACCC AAAACTCATT 420
GAATATTCCC AACAAGATTT CTACCCCCTA AAAAATGGGG ATATTATCAT GAGTAAAGAA 480
GGGGATCAAT GGTTGATAGA AATCCAATCC AAAGCCTTGA AGCGTTTTTT AAAAGATCAA 540
AACGATAAAG ATCGCCAGAT CCAAACTTTC ACTTTTAATG ACACTAAAAC GCAAATCGCG 600
CAAATTAAGG GCAAAATTTC TTCGTATGTT TATACCACCA ATAACGGTAG CTTGAGTTTA 660 AGGCCTTTTT ATGAATCGTT TTTGTTAGAA AAAAAGAGCG ATAATGTTTA TACGATAGAG 720
AATAAGGCTT TAGATACTAT GGAGATTTCA AAGTGTCAAA TGGTGTTAAA AAAGCATTCA 780
ACCGATAAAT TAGACAGCCA GCATAAAGCC ATCAGTATTG ATTTGGATTT TAAAAAAGAG 840
CGCTTTAAGA GCGATACGGA ACTCTTTTTA GAATGTCTTA AGGAAAGTTA G 891 (2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 747 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...747
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
GTGAGCTATG ACAACACCGA TGATTATTAT TTCCCTAGAA ATGGGGTTAT CTTTAGTTCC 60
TATGCGACAA TGTCTGGTTT GCCAAGCTCT GGCACGCTCA ATTCTTGGAA CGGGTTAGGC 120 GGGAATGTCC GTAACACCAA AGTTTATGGT AAATTCGCCG CTTACCACCA TTTGCAAAAA 180
TATTTATTGA TAGATTTGAT CGCTCGTTTT AAAACGCAAG GGGGCTATAT CTTTAGGTAT 240
AACACCGATG ATTACTTGCC CTTAAACTCC ACTTTCTACA TGGGGGGCGT AACCACGGTG 300
AGAGGCTTTA GGAACGGCTC AATCACACCT AAAGATGAGT TTGGCTTGTG GCTTGGAGGC 360
GATGGGATTT TTACCGCTTC TACTGAATTG AGCTATGGGG TGTTAAAAGC GGCTAAAATG 420 CGTTTAGCGT GGTTTTTTGA CTTTGGTTTC TTAACCTTTA AAACCCCAAC TAGGGGGAGT 480
TTCTTCTATA ACGCTCCCAC CACGACGGCG AATTTTAAAG ATTATGGCGT TGTAGGGGCT 540
GGGTTTGAAA GGGCGACTTG GAGGGCTTCT ACAGGCTTAC AGATTGAATG GATTTCGCCC 600
ATGGGGCCTT TGGTGTTGAT TTTCCCTATA GCGTTTTTCA ACCAATGGGG CGATGGCAAT 660
GGCAAAAAAT GTAAAGGGCT GTGCTTTAAC CCTAACATGA ACGATTACAC GCAACATTTT 720 GAATTTTCTA TGGGAACAAG GTTTTAA 747
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1008 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...1008
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: GTGCAACACT TCAATTTCCT CTATAAAGAT TCTTTATTTT CTATCGCTTT ATTCACTTTC 60 ATTATCGCTC TTGTGATTTT ATTAGAACAG GCTAGAGCGT ATTTCACCCG AAAGAGAAAC 120
AAAAAATTTT TGCAAAAATT CGCCCAAAAT CAAAACGCCT ATGCGAGCAG CGAGAATTTA 180
GACGAGCTTT TAAAGCATGC TAAAATTTCC AGTTTGATGT TTTTAGCTAG GGCGTATTCT 240
AAAGCGGATG TGGAAATGAG CATTGAAATC TTAAAAGGGC TTTTGAATCG CCCCTTAAAA 300 GATGAAGAAA AAATCGCTGT TTTAGATTTA TTGGCTAAAA ATTATTTTAG CGTGGGGTAT 360
TTGCAGAAAA CAAAAGACAC CGTGAAAGAA ATTTTGCGCT TTTCCCCAAG GAATGTGGAA 420
GCGTTGTTGA AGCTTTTGCA TGCGTATGAA TTAGAAAAAG ATTATTCAAA GGCTTTAGAA 480
ACTTTGGAAT GTTTGGAAGA ATTAGAGGTG CCTAAAATTG AAACGATTAA AAATTACCTC 540
TATTTAATGC ATTTAATAGA GAATAAGGAA GATGCGGCTA AAATCTTGCA TGTTTCAAAA 600 GCGTCGTTAG ATTTGAAAAA AATCGCTCTG AATCACTTAA AATCGCATGA TGAAAATCTT 660
TTTTGGCAAG AAATTGATAC AACCGAACGG CTAGAAAATG TGATCGATCT TTTATGGGAT 720
ATGAATATCC CTGCTTTTAT TTTAGAAAAA CATGCCCTTT TGCAGGACAT CGCGCGATCT 780
CAAGGGTTGC TTTTGGATCA CAAACCTTGC CAAATTTTTG AATTAGAGGT TTTACGCGCT 840
CTATTGCATA GCCCTATAAA AGCGAGTCTG ACTTTTGAAT ACCGCTGCAA GCATTGCAAA 900 CAAATCTTTC CTTTTGAAAG CCATAGGTGT CCTGTGTGTT ACCAGTTAGC GTTTATGGAT 960
ATGGTGCTTA AAATCTCTAA AAAAACGCAT GCTATGGGAG TGGATTAA 1008
(2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1242 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1242
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
ATGAGGAAAA TTTTTTCTTA TATTTCTAAG GTTCTATTAT TTATTGGGGT GGTTTATGCA 60
GAGCCTGATT CTAAAGTGGA AGCCTTAGAA GGGAGGAAGC AAGAGTCTTC TTTGGATAAA 120
AAAATCCGCC AAGAATTGAA GAGTAAGGAA TTGAAGAATA AGGAATTAAA GAATAAGGAT 180
TTGAAAAATA AAGAAGAAAA GAAAGAAACA AAAGCCAAGA GAAAACCCAG AGCAGAAGTC 240 CATCATGGGG ACGCCAAAAA TCCCACTCCA AAGATCACGC CTCCTAAAAT CAAAGGGAGT 300
AGTAAGGGCG TTCAAAATCA AGGCGTTCAA AACAACGCGC CAAAACCTGA AGAAAAAGAT 360
ACAACCCCTC AAGCTACTGA AAAAAATAAG GAAACAAGCC CTAGCTCTCA ATTCAATTCC 420
ATTTTTGGTA ATCCTAATAA CGCTACCAAC AACACCCTTG AAGATAAGGT CGTAGGGGGC 480
ATTTCATTGC TTGTTAATGG TTCGCCTATC ACGCTGTATC AAATCCAAGA AGAGCAAGAA 540 AAATCTAAAG TGAGTAAGGC TCAAGCTAGG GATCGTTTGA TCGCTGAACG CATTAAAAAC 600
CAAGAAATTG AGCGCTTAAA AATCCATGTA GATGATGACA AGCTAGACCA AGAAATGGCG 660
ATGATGGCGC AACAACAAGG CATGGATTTA GACCATTTCA AACAGATGCT TATGGCTGAG 720
GGGCATTATA AACTCTATAG AGATCAACTT AAAGAGCATT TAGAAATGCA AGAATTGTTG 780
CGTAATATTT TGCTCACGAA TGTGGATACC AGCTCTGAAA CCAAAATGCG CGAATATTAC 840 AACAAACACA AGGAGCAATT CAGTATCCCC ACAGAAATAG AAACCGTGCG CTACACTTCC 900 ACCAATCAAG AGGATTTAGA AAGGGCTATG GCAGACCCTA ATTTGGAAGT CCCAGGGGTG 960
AGTAAGGCCA ATGAAAAAAT AGAGATGAAA ACCCTAAACC CTCAAATCGC CCAAGTCTTT 1020
ATTTCGCATG AGCAAGGCTC TTTCACGCCC GTTATGAATG GGGGTGGGGG GCAGTTCATC 1080
ACCTTTTATA TCAAGGAAAA AAGGGGTAAA AATGAAGTGA GCTTCAGTCA GGCCAAGCAA 1140 TTCATCGCCC AAAAATTAGT GGAAGAATCT AAGGATAAGA TTTTAGAAGA GCATTTTGAA 1200
AAATTGCGCG TTAAGTCTAG GATTGTGATG ATCAGAGAGT GA 1242
(2) INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 561 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...561
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
ATGATTAAAA GAATTGCTTG TATTTTAAGC TTGAGCGCGA GTTTAGCGTT AGCTGGCGAA 60
GTGAATGGGT TTTTCATGGG TGCGGGTTAT CAACAAGGTC GTTATGGCCC TTATAACAGC 120
AATTACTCTG ATTGGCGTCA TGGCAATGAC CTTTATGGTT TGAATTTCAA ATTAGGTTTT 180
GTAGGCTTTG CCAATAAATG GTTTGGGGCT AGGGTGTATG GCTTTTTAGA TTGGTTTAAC 240 ACTTCAGGGA CTGAACACAC CAAAACCAAT TTGCTCACCT ATGGCGGCGG TGGCGATTTG 300
ATTGTCAATC TCATTCCTTT GGATAAATTC GCTCTAGGTC TCATTGGTGG CGTTCAATTA 360
GCCGGAAACA CTTGGATGTT CCCTTATGAT GTCAATCAAA CCAGATTCCA GTTCTTATGG 420
AATTTAGGCG GAAGAATGCG TGTTGGGGAT CGCAGTGCGT TTGAAGCGGG CGTGAAATTC 480
CCTATGGTTA ATCAGGGTAG CAAAGATGTA GGGCTTATCC GCTACTATTC TTGGTATGTG 540 GATTATGTCT TCACTTTCTA G 561
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 729 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...729
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: ATGAAAAAAT TTTTTTCTCA ATCTTTGTTA GCTCTTATTA TCTCTATGAA TGCGGTATCT 60
GGCATGGATG GTAATGGCGT TTTTTTAGGG GCGGGTTATT TGCAAGGACA GGCGCAAATG 120
CATGCGGATA TTAATTCTCA AAAACAAGCC ACCAACGCTA CGATCAAAGG CTTTGACGCG 180
CTCTTGGGGT ATCAATTTTT CTTTGAAAAA CACTTTGGCT TACGCCTTTA TGGGTTTTTT 240
GACTACGCTC ATGCCAATTC TATTAAGCTT AAAAACCCTA ACTATAATAG CGAAGCGGCG 300 CAAGTGGCTA GTCAAATTCT TGGGAAACAA GAAATCAATC GTTTAACAAA CATTGCCGAT 360
CCCAGAACTT TTGAGCCGAA CATGCTCACT TATGGGGGGG CTATGGACGT GATGGTTAAT 420
GTCATCAATA ACGGCATCAT GAGTTTGGGG GCTTTTGGCG GGATACAATT GGCCGGCAAT 480
TCATGGCTTA TGGCGACACC GAGCTTTGAG GGCATTTTAG TGGAACAAGC CCTTGTGAGC 540
AAGAAAGCCA CTTCTTTCCA ATTTTTATTC AATGTGGGGG CTCGCTTAAG GATCTTAAAA 600 CATTCTAGCA TTGAAGCGGG CGTGAAATTC CCCATGCTAA AGAAAAACCC CTACATCACT 660
GCAAAAAATT TGGATATAGG GTTTAGGCGC GTGTATTCGT GGTATGTGAA TTACGTGTTC 720
ACTTTCTAG 729
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 771 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...771 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
ATGGGATACG CAAGCAAATT AGCTTTAAAG ATTTGTTTGG TAGGTTTATG TTTATTTAGC 60
ACCCTTGGTG CAGAACACCT TGAGCAAAAA GGGAATTATA TTTATAAGGG AGAGGAGGCT 120
TATAATAATA AGGAATATGA GCGAGCGGCT TCTTTTTATA AGAGCGCTAT TAAAAATGGT 180 GAGTCGCTTG CTTATATTCT TTTAGGGATC ATGTATGAAA ATGGTAGGGG TGTACCTAAA 240
GATTACAAGA AAGCGGTTGA ATATTTCCAA AAAGCTGTTG ATAACGATAT ACCTAGAGGG 300
TATAACAATT TGGGCGTGAT GTATAAAGAG GGTAAGGGAG TTCCTAAAGA TGAAAAGAAA 360
GCGGTGGAAT ATTTTAGAAT AGCTACAGAG AAAGGTTATA CTAACGCTTA TATCAACTTA 420
GGCATCATGT ATATGGAGGG CAGGGGAGTT CCAAGTAACT ATGCGAAAGC GACAGAATGT 480 TTTAGAAAAG CGATGCATAA GGGCAATGTG GAAGCTTATA TTCTCCTAGG GGATATTTAT 540 TATAGCGGGA ATGATCAATT GGGTATTGAG CCGGACAAAG ATAAGGCTGT TGTCTATTAT 600
AAAATGGCGG CTGATGTGAG TTCTTCTAGA GCTTATGAAG GGTTGTCAGA GTCTTATCGG 660
TATGGGTTAG GCGTGGAAAA AGATAAAAAA AAGGCTGAAG AATACATGCA AAAAGCATGC 720
GATTTTGACA TTGATAAAAA TTGTAAGAAA AAGAACACTT CAAGCCGATA A 771
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1974 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...1974
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
ATGAGAAAAC TATTCATCCC ACTTTTATTA TTCAGCGCTT TAGAAGCGAA CGAGAAAAAC 60 GGCTTTTTCA TAGAAGCCGG CTTTGAAACT GGGCTATTAG AAGGCACACA AACGCAAGAA 120
AAAAGACACA CCACCACAAA AAACACTTAC GCAACTTACA ATTATTTACC CACAGACACG 180
ATTTTAAAAA GAGCGGCTAA TTTATTCACC AATGCCGAAG CGATTTCAAA ATTAAAATTC 240
TCATCTTTAT CCCCTGTTAG AGTGTTGTAT ATGTATAATG GTCAATTAAC TATAGAAAAC 300
TTCTTGCCTT ATAATTTAAA TAATGTTAAG CTTAGTTTTA CAGACGCTCA AGGCAATGTG 360 ATCGATCTAG GCGTGATAGA GACTATCCCC AAACACTCTA AGATTGTTTT GCCCGGAGAG 420
GCATTTGATA GTCTAAAAAT TGACCCCTAT ACTTTATTTC TTCCAAAAAT TGAAGCCACT 480
AGCACTTCTA TTTCTGACGC TAACACGCAG AGGGTGTTTG AAACGCTCAA TAAGATTAAG 540
ACAAATTTGG TCGTAAATTA TAGGAATGAA AACAAATTTA AAGATCACGA AAATCATTGG 600
GAAGCCTTTA CCCCACAAAC CGCAGAAGAA TTCACTAATT TAATGTTGAA CATGATCGCT 660 GTTTTAGACT CCCAATCTTG GGGCGATGCG ATCTTAAACG CTCCTTTTGA GTTCACTAAC 720
AGCCCAACAG ATTGCGATAA TGATCCTTCA AAATGCGTAA ATCCTGGGAC AAACGGGCTT 780
GTCAATTCTA AAGTCGATCA AAAATATGTG TTAAACAAAC AAGACATTGT CAATAAATTT 840
AAAAACAAAG CGGATCTTGA TGTAATTGTT TTAAAGGATT CAGGGGTTGT AGGGCTTGGG 900
AGTGATATTA CCCCTAGCAA CAATGATGAT GGCAAGCATT ATGGCCAGTT AGGGGTAGTA 960 GCTTCTGCTT TAGATCCTAA AAAACTCTTT GGCGATAACC TTAAGACTAT CAATTTAGAG 1020
GATTTAAGAA CCATCTTGCA TGAATTCAGC CACACTAAAG GCTATGGGCA TAACGGGAAT 1080
ATGACCTATC AAAGAGTGCC GGTAACGAAA GATGGTCAAG TGGAAAAGGA TAGTAATGGC 1140
AAGCCAAAAG ATTCTGATGG CCTCCCCTAT AATGTGTGTT CGCTTTATGG GGGATCCAAT 1200
CAGCCCGCTT TCCCTAGCAA CTACCCTAAT TCCATCTATC ACAATTGTGC GGATGTCCCG 1260 GCTGGCTTTT TAGGGGTAAC AGCAGCGGTT TGGCAGCAGC TCATCAATCA AAACGCCTTG 1320
CCGATCAACT ACGCTAACTT GGGGAGTCAA ACAAACTACA ACCTAAACGC TAGTTTAAAC 1380
ACGCAAGATT TAGCCAATTC CATGCTCAGC ACCATCCAAA AAACCTTTGT AACTTCTAGC 1440
GTTACCAACC ACCATTTTTC AAACGCATCG CAAAGTTTTA GAAGCCCTAT TTTAGGGGTT 1500
AACGCTAAAA TAGGCTATCA AAACTACTTT AATGATTTCA TAGGGTTGGC TTATTATGGC 1560 ATCATCAAAT ACAATTACGC TAAAGCTGTT AATCAAAAAG TCCAGCAATT GAGCTATGGT 1620 GGGGGGATAG ATTTGTTATT GGATTTCATC ACCACTTACT CCAATAAAAA TAGCCCTACA 1680
GGCATTCAAA CCAAAAGGAA TTTTTCTTCA TCTTTTGGTA TCTTTGGGGG GTTAAGGGGC 1740
TTGTATAACA GCTATTATGT GTTGAACAAA GTCAAAGGAA GCGGCAATTT AGATGTGGCT 1800
ACCGGGTTGA ACTACCGCTA TAAGCATTCT AAATATTCTG TAGGGATTAG CATCCCTTTA 1860 ATCCAAAGAA AAGCTAGCGT CGTTTCTAGC GGTGGCGATT ATACGAACTC TTTTGTTTTC 1920
AATGAAGGGG CTAGCCACTT TAAGGTGTTT TTCAATTACG GGTGGGTGTT TTAG 1974
(2) INFORMATION FOR SEQ ID NO: 46: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 504 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...504
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
ATGAAATTGG TGAGTCTTAT TGTAGCGTTA GTTTTTTGTT GTTTTTTAGG GGCTGTAGAG 60
TTGCCTGGAG TTTATCAAAC TCAAGAATTT TTATACATGA AAAGCTCTTT TGTGGAGTTT 120
TTTGAGCATA ACGGGAAGTT CTATGCCTAT GGTATTTCTG ATGTGGATGG CTCTAAAGCC 180
AAAAAAGACA AACTCAATCC TAACCCAAAG CTAAGGAATC GCAGCGATAA AGGCGTGGTG 240 TTTTTAAGCG ATTTGATTAA GGTTGGGGAA CAATCTTATA AAGGCGGTAA GGCGTATAAT 300
TTTTATGACG GCAAGACCTA CCATGTGAGA GTCACTCAAA ATTCAAACGG GGATTTGGAA 360
TTCACTTCAA GCTATGACAA ATGGGGGTAT GTGGGCAAAA CCTTCACCTG GAAACGCCTG 420
AGCGATGAAG AAATCAAAAA TCTAAAGCTC AAGCGTTTTA ACTTGGACGA AGTCCTTAAA 480
ACCCTCAAAG ATAGCCCTAT TTAA 504
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 885 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...885
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
ATGAGTAATC AAGCGAGCCA TTTGGATAAT TTTATGAACG CTAAAAATCC CAAAAGTTTT 60 TTTGATAATA AGGGGAATAC CAAATTCATC GCTATCACAA GCGGTAAGGG GGGCGTGGGG 120
AAATCCAACA TTAGCGCTAA TTTAGCTTAC TCTTTATACA AGAAAGGTTA TAAGGTAGGG 180
GTATTTGATG CGGATATTGG TTTAGCGAAT TTAGATGTCA TTTTTGGGGT GAAAACCCAT 240
AAAAATATCT TGCATGCCTT AAAAGGCGAA GCCAAATTGC AAGAAATCAT TTGCGAGATT 300
GAACCCGGGC TTTGCTTAAT CCCTGGGGAT AGCGGCGAAG AAATTTTAAA ATACATCAGC 360 GGCGCGGAAG CTTTGGATCG ATTCGTAGAT GAAGAGGGGG TTTTAAGCTC TTTAGATTAT 420
ATTGTGATTG ATACGGGTGC TGGGATTGGG GCCACTACGC AAGCGTTTTT GAATGCGAGC 480
GATTGCGTGG TGATTGTTAC CACACCCGAT CCTTCAGCGA TTACCGATGC GTATGCATGC 540
ATTAAAATCA ACTCCAAGAA TAAAGATGAA TTGTTCCTTA TCGCTAACAT GGTAGCCCAA 600
CCTAAAGAAG GCAGGGCGAC TTATGAAAGG CTATTCAAGG TGGCTAAAAA CAATATCGCT 660 TCATTAGAAT TGCACTATTT AGGGGCGATT GAAAACAGCT CCTTATTGAA ACGCTATGTG 720
AGGGAGCGAA AGATTTTGAG GAAAATAGCC CCTAACGATT TGTTTTCGCA ATCCATTGAC 780
CAGATAGCGA GCCTTTTAGT TTCTAAACTA GAAACCGGCA CTTTAGAAAT ACCAAAAGAA 840
GGTTTAAAAA GCTTTTTTAA AAGGCTTTTG AAGTATTTGG GGTAG 885 (2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1119 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1119
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48
TTGGAACCTT CAAGAAATCG CCTAAAACAT GCCGCCTTTT TTGTGGGGCT TTTTATCGTT 60
TTGTTTTTAA TTATAATGAA GCACCAAACC TCCCCCTATG CTTTCACGCA TAATCAAGCC 120 CTTGTCACTC AAACCCCCCC CTATTTCACG CAACTCACTA TCCCTAAACC AAATGACGCT 180
TTAAGCGCGC ATGCGAGCTC TTTAATCAGC TTGCCTAACG ACAATCTTTT GAGCGCTTAT 240
TTTAGCGGCA CTAAAGAAGG GGCAAGGGAT GTGAAAATCA GCGCGAATCT TTTTGACAGC 300
AAGACTAATC GCTGGAGCGA AGCCTTCATT CTTTTAACCA AAGAAGAGCT TTCTCATCAT 360
TCGCATGAAT ACATCAAAAA ATTAGGTAAC CCCTTGCTTT TTTTGCATGA TAATAAAATT 420 TTGTTGTTTG TCGTAGGGGT GAGCATGGGC GGGTGGGCCA CTTCTAAAAT CTATCAATTT 480 GAAAGCGCTT TAGAGCCGAT TCATTTTAAG TTTGCGCGAA AACTCTCTTT AAGCCCTTTT 540
TTAAATTTGA GCCATTTAGT AAGGAATAAG CCTTTAAACA CCACTGATGG CGGGTTTATG 600
CTACCACTCT ATCACGAATT AGCCACCCAA TACCCCTTGT TGTTGAAATT TGACCAACAA 660
AATAACCCAA GAGAGCTTTT AAGGCCTAAT ACCTTAAACC ACCAGCTCCA ACCAAGCTTA 720 ACCCCCTTTA AAGACTGCGC TGTCATGGCG TTTAGAAACC ATTCTTTTAA AGATAGCCTC 780
ATGCTAGAAA CCTGTAAAAC CCCCACTGAT TGGCAAAAAC CCATTTCTAC AAATCTTAAA 840
AACTTAGATG ATTCTTTAAA TTTACTCAAT TTAAATGGAA TATTGTATTT GATCCACAAC 900
CCTAGCGATT TATCACTGCG TCGTAAAGAA CTTTGGCTTT CTAAATTAGA AAACTCCAAC 960
TCGTTTAAAA CCTTAAAAGT TTTGGATAAA GCGAATGAAG TGAGTTACCC AAGCTATAGC 1020 CTTAATCCGC ATTTTATAGA TATTGTCTAT ACTTACAACC GCTCTCATAT CAAACACATC 1080
CGTTTCAATA TGGCTTATTT AAATTCCCTT CTCAAGTGA 1119
(2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2937 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...2937
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
ATGAAGAAAA GAAAACATGT ATCCAAGAAA GTGTTTAATG TCATTATCTT GTTTGTGGCA 60
GTATTCACTC TTTTAGTCGT CATTCACAAA ACCCTTTCAA ACGGCATTCA CATACAAAAT 120
TTAAAAATTG GAAAACTTGG CATTTCTGAA TTATACTTAA AACTCAATAA CAAGCTTTCT 180
TTGGAAGTTG AGCGGGTTGA TCTCTCTTCT TTCTTCCATC AAAAACCCAC TAAAAAGCGT 240 TTAGAAGTTT CTGATTTGAT TAAAAATATC CGTTATGGCA TTTGGGCGGT GTCTTATTTT 300
GAAAAACTTA AAGTCAAAGA AATCATTTTA GACGATAAAA ATAAAGCCAA TATCTTTTTT 360
GATGGGAATA AATACGAGTT AGAATTTCCA GGAATCAAAG GGGAATTTTC CCTAGAAGAC 420
GATAAAAATA TCAAGCTTAA AATCATCAAT TTGCTTTTTA AAGATGTTAA AGTCCAAGTG 480
GATGGCAACG CCCACTATTC ACCCAAAGCC AGGAAAATGG CGTTCAATTT GATTGTCAAG 540 CCCTTAGTTG AACCCAGCGC TGCAATTTAT TTGCAAGGGC TAACCGATTT AAAAACCATA 600
GAATTAAAAA TTAACACTTC TCCAATGAAA AGCCTAGCGT TTTTAAAGCC TCTTTTCCAA 660
CGCCAATCGC AAAAAAATTT AAAAACGTGG ATTTTTGACA AGATCCAATT TGCCAGCTTT 720
AAGATTGATA ACGCTTTAAT CAAGGCTAAT TTCACTCCTA GCGAGTTTAT CCCATCGCTT 780
TTGGAAAATT CTGTAGTTAA AGCCACTTTG ATTAAGCCTT CAGTCGTTTT TAATGATGGC 840 TTATCGCCCA TTAAAATGGA TAAAACCGAA TTGATTTTCA AAAACAAACA GCTCCTCATA 900
CAGCCCCAAA AAATCACTTA TGAAACCATG GAATTAACCG GCTCTTACGC CACTTTTTCC 960
AATTTGTTAG AAGCCCCTAA GTTGGAGGTT TTTTTAAAAA CGACCCCTAA TTATTATGGC 1020
GATAGCATTA AGGATTTATT GAGCGCTTAT AAAGTCGTTT TACCTTTGGA TAAAATCAGC 1080
ATGCCATCTA GCGCGGATTT GAAGCTCACT TTGCAATTCT TAAAAAACAC CGCCCCCTTA 1140 TTTAGCGTTC AAGGCAGCGT TAATTTGCAA GAAGGCACTT TCTCGCTCTA TAATATCCCC 1200 CTTTACACGC AAAGCGCTCA AATCAATTTG GACATCGCCC AAGAATACCA ATACATCTAC 1260
ATAGACACGA TCCACACGCG CTATGCAAAC ATGCTGGATT TAGACGCTAA AATCGCTTTA 1320
GATTTAGGTC AAAAAAACCT TTCTTTGGAT TCTTTAGTCC ATAAAATCCA AGTCAATACC 1380
AATAACAATA TCAACATGCG CTCTTATGAT CCCAATAACA CTCAAGAAGA TCCGCAAACT 1440 AACTTTACTT TGGATCTAAA AAGCTTGCAT TCTATCATTC AAGAGGGTGA AAATTCAGAA 1500
GTTTTTAGAA GAAAAATCAT AGACACCATT AAAGCCCAAA GCGAAGATAA ATTCACTAAA 1560
GATGTTTTTT ACGCCACAGG AGACACTCTC AAAAGCCTGT CGTTGAGTTT TGATTTTTCC 1620
AACCCCGATC ACATACAATG GAGCGTGCCA CAACTCTTAT TAGAAGGCGA ATTTAAAGAT 1680
AACGCCTATA CTTTTAAGAT CAAAGATTTG AAAAAGATCA AGCCCTATTC CCCCATTATG 1740 GACTATATTG CCCTAAAAGA CGGCTCTTTA GAGGTTTCTA CGAGCGATTT TGTCAATATT 1800
GATTTTTTTG CTAAAGATTT GAAAATCAAC CTCCCCATTT ATAGGAGCGA TGGATCGCAT 1860
TTTGATTCTT TTTCTTTATT TGGCTCTATC AATAAAGATG AAATTTCTGT CTATACTCCA 1920
AGCAAAAGCA TATCCATAAA AGTTAAGGGG GATCAAAAGG ATATTACCCT TAATAACATT 1980
GATTTGAGTA TTGATGATTT CTTGGATAGT AAAATGCCAG CTATTGCGGG ATTATTCTCA 2040 AAAGAACGAA AAGAAAAGCC TAGCTCTAAA GAAATCCAAG ATGAAGATGT TTTCATTAGC 2100
GCCAAACAAC GCTATGAAAA AGCCCACAAA ATTATCCCCA TCTCTACACG CATCCATGCT 2160
AAAGATGTCG TGCTGATCTA TAAAAAAATG CCTTTTCCTT TAGAAAATCT TGATATTGTC 2220
GCTCAAGACG ATAGGGTGAA AATTGATGGC AATTATAAAA ACGCCATGAT CATGGCGGAT 2280
TTAGTGCATG GGGCTTTGTA TCTTAAGGCT CATAATTTTA GCGGGGATTA TATCAACACC 2340 ATTCTTCAAA AAGATTTCGT AGAAGGAGGC TTATTCACGC TTATTGGGGC TCTTGAAGAT 2400
CAGGTTTTCA ATGGCGAATT GAAATTCCAA AACACAAGCT TAAAGAATTT CGCCCTCATG 2460
CAAAACATGG TCAATCTCAT CAACACCATT CCCTCCCTCA TTGTCTTTAG AAACCCTCAT 2520
TTAGGGGCTA ATGGCTATCA AATCAAAACC GGCTCCGTTG TGTTTGGGAT CACTAAAGAA 2580
TATTTAGGGT TAGAAAAAAT TGATCTTGTC GGCAAAACGC TTGATATTGC TGGCAATGGA 2640 ATCATTGAAT TAGACAAAAA CAAATTAGAT TTAAACTTAG AAGTTTCCAC TATCAAGGCT 2700
TTGAGTAATG TCTTAAATAA AATCCCTATC GTGGGCTATC TCGTTTTAGG AAAAGGAGGT 2760
AAAATCACCA CTAACGTGAA TGTCAAAGGC ACGTTGGATA AGCCTAAAAC CCAAGTAACT 2820
TTAGCGTCAG ATATTATCCA AGCGCCTTTT AAAATCTTAC GCCGTATTTT CACGCCTATT 2880
GACATCATCG TGGATGAAGT CAAGAAAAAC ATTGATTCAA AAAGGAAATT AAAATGA 2937
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1434 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...1434
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
ATGAATACTA TTATAAGATA TGCGAGTTTA TGGGGCTTGT GTATTACTCT AACTCTAGCG 60 CAAACCCCCT CTAAAACCCC TGATGAAATC AAGCAAATCC TTAACAATTA TAGCCATAAG 120 AATTTAAAGC TCATTGATCC GCCGACAAGT TCTTTAGAAG CGACACCGGG TTTTTTACCC 180
TCGCCTAAAG AAACAGCGAC CACGATCAAT CAAGAGATCG CTAAATACCA TGAAAAAAGC 240
GATAAAGCCG CTTTGGGGCT TTATGAATTG CTAAAGGGGG CTACCACCAA TCTCAGTTTG 300
CAAGCGCAAG AACTCAGTGT CAAGCAAGCG ATGAAGAACC ACACCATCGC CAAAGCGATG 360 TTTTTGCCTA CTTTGAACGC GAGTTATAAT TTTAAAAATG AAGCTAGGGA TACTCCAGAA 420
TATAAGCATT ATAACACCCA ACAACTCCAA GCTCAAGTCA CATTGAATGT GTTTAATGGC 480
TTTAGCAATG TGAATAATGT CAAAGAAAAG TCTGCGACTT ACCGATCCAC TGTGGCTAAT 540
TTAGAATATA GCCGCCAAAG CGTGTATTTG CAAGTGGTGC AACAATACTA CGAGTATTTT 600
AACAATCTCG CTCGCATGAT CGCTTTGCAA AAGAAATTAG AGCAAATCCA AACGGACATT 660 AAAAGGGTTA CTAAGCTCTA TGACAAAGGG CTGACCACGA TTGATGATTT ACAAAGCTTA 720
AAAGCGCAAG GGAATTTGAG CGAATACGAT ATTTTGGACA TGCAATTTGC TTTGGAGCAA 780
AACCGCTTGA CTTTAGAATA CCTCACTAAC CTCAGTGTGA AAAATTTGAA AAAGACCACG 840
ATTGATGCGC CTAATTTGCA ATTAAGAGAA AGGCAGGATT TGGTTTCTTT AAGGGAGCAG 900
ATTTCTGCAC TCAGATACCA AAACAAGCAA CTCAATTATT ACCCCAAGAT AGATGTGTTT 960 GACTCATGGC TTTTTTGGAT CCAAAAACCC GCTTATGCCA CAGGGCGTTT TGGGAATTTC 1020
TACCCAGGTC AGCAAAATAC GGCTGGGGTT ACTGCGACTT TGAATATTTT TGATGATATA 1080
GGGTTGAGCT TGCAAAAACA ATCCATCATG CTAGGCCAAT TAGCGAATGA AAAGAATTTA 1140
GCGTATAAAA AATTGGAGCA AGAAAAAGAC GAACAGCTTT ACAGAAAGTC GCTTGATATT 1200
GCCAGAGCTA AGATTGAATC TTCAAAGGCT AGTTTGGATG CGGCCAATCT TTCTTTTGCC 1260 AATATTAAAA GGAAATACGA CGCTAATTTA GTGGATTTCA CTACCTATTT AAGGGGCTTA 1320
ACCACGCGCT TTGATGCAGA AGTGGCTTAC AATTTAGCGC TCAACAATTA CGAAGTGCAA 1380
AAAGCCAATT ACATTTTTAA CAGCGGGCAT AAAATAGACG ACTATGTGCA TTAA 1434
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1239 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1239 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
ATGCTATCTT TTATAAGCGC GTTTGATAAA AGGGGCGTTT CAATACGCCT TCTAACAGCC 60
TTGTTACTGC TTTTTAGTTT GGGTTTGGCT AAAGATTTAG AAATCCAAAC TTTTGTGGCT 120
AAATACCTTT CTAAAAATCA AAAAATACAA GCCCTACAGG AGCAAATTGA CGCTTTAGAT 180 TCTCAAGAAA AAGTCGTTAG CAAATGGGAT AACCCTATTT TGTATTTAGG CTATAACAAC 240
GCTAACGTGA GCGATTTTTT CAGGCTGGAT AGCACCTTAA TGCAAAACAT GAGCTTGGGT 300
TTGTCTCAAA AAGTGGATTT AAATGGTAAA AAACTCACGC AGTCTAAAAT GATCAATTTA 360
GAAAAACAAA AAAAAATATT AGAGCTTAAA AAAACCAAGC AGCAATTGGT GATTAATTTA 420
ATGATAAACG GCATTGAAAA CTATAAAAAC CAACAAGAAA TAGAGCTTTT AAACACAGCG 480 ATTAAAAATT TAGAAAACAC CCTCTATCAA GCCAACCATT CCAGTTCGCC CGATTTAATA 540 GCGATCGCCA AGTTAGAAAT TTTAAAATCG CTATTAGAAA TCCAAAAAAA CGATTTAGAA 600
GTAGCGCTCT CTAGCAGCCA TTATTCCATG GGCGAATTGA CTTTTAAAGA AAACGAGATT 660
TTAAGCATTG CCCCTAAAAA TTTTGAATTC AATAACGAGC AAGAGCTGCA TAACATTAGC 720
GCCACTAATT ACGATATTGC GATCGCCAGG CTTGATGAAG AAAAAGCACA AAAAGACATC 780 ACTCTGGCTA AAAAAAGCTT TTTAGAAGAC ATAAACGTTA CCGGGGTGTA TTATTTCCGC 840
TCCAAACAAT ACTATAACTA CGACATGTTT AGCGTCGCTT TGTCTATCCC TTTACCTCTT 900
TATGGCAAGC AGGCTAAATT AGTGGAGCAA AAGAAAAAAG AAAGCTTGGC GTTTAAAAGC 960
GAAGTGGAAA ACGCCAAAAA CAAAACGCGC CACCTGGCCC TAAAACTCCT TAAAAAATTA 1020
GAAACCTTGC AAAAAAACCT GGAATCGATC AATAAAATCA TCAAACAGAA TGAAAAAATC 1080 GCGCAAATTT ATGCGCTTGA TTTGAAAACT AATGGCGATT ACAACGCTTA TTACAACGCC 1140
TTGAATGACA AAATCACTAT TCAAATCACC CAGCTTGAAA CCTTAAGCGC TCTAAATAGT 1200
GCTTATTTGT CCTTACAAAA TCTCAAAGGA TTAGAATGA 1239
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 414 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...414 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
ATGCGTATAG TTAGAAATTT ATTTCTTGTA TCGTTTGTGG CGTATAGTAG TGCGTTCGCA 60
GCGGATTTAG AAACCGGAAC CAAAAACGAC AAAAAGAGCG GTAAAAAATT TTACAAACTC 120
CATAAAAACC ATGGCTCAGA AACCGAGACT AAAAACGATA AAAAGCTTTA TGATTTCACT 180 AAAAATAGCG GATTAGAAGG CGTGGATTTA GAAAAAAGCC CTAACCTTAA AAGCCATAAA 240
AAAAGCGATA AAAAGTTTTA TAAACAACTC GCTAAAAACA ATATCGCTGA AGGGGTGAGC 300
ATGCCGATTG TGAATTTCAA TAAAGCCCTA TCTTTTGGGC CTTATTTTGA AAGGACTAAA 360
AGCAAAAAAA CCCAATACAT GGACGGCGGG TTGATGATGC ACATCCGTTT TTAA 414 (2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 930 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO ( iv) ANTI - SENSE : NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...930
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
TTGATGCCAC AAAACCAGCT TGTGATCACC ATCATTGATG AATCAGGCTC TAAGCAACTC 60
AAATTTTCTA AAAATTTAAA ACGCAACCTC ATCATTTCTG TTGTCATTCT TTTATTGATC 120 GTGGGGCTTG GCGTGGGGTT TTTAAAATTT TTAATCGCTA AAATGGATAC GATGACAAGC 180
GAGAGGAATG CGGTTTTAAG GGATTTTAGG GGTTTGTATC AAAAAAATTA CGCCCTAGCG 240
AAAGAGATTA AAAACAAGCG AGAAGAGCTT TTTATTGTGG GGCAAAAGAT CCGTGGGCTA 300
GAATCCTTGA TTGAAATCAA AAAGGGGGCT AATGGGGGAG GGCATCTCTA TGATGAAGTG 360
GATTTAGAAA ATTTGAGCTT AAATCAAAAA CATTTAGCAC TCATGCTCAT TCCTAATGGC 420 ATGCCCCTAA AAACTTATAG CGCTATCAAA CCCACTAAAG AAAGGAACCA CCCCATTAAA 480
AAGATTAAGG GCGTTGAATC CGGGATCGAT TTTATCGCGC CATTGAACAC GCCTGTGTAT 540
GCGAGCGCTG ATGGGATTGT GGATTTTGTG AAGACTCGTT CTAATGCGGG GTATGGGAAC 600
TTGGTGCGCA TTGAACATGC GTTTGGTTTC AGCTCCATTT ATACGCACTT AGATCATGTC 660
AATGTGCAGC CTAAAAGCTT CATCCAAAAA GGGCAGTTGA TTGGCTATAG CGGGAAGAGC 720 GGTAATAGCG GCGGCGAAAA ATTGCATTAT GAAGTGCGGT TTTTGGGTAA AATTTTAGAC 780
GCAGAAAAAT TCCTAGCATG GGATTTGGAT CATTTTCAAA GCGCTTTAGA AGAAAATAAA 840
TTTATTGAAT GGAAGAATCT GTTTTGGGTT TTAGAAGACA TCGTCCAGCT CCAAGAGCAT 900
GTGGATAAAG ACACCTTAAA AGGTCAGTAG 930 (2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 999 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...999
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 54 :
GTGCTATATT TTTTAACCAG TTTATTTATT TGCTCTTTGA TTGTTTTGTG GTCTAAAAAA 60
TCCATGCTCT TTGTGGATAA CGCTAATAAA ATCCAAGGCT TCCATCATGC AAGAACCCCA 120 CGAGCCGGGG GGCTTGGGAT CTTTCTTTCT TTTGCGTTGG CTTGTTATCT TGAACCTTTT 180 GAGATGCCTT TTAAGGGGCC TTTTGTTTTC TTAGGGCTAT CGCTAGTGTT TTTGAGCGGT 240
TTTTTAGAAG ACATTAACCT TTCATTAAGC CCCAAAATAC GCCTTATTTT GCAAGCTGTA 300
GGGGTCGTTT GCATCATTTC ATCAACGCCT TTAGTGGTGA GCGATTTTTC GCCCCTTTTT 360
AGCTTGCCTT ATTTCATCGC TTTTTTATTC GCTATTTTTA TGCTGGTGGG TATCAGTAAC 420 GCTATTAATA TCATTGACGG GTTTAACGGG CTTGCATCTG GGATTTGCGC GATCGCGCTT 480
TTAGTCATTC ATTATATAGA CCCTAGCAGT TTGTCTTGTT TGCTCGCTTA CATGGTGCTT 540
GGGTTTATGG TGTTAAATTT CCCTTCAGGA AAGATTTTTT TAGGCGATGG GGGGGCGTAT 600
TTTTTGGGTT TGGTGTGCGG GATTTCTCTC TTGCATTTGA GTTTGGAGCA AAAAATCAGC 660
GTGTTTTTTG GGCTCAATTT AATGCTTTAT CCGGTCATAG AGGTGCTTTT TAGTATCCTT 720 AGGCGCAAAA TAAAACGCCA GAAAGCCACC ATGCCGGATA ATTTGCATTT GCACACCCTT 780
TTATTTAAAT TCTTGCAACA ACGCTCTTTC AATTACCCTA ACCCTTTATG CGCGTTTATC 840
CTTATTCTAT GCAACCTGCC TTTTATTTTA ATAAGCGTTT TGTTTCGCTT GGACGCTTAT 900
GCGCTCATTG TGATTAGCCT AGTCTTTATC GCATGCTATT TAATAGGCTA TGCTTATTTG 960
AATAGGCAAG TTTGCGCTTT AGAAAAGCGG GCGTTTTAA 999
(2) INFORMATION FOR SEQ ID NO: 55:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 816 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: mιsc_feature (B) LOCATION 1...816
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
ATGAACATAT TCAAGCGTAT TATTTGCGTA ACCGCTATTG TTTTAGGTTT TTTTAACCTT 60 TTAGACGCCA AACACCACAA AGAAAAAAAA GAAGACCACA AAATCACTCG TGAGCTTAAA 120
GTGGGCGCTA ACCCTGTGCC GCATGCGCAA ATCTTGCAAT CAGTTGTGGA TGATTTGAAA 180
GAGAAAGGGA TCAAATTAGT GATCGTGTCT TTTACGGATT ATGTGTTGCC TAATTTAGCG 240
CTCAATGACG GCTCTTTAGA CGCGAATTAC TTCCAGCACC GCCCTTATTT GGATCGGTTT 300
AATTTGGACA GAAAAATGCA CCTTGTTGGT TTGGCCAATA TCCATGTGGA GCCTTTAAGA 360 TTTTATTCTC AAAAAATCAC AGACATTAAA AACCTTAAAA AAGGCTCAGT GATTGCTGTG 420
CCAAATGATC CGGCCAATCA AGGCAGGGCG TTGATTTTAC TCCATAAACA AGGCCTTATC 480
GCTCTCAAAG ACCCAAGCAA TCTATACGCT ACGGAGTTTG ATATTGTCAA AAATCCTTAC 540
AACATCAAAA TCAAACCCCT AGAAGCTGCG TTATTGCCTA AGGTTTTAGG GGATGTGGAT 600
GGGGCTATCA TAACAGGGAA TTATGCCTTG CAAGCAAAAC TCACCGGAGC CTTATTTTCA 660 GAAGATAAGG ACTCGCCTTA TGCTAATCTT GTAGCCTCTC GTGAGGATAA TGCGCAAGAT 720
GAAGCGATAA AAGCGTTGAT TGAAGCCTTA CAGAGCGAAA AGACCAGGAA ATTCATTTTG 780
GATACCTATA AGGGGGCGAT TATCCCGGCT TTTTAA 816
(2) INFORMATION FOR SEQ ID NO: 56: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 951 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...951 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
ATGCAAGAAT TCAGTTTGTG GTGCGATTTT ATAGAAAGGG ATTTTTTAGA AAACGATTTT 60
TTAAAGCTCA TCAATAAGGG GGCTATTTGC GGGGCGACGA GTAACCCTAG TTTGTTTTGC 120
GAAGCGATCA CAAAAAGCGC GTTTTATCAA GATGAAATCG CTAAACTCAA AGGCAAAAAA 180 GCTAAAGAAA TTTATGAAAC TCTGGCACTA AAGGATATTT TACAAGCCTC TAGCGCGTTA 240
ATGCCTTTGT ATGAAAAAGA CCCTAACAAC GGCTACATCA GCCTAGAAAT TGACCCCTTT 300
TTAGAAGACG ATGCGATTAA AAGCATTGAT GAAGCCAAGC GGTTATTCAA AACATTAAAC 360
CGCCCCAATG TGATGATTAA AGTCCCGGCG AGTGAAAGCG CTTTTGAAGT CATTAGCGCT 420
CTGGCTCAAG CCTCTATCCC CATTAATGTA ACTTTAGTCT TTTCGCCTAA AATTGCCGGT 480 GAAATCGCTC AAATCTTAGC CAAAGAAGCA CGAAAAAGAG CGGTCATTAG CGTGTTTGTC 540
TCACGATTTG ACAAAGAAAT AGACCCACTA GTGCCACAAA ATTTGCAAGC TCAAAGTGGG 600
ATCATGAACG CTACCGAGTG TTATTATCAA ATCAACCAGC ATGCTAATAA GCTAATAAGC 660
ACCCTTTTTG CATCCACCGG CGTTAAATCT AATTCTTTAG CTAAAGATTA CTACATTAAA 720
GCGCTGTGTT TTAAAAACTC TATCAACACA GCCCCCCTAG ACGCCCTAAA CGCTTATTTG 780 CTTGACCCAA ACACCGAGTG TCAAACCCCT TTAAAAATCA CAGAAATTGA AGCGTTCAAA 840
AAAGAATTAA AAACGCACAA TATTGATTTA GAAAACACCG CCCAAAAACT CCTTAAAGAA 900
GGCTTGATAG CGTTCAAACA ATCCTTTGAA AAGCTTTTAA GCAGTTTTTG A 951
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 783 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori ( ix) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION 1...783 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
ATGAAAACAA ATGGTCATTT TAAGGATTTT GCATGGAAAA AATGCTTTTT AGGCGCGAGC 60
GTGGTGGCTT TATTAGTGGG GTGTAGCCCG CATATTATTG AAACCAATGA AGTTGCTTTG 120
AAATTGAATT ACCATCCAGC TAGCGAGAAA GTTCAAGCGT TAGATGAAAA GATTTTACTT 180 TTAAGGCCAG CTTTCCAATA CAGCGATAAT ATTGCTAAAG AGTATGAAAA CAAATTCAAG 240
AATCAAACCA CGCTTAAAGT TGAAGAGATC TTGCAAAATC AGGGCTATAA GGTTATTAAT 300
GTGGATAGCA GCGATAAAGA CGATTTTTCT TTTGCGCAAA AAAAAGAAGG GTATTTGGCT 360
GTCGCTATGA ATGGCGAAAT TGTTTTACGC CCCGATCCTA AAAGGACCAT ACAGAAAAAA 420
TCAGAACCCG GGTTATTATT CTCCACTGGT TTGGATAAAA TGGAAAGGGT TTTAATCCCG 480 GCTGGGTTTG TCAAGGTTAC CATACTAGAG CCTATGAGTG GGGAATCTTT GGATTCTTTT 540
ACGATGGATT TGAGCGAGTT GGACATCCAA GAAAAATTCT TAAAAACCAC CCATTCAAGC 600
CATAGCGGAG GGTTAGTTAG CACTATGGTT AAGGGGACGG ATAATTCTAA TGACGCAATT 660
AAGAGCGCTT TGAATAAGAT TTTTGCAAGT ATCATGCAAG AAATGGATAA GAAACTCACT 720
CAAAGGAATT TAGAATCTTA TCAAAAAGAC GCCAAGGAAT TAAAAAACAA GAGAAACCGA 780 TAA 783
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4149 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...4149
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: TTGAATTTTA ATAACCTTAC GGCTAATGGG GCGTTAAATT TTAATGGTTA TGCGCCCTCT 60
TTAACTAAGG CTTTAATGAA TGTCAGCGGG CAGTTTGTTT TAGGGAATAA TGGGGATATT 120
AATTTATCTG ACATCAATAT CTTTGACAAC ATCACAAAAT CTGTAACTTA CAACATCTTA 180
AACGCTCAAA AAGGGATTAC TGGCATTAGT GGGGCTAATG GCTATGAAAA AATCCTTTTT 240
TATGGCATGA AAATCCAAAA CGCTACCTAT AGCGATAATA ACAACATCCA AACTTGGTCG 300 TTTATAAACC CTCTCAATTC TTCTCAAATC ATTCAAGAGA GCATTAAAAA TGGGGATCTA 360
ACCATAGAAG TTTTAAATAA CCCTAACTCG GCTTCCAACA CTATTTTTAA TATCGCTCCT 420
GAGCTTTATA ATTACCAAGA TTCTAAGCAA AATCCTACCG GCTATAGCTA TGATTATAGC 480
GACAATCAAG CAGGCACTTA TTACTTGACA AGCAACATTA AAGGTCTTTT CACCCCTAAA 540
GGCTCTCAAA CGCCTCAAAC CCCAGGCACT TATAGCCCAT TTAACCAGCC TTTGAATAGT 600 TTGAATATCT ACAATAAGGG TTTTTCTAGC GAGAATTTAA AAACGCTTTT AGGGATCCTT 660 TCTCAAAATT CCGCCACCTT AAAAGAAATG ATTGAATCCA ACCAACTAGA CAATATCACT 720
AACATTAATG AAGTGTTGCA ACTCTTAGAT AAGATTAAAA TCACCCAAGC GCAAAAGCAA 780
GCGCTCCTAG AAACGATCAA CCATTTGACT GACAACATCA ATCAAACCTT TAATAACGGG 840
AATCTCGTTA TAGGCGCTAC CCAAGATAAT GTTACAAACT CTACTAGCTC TATATGGTTT 900 GGGGGCAATG GCTATAGCAG CCCTTGCGCG CTAGATAGCG CCACTTGTTC TTCTTTTAGA 960
AACACTTACT TGGGGCAATT ATTAGGCTCA ACTTCCCCTT ATTTAGGCTA CATTAACGCT 1020
GATTTTAAAG CTAAAAGCAT TTATATTACC GGGACAATTG GAAGTAGTAA CGCTTTTGAA 1080
AGCGGAGGGA GCGCGGATGT AACCTTTCAA AGCGCTAATA ACTTAGTGTT GAATAAAGCT 1140
AACATAGAAG CTCAAGCCAC AGACAATATC TTTAATCTTT TGGGTCAAGA AGGGATTGAT 1200 AAAATCTTTA ATCAGGGGAA TTTAGCGAAT GTTCTTAGTC AAATGGCTAT GGAAAAAATC 1260
AAGCAAGCCG GCGGTTTAGG GAACTTTATA GAAAACGCTC TAAGCCCTTT GAGTAAGGAA 1320
TTACCCGCTA GCTTGCAAGA TGAAACCTTA GGCCAACTTA TAGGTCAAAA TAACTTAGAT 1380
GATTTATTGA ATAATAGTGG AGTCATGAAT GAAATCCAAA ACATTATCAG TCAAAAACTA 1440
AGCATTTTTG GCAATTTTGT TACCCCATCC ATCATAGAAA ACTACCTTGC TAAGCAGTCT 1500 TTAAAAAGCA TGCTAGACGA TAAAGGGCTT TTGAATTTTA TCGGTGGGTA TATAGACGCT 1560
TCTGAATTAA GCTCTATTTT AGGCGTGATT TTAAAGGATA TTACTAACCC CCCTACAAGC 1620
CTGCAAAAAG ACATTGGTGT GGTAGCGAAC GACTTGTTGA ACGAGTTTTT AGGACAAGAT 1680
GTTGTCAAAA AGCTAGAAAG TCAAGGCTTG GTGAGTAATA TCATCAATAA TGTTATTTCT 1740
CAAGGCGGGT TGAGCGGCGT TTATAATCAA GGTTTAGGGA GCGTGTTGCC GCCCTCTTTA 1800 CAAAACGCGC TCAAAGAAAA CGATTTAGGC ACTCTTTTAT CGCCTAGAGG CTTGCATGAT 1860
TTTTGGCAAA AAGGGTATTT TAACTTTTTA AGCAATGGCT ATGTTTTTGT CAATAACAGC 1920
TCTTTTAGTA ACGCTACTGG GGGTAGTTTG AATTTTGTCG CCAACAAGTC TATTATCTTT 1980
AATGGCGATA ATACGATTGA CTTTAGCAAG TATCAAGGCG CATTGATTTT TGCTTCTAAT 2040
GGTGTTTCTA ATATCAATAT CACCACCCTA AACGCCACTA ATGGCTTAAG CCTTAATGCG 2100 GGTTTGAATA ATGTGAGCGT TCAAAAAGGA GAAATTTGTA TCAATTTAGC CAATTGCCCT 2160
ACAACCAAAA ACAGCTCTCC TGCAAACTCT AGCGTAACCC CCACTAATGA GTCTTTAAGC 2220
GTGCACGCTA ATAATTTCAC TTTCTTAGGC ACAATCATCT CTAATGGGGC TATTGATTTG 2280
TCTCAAGTAA CAAATAATAG CGTTATAGGC ACGCTCAATC TCAATGAAAA TGCGACCTTG 2340
CAAGCTAATA ATTTAACGAT CACCAACGCT TTTAACAACG CCTCTAACTC TACGGCTAAT 2400 ATTGATGGTA ATTTCACCTT AAACCAACAA GCGACTTTAA GCACTAACGC TAGTGGTTTG 2460
AATGTCATGG GGAATTTTAA TAGCTATGGC GATTTGGTGT TTAACCTCAG TCATTCAGTT 2520
AGTCATGCTA TTATCAATAC TCAAGGCACA GCGACGATCA TGGCCAATAA TAACCCTTTG 2580
ATCCAATTCA ACGCTTCTTC AAAAGAAGTG GGTACTTACA CGCTGATTGA TAGCGCTAAA 2640
GCCATTTATT ACGGGTATAA CAACCAAATC ACAGGAGGCA GTAGCCTGGA TAATTACCTT 2700 AAGCTTTATG CGCTCATTGA TATTAATGGC AAGCACATGG TGATGACTGA CAACGGCTTA 2760
ACCTATAACG GGCAAGCCGT GAGCGTTAAA GATGGCGGTT TAGTTGTAGG CTTTAAGGAC 2820
TCTCAAAATC AATACATTTA CACTTCCATT CTTTATAATA AAGTGAAAAT CGCTGTTTCT 2880
AATGATCCTA TCAATAACCC ACAAGCCCCC ACTTTAAAAC AATATATCGC TCAAATTCAG 2940
GGCGTTCAAA GCGTGGATAG CATCGATCAA GCTGGGGGAA ATCAAGCGAT TAATTGGCTC 3000 AATAAAATCT TTGAAACTAA AGGAAGCCCT TTATTCGCTC CCTATTATCT AGAGAGCCAC 3060
TCCACAAAAG ATTTAACCAC GATCGCTGGA GATATTGCTA ACACTTTAGA AGTCATCGCT 3120
AACCCTAATT TTAAAAATGA CGCCACTAAT ATTTTACAGA TCAACACCTA CACGCAGCAA 3180
ATGAGTCGTT TAGCCAAGCT CTCTGACACT TCAACTTTCG CCCGTTCTGA TTTCTTAGAA 3240
CGCTTAGAAG CCCTTAAAAA CAAGCGATTC GCTGATGCGA TCCCTAACGC TATGGATGTG 3300 ATTTTAAAAT ACTCTCAAAG GAATAGAGTT AAAAATAATG TGTGGGCGAC AGGAGTTGGA 3360
GGGGCTAGTT TCATTAGTGG AGGTACTGGA ACTTTATATG GTATCAATGT AGGGTATGAT 3420
AGGTTTATTA AGGGCGTGAT TGTGGGAGGT TATGCCGCTT ATGGGTATAG CGGGTTCCAT 3480
GCAAACATCA CTCAATCAGG CTCTAGCAAT GTCAATGTGG GCGTTTATAG CCGAGCGTTT 3540
ATCAAAAGAA GCGAGCTAAC CATGAGCTTG AATGAGACTT GGGGATACAA TAAAACTTTC 3600 ATCAACTCCT ATGACCCCCT ACTCTCAATC ATCAATCAGT CTTACAGATA CGACACTTGG 3660
ACGACTGACG CTAAAATCAA TTATGGCTAT GATTTCATGT TTAAAGATAA AAGCGTTATT 3720
TTTAAACCCC AAGTAGGCTT AAGCTATTAT TACATTGGTT TGTCTGGTTT AAGGGGCATT 3780
ATGGATGATC CTATTTACAA CCAATTCAGA GCCAATGCTG ACCCTAATAA AAAATCCGTT 3840
CTAACGATCA ATTTTGCCCT AGAAAGTCGG CATTATTTCA ATAAAAACTC TTATTATTTT 3900 GTGATTGCGG ATGTGGGCAG AGACTTATTC ATTAATTCTA TGGGGGATAA AATGGTGCGT 3960 TTCATCGGTA ATAACACCCT AAGCTATAGA GATGGTGGCA GATACAACAC TTTTGCTAGC 4020
ATTATCACAG GCGGGGAGAT AAGATTGTTC AAAACCTTTT ATGTGAATGC GGGCATAGGG 4080
GCTAGGTTTG GGCTTGATTA TAAAGATATT AATATTACCG GAAATATTGG TATGCGCTAT 4140
GCTTTTTAA 4149
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 789 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...789
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
ATGAAAAAAA TTGGTTTGAG CTTGTGTTTG GTTTTGAGTT TGGGTTTTTT AAAAGCCCAT 60 GAAGTGAGCG CTGAAGAGAT TGCGGATATT TTCTACAAAC TCAACGCCAA AGAGCCTAAA 120
ATGAAAATCA ACCACACGAA GGGGTTTTGC GCTAAAGGCG TGTTCCTCCC TAACCCGCAA 180
GCAAGAGAGG ATTTAGAGGT GCCACTACTC AATGAAAAAG AAATCCCTGC GTCTGTAAGG 240
TATTCTTTAG GGGGCGTGGC GATGGACGAT AAAAGCAAGG TTAGGGGAAT GGCGTTAAAA 300
CTAGAAAATC AAAACGCTAG TTGGACAATG GTGATGCTCA ATACAGAAAT CAATTTTGCC 360 AAAAACCCTG AAGAATTCGC CCAATTTTTT GAAATGAGAC TTCCTAAAAA TGGCAAGGTA 420
GATGAAGCAA GAATCAAAAA GCTTTACGAA GAAGTCCCCT CTTATAGGAA TTTTGCCGCC 480
TATATGAAAA CGATAGGGAT TAGCTCAAGC GTGGCTAATA CGCCTTATTA TAGCGTGCAT 540
GCGTTCAAGT TTAAAGATAA GAAAGAAAAA TTATTGCCTG CGAGGTGGAA ATTTGTGCCT 600
AAAGAGGGCG TTAAATACTT AAATCCTCAA GAATTAAAGC AAAAAGATTC AAATTATCTG 660 CTCTCTTCAT TCCAACAACA CCTTAAAAAT AAACCCATAG AATACCAAAT GTATTTGGTG 720
TTTGCGAATC AAAATGATGC CACCAACGAC ACGACCGCGC TTTGGAAAGG CAGCATAAGG 780
AATTATTAG 789
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 741 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO ( iv) ANTI - SENSE : NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...741 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
ATGAAACAAT TTAAAAAGAA ACCAAAAAAG ATAAAACGAT CGCATCAAAA TCAAAAAACA 60
ATCTTAAAGC GTCCTTTATG GCTTATGCCT TTACTGATTG GCGGGTTTGC TAGTGGGGTG 120
TATGCGGATG GAACAGACAT TTTGGGGCTT AGTTGGGGGG AAAAAAGCCA AAAGGTATGC 180 GTGCATCGTC CATGGTATGC TATATGGAGT TGCGATAAAT GGGAGGAAAA AACACAACAA 240
TTTACAGGAA ACCAACTCAT CACAAAAACT TGGGCAGGGG GTAATGCGGC TAACTACTAC 300
CACTCTCAAA ACAACCAAGA CATCACAGCC AATTTAAAAA ATGATAACGG CACTTATTTT 360
TTAAGCGGTC TGTATAACTA CACCGGAGGG GAATATAATG GGGGGAATTT AGACATTGAA 420
TTAGGCAGTA ACGCTACTTT TAATCTAGGT GCGAGTAGTG GGAATAGCTT CACTTCTTGG 480 TATCCTAATG GGCATACTGA TGTTACTTTT AGCGCTGGGA CTATCAATGT GAATAACAGC 540
GTAGAAGTGG GCAATCGTGT GGGATCGGGA GCTGGCACGC ACACCGGCAC AGCCACTTTA 600
AACTTGAACG CTAATAAGGT TACTATCAAT TCCAATATCA GCGCGTATAA AACTTCGCAA 660
GTGAATGTAG GCAATGCTAA CAGCGTTATT ACCATTAATT CGGTTTCTTT AAATGGGGAA 720
TACTTGCAGT TCTTTAGCTA G 741
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 738 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...738
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
ATGATAAAAA AGACCCTTGC ATCGGTTTTA TTAGGATTGA GTTTGATGAG TGTGTTAAAT 60 GCCAAAGAAT GCGTTTCGCC CATAACAAGA AGCGTTAAGT ATCATCAGCA AAGTGCTGAG 120
ATCAGAGCCT TGCAATTACA AAGTTACAAA ATGGCGAAAA TGGCGCTAGA CAATAACCTT 180
AAGCTCGTTA AAGACAAAAA GCCAGCCGTC ATCTTGGATT TAGATGAAAC CGTTTTGAAC 240
ACTTTTGATT ATGCGGGCTA TTTAGTCAAA AACTGCATTA AATACACCCC AGAAACTTGG 300
GATAAATTTG AAAAAGAAGG CTCTCTTACG CTCATTCCTG GAGCGCTAGA CTTTTTAGAA 360 TACGCTAATT CTAAGGGCGT TAAGATTTTT TACATTTCTA ACCGCACCCA AAAAAATAAG 420 GCATTCACTT TAAAAACGCT CAAAAGCTTT AAGCTCCCCC AAGTGAGTGA AGAATCCGTT 480
TTGTTAAAGG AAAAAGGCAA GCCTAAAGCC GTTAGGCGGG AGTTAGTCGC TAAGGATTAT 540
GCGATTGTTT TACAAGTGGG CGACACTTTG CATGATTTTG ACGCCATTTT TGCTAAAGAC 600
GCTAAAAACA GCCAAGAACA ACAAGCCAAA GTCTTGCAAA ACGCTCAAAA ATTCGGCACA 660 GAATGGATCA TTTTACCCAA CTCTCTTTAT GGCACATGGG AAGATGGGCC TATAAAAGCA 720
TGGCAAAATA AAAAATAA 738
(2) INFORMATION FOR SEQ ID NO:62: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 867 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...867
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
TTGTGGTGTT TAAAAACCCC TATCATAGGG CATGGCATGA AGAAAAAAGC AAAAGTCTTT 60
TGGTGTTGTT TTAAAATGAT TCGTTGGTTG TATTTGGCGG TCTTTTTTTT GTTGAGCGTA 120
TCAGACGCTA AAGAAATCGC TATGCAACGA TTTGACAAAC AAAACCATAA GATTTTTGAA 180
ATCCTTGCGG ATAAAGTGAG CGCCAAAGAC AATGTGATAA CCGCCTCAGG GAATGCGATC 240 CTATTGAATT ATGACGTGTA TATTCTAGCG GATAAGGTGC GTTATGACAC CAAGACTAAA 300
GAAGCGTTAT TAGAAGGCAA TATTAAGGTT TATAGGGGCG AGGGCTTGCT CGTTAAAACC 360
GATTATGTGA AATTGAGTTT GAACGAAAAA TATGAGATCA TTTTCCCCTT TTATGTCCAA 420
GACAGCGTGA GCGGGATTTG GGTGAGCGCG GATATTGCTA GCGGGAAGGA TCAAAAATAT 480
AAGATTAAAA ACATGAGCGC TTCAGGGTGC AGCATTGACA ACCCCATTTG GCATGTCAAT 540 GCGACTTCAG GCTCATTTAA CATGCAAAAA TCGCATTTGT CAATGTGGAA TCCTAAGATT 600
TATGTCGGCG ATATTCCTGT ATTGTATTTG CCCTATATTT TCATGTCCAC GAGCAATAAA 660
AGAACTACCG GGTTTTTATA CCCTGAGTTT GGCACTTCCA ACTTAGACGG CTTTATTTAT '720
TTGCAACCCT TTTATTTAGC CCCCAAAAAC TCATGGGATA TGACCTTTAC CCCACAAATC 780
CGTTACAAAA GGGGTTTTGG CTTGAATTTT GAAGCGCGCT ACATCAACTC TAAGACGCAG 840 GTTTTTATTC AATGCGCGCT ATTTTAG 867
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 387 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...387
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: TTGATGTTTA AAAAAATGTG TTTGAGCCTG CTAATGATAA GCGGTGTTTG TGTGGGGGCA 60
AAGGATTTGG ATTTCAAGCT GGATTATCGC GCGACTGGGG GGAAATTCAT GGGGAAAATG 120
ACGGACTCTA GTCTTTTAAG TATCACTTCT ATGAACGATG AACCGGTGGT GATTAAAAAC 180
CTTATTGTCA ATAGGGGAAA TTCATGCGAA GCGACTAAAA AAGTAGAACC CAAATTTGGC 240
GATAAGTTTA AAAAAGAAAA ACTCTTTGAT CATGAATTAA AATACTCGCA ACAGATATTT 300 TACCGCCTGG ATTGCAAGCC TAACCAATTG TTAGAAGTTA AAATCATCAC GGACAAGGGC 360
GAATATTACC ATAAATTTTC CAAATAG 387
(2) INFORMATION FOR SEQ ID NO: 64: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 510 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...510
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
ATGCAAGCGT TAAAATCATT GCTTGAAGTG ATTACAAAAC TCCAGAATCT AGGCGGCTAT 60
TTGATGCATA TAGCTATTTT CATCATTTTT ATTTGGATTG GAGGGCTTAA GTTTGTGCCT 120
TACGAAGCTG AAGGGATCGC CCCTTTTGTG GCCAACTCCC CTTTCTTTTC TTTCATGTAT 180
AAATTTGAAA AACCTGCATA CAAACAACAC AAAATGTCTG AATCCCAATC CATGCAAGAA 240 GAAATGCAAG ATAACCCTAA AATCGTTGAA AACAAAGAAT GGCATAAAGA AAACCGCACT 300
TATTTAGTGG CTGAAGGTTT AGGGATTACG ATCATGATCC TAGGCATTTT GGTGCTTTTG 360
GGGCTTTGGA TGCCTTTAAT GGGCGTAGTT GGGGGCTTGC TTGTCGCTGG AATGACGATC 420
ACCACCCTAT TCTTTTTTAT TCACAACGCC AGAAGTGTTT GTCAATCAGC ATTTCCCATG 480
GCTTTCTGGG GCTGGAAGGC TAGTGGTTAA 510 (2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1464 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...1464
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
ATGATTGAAT GGATGCAAAA TCATAGAAAG TATTTAGTGG TTACGATATG GATAAGCACG 60 ATCGCTTTTA TTGCCGCCGG AATGATAGGT TGGGGGCAAT ACAGCTTTTC TTTAGATAGC 120
GATAGCGCTG CCAAAGTGGG ACAGATTAAG ATTTCTCAAG AAGAATTAGC CCAAGAATAC 180
CGCCGCCTTA AAGACGCCTA TGCTGAGTCT ATCCCTGATT TTAAAGAACT CACCGAAGAT 240
CAAATCAAAG CCATGCATTT AGAAAAAAGC GCGCTAGATT CGCTCATCAA TCAAGCTTTA 300
TTGAGGAATT TCGCTTTAGA TTTAGGGCTT GGTGCTACCA AGCAAGAAGT GGCCAAAGAG 360 ATCAGAAAAA CGAACGTTTT TCAAAAAGAT GGCGTTTTTG ATGAAGAATT GTATAAAAAT 420
ATCTTAAAAC AAAGCCATTA CCGCCCCAAG CATTTTGAAG AAAGCGTTGA AAGGCTTTTA 480
ATCCTTCAAA AAATCAGCGC TCTATTCCCC AAAACCACCA CCCCTTTGGA GCAATCCAGT 540
CTATCGCTTT GGGCAAAATT GCAAGACAAA TTAGACATTC TTATCCTAAA TCCTAATGAT 600
GTTAAAATCT CTCTCAATGA AGAAGAGATG AAAAAATATT ATGAAAACCA TAGAAAGGAT 660 TTTAAAAAGC CCACAAGCTT TAAAACACGC TCTTTATATT TTGACGCTAG TTTAGAAAAA 720
ACTGATTTGA AAGAGTTGGA GGAATACTAC CATAAAAACA AGGTGTCTTA TTTGGACAAA 780
GAGGGGAAAT TACAGGATTT TAAAAGCGTT CAAGAGCAAG TCAAGCATGA TTTAAACATG 840
CAAAAGGCGA ATGAAAAAGC CTTAAGGAGC TATATCGCTC TAAAAAAGGG GAACGCACAA 900
AACTACACCA CGCAAGATTT TGAAAAAAAC AACTCCCCCT ATACTGCTGA AATCACGCAA 960 AAACTCACCG CTCTCAAGCC CCTTGAAGTC CTAAAACCAG AGCCTTTTAA AGATGGTTTT 1020
ATCGTGGTGC AGCTTGTCTC TCAAATTAAA GACGAATTGC AAAATTTTGA TGAAGCCAAA 1080
AGCGCTCTTA AAACCCGTCT GACTCAAGAA AAAACCCTTA TGGCGTTGCA AACTTTAGCT 1140
AAAGAAAAGC TTAAGGATTT TAAAGGGAAA AGCGTGGGTT ATGTAAGCCC TAATTTTGGA 1200
GGCACTATCA GTGAACTTAA CCAAGAAGAG AGCGCGAAGT TTATCAACAC CCTTTTTAAC 1260 CGCCAGGAAA AAAAAGGGTT TGTAACCATA GGTAATAAAG TGGTGCTTTA TCAAATCACA 1320
GAGCAAAATT TCAATCACCC CTTTAGTGCA GAAGAAAACC AATACATGCA GCGTTTAGTC 1380
AATAACACTA AAACGGATTT TTTTGATAAA GCGTTGATAG AAGAATTGAA AAAACGCTAT 1440
AAGATAGTCA AATACATTCA ATAA 1464 (2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 429 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...429
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
ATGAAAACGA ACTTTTATAA AATTAAATTA CTATTTGCTT GGTGTCTTAT CATTGGCATG 60
TTTAACGCTC CGCTTAACGC TGACCAAAAC ACGGATATAA AAGATATTAG TCCTGAAGAT 120 ATGGCGCTAA ATAGCGTGGG GCTTGTTTCT AGAGATCAGC TAAAAATAGA GATCCCTAAA 180
GAAACCCTAG AGCAAAAAGT GGCCATACTC AATGACTATA ATGATAAGAA TGTTAATATC 240
AAGTTTGACG ACATAAGTTT AGGGAGTTTC CAACCTAATG ATAATCTAGG TATCAATGCG 300
ATGTGGGGCA TTCAAAATCT TCTCATGAGC CAAATGATGA GCAATTACGG TCCAAACAAT 360
TCTTTCATGT ATGGCTATGC GCCAACATAC TCAGATTCAT CGTTTTTACC ACCGATCTTA 420 GGGTATTAA 429
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 627 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...627
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: TTGATCAACA ATAATAATAA CAATAAAAAA CTGAGAGGCT TTTTTTTGAA AGTTCTCTTA 60
AGTCTCGTTG TTTTCAGTTC GTATGGGTCA GCAAATGACG ATAAAGAAGC CAAAAAAGAA 120
GCGCTAGAAA AAGAAAAAAA CACTCCCAAT GGGCTTGTTT ATACGAATTT AGATTTTGAT 180
AGTTTTAAAG CGACTATCAA AAATTTGAAA GACAAGAAAG TAACTTTCAA AGAAGTCAAT 240
CCCGATATTA TCAAAGATGA AGTTTTTGAC TTCGTGATTG TCAATAGAGT CCTTAAAAAA 300 ATAAAGGATT TGAAGCATTA CGATCCAGTT ATTGAAAAAA TCTTTGATGA AAAGGGTAAA 360 GAAATGGGAT TGAATGTAGA ATTACAGATC AATCCTGAAG TGAAAGACTT TTTTACTTTC 420
AAAAGCATCA GCACGACCAA CAAACAACGC TGCTTTCTAT CATTGCACGG AGAAACAAGA 480
GAAATTTTAT GCGATGATAA GCTATATAAT GTTTTATTGG CCGTATTCAA TTCTTATGAT 540
CCTAATGATC TTTTGAAACA CATTAGCACC ATAGAGTCTC TCAAAAAAAT CTTTTATACG 600 ATTACATGTG AAGCGGTATA TCTATAA 627
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 738 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...738
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: ATGGCAGGCA CACAAGCTAT ATATGAATCA TCTTCTGCAG GATTCTTATC GCAAGTCTCC 60
TCAATCATCT CAAGCACAAG TGGTGTCGCA GGGCCATTTG CAGGAATAGT AGCGGGCGCT 120
ATGACAGCAG CGATTATTCC TATTGTTGTG GGATTTACTA ATCCGCAAAT GACCGCTATC 180
ATGACCCAAT ACAATCAAAG CATCGCTGAA GCTGTAAGCG TGCCTATGAA AGCCGCTAAC 240
CAACAATACA ACCAATTGTA TCAAGGTTTT AACGATCAAA GCATGGCTGT GGGGAACAAT 300 ATCTTAAATA TCAGCAAATT AACAGGGGAA TTTAACGCGC AAGGCAACAC GCAAAGCGCG 360
CAAATTAGTG CTGTCAATAG TCAGATTGCA AGCATTTTAG CGAGTAACAC TACCCCTAAA 420
AATCCTAGCG CTATTGAAGC TTATGCGACG AATCAAATCG CTGTTCCTAG CGTGCCAACA 480
ACGGTTGAAA TGATGAGCGG TATATTAGGC AATATTACAA GCGCAGCACC AAAATACGCC 540
CTAGCTCTAC AAGAGCAACT GCGTTCTCAA GCAAGCAACA GCTCAATGAA TGATACAGCC 600 GATTCCCTTG ATAGCTGTAC CGCTTTAGGC GCACTTGTTG GCTCATCAAA AGTGTTTTTC 660
AGTTGCATGC AAATTTCTAT GACTCCTATG AGTGTTTCTA TGCCCACTGT TATGCCAAAT 720
ACCAGCGGTT GCCACTAA 738
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1104 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO ( iv) ANTI - SENSE : NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1104 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
ATGATTAAAA GCGTAGAGAT TGAAAATTAC AAAAATTTTG AGCACCTTAA AATGGAAAAT 60
TTTAAACTCA TCAACTTTTT TACCGGTCAA AACGATGCGG GTAAAACCAA TCTTTTAGAA 120
GCTCTTTATA CCAACACAGG CCTTTGTGAT CCTACTGCCA ATCAAGTCAG TCTTCCTCCT 180 GAACATGCCG TGAATATTAG TGAATTCAGA AAAATCAAAC TCGATGCCGA CAACCTAAAA 240
ACCTTTTTTT ATCAAGGAAA CACCGCTAAT CCCATTAGTA TCCGCACTGA ATTTGAACAT 300
GCTACTATCC CTCTTACTAT CCAATACCCC ACACAAACCA GTTACAGCAA AGACATCAAT 360
TTGAATAGCG ATGATGCTCA TATGACAAAC CTTATAAACA CAACAATAAC GAAGCCACAG 420
CTCCAATTTT CCTACAATCC ATCCCTTTCC CCCATGACAA TGACTTATGA ATTTGAAAGG 480 CAAAACCTAG GTTTAATCCA TTCTAATTTA GATAAAATCG CTCAAACCTA TAAAGAAAAT 540
GCGATGTTTA TTCCTATAGA ATTATCTATT GTTAATTCTC TTAAAGCATT GGAAAATTTA 600
CAATTAGCAA GCAAAGAAAA AGAATTGATT GAAATCCTAC AATGTTTCAA CCCTAATATT 660
TTAAATGCTA ATACAATAAG AAAGTCTGTC TATATCCAAA TCAAAGATGA AAACACACCG 720
CTAGAAGAAA GTCCCAAAAG GCTTTTAAAT TTGTTTGGTT GGGGTTTTAT CAAATTCTTT 780 ATTATGGTGA GCATTCTTAT AGACAATCGT GTCAAGTATC TTTTTATTGA TGAAATAGAA 840
AGCGGTTTGC ACCATACAAA AATGCAAGAG TTTTTAAAAG CTCTGTTTAA GTTAGCTCAA 900
AAATTACAGA TTCAAATTTT TGCCACCACG CACAATAAGG AATTTTTATT AAACGCCATC 960
AACACGATAT CCGATAATGA AACGGGAGTT TTTAAAGACA TAGCCTTGTT TGAGCTTGAA 1020
AAAGAAAGCG CTTCTGGCTT TATCAGACAC AGCTATTCTA TGCTAGAAAA AGCGCTTTAT 1080 AGGGGTATGG AGGTTAGAGG CTGA 1104
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1230 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...1230
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: ATGTCCTTGA TTAGAGTGAA TGGGGAAGCT TTTAAACTCT CTTTGGAAAG TTTAGAAGAA 60 GATCCTTTTG AAACTAAAGA AACGCTAGAA ACGCTAGAAA CGCTTATCAA ACAAACGAGC 120 GTTGTTTTAT TGGCCGCTGG GGAGTCTAAG CGTTTTTCTC GTGCGATTAA AAAGCAGTGG 180 CTACGCTCTC ACCACACCCC CTTATGGCTC AGCGTGTATG AAAGCTTTAA AGAAGCCCTA 240 GACTTTAAGG AAGTCATTCT AGTTGTAAGC GAATTGGATT ATGTTTATAT CCAACGCCAT 300 TACCCCAAAA TCAAGCTTGT AAAAGGCGGG GCATCAAGGC AAGAATCCGT GCGTAACGCT 360 TTGAAAGTAA TTGATAGCAC TTACACGATC ACCAGCGATG TGGCTAGGGG TTTAGCGAAT 420 ATGGAAGCGC TTAAAAGCTT GTTTTTAACC CTCCAACAAA CGAGCCATTA TTGCATCGCC 480 CCTTACTTGC CTTGCTATGA CACAGCGATC TATTATAACG AGGCTTTAGA TAGAGAAGCG 540 ATCAAACTCA TTCAAACCCC GCAATTAAGC CACACCAAAA CGCTCCAATC AGCCCTAAAC 600 CAAGGGGGTT TTAAAGATGA AAGCAGCGCG ATTTTACAAG CTTTCCCTAA CTCTGTGAGC 660 TATATTGAAG GCAGTAAGGA TTTGCACAAA CTCACCACAA GCGGCGATTT AAAGTTTTTT 720 ACGCCTTTTT TTAACCCAGC AAAGGACACT TTTATAGGCA TGGGTTTTGA TACGCATGCG 780 TTCATTAAAG ATAAGCCTAT GGTTTTAGGG GGGGTTGTTT TGGATTGCGA GTTTGGGTTA 840 AAGGCTCATA GCGATGGCGA TGCTTTATTG CATGCGGTTA TTGATGCGAT TTTAGGAGCG 900 ATTAAAGGGG GGGATATTGG CGAATGGTTC CCTGATAATG ACCCCAAATA CAAAAACGCC 960
TCTTCTAAAG AGCTTTTAAA AATCGTGTTG GATTTTTCTC AAAGCATTGG GTTTGAATTG 1020
CTTGAAATGG GAGCGACCAT CTTTAGCGAA ATCCCTAAAA TCACTCCTTA CAAACCGGCG 1080
ATTTTAGAGA ATTTGAGCCA ACTTTTGGGT TTAGAAAAAT CTCAAATCAG CTTGAAAGCC 1140
ACTACAATGG AAAAAATGGG GTTCATTGGC AAACAAGAAG GGCTGTTAGT CCAAGCGCAT 1200 GTGAGCATGC GTTATAAACA AAAACTTTAA 1230
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 813 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...813
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: ATGAAAAAGT TTGTAGCTTT AGGGCTTCTA TCCGCGGTTT TAAGCTCTTC GTTGTTAGCC 60
GAAGGTGATG GTGTTTATAT AGGGACTAAT TATCAGCTTG GACAAGCCCG TTTGAATAGC 120
AATATTTATA ATACAGGGGA TTGCACAGGG AGTGTTGTAG GTTGCCCCCC AGGTCTTACC 180
GCTAATAAGC ATAATCCAGG AGGCACCAAT ATCAATTGGC ACTCCAAATA CGCTAATGGG 240
GCTTTGAATG GTTTTGGGTT GAATGTGGGT TATAAGAAAT TCTTCCAATT CAAGTCGCTA 300 GATATGACAA GCAAGTGGTT TGGTTTTAGA GTGTATGGGC TTTTTGATTA CGGGCATGCC 360
GATTTAGGTA AACAAGTTTA TGCACCTAAT AAAATCCAGT TGGATATGGT CTCTTGGGGT 420
GTGGGGAGCG ATTTGTTAGC TGATATTATT GATAAAGACA ACGCTTCTTT TGGTATTTTT 480
GGTGGGGTCG CTATCGGCGG TAACACTTGG AAAAGCTCTG CAGCAAACTA TTGGAAAGAG 540
CAAATCATTG AAGCCAAAGG TCCTGATGTT TGTACCCCTA CTTATTGTAA CCCTAATGCC 600 CCTTATAGCA CCAACACTTC AACCGTCGCT TTTCAAGTGT GGTTGAATTT TGGGGTGAGA 660 GCCAATATCT ACAAGCATAA TGGCGTGGAA TTTGGCGTGA GAGTGCCGCT ACTCATCAAT 720 AAATTTTTGA GCGCGGGTCC TAACGCTACT AACCTTTATT ACCATTTGAA ACGGGATTAT 780 TCGCTTTATT TGGGGTATAA CTACACTTTT TAA 813 (2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1317 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...1317
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
ATGGCTTACA AACCTAACAA AAAGAAGTTA AAAGAATTAA GAGAGCAACC GAATTTATTT 60
AGCATCTTAG ATAAGGGCGA TGTTGCAACA AACAATCCTG TTGAAGAGTC AGACAAGGCC 120 AATAAAATAC AAGAGCCACT CCCTTATGTC GTGAAAACGC AAATCAATAA AGCAAGCATG 180
ATTTCTAGAG ATCCTATTGA ATGGGCAAAG TATTTAAGCT TTGAAAAACG AGTCTATAAG 240
GATAATAGTA AAGAAGATGT CAATTTCTTT GCCAATGGTG AGATAAAAGA AAGTTCTCGT 300
GTTTATGAAG CGAATAAAGA AGGGTTTGAA AGGCGCATCA CTAAAAGATA CGATCTGATT 360
GATAGAAATA TTGATAGAAA TAGAGAATTT TTTATAAAAG AAATTGAAAT TCTAACCCAC 420 ACAAACAGCT TAAAAGAATT GAAAGAGCAA GGGTTAGAAA TCCAATTGAC CCACCATAAT 480
GAAACGCATA AGAAAGCCTT AGAAAATGGC AATGAAATCG TTAAAGAATA CGACCATCTT 540
AAAGATATTT ACCAAGAAGT AGAAAGAACA AAAGATGGTG GATTGGTAAG AGAAATAATC 600
CCCAGTATTT CTAGCGCTGA GTATTTCAAG CTTTACAACA AACTGCCTTT TGAATCAATA 660
AACAATGAAA ATACCAAACT GAATACTAAC GACAATGAAG AAGTTAAAAA ACTAGAATTT 720 GAATTAGCTA AAGAAGTGCA TATTTTAATC CTAGAGCAAC AATTGCTTTC AGCAACAAAT 780
TATTATTCTT GGATAGATAA AGATGATAAT GCGAATTTTG CTTGGAAAAT GCATAGGCTT 840
ATCAATGAAA ATAAACTCAA AGAAAACCAT CTCAGCGCCA ATAACGCTAA TAAGATTAAG 900
CAATTTTTCT TTAATAATGG TTCTATTTTA GGCTGGACTA AAGAAGAACA AAGCGCTATA 960
CAAGAAAACA GAGATTATTC TTTAAGAAGC GCTCTTTTAA GTTTAGAAGA AATCGCTCAA 1020 GCAAAAATTG AATTGCAAAA ATACTATGAA AGCGTTTATG TTAATGGTGA TGGGAATAAA 1080
AGAGAAATCA AGCCTTTTAA AGAAATTTTA AGAGACACCA ACAATTTTGA AAAAGCTTAT 1140
AAGGAGCGTT ATGACAAATT GGTAAGCTTG AGTGCAGCAA TCATTCAAGC TAAAGAGGGT 1200
GGTAATGAGC GACCAAATTC TAGTGCAAAT AACAATAACC CTATTAAAAA TACAATAGAG 1260
ACTAATACTT CTAACAATAT TATTCAAAAT AATGATAATA TAATCATCCA AATTTAA 1317
(2) INFORMATION FOR SEQ ID NO: 73
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 648 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...648
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
ATGCAAGCGT TAAAATCATT GCTTGAAGTG ATTACAAAAC TCCAGAATCT AGGCGGCTAT 60 TTGATGCATA TAGCTATTTT CATCATTTTT ATTTGGATTG GAGGGCTTAA GTTTGTGCCT 120
TACGAAGCTG AAGGGATCGC CCCTTTTGTG GCCAACTCCC CTTTCTTTTC TTTCATGTAT 180
AAATTTGAAA AACCTGCATA CAAACAACAC AAAATGTCTG AATCCCAATC CATGCAAGAA 240
GAAATGCAAG ATAACCCTAA AATCGTTGAA AACAAAGAAT GGCATAAAGA AAACCGCACT 300
TATTTAGTGG CTGAAGGTTT AGGGATTACG ATCATGATCC TAGGCATTTT GGTGCTTTTG 360 GGGCTTTGGA TGCCTTTAAT GGGCGTAGTT GGGGGCTTGC TTGTCGCTGG AATGACGATC 420
ACCACCCTAT CTTTTTTATT CACAACGCCA GAAGTGTTTG TCAATCAGCA TTTCCCATGG 480
CTTTCTGGGG CTGGAAGGCT AGTGGTTAAA GACTTGGCGT TATTTGCTGG AGGCTTGTTT 540
GTGGCCGGAT TTGATGCGAA ACGCTATTTG GAGGGTAAAG GGTTTTGCTT GATGGACCGC 600
TCATCGGTAG GGATTAAAAC TAAATGCTCT AGCGGGTGTT GCTCTTAA 648
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 186 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...186
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
Met lie Lys Arg lie Ala Cys lie Leu Ser Leu Ser Ala Ser Leu Ala 1 5 10 15
Leu Ala Gly Glu Val Asn Gly Phe Phe Met Gly Ala Gly Tyr Gin Gin 20 25 30 Gly Arg Tyr Gly Pro Tyr Asn Ser Asn Tyr Ser Asp Trp Arg His Gly 35 ' 40 45
Asn Asp Leu Tyr Gly Leu Asn Phe Lys Leu Gly Phe Val Gly Phe Ala
50 55 60
Asn Lys Trp Phe Gly Ala Arg Val Tyr Gly Phe Leu Asp Trp Phe Asn 65 70 75 80
Thr Ser Gly Thr Glu His Thr Lys Thr Asn Leu Leu Thr Tyr Gly Gly
85 90 95
Gly Gly Asp Leu lie Val Asn Leu lie Pro Leu Asp Lys Phe Ala Leu 100 105 110 Gly Leu lie Gly Gly Val Gin Leu Ala Gly Asn Thr Trp Met Phe Pro 115 120 125
Tyr Asp Val Asn Gin Thr Arg Phe Gin Phe Leu Trp Asn Leu Gly Gly
130 135 140
Arg Met Arg Val Gly Asp Arg Ser Ala Phe Glu Ala Gly Val Lys Phe 145 150 155 160
Pro Met Val Asn Gin Gly Ser Lys Asp Val Gly Leu lie Arg Tyr Tyr
165 170 175
Ser Trp Tyr Val Asp Tyr Val Phe Thr Phe 180 185
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 116 ammo acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...116
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
Leu Met Arg lie lie lie Arg Leu Leu Ser Phe Lys Met Asn Ala Phe 1 5 10 15
Leu Lys Leu Ala Leu Ala Ser Leu Met Gly Gly Leu Trp Tyr Ala Phe 20 25 30 Asn Gly Glu Gly Ser Glu He Val Ala He Gly He Phe Val Leu He 35 40 45
Leu Phe Val Phe Phe He Arg Pro Val Ser Phe Gin Asp Pro Glu Lys
50 55 60
Arg Glu Glu Tyr He Glu Arg Leu Lys Lys Asn His Glu Arg Lys Met 65 70 75 80
He Leu Gin Asp Lys Gin Lys Glu Glu Gin Met Arg Leu Tyr Gin Ala
85 90 95
Lys Lys Glu Arg Glu Ser Arg Gin Lys Gin Asp Leu Lys Glu Gin Met 100 105 110 Lys Lys Tyr Ser 115 (2) INFORMATION FOR SEQ ID NO:76: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 345 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...345
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
Met Val Lys His Tyr Leu Phe Met Ala Val Ser Gin Val Phe Phe Ser 1 5 10 15 Phe Phe Leu Val Leu Phe Phe He Ser Ser He Val Leu Leu He Ser 20 25 30
He Ala Ser Val Thr Leu Val He Lys Val Ser Phe Leu Asp Leu Val
35 40 45
Gin Leu Phe Leu Tyr Ser Leu Pro Gly Thr He Phe Phe He Leu Pro 50 55 60
He Thr Phe Phe Ala Ala Cys Ala Leu Gly Leu Ser Arg Leu Ser Tyr 65 70 75 80
Asp His Glu Leu Leu Val Phe Phe Ser Leu Gly Val Ser Pro Lys Lys 85 90 95 Met Thr Lys Ala Phe Val Pro Leu Ser Leu Leu Val Ser Ala He Leu 100 105 110
Leu Ala Phe Ser Leu He Leu He Pro Thr Ser Lys Ser Ala Tyr Tyr
115 120 125
Gly Phe Leu Arg Gin Lys Lys Asp Lys He Asp He Asn He Arg Ala 130 135 140
Gly Glu Phe Gly Gin Lys Leu Gly Asp Trp Leu Val Tyr Val Asp Lys
145 150 155 160
Thr Glu Asn Asn Ser Tyr Asp Asn Leu Val Leu Phe Ser Asn Lys Ser
165 170 175 Leu Ser Gin Glu Ser Phe He Leu Ala Gin Lys Gly Asn He Asn Asn
180 185 190
Gin Asn Gly Val Phe Glu Leu Asn Leu Tyr Asn Gly His Ala Tyr Phe
195 200 205
Thr Gin Gly Asp Lys Met Arg Lys Val Asp Phe Glu Glu Leu His Leu 210 215 220
Arg Asn Lys Leu Lys Ser Phe Asn Ser Asn Asp Ala Ala Tyr Leu Gin
225 230 235 240
Gly Thr Asp Tyr Leu Gly Tyr Trp Lys Lys Ala Phe Gly Lys Asn Ala
245 250 255 Asn Lys Asn Gin Lys Arg Arg Phe Ser Gin Ala He Leu Val Ser Leu 260 265 270
Phe Pro Leu Ala Ser Val Phe Leu He Pro Leu Phe Gly He Ala Asn
275 280 285
Pro Arg Phe Lys Thr Asn Trp Ser Tyr Phe Tyr Val Leu Gly Ala Val 290 295 300
Gly Val Tyr Phe Leu Met Val His Val He Ser Thr Asp Leu Phe Leu 305 310 315 320
Met Thr Phe Phe Phe Pro Phe He Trp Ala Phe He Ser Tyr Leu Leu 325 330 335 Phe Arg Lys Phe He Leu Lys Arg Tyr 340 345
(2) INFORMATION FOR SEQ ID NO: 77: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 276 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...276
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
Met Lys Lys Lys Ala Lys Val Phe Trp Cys Cys Phe Lys Met He Arg 1 5 10 15 Trp Leu Tyr Leu Ala Val Phe Phe Leu Leu Ser Val Ser Asp Ala Lys 20 25 30
Glu He Ala Met Gin Arg Phe Asp Lys Gin Asn His Lys He Phe Glu
35 40 45
He Leu Ala Asp Lys Val Ser Ala Lys Asp Asn Val He Thr Ala Ser 50 55 60
Gly Asn Ala He Leu Leu Asn Tyr Asp Val Tyr He Leu Ala Asp Lys 65 70 75 80
Val Arg Tyr Asp Thr Lys Thr Lys Glu Ala Leu Leu Glu Gly Asn He 85 90 95 Lys Val Tyr Arg Gly Glu Gly Leu Leu Val Lys Thr Asp Tyr Val Lys 100 105 110
Leu Ser Leu Asn Glu Lys Tyr Glu He He Phe Pro Phe Tyr Val Gin
115 120 125
Asp Ser Val Ser Gly He Trp Val Ser Ala Asp He Ala Ser Gly Lys 130 135 140
Asp Gin Lys Tyr Lys He Lys Asn Met Ser Ala Ser Gly Cys Ser He
145 150 155 160
Asp Asn Pro He Trp His Val Asn Ala Thr Ser Gly Ser Phe Asn Met
165 170 175 Gin Lys Ser His Leu Ser Met Trp Asn Pro Lys He Tyr Val Gly Asp 180 185 190
He Pro Val Leu Tyr Leu Pro Tyr He Phe Met Ser Thr Ser Asn Lys
195 200 205
Arg Thr Thr Gly Phe Leu Tyr Pro Glu Phe Gly Thr Ser Asn Leu Asp 210 215 220
Gly Phe He Tyr Leu Gin Pro Phe Tyr Leu Ala Pro Lys Asn Ser Trp 225 230 235 240
Asp Met Thr Phe Thr Pro Gin He Arg Tyr Lys Arg Gly Phe Gly Leu 245 250 255 Asn Phe Glu Ala Arg Tyr He Asn Ser Lys Thr Gin Val Phe He Gin 260 265 270
Cys Ala Leu Phe 275 (2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 224 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...224
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: Met He Arg Leu Lys Gly Leu Asn Lys Thr Leu Lys Thr Ser Leu Leu 1 5 10 15
Ala Gly Val Leu Leu Gly Ala Thr Ala Pro Leu Met Ala Lys Pro Leu
20 25 30
Leu Ser Asp Glu Asp Leu Leu Lys Arg Val Lys Leu His Asn He Lys 35 40 45
Glu Asp Thr Leu Thr Ser Cys Asn Ala Lys Val Asp Gly Ser Gin Tyr
50 55 60
Leu Asn Ser Gly Trp Asn Leu Ser Lys Glu Phe Pro Gin Glu Tyr Arg 65 70 75 80 Glu Lys He Phe Glu Cys Val Glu Glu Glu Lys His Lys Gin Ala Leu
85 90 95
Asn Leu He Asn Lys Glu Asp Thr Lys Asp Lys Glu Glu Leu Ala Lys
100 105 110
Lys He Lys Glu He Lys Glu Lys Ala Lys Val Leu Arg Gin Lys Phe 115 120 125
Met Ala Phe Glu Met Lys Glu His Ser Lys Glu Phe Pro Asn Lys Lys
130 135 140
Gin Leu Gin Thr Met Leu Glu Asn Ala Phe Asp Asn Gly Ala Glu Ser 145 150 155 160 Phe He Asp Asp Trp His Glu Arg Phe Gly Gly He Ser Arg Glu Asn 165 170 175
Thr Tyr Lys Ala Leu Gly He Lys Glu Tyr Ser Asp Glu Gly Lys He
180 185 190
Leu Pro Leu Ala Lys Glu Val He Leu Asp Asn He Lys Lys He Leu 195 200 205
Lys Lys Ala Leu Met He Leu Asp Asn Pro Tyr Leu Leu Trp Leu Val 210 215 220
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 429 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...429
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
Met Pro Tyr Ala Leu Arg Lys Arg Phe Phe Lys Arg Leu Leu Leu Phe 1 5 10 15
Phe Leu He Val Cys Met He Asn Leu His Ala Lys Ser Tyr Leu Phe
20 25 30
Ser Pro Leu Pro Pro Ala His Gin Gin He He Lys Thr Glu Pro Cys 35 40 45 Ser Leu Glu Cys Leu Lys Asp Leu Met Leu Gin Asn Gin He Phe Ser 50 55 60
Phe Val Ser Gin Tyr Asp Asp Asn Asn Gin Asp Glu Ser Leu Lys Thr 65 70 75 80
Tyr Tyr Lys Asp He Leu Asn Lys Leu Asn Pro Val Phe He Ala Ser 85 90 95
Gin Thr Pro Ala Lys Glu Ser Tyr Glu Pro Lys He Glu Leu Ala He
100 105 110
Leu Leu Pro Lys Lys Val Val Gly Arg Tyr Ala He Leu Val Met Asn 115 120 125 Thr Leu Leu Ala Tyr Leu Asn Thr Arg Asn Asn Asp Phe Asn He Gin 130 135 140
Val Phe Asp Ser Asp Glu Glu Ser Pro Glu Lys Leu Glu Glu Thr Tyr 145 150 155 160
Lys Glu He Glu Lys Glu Lys Phe Pro Phe He He Ala Leu Leu Thr 165 170 175
Lys Glu Gly Val Glu Asn Leu Leu Gin Asn Thr Thr He Asn Thr Pro
180 185 190
Thr Tyr Val Pro Thr Val Asn Lys Thr Gin Leu Glu Asn His Thr Glu 195 200 205 Leu Ser Leu Ser Glu Arg Leu Tyr Phe Gly Gly He Asp Tyr Lys Glu 210 215 220
Gin Leu Gly Met Leu Ala Thr Phe He Ser Pro Asn Ser Pro Val He 225 230 235 240
Glu Tyr Asp Asp Asp Gly Leu He Gly Glu Arg Leu Arg Gin He Thr 245 250 255
Glu Ser Leu Asn Val Glu Val Lys His Gin Glu Asn He Ser Tyr Lys
260 265 270
Gin Ala Thr Ser Phe Ser Lys Asn Phe Arg Lys His Asp Ala Phe Phe 275 280 285 Lys Asn Ser Thr Leu He Leu Asn Thr Pro Thr Thr Lys Ser Gly Leu 290 295 300
He Leu Ser Gin He Gly Leu Leu Glu Tyr Lys Pro Leu Lys He Leu 305 310 315 320
Ser Thr Gin He Asn Phe Asn Pro Ser Leu Leu Leu Leu Thr Gin Pro 325 330 335
Lys Asp Arg Lys Asn Leu Phe He Val Asn Ala Leu Gin Asn Ser Asp
340 345 350
Glu Thr Leu He Glu Tyr Ala Ser Leu Leu Glu Ser Asp Leu Arg His 355 360 365 Asp Trp Val Asn Tyr Ser Ser Ala He Gly Leu Glu Met Phe Leu Asn 370 375 380
Thr Leu Asp Pro His Phe Lys Lys Ser Phe Gin Glu Ser Leu Glu Asp 385 390 395 400
Asn Gin Val Arg Tyr His Asn Gin He Tyr Gin Ala Leu Gly Tyr Ser 405 410 415
Phe Glu Pro He Lys Asn Glu Ser Glu Thr Lys Lys Glu 420 425
(2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 455 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...455
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
Val Leu Lys Phe Gin Lys Leu Pro Leu Leu Phe Val Ser He Leu Tyr 1 5 10 15
Asn Gin Ser Pro Leu Leu Ala Phe Asp Tyr Lys Phe Ser Gly Val Ala
20 25 30
Glu Ser Val Ser Lys Val Gly Phe Asn His Ser Lys Leu Asn Ser Lys 35 40 45 Glu Gly He Phe Pro Thr Ala Thr Phe Val Thr Ala Thr He Lys Leu 50 55 60
Gin Val Asp Ser Asn Leu Leu Pro Lys Asn He Glu Lys His Ser Leu 65 70 75 80
Lys He Gly Val Gly Gly He Leu Gly Ala Leu Ala Tyr Asp Ser Thr 85 90 95
Lys Thr Leu He Asp Gin Ala Thr His Gin He Tyr Gly Ser Glu Leu
100 105 110
Phe Tyr Leu He Gly Arg Trp Trp Gly Phe Leu Gly Asn Ala Pro Trp 115 120 125 Lys Asp Ser Leu He Glu Ser Asp Ala His Thr Arg Asn Tyr Val Leu 130 135 140
Tyr Asn Ser Tyr Leu Phe Tyr Ser Tyr Gly Asp Lys Phe His Leu Lys 145 150 155 160
Leu Gly Arg Tyr Leu Ser Asn Met Asp Phe Met Ser Ser Tyr Thr Gin 165 170 175
Gly Phe Glu Leu Asp Tyr Lys He Asn Ser Lys He Ala Leu Lys Trp
180 185 190
Phe Ser Ser Phe Gly Arg Ala Leu Ala Phe Gly Gin Trp He Arg Asp 195 200 205 Trp Tyr Ala Pro He Val Thr Glu Asp Gly Arg Lys Glu Val Tyr Asp 210 215 220
Gly He His Ala Ala Gin Leu Tyr Phe Ser Ser Lys His Val Gin Val 225 230 235 240
Met Pro Phe Ala Tyr Phe Ser Pro Lys He Tyr Gly Ala Pro Gly Val 245 250 255
Lys He His He Asp Ser Asn Pro Lys Phe Lys Gly Leu Gly Leu Arg
260 265 270
Ala Gin Thr Thr He Asn Val He Phe Pro Val Tyr Ala Lys Asp Leu 275 280 285 Tyr Asp Val Tyr Trp Arg Asn Ser Lys He Gly Glu Trp Gly Ala Ser 290 295 300
Leu Leu He His Gin Arg Phe Asp Tyr Asn Glu Phe Asn Phe Gly Phe 305 310 315 320
Gly Tyr Tyr Gin Asn Phe Gly Asn Ala Asn Ala Arg He Gly Trp Tyr 325 330 335
Gly Asn Pro He Pro Phe Asn Tyr Arg Asn Asn Ser Val Tyr Gly Gly
340 345 350
Val Phe Ser Asn Ala He Thr Ala Asp Ala Val Ser Gly Tyr Val Phe 355 360 365 Gly Gly Gly Val Tyr Arg Gly Phe Leu Trp Gly He Leu Gly Arg Tyr 370 375 380
Thr Tyr Ala Thr Arg Ala Ser Glu Arg Ser He Asn Leu Asn Leu Gly 385 390 395 400
Tyr Lys Trp Gly Ser Phe Ala Arg Val Asp Val Asn Leu Glu Tyr Tyr 405 410 415
Val Val Ser Met His Asn Gly Tyr Arg Leu Asp Tyr Leu Thr Gly Pro
420 425 430
Phe Asn Lys Ala Phe Lys Ala Asp Ala Gin Asp Arg Ser Asn Leu Met 435 440 445 Val Ser Met Lys Phe Phe Phe 450 455
(2) INFORMATION FOR SEQ ID NO: 81: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 282 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...282
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
Met Gly Cys Ser Phe He Phe Lys Lys Val Arg Val Tyr Ser Lys Met 1 5 10 15 Leu Val Ala Leu Gly Leu Ser Ser Val Leu He Gly Cys Ala Met Asn 20 25 30
Pro Ser Ala Glu Thr Lys Lys Pro Asn Asp Ala Lys Asn Gin Gin Pro
35 40 45
Val Gin Thr His Glu Arg Met Thr Thr Ser Ser Glu His Val Thr Pro 50 55 60
Leu Asp Phe Asn Tyr Pro Val His He Val Gin Ala Pro Gin Asn His 65 70 75 80
His Val Val Gly He Leu Met Pro Arg He Gin Val Ser Asp Asn Leu 85 90 95 Lys Pro Tyr He Asp Lys Phe Gin Asp Ala Leu He Asn Gin He Gin 100 105 110
Thr He Phe Glu Lys Arg Gly Tyr Gin Val Leu Arg Phe Gin Asp Glu
115 120 125
Lys Ala Leu Asn Val Gin Asp Lys Lys Lys He Phe Ser Val Leu Asp 130 135 140
Leu Lys Gly Trp Val Gly He Leu Glu Asp Leu Lys Met Asn Leu Lys
145 150 155 160
Asp Pro Asn Ser Pro Asn Leu Asp Thr Leu Val Asp Gin Ser Ser Gly
165 170 175 Ser Val Trp Phe Asn Phe Tyr Glu Pro Glu Ser Asn Arg Val Val His
180 185 190
Asp Phe Ala Val Glu Val Gly Thr Phe Gin Ala He Thr Tyr Thr Tyr
195 200 205
Thr Ser Thr Asn Asn Ala Ser Gly Gly Phe Asn Ser Ser Lys Ser Val 210 215 220
He His Glu Asn Leu Asp Lys Asn Arg Glu Asp Ala He His Lys He 225 230 235 240
Leu Asn Arg Met Tyr Ala Val Val Met Lys Lys Ala Val Thr Glu Leu 245 250 255 Thr Lys Glu Asn He Ala Lys Tyr Arg Asp Ala He Asp Arg Met Lys 260 265 270
Gly Phe Lys Ser Ser Met Pro Gin Lys Lys 275 280 (2) INFORMATION FOR SEQ ID NO: 82: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 280 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
[iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...280
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: Met Lys Leu Arg Ala Ser Val Leu He Gly Val Ala He Leu Cys Leu 1 5 10 15
He Leu Ser Ala Cys Ser Asn Tyr Ala Lys Lys Val Val Lys Gin Lys
20 25 30
Asn His Val Tyr Thr Pro Val Tyr Asn Glu Leu He Glu Lys Tyr Ser 35 40 45
Glu He Pro Leu Asn Asp Lys Leu Lys Asp Thr Pro Phe Met Val Gin
50 55 60
Val Lys Leu Pro Asn Tyr Lys Asp Tyr Leu Leu Asp Asn Lys Gin Val 65 70 75 80 Val Leu Thr Phe Lys Leu Val His His Ser Lys Lys He Thr Leu He
85 90 95
Gly Asp Ala Asn Lys He Leu Gin Tyr Lys Asn Tyr Phe Gin Ala Asn
100 105 110
Gly Ala Arg Ser Asp He Asp Phe Tyr Leu Gin Pro Thr Leu Asn Gin 115 120 125
Lys Gly Val Val Met He Ala Ser Asn Tyr Asn Asp Asn Pro Asn Asn
130 135 140
Lys Glu Lys Pro Gin Thr Phe Asp Val Leu Gin Gly Ser Gin Pro Met 145 150 155 160 Leu Gly Ala Asn Thr Lys Asn Leu His Gly Tyr Asp Val Ser Gly Ala
165 170 175
Asn Asn Lys Gin Val He Asn Glu Val Ala Arg Glu Lys Ala Gin Leu
180 185 190
Glu Lys He Asn Gin Tyr Tyr Lys Thr Leu Leu Gin Asp Lys Glu Gin 195 200 205
Glu Tyr Thr Thr Arg Lys Asn Asn Gin Arg Glu He Leu Glu Thr Leu
210 215 220
Ser Asn Arg Ala Gly Tyr Gin Met Arg Gin Asn Val He Ser Ser Glu 225 230 235 240 He Phe Lys Asn Gly Asn Leu Asn Met Gin Ala Lys Glu Glu Glu Val
245 250 255
Arg Glu Lys Leu Gin Glu Glu Arg Glu Asn Glu Tyr Leu Arg Asn Gin
260 265 270
He Arg Ser Leu Leu Ser Gly Lys 275 280 (2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 393 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...393 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
Met Arg Lys Leu Phe He Pro Leu Leu Leu Phe Ser Ala Leu Glu Ala 1 5 10 15
Asn Glu Lys Asn Gly Phe Phe He Glu Ala Gly Phe Glu Thr Gly Leu 20 25 30
Leu Glu Gly Thr Gin Thr Gin Glu Lys Arg His Thr Thr Thr Lys Asn
35 40 45
Thr Tyr Ala Thr Tyr Asn Tyr Leu Pro Thr Asp Thr He Leu Lys Arg 50 55 60 Ala Ala Asn Leu Phe Thr Asn Ala Glu Ala He Ser Lys Leu Lys Phe 65 70 75 80
Ser Ser Leu Ser Pro Val Arg Val Leu Tyr Met Tyr Asn Gly Gin Leu
85 90 95
Thr He Glu Asn Phe Leu Pro Tyr Asn Leu Asn Asn Val Lys Leu Ser 100 105 110
Phe Thr Asp Ala Gin Gly Asn Val He Asp Leu Gly Val He Glu Thr
115 120 125
He Pro Lys His Ser Lys He Val Leu Pro Gly Glu Ala Phe Asp Ser
130 135 140 Leu Lys He Asp Pro Tyr Thr Leu Phe Leu Pro Lys He Glu Ala Thr
145 150 155 160
Ser Thr Ser He Ser Asp Ala Asn Thr Gin Arg Val Phe Glu Thr Leu
165 170 175
Asn Lys He Lys Thr Asn Leu Val Val Asn Tyr Arg Asn Glu Asn Lys 180 185 190
Phe Lys Asp His Glu Asn His Trp Glu Ala Phe Thr Pro Gin Thr Ala
195 200 205
Glu Glu Phe Thr Asn Leu Met Leu Asn Met He Ala Val Leu Asp Ser
210 215 220 Gin Ser Trp Gly Asp Ala He Leu Asn Ala Pro Phe Glu Phe Thr Asn
225 230 235 240
Ser Pro Thr Asp Cys Asp Asn Asp Pro Ser Lys Cys Val Asn Pro Gly
245 250 255
Thr Asn Gly Leu Val Asn Ser Lys Val Asp Gin Lys Tyr Val Leu Asn 260 265 270 Lys Gin Asp He Val Asn Lys Phe Lys Asn Lys Ala Asp Leu Asp Val
275 280 285
He Val Leu Lys Asp Ser Gly Val Val Gly Leu Gly Ser Asp He Thr
290 295 300 Pro Ser Asn Asn Asp Asp Gly Lys His Tyr Gly Gin Leu Gly Val Val
305 310 315 320
Ala Ser Ala Leu Asp Pro Lys Lys Leu Phe Gly Asp Asn Leu Lys Thr
325 330 335
He Asn Leu Glu Asp Leu Arg Thr He Leu His Glu Phe Ser His Thr 340 345 350
Lys Gly Tyr Gly His Asn Gly Asn Met Thr Tyr Gin Arg Val Pro Val
355 360 365
Thr Lys Asp Gly Gin Val Glu Lys Asp Ser Asn Gly Lys Pro Lys Asp 370 375 380 Ser Asp Gly Leu Pro Tyr Asn Val Cys 385 390
(2) INFORMATION FOR SEQ ID NO: 84: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 270 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...270
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
Met Lys Lys Phe Val Ala Leu Gly Leu Leu Ser Ala Val Leu Ser Ser 1 5 10 15 Ser Leu Leu Ala Glu Gly Asp Gly Val Tyr He Gly Thr Asn Tyr Gin 20 25 30
Leu Gly Gin Ala Arg Leu Asn Ser Asn He Tyr Asn Thr Gly Asp Cys
35 40 45
Thr Gly Ser Val Val Gly Cys Pro Pro Gly Leu Thr Ala Asn Lys His 50 55 60
Asn Pro Gly Gly Thr Asn He Asn Trp His Ser Lys Tyr Ala Asn Gly 65 70 75 80
Ala Leu Asn Gly Phe Gly Leu Asn Val Gly Tyr Lys Lys Phe Phe Gin 85 90 95 Phe Lys Ser Leu Asp Met Thr Ser Lys Trp Phe Gly Phe Arg Val Tyr 100 105 110
Gly Leu Phe Asp Tyr Gly His Ala Asp Leu Gly Lys Gin Val Tyr Ala
115 120 125
Pro Asn Lys He Gin Leu Asp Met Val Ser Trp Gly Val Gly Ser Asp 130 135 140 Leu Leu Ala Asp He He Asp Lys Asp Asn Ala Ser Phe Gly He Phe
145 150 155 160
Gly Gly Val Ala He Gly Gly Asn Thr Trp Lys Ser Ser Ala Ala Asn
165 170 175 Tyr Trp Lys Glu Gin He He Glu Ala Lys Gly Pro Asp Val Cys Thr
180 185 190
Pro Thr Tyr Cys Asn Pro Asn Ala Pro Tyr Ser Thr Asn Thr Ser Thr
195 200 205
Val Ala Phe Gin Val Trp Leu Asn Phe Gly Val Arg Ala Asn He Tyr 210 215 220
Lys His Asn Gly Val Glu Phe Gly Val Arg Val Pro Leu Leu He Asn 225 230 235 240
Lys Phe Leu Ser Ala Gly Pro Asn Ala Thr Asn Leu Tyr Tyr His Leu 245 250 255 Lys Arg Asp Tyr Ser Leu Tyr Leu Gly Tyr Asn Tyr Thr Phe 260 265 270
(2) INFORMATION FOR SEQ ID NO: 85: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 140 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...140
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:
Met His Pro He Met Phe Ala Tyr He Ala Asn Ala Leu Ala Gin Ala 1 5 10 15 Arg Lys He Asn Gly Thr Leu Cys Met Ala Phe Gin Lys He Ser Gin 20 25 30
Val Lys Glu Leu Gly He Asp Lys Ala Lys Ser Leu He Gly Asn Leu
35 40 45
Ser Gin Val He He Tyr Pro Thr Lys Asp Thr Asp Glu Leu He Glu 50 55 60
Cys Gly Val Pro Leu Ser Asp Ser Glu He Asn Phe Leu His Asn Thr 65 70 75 80
Asp Met Arg Ala Arg Gin Val Leu Val Lys Asn He Val Thr Asn Ala 85 90 95 Ser Ala Phe He Glu He Asp Leu Lys Lys He Cys Lys Asn Tyr Phe 100 105 110
He Phe Leu He Ala Met Leu Val He Glu Lys Ser Ser Met He Leu
115 120 125
Lys Lys Gin Thr Lys Lys Leu He Arg Lys Ser He 130 135 140 (2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 256 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...256 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
Met Leu Gly Ser Val Lys Lys Ala Val Phe Arg Val Leu Cys Leu Gly 1 5 10 15
Ala Leu Cys Leu Cys Gly Gly Leu Met Ala Glu Gin Asp Pro Lys Glu 20 25 30
Leu He Phe Ser Gly He Thr He Tyr Thr Asp Lys Asn Phe Thr Arg
35 40 45
Ala Lys Lys Tyr Phe Glu Lys Ala Cys Lys Ser Asn Asp Ala Asp Gly 50 55 60 Cys Ala He Leu Arg Glu Val Tyr Ser Ser Gly Lys Ala He Ala Arg 65 70 75 80
Glu Asn Ala Arg Glu Ser He Glu Lys Ala Leu Glu His Thr Ala Thr
85 90 95
Ala Lys Val Cys Lys Leu Asn Asp Ala Glu Lys Cys Lys Asp Leu Ala 100 105 110
Glu Phe Tyr Phe Asn Val Asn Asp Leu Lys Asn Ala Leu Glu Tyr Tyr
115 120 125
Ser Lys Ser Cys Lys Leu Asn Asn Val Glu Gly Cys Met Leu Ser Ala
130 135 140 Thr Phe Tyr Asn Asp Met He Lys Gly Leu Lys Lys Asp Lys Lys Asp
145 150 155 160
Leu Glu Tyr Tyr Ser Lys Ala Cys Glu Leu Asn Asn Gly Gly Gly Cys
165 170 175
Ser Lys Leu Gly Gly Asp Tyr Phe Phe Gly Glu Gly Val Thr Lys Asp 180 185 190
Phe Lys Lys Ala Phe Glu Tyr Ser Ala Lys Ala Cys Glu Leu Asn Asp
195 200 205
Ala Lys Gly Cys Tyr Ala Leu Ala Ala Phe Tyr Asn Glu Gly Lys Gly 210 215 220 Val Ala Lys Asp Glu Lys Gin Thr Thr Glu Asn Leu Glu Lys Ser Cys 225 230 235 240
Lys Leu Gly Leu Lys Glu Ala Cys Asp He Leu Lys Glu Gin Lys Gin 245 250 255 (2) INFORMATION FOR SEQ ID NO: 87: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...242
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: Met Lys Lys Phe Phe Ser Gin Ser Leu Leu Ala Leu He He Ser Met 1 5 10 15
Asn Ala Val Ser Gly Met Asp Gly Asn Gly Val Phe Leu Gly Ala Gly
20 25 30
Tyr Leu Gin Gly Gin Ala Gin Met His Ala Asp He Asn Ser Gin Lys 35 40 45
Gin Ala Thr Asn Ala Thr He Lys Gly Phe Asp Ala Leu Leu Gly Tyr
50 55 60
Gin Phe Phe Phe Glu Lys His Phe Gly Leu Arg Leu Tyr Gly Phe Phe 65 70 75 80 Asp Tyr Ala His Ala Asn Ser He Lys Leu Lys Asn Pro Asn Tyr Asn
85 90 95
Ser Glu Ala Ala Gin Val Ala Ser Gin He Leu Gly Lys Gin Glu He
100 105 110
Asn Arg Leu Thr Asn He Ala Asp Pro Arg Thr Phe Glu Pro Asn Met 115 120 125
Leu Thr Tyr Gly Gly Ala Met Asp Val Met Val Asn Val He Asn Asn
1,30 135 140
Gly He Met Ser Leu Gly Ala Phe Gly Gly He Gin Leu Ala Gly Asn 145 150 155 160 Ser Trp Leu Met Ala Thr Pro Ser Phe Glu Gly He Leu Val Glu Gin
165 170 175
Ala Leu Val Ser Lys Lys Ala Thr Ser Phe Gin Phe Leu Phe Asn Val
180 185 190
Gly Ala Arg Leu Arg He Leu Lys His Ser Ser He Glu Ala Gly Val 195 200 205
Lys Phe Pro Met Leu Lys Lys Asn Pro Tyr He Thr Ala Lys Asn Leu
210 215 220
Asp He Gly Phe Arg Arg Val Tyr Ser Trp Tyr Val Asn Tyr Val Phe 225 230 235 240 Thr Phe
(2) INFORMATION FOR SEQ ID NO: 88: (i) SEQUENCE CHARACTERISTICS; (A) LENGTH: 267 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...267
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
Met Asn Tyr Pro Asn Leu Pro Asn Ser Ala Leu Glu He Ser Glu Gin 1 5 10 15 Pro Glu Val Lys Glu He Thr Asn Glu Leu Leu Lys Gin Leu Gin Asn 20 25 30
Ala Leu Arg Ser Asn Ala His Phe Ser Glu Gin Val Glu Leu Ser Leu
35 40 45
Lys Cys He Val Arg He Leu Glu Val Leu Leu Ser Leu Asp Phe Phe 50 55 60
Lys Asn Ala Asn Glu He Asp Ser Ser Leu Arg Asn Ser He Glu Trp 65 70 75 80
Leu Thr Asn Ala Gly Glu Ser Leu Lys Leu Lys Met Lys Glu Tyr Glu 85 90 95 Arg Phe Phe Ser Glu Phe Asn Thr Ser Met His Ala Asn Glu Gin Glu 100 105 110
Val Thr Asn Thr Leu Asn Ala Asn Ala Glu Asn He Lys Ser Glu He
115 120 125
Lys Lys Leu Glu Asn Gin Leu He Glu Thr Thr Thr Arg Leu Leu Thr 130 135 140
Ser Tyr Gin He Phe Leu Asn Gin Ala Arg Asp Asn Ala Asn Asn Gin
145 150 155 160
He Thr Lys Asn Lys Thr Gin Ser Leu Glu Ala He Thr Gin Ala Lys
165 170 175 Asn Asn Ala Asn Asn Glu He Ser Asn Asn Gin Thr Gin Ala He Thr
180 185 190
Asn He Thr Glu Ala Lys Thr Asn Ala Asn Asn Glu He Ser Asn Asn
195 200 205
Gin Thr Gin Ala He Thr Asn He Asn Glu Ala Lys Glu Ser Ala Thr 210 215 220
Thr Gin He Asn Ala Asn Lys Gin Glu Ala He Asn Asn He Thr Gin 225 230 235 240
Glu Lys Thr Gin Ala Thr Ser Glu He Thr Glu Ala Lys Lys Thr Asp 245 250 255 His Tyr Gin Asn He Asp Phe Phe Glu Phe Glu 260 265
(2) INFORMATION FOR SEQ ID NO: 89: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 544 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...544
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
Val He Glu Thr He Pro Lys His Ser Lys He Val Leu Pro Gly Glu 1 5 10 15 Ala Phe Asp Ser Leu Lys Glu Ala Phe Asp Lys He Asp Pro Tyr Thr 20 25 30
Phe Phe Phe Pro Lys Phe Glu Ala Thr Ser Thr Ser He Ser Asp Thr
35 40 45
Asn Thr Gin Arg Val Phe Glu Thr Leu Asn Asn He Lys Thr Asn Leu 50 55 60
He Met Lys Tyr Ser Asn Glu Asn Pro Asn Asn Phe Asn Thr Cys Pro 65 70 75 80
Tyr Asn Asn Asn Gly Asn Thr Lys Asn Asp Cys Trp Gin Asn Phe Thr 85 90 95 Pro Gin Thr Ala Glu Glu Phe Thr Asn Leu Met Leu Asn Met He Ala 100 105 110
Val Leu Asp Ser Gin Ser Trp Gly Asp Ala He Leu Asn Ala Pro Phe
115 120 125
Glu Phe Thr Asn Ser Ser Thr Asp Cys Asp Ser Asp Pro Ser Lys Cys 130 135 140
Val Asn Pro Gly Val Asn Gly Arg Val Asp Thr Lys Val Asp Gin Gin
145 150 155 160
Tyr He Leu Asn Lys Gin Gly He He Asn Asn Phe Arg Lys Lys He
165 170 175 Glu He Asp Ala Val Val Leu Lys Asn Ser Gly Val Val Gly Leu Ala
180 185 190
Asn Gly Tyr Gly Asn Asp Gly Glu Tyr Gly Thr Leu Gly Val Glu Ala
195 200 205
Tyr Ala Leu Asp Pro Lys Lys Leu Phe Gly Asn Asp Leu Lys Thr He 210 215 220
Asn Leu Glu Asp Leu Arg Thr He Leu His Glu Phe Ser His Thr Lys
225 230 235 240
Gly Tyr Gly His Asn Gly Asn Met Thr Tyr Gin Arg Val Pro Val Thr
245 250 255 Lys Asp Gly Gin Val Glu Lys Asp Ser Asn Gly Lys Pro Lys Asp Ser
260 265 270
Asp Gly Leu Pro Tyr Asn Val Cys Ser Leu Tyr Gly Gly Ser Asn Gin
275 280 285
Pro Ala Phe Pro Ser Asn Tyr Pro Asn Ser He Tyr His Asn Cys Ala 290 295 300 Asp Val Pro Ala Gly Phe Leu Gly Val Thr Ala Ala Val Trp Gin Gin
305 310 315 320
Leu He Asn Gin Asn Ala Leu Pro He Asn Tyr Ala Asn Leu Gly Ser
325 330 335 Gin Thr Asn Tyr Asn Leu Asn Ala Ser Leu Asn Thr Gin Asp Leu Ala
340 345 350
Asn Ser Met Leu Ser Thr He Gin Lys Thr Phe Val Thr Ser Ser Val
355 360 365
Thr Asn His His Phe Ser Asn Ala Ser Gin Ser Phe Arg Ser Pro He 370 375 380
Leu Gly Val Asn Ala Lys He Gly Tyr Gin Asn Tyr Phe Asn Asp Phe
385 390 395 400
He Gly Leu Ala Tyr Tyr Gly He He Lys Tyr Asn Tyr Ala Lys Ala
405 410 415 Val Asn Gin Lys Val Gin Gin Leu Ser Tyr Gly Gly Gly He Asp Leu
420 425 430
Leu Leu Asp Phe He Thr Thr Tyr Ser Asn Lys Asn Ser Pro Thr Gly
435 440 445
He Gin Thr Lys Arg Asn Phe Ser Ser Ser Phe Gly He Phe Gly Gly 450 455 460
Leu Arg Gly Leu Tyr Asn Ser Tyr Tyr Val Leu Asn Lys Val Lys Gly
465 470 475 480
Ser Gly Asn Leu Asp Val Ala Thr Gly Leu Asn Tyr Arg Tyr Lys His
485 490 495 Ser Lys Tyr Ser Val Gly He Ser He Pro Leu He Gin Arg Lys Ala
500 505 510
Ser Val Val Ser Ser Gly Gly Asp Tyr Thr Asn Ser Phe Val Phe Asn
515 520 525
Glu Gly Ala Ser His Phe Lys Val Phe Phe Asn Tyr Gly Gly Cys Phe 530 535 540
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 356 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...356 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
Leu Met Lys Ser He Leu Leu Phe Met He Phe Val Val Cys Gin Leu 1 5 10 15
Glu Gly Lys Lys Phe Ser Gin Asp Asn Phe Lys Val Asp Tyr Asn Tyr 20 25 30 Tyr Leu Arg Lys Gin Asp Leu His He He Lys Thr Gin Asn Asp Leu
35 40 45
Ser Asn Ala Trp Tyr Leu Pro Pro Gin Lys Ala Pro Lys Glu His Ser 50 55 60 Trp Val Asp Phe Ala Lys Lys Tyr Leu Asn Met Met Asp Tyr Leu Gly 65 70 75 80
Thr Tyr Phe Leu Pro Phe Tyr His Ser Phe Thr Pro He Phe Gin Trp
85 90 95
Tyr His Pro Asn He Asn Pro Tyr Gin Arg Asn Glu Phe Lys Phe Gin 100 105 110
He Ser Phe Arg Val Pro Val Phe Arg His He Leu Trp Thr Lys Gly
115 120 125
Thr Leu Tyr Leu Ala Tyr Thr Gin Thr Asn Trp Phe Gin He Tyr Asn
130 135 140 Asp Pro Gin Ser Ala Pro Met Arg Met He Asn Phe Met Pro Glu Leu
145 150 155 160
He Tyr Val Tyr Pro He Asn Phe Lys Pro Phe Gly Gly Lys He Gly
165 170 175
Asn Phe Ser Glu He Trp He Gly Trp Gin His He Ser Asn Gly Val 180 185 190
Gly Gly Ala Gin Cys Tyr Gin Pro Phe Asn Lys Glu Gly Asn Pro Glu
195 200 205
Asn Gin Phe Pro Gly Gin Pro Val He Val Lys Asp Tyr Asn Gly Gin
210 215 220 Lys Asp Val Arg Trp Gly Gly Cys Xaa Ser Val Xaa Xaa Gly Asn Xaa
225 230 235 240
Leu Cys Phe Val Leu Val Trp Glu Lys Gly Gly Leu Lys He Met Val
245 250 255
Ala Tyr Trp Pro Tyr Val Pro Tyr Asp Gin Ser Asn Pro Gin Leu He 260 265 270
Asp Tyr Met Gly Tyr Gly Asn Ala Lys He Asp Tyr Arg Arg Gly Arg
275 280 285
His His Phe Glu Leu Gin Leu Tyr Asp He Phe Thr Gin Tyr Trp Arg
290 295 300 Tyr Asp Arg Trp His Gly Ala Phe Arg Leu Gly Tyr Thr Tyr Arg He
305 310 315 320
Asn Pro Phe Val Gly He Tyr Ala Gin Trp Phe Asn Gly Tyr Gly Asp
325 330 335
Gly Leu Tyr Glu Tyr Asp Val Phe Ser Asn Arg He Gly Val Gly He 340 345 350
Arg Leu Asn Pro 355
(2) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 675 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...675
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
Leu Ser Lys Gly Leu Ser He Gly Asn Lys He He Leu Cys Val Ala 1 5 10 15
Leu He Val He Val Cys Val Ser He Leu Gly Val Ser Leu Asn Ser
20 25 30
Arg Val Lys Glu He Leu Lys Glu Ser Ala Leu His Ser Met Gin Asp 35 40 45 Ser Leu His Phe Lys Val Lys Glu Val Gin Ser Val Leu Glu Asn Thr 50 55 60
Tyr Thr Ser Met Gly He Val Lys Glu Met Leu Pro Glu Asp Thr Lys 65 70 75 80
Arg Glu He Lys He Gin Leu Leu Lys Asn Phe He Leu Ala Asn Ser 85 90 95
His Val Ala Gly Val Ser Met Phe Phe Lys Asp Arg Glu Asp Leu Arg
100 105 110
Leu Thr Leu Leu Arg Asp Asn Asp Thr He Lys Leu Met Glu Asn Pro 115 120 125 Ser Leu Gly Ser Asn Pro Leu Ala Gin Lys Ala Met Lys Asn Lys Glu 130 135 140
He Ser Lys Ser Leu Pro Tyr Tyr Arg Lys Met Pro Asn Gly Ala Glu 145 150 155 160
Val Tyr Gly Val Asp He Leu Leu Pro Leu Phe Lys Glu Asn Thr Gin 165 170 175
Glu Val Val Gly Val Leu Met He Phe Phe Ser He Asp Ser Phe Ser
180 185 190
Asn Glu He Thr Lys Asn Arg Ser Asp Leu Phe Leu He Gly Val Lys 195 200 205 Gly Lys Val Leu Leu Ser Ala Asn Lys Ser Leu Gin Asp Lys Ser He 210 215 220
Thr Glu He Tyr Lys Ser Val Pro Lys Ala Thr Asn Glu Val Met Ala 225 230 235 240
He Leu Glu Asn Gly Ser Lys Ala Thr Leu Glu Tyr Leu Asp Pro Phe 245 250 255
Ser His Lys Glu Asn Phe Leu Ala Val Glu Thr Phe Lys Met Leu Gly
260 265 270
Lys Thr Glu Ser Lys Asp Asn Leu Asn Trp Met He Ala Leu He He 275 280 285 Glu Lys Asp Lys Val Tyr Glu Gin Val Gly Ser Val Arg Phe Val Val 290 295 300
Val Ala Ala Ser Ala He Met Val Leu Ala Leu He He Ala He Thr 305 310 315 320
Leu Leu Met Arg Ala He Val Ser Asn Arg Leu Glu Val Val Ser Ser 325 330 335
Thr Leu Ser His Phe Phe Lys Leu Leu Asn Asn Gin Ala His Ser Ser
340 345 350
Asp He Lys Leu Val Glu Ala Arg Ser Asn Asp Glu Leu Gly Arg Met 355 360 365 Gin Thr Ala He Asn Lys Asn He Leu Gin Thr Gin Lys Thr Met Gin 370 375 380
Glu Asp Arg Gin Ala Val Gin Asp Thr He Lys Val Val Ser Asp Val 385 390 395 400
Lys Ala Gly Asn Phe Ala Val Arg He Thr Ala Glu Pro Ala Ser Pro 405 410 415
Asp Leu Lys Glu Leu Arg Asp Ala Leu Asn Gly He Met Asp Tyr Leu
420 425 430
Gin Glu Ser Val Gly Thr His Met Pro Ser He Phe Lys He Phe Glu 435 440 445 Ser Tyr Ser Gly Leu Asp Phe Arg Gly Arg He Gin Asn Ala Ser Gly 450 455 460
Arg Val Glu Leu Val Thr Asn Ala Leu Gly Gin Glu He Gin Lys Met 465 470 475 480
Leu Glu Thr Ser Ser Asn Phe Ala Lys Asp Leu Ala Asn Asp Ser Ala 485 490 495
Asn Leu Lys Glu Cys Val Gin Asn Leu Glu Lys Ala Ser Asn Ser Gin
500 505 510
His Lys Ser Leu Met Glu Thr Ser Lys Thr He Glu Asn He Thr Thr 515 520 525 Ser He Gin Gly Val Ser Ser Gin Ser Glu Ala Met He Glu Gin Gly 530 535 540
Lys Asp He Lys Ser He Val Glu He He Arg Asp He Ala Asp Gin 545 550 555 560
Thr Asn Leu Leu Ala Leu Asn Ala Ala He Glu Ala Ala Arg Ala Gly 565 570 575
Glu His Gly Arg Gly Phe Ala Val Val Ala Asp Glu Val Arg Lys Leu
580 585 590
Ala Glu Arg Thr Gin Lys Ser Leu Ser Glu He Glu Ala Asn He Asn 595 600 605 He Leu Val Gin Ser He Ser Asp Thr Ser Glu Ser He Lys Asn Gin 610 615 620
Val Lys Glu Val Glu Glu He Asn Ala Ser He Glu Ala Leu Arg Ser 625 630 635 640
Val Thr Glu Gly Asn Leu Lys He Ala Ser Asp Ser Leu Glu He Ser 645 650 655
Gin Glu He Asp Lys Val Ser Asn Asp He Leu Glu Asp Val Asn Lys
660 665 670
Lys Gin Phe 675
(2) INFORMATION FOR SEQ ID NO : 92 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...271
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
Met Asn He Phe Lys Arg He He Cys Val Thr Ala He Val Leu Gly 1 5 10 15
Phe Phe Asn Leu Leu Asp Ala Lys His His Lys Glu Lys Lys Glu Asp 20 25 30 His Lys He Thr Arg Glu Leu Lys Val Gly Ala Asn Pro Val Pro His 35 40 45
Ala Gin He Leu Gin Ser Val Val Asp Asp Leu Lys Glu Lys Gly He
50 55 60
Lys Leu Val He Val Ser Phe Thr Asp Tyr Val Leu Pro Asn Leu Ala 65 70 75 80
Leu Asn Asp Gly Ser Leu Asp Ala Asn Tyr Phe Gin His Arg Pro Tyr
85 90 95
Leu Asp Arg Phe Asn Leu Asp Arg Lys Met His Leu Val Gly Leu Ala 100 105 110 Asn He His Val Glu Pro Leu Arg Phe Tyr Ser Gin Lys He Thr Asp 115 120 125
He Lys Asn Leu Lys Lys Gly Ser Val He Ala Val Pro Asn Asp Pro
130 135 140
Ala Asn Gin Gly Arg Ala Leu He Leu Leu His Lys Gin Gly Leu He 145 150 155 160
Ala Leu Lys Asp Pro Ser Asn Leu Tyr Ala Thr Glu Phe Asp He Val
165 170 175
Lys Asn Pro Tyr Asn He Lys He Lys Pro Leu Glu Ala Ala Leu Leu 180 185 190 Pro Lys Val Leu Gly Asp Val Asp Gly Ala He He Thr Gly Asn Tyr 195 200 205
Ala Leu Gin Ala Lys Leu Thr Gly Ala Leu Phe Ser Glu Asp Lys Asp
210 215 220
Ser Pro Tyr Ala Asn Leu Val Ala Ser Arg Glu Asp Asn Ala Gin Asp 225 230 235 240
Glu Ala He Lys Ala Leu He Glu Ala Leu Gin Ser Glu Lys Thr Arg
245 250 255
Lys Phe He Leu Asp Thr Tyr Lys Gly Ala He He Pro Ala Phe 260 265 270
(2) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 161 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...161
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:
Met Phe Phe Lys Thr Tyr Gin Lys Leu Leu Gly Ala Ser Cys Leu Ala 1 5 10 15
Leu Tyr Leu Val Gly Cys Gly Asn Gly Gly Gly Gly Glu Ser Pro Val 20 25 30 Glu Met He Ala Asn Ser Glu Gly Thr Phe Gin He Asp Ser Lys Ala 35 40 45
Asp Ser He Thr He Gin Gly Val Lys Leu Asn Arg Gly Asn Cys Ala
50 55 60
Val Asn Phe Val Pro Val Ser Glu Thr Phe Gin Met Gly Val Leu Ser 65 70 75 80
Gin Val Thr Pro He Ser He Gin Asp Phe Lys Asp Met Ala Ser Thr
85 90 95
Tyr Lys He Phe Asp Gin Lys Lys Gly Leu Ala Asn He Ala Asn Lys 100 105 110 He Ser Gin Leu Glu Gin Lys Gly Val Met Met Glu Pro Gin Thr Leu 115 120 125
Asn Phe Gly Glu Ser Leu Lys Gly He Ser Gin Gly Cys Asn He He
130 135 140
Glu Ala Glu He Gin Thr Asp Lys Gly Ala Trp Thr Phe Asn Phe Asp 145 150 155 160
Lys
(2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 337 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1.-..337
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
Met He Arg Leu Lys Gly Leu Asn Lys Thr Leu Lys Thr Ser Leu Leu 1 5 10 15
Ala Gly Val Leu Leu Gly Ala Thr Ala Pro Leu Met Ala Lys Pro Leu
20 25 30
Leu Ser Asp Glu Asp Leu Leu Lys Arg Val Lys Leu His Asn He Lys 35 40 45 Glu Asp Thr Leu Thr Ser Cys Asn Ala Lys Val Asp Gly Ser Gin Tyr 50 55 60
Leu Asn Ser Gly Trp Asn Leu Ser Lys Glu Phe Pro Gin Glu Tyr Arg 65 70 75 80
Glu Lys He Phe Glu Cys Val Glu Glu Glu Lys His Lys Gin Ala Leu 85 90 95
Asn Leu He Asn Lys Glu Asp Thr Glu Asp Lys Glu Glu Leu Ala Lys
100 105 110
Lys He Lys Glu He Lys Glu Lys Ala Lys Val Leu Arg Gin Lys Phe 115 120 125 Met Ala Phe Glu Met Lys Glu His Ser Lys Glu Phe Pro Asn Lys Lys 130 135 140
Gin Leu Gin Thr Met Leu Glu Asn Ala Phe Asp Asn Gly Ala Glu Ser 145 150 155 160
Phe He Asp Asp Trp His Glu Arg Phe Gly Gly He Ser Arg Glu Asn 165 170 175
Thr Tyr Lys Ala Leu Gly He Lys Glu Tyr Ser Asp Glu Gly Lys He
180 185 190
Leu Ala Phe Gly Glu Arg Ser Tyr He Arg Gin Tyr Lys Lys Asp Phe 195 200 205 Glu Glu Ser Thr Tyr Asp Thr Arg Gin Thr Leu Ser Ala Met Ala Asn 210 215 220
Met Ser Gly Glu Asn Asp Tyr Lys He Thr Trp Leu Lys Pro Lys Tyr 225 230 235 240
Gin Leu His Ser Ser Asn Asn He Lys Pro Leu Met Ser Asn Thr Glu 245 250 255
Leu Leu Asn Met He Glu Leu Thr Asn He Lys Lys Glu Tyr Val Met
260 265 270
Gly Cys Asn Met Glu He Asp Gly Ser Lys Tyr Pro He His Lys Asp 275 280 285 Trp Gly Phe Phe Gly Lys Ala Lys Val Pro Glu Thr Trp Arg Asn Lys 290 295 300
He Trp Glu Cys He Lys Asn Lys Val Lys Ser Tyr Asp Asn Thr Thr 305 310 315 320
Ala Glu He Gly He Val Trp Lys Lys Asn Thr Tyr Ser He Ser His 325 330 335
His
(2) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 416 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...416 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
Met Lys Lys Leu Val Phe Ser Met Leu Leu Cys Cys Lys Ser Val Phe 1 5 10 15
Ala Glu Gly Glu Thr Pro Leu He Val Asn Asp Pro Glu Thr His Val
20 25 30
Ser Gin Ala Thr He He Gly Lys Met Val Asp Ser He Lys Arg Tyr 35 40 45 Glu Glu He He Ser Lys Ala Gin Ala Gin Val Asn Gin Leu Gin Lys 50 55 60
Val Asn Asn Met He Asn Thr Thr Asn Ser Leu He Ser Ser Ser Ala 65 70 75 80
He Thr Leu Ala Asn Pro Met Gin Val Leu Gin Asn Ala Gin Tyr Gin 85 90 95
He Glu Ser He Arg Tyr Asn Tyr Glu Asn Leu Lys Gin Ser He Glu
100 105 110
Asn Trp Asn Ala Gin Asn Leu Leu Arg Asn Lys Tyr Leu Gin Gin Gin 115 120 125 Cys Pro Trp Leu Asn Val Asn Ala Leu Thr Asn Asn Lys He Val Asn 130 135 140
Leu Lys Asp Leu Asn Asn Leu He Thr Lys Asn Gly Glu Gin Thr Gin 145 150 155 160
Thr Ala Arg Asp Val Gin Asn Leu He Gin Ser He Ser Gly Ser Gly 165 170 175
Tyr Gly Asn Met Gin Ser Leu Ala Gly Glu Leu Ser Gly Arg Ala Trp
180 185 190
Gly Glu Met Leu Cys Lys Met Val Asn Asp Ser Asn Tyr Glu Ser Glu 195 200 205 Gin Ala Leu Leu Ala Thr Gly Asn Asn Pro Glu Glu Gin Lys Arg Arg 210 215 220
Phe Leu Leu Arg Val Lys Lys Lys Val Asn Asp Asn Lys Gin Leu Lys 225 230 235 240
Asp Lys Leu Asp Pro Phe Leu Lys Arg Leu Asp Val Leu Gin Thr Glu 245 250 255
Phe Gly Val Thr Asp Pro Thr Ala Asn His Asn Lys Gin Gly He His
260 265 270
Tyr Cys Thr Glu Asn Lys Glu Thr Gly Lys Cys Asp Pro He Lys Asn 275 280 285 Val Phe Arg Thr Thr Arg Leu Asp Asn Glu Leu Glu Gin Glu He Gin 290 295 300
Thr Leu Thr Leu Asp Leu He Lys Ala Ser Asn Lys Asp Ala Gin Ser 305 310 315 320
Gin Ala Tyr Ala Asn Phe Asn Gin Arg He Lys Leu Leu Thr Leu Lys 325 330 335
Tyr Leu Lys Glu He Thr Asn Gin Met Leu Phe Leu Asn Gin Thr Met
340 345 350
Ala Met Gin Ser Glu He Met Thr Asp Asp Tyr Phe Arg Gin Asn Asn 355 360 365 Asp Gly Phe Gly Glu Lys Glu Asn His He Asp Lys Gin Leu Thr Gin 370 375 380
Lys Arg He Asn Glu Arg Glu Arg Ala Arg He Tyr Phe Gin Asn Pro 385 390 395 400
Asn Val Lys Phe Asp Gin Phe Gly Phe Pro He Phe Ser He Trp Asp 405 410 415 (2) INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 376 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...376 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
Val Asn Lys Trp He Lys Gly Ala Val Val Phe Val Gly Gly Phe Ala 1 5 10 15
Thr He Thr Thr Phe Ser Leu He Tyr His Gin Lys Pro Lys Ala Pro 20 25 30
Leu Asn Asn Gin Pro Ser Leu Leu Asn Asp Asp Glu Val Lys Tyr Pro
35 40 45
Leu Gin Asp Tyr Thr Phe Thr Gin Asn Pro Gin Pro Thr Asn Thr Glu 50 55 60 Ser Ser Lys Asp Ala Thr He Lys Ala Leu Gin Glu Gin Leu Lys Ala 65 70 75 80
Ala Leu Lys Ala Leu Asn Ser Lys Glu Met Asn Tyr Ser Lys Glu Glu
85 90 95
Thr Phe Thr Ser Pro Pro Met Asp Pro Lys Thr Thr Pro Pro Lys Lys 100 105 110
Asp Phe Ser Pro Lys Gin Leu Asp Leu Leu Ala Ser Arg He Thr Pro
115 120 125
Phe Lys Gin Ser Pro Lys Asn Tyr Glu Glu Asn Leu He Phe Pro Val
130 135 140 Asp Asn Pro Asn Gly He Asp Ser Phe Thr Asn Leu Lys Glu Lys Asp
145 150 155 160
He Ala Thr Asn Glu Asn Lys Leu Leu Arg Thr He Thr Ala Asp Lys
165 170 175
Met He Pro Ala Phe Leu He Thr Pro He Ser Ser Gin He Ala Gly 180 185 190
Lys Val He Ala Gin Val Glu Ser Asp He Phe Ala Ser Met Gly Lys
195 200 205
Ala Val Leu He Pro Lys Gly Ser Lys Val He Gly Tyr Tyr Ser Asn
210 215 220 Asn Asn Lys Met Gly Glu Tyr Arg Leu Asp He Val Trp Ser Arg He
225 230 235 240
He Thr Pro His Gly He Asn He Met Leu Thr Asn Ala Lys Gly Ala
245 250 255
Asp He Lys Gly Tyr Asn Gly Leu Val Gly Glu Leu He Glu Arg Asn 260 265 270 Phe Gin Arg Tyr Gly Val Pro Leu Leu Leu Ser Thr Leu Thr Asn Gly
275 280 285
Leu Leu He Gly He Thr Ser Ala Leu Asn Asn Arg Gly Asn Lys Glu
290 295 300 Glu Val Thr Asn Phe Phe Gly Asp Tyr Leu Leu Leu Gin Leu Met Arg
305 310 315 320
Gin Ser Gly Met Gly He Asn Gin Val Val Asn Gin He Leu Arg Asp
325 330 335
Lys Ser Lys He Ala Pro He Val Val He Arg Glu Gly Ser Arg Val 340 345 350
Phe He Ser Pro Asn Thr Asp He Phe Phe Pro He Pro Arg Glu Asn
355 360 365
Glu Val He Ala Glu Phe Leu Lys 370 375
(2) INFORMATION FOR SEQ ID NO: 97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 916 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...916
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:
Val Asp Leu Arg He Gin Ser Lys Glu Val Ser His Asn Leu Lys Glu 1 5 10 15
Leu Ser Lys Thr Leu He Ser Tyr Pro Phe Glu Lys His Val Glu Ala 20 25 30 Leu Gly Glu Gin Cys Ser Asn Phe Val Ser He Pro He Asn Asn Asp 35 40 45
Asp Tyr Ser Asn He Cys Thr Phe Val Ser Asp Phe He Asn Leu He
50 55 60
Ala Ser Tyr Asn Leu Leu Glu Ser Phe Leu Asp Phe Tyr Lys Asp Lys 65 70 75 80
Leu Lys Leu Ser Glu Leu Val Thr Glu Tyr Ala Asn Val Thr Asn Asn
85 90 95
Leu Leu Phe Lys Lys Leu He Lys His Leu Ser Gly Asn Asn Gin Leu 100 105 110 Val Lys Asn Phe Tyr Gin Cys He Arg Glu He He Lys Tyr Asn Ala 115 120 125
Pro Asn Lys Glu Tyr Lys Pro Asn Gin Phe Phe He He Gly Lys Gly
130 135 140
Lys Gin Lys Gin Leu Ala Lys He Tyr Ser His Leu Lys Glu Leu Ser 145 150 155 160 Ala Ser Glu He Lys Pro Gin Asp Met Glu Asp He Leu Lys Lys Leu
165 170 175
Glu Glu Leu Asp Lys He Phe Lys Thr Thr Asp Phe Thr Lys Phe Thr 180 185 190 Pro Lys Thr Glu He Lys Asp He He Lys Glu He Asp Glu Lys Tyr 195 200 205
Pro He Asn Glu Asn Phe Lys Arg Gin Phe Asn Glu Phe Glu Ser Asn
210 215 220
He Glu Lys His Asp Glu He Lys Lys Asp Phe Glu Arg Asn Lys Glu 225 230 235 240
Ser Leu He Arg Glu He Glu Asn His Cys Lys Asn Glu Cys Asn Ser
245 250 255
Glu Glu Glu Pro Glu Tyr Lys He Asn Asp Leu Leu Lys Asn He Gin 260 265 270 Gin He Cys Lys Asn Tyr He Glu Ser His Ala Val Asn Asp Val Ser 275 280 285
Lys Asp He Lys Ser Met Met Cys Gin Phe Tyr Leu Lys Gin He Asp
290 295 300
Leu Leu Val Asn Ser Glu He Val Arg Tyr Arg Tyr Ser Asn Leu Phe 305 310 315 320
Glu Pro He Gin Arg Ser Leu Trp Glu Ser He Lys He Leu Asp Asn
325 330 335
Glu Ser Gly He Tyr Leu Phe Pro Lys Asn He Gly Glu He Lys Asp 340 345 350 Lys Phe Glu Ala Asn Lys Glu Lys Phe Lys Gin Ser Lys Asn Val Ser 355 360 365
Glu Phe Ala Glu Tyr Cys Arg Glu Cys Asn Pro Tyr Thr Ala Phe Asn
370 375 380
Phe His Leu Asn He Asn Asn Gly Leu Ser His Gin Phe Glu Lys Phe 385 390 395 400
Val Pro He Met Lys Glu Tyr Lys Glu Pro Lys He Thr Asp Asn Asp
405 410 415
Leu Glu Ala He Ser Thr Lys Glu Thr Gly Leu Ala Ser Gin Leu Ser 420 425 430 Gly His Trp Phe Phe Gin Leu Ser Leu Phe Asn Lys Thr Asn Phe Asn 435 440 445
Pro Asn Lys He Trp He Pro Leu Glu Phe Asn Lys Arg Ser Lys He
450 455 460
Lys Phe Asp Lys Asp Leu Glu He Tyr Phe Asp Ser His Glu Ser Phe 465 470 475 480
Asn He Ser Lys Lys Tyr Leu Gin Glu He Asp Gin Glu Ser Leu Lys
485 490 495
Lys He Lys Gin Ser Lys Asp Phe Phe Ser He Gin Lys He Glu Ser 500 505 510 Lys His Asp Asn Asn Asp He Leu Gin Leu Glu Phe Phe Glu Asn Asp 515 520 525
Thr Ser Phe Leu Phe Ala Lys Gly Ser Phe Ala Glu He Leu Glu Tyr
530 535 540
Asn Met Gin Leu Lys He Asp Ser Leu He Thr Lys Glu Phe Asn Lys 545 550 555 560
Leu Leu Ala He Val Gin Asp Ser Pro Gin Asp Ser Tyr Gin Leu Lys
565 570 575
He Arg Val Arg His Asn Asn Lys Leu Pro Arg Glu Lys Tyr Thr Glu 580 585 590 His Glu He Lys Leu Glu Val Tyr Asp Cys Arg Lys Ser His Asp His 595 600 605
Asn Glu Pro He He Leu Ser Gin Gin Ser Thr Gly Phe Gin Trp Ala
610 615 620
Phe Asn Phe Met Phe Gly Phe Leu Tyr Asn Val Gly Ser His Phe Ser 625 630 635 640
Phe Asn His Asn He He Tyr Val Met Asp Glu Pro Ala Thr His Leu
645 650 655
Ser Val Pro Ala Arg Lys Glu Phe Arg Lys Phe Leu Lys Glu Tyr Ala 660 665 670 His Lys Asn His Val Thr Phe Val Leu Ala Thr His Asp Pro Phe Leu 675 680 685
Val Asp Thr Asp His Leu Asp Glu He Arg He Val Glu Lys Glu Thr
690 695 700
Glu Gly Ser Val He Lys Asn His Phe Asn Tyr Pro Leu Asn Asn Ala 705 710 715 720
Ser Lys Asp Ser Asp Ala Leu Asp Lys He Lys Arg Ser Leu Gly Val
725 730 735
Gly Gin His Val Phe His Asn Pro Gin Lys His Arg He He Phe Val 740 745 750 Glu Gly He Thr Asp Tyr Cys Tyr Leu Ser Ala Phe Lys Leu Tyr Leu 755 760 765
Arg Tyr Lys Glu Tyr Lys Asp Asn Pro He Pro Phe Thr Phe Leu Pro
770 775 780
He Ser Gly Leu Lys Asn Asp Ser Asn Asp Met Lys Glu Thr He Glu 785 790 795 800
Lys Leu Cys Glu Leu Asp Asn His Pro He Val Leu Thr Asp Asp Asp
805 810 815
Arg Lys Cys Val Phe Asn Gin Gin Ala Thr Ser Glu Arg Phe Lys Arg 820 825 830 Ala Asn Glu Glu Met His Asp Pro He Thr He Leu Gin Leu Ser Asp 835 840 845
Cys Asp Arg His Phe Lys Gin He Glu Asp Cys Phe Ser Ala Asn Asp
850 855 860
Arg Asn Lys Tyr Ala Lys Asn Lys Gin Met Glu Leu Ser Met Ala Phe 865 870 875 880
Lys Thr Arg Leu Leu Tyr Gly Gly Glu Asp Ala He Glu Lys Gin Thr
885 890 895
Lys Arg Asn Phe Leu Lys Leu Phe Lys Trp He Ala Trp Ala Thr Asn 900 905 910 Leu He Lys Asn 915
(2) INFORMATION FOR SEQ ID NO: 98: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 176 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori ( ix) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION 1...176
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:
Met Thr Ala Met Met Arg Tyr Phe His He Tyr Ala Thr Thr Phe Phe 1 5 10 15 Phe Pro Leu Ala Leu Leu Phe Ala Val Ser Gly Leu Ser Leu Leu Phe 20 25 30
Lys Ala Arg Gin Asp Thr Gly Ala Lys He Lys Glu Trp Val Leu Glu
35 40 45
Lys Ser Leu Lys Lys Glu Glu Arg Leu Asp Phe Leu Lys Gly Phe He 50 55 60
Lys Glu Asn His He Ala Met Pro Lys Lys He Glu Pro Arg Glu Tyr 65 70 75 80
Arg Gly Ala Leu Val He Gly Thr Pro Leu Tyr Glu He Asn Leu Glu 85 90 95 Thr Lys Gly Thr Gin Thr Lys He Lys Thr He Glu Arg Gly Phe Leu 100 105 110
Gly Ala Leu He Met Leu His Lys Ala Lys Val Gly He Val Phe Gin
115 120 125
Ala Leu Leu Gly He Phe Cys Val Phe Leu Leu Leu Phe Tyr Leu Ser 130 135 140
Ala Phe Leu Met Val Ala Phe Lys Asp Thr Lys Arg Met Phe He Ser 145 150 155 160
Val Leu He Gly Ser Val Val Phe Phe Gly Ala He Tyr Trp Ser Leu 165 170 175
(2) INFORMATION FOR SEQ ID NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 222 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...222
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
Met Phe Lys Asn Ala Leu Asn He Gin Asp Phe Ser Phe Lys Asn His 1 5 10 15
Thr Ser Thr Ala He He Gly Thr Asn Gly Ala Gly Lys Ser Thr Leu
20 25 30 He Asn Thr He Leu Gly He Arg Ser Asp Tyr Asn Phe Lys Ala Gin 35 40 45
Asn Asn Asn He Pro Tyr His Asp Asn Val He Pro Gin Arg Lys Gin
50 55 60
Leu Gly Val Val Ser Asn Leu Phe Asn Tyr Pro Pro Gly Leu Asn Ala 65 70 75 80
Asn Asp Leu Phe Lys Phe Tyr Gin Phe Phe His Lys Asn Cys Thr Leu
85 90 95
Asp Leu Phe Glu Lys Asn Leu Leu Asn Lys Thr Tyr Glu His Leu Ser 100 105 110 Asp Gly Gin Lys Gin Arg Leu Lys He Asp Leu Ala Leu Ser His His 115 120 125
Pro Gin Leu Val He Met Asp Glu Pro Glu Thr Ser Leu Glu Gin Asn
130 135 140
Ala Leu He Arg Leu Ser Asn Leu He Ser Leu Arg Asn Thr Gin Gin 145 150 155 160
Leu Thr Ser He He Ala Thr His Asp Pro He Val Leu Asp Ser Cys
165 170 175
Glu Trp Val Leu Leu Leu Lys Asn Gly Asn He Ala Gin Tyr Lys Pro 180 185 190 Leu Asn Ser He Leu Lys Ser Val Ala Lys Thr Phe Asn Phe Lys Glu 195 200 205
Lys Pro Thr Thr Lys Asp Leu Leu Ala Leu Leu Lys Asp He 210 215 220 (2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 406 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...406
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: Met Tyr Ala Ala His Pro He Lys Pro He Lys Ala Pro Lys Leu Lys 1 5 10 15
Ser Gin Phe Leu Arg Arg Val Phe Val Gly Ala Ser He Arg Arg Trp
20 25 30
Asn Asp Gin Ala Cys Pro Leu Glu Phe Val Glu Leu Asp Lys Gin Ala 35 40 45
His Lys Ala Met He Ala Tyr Leu Leu Ala Lys Asp Leu Lys Asp Arg
50 55 60
Gly Lys Asp Leu Asp Leu Asp Leu Leu He Lys Tyr Phe Cys Phe Glu 65 70 75 80 Phe Leu Glu Arg Leu Val Leu Thr Asp He Lys Pro Pro He Phe Tyr 85 90 95
Ala Leu Gin Gin Thr His Ser Lys Glu Leu Ala Ser Tyr Val Ala Gin
100 105 110
Ser Leu Gin Asp Glu He Ser Ala Tyr Phe Ser Leu Glu Glu Leu Lys 115 120 125
Glu Tyr Leu Ser His Arg Pro Gin He Leu Glu Thr Gin He Leu Glu
130 135 140
Ser Ala His Phe Tyr Ala Ser Lys Trp Glu Phe Asp He He Tyr His 145 150 155 160 Phe Asn Pro Asn Met Tyr Gly Val Lys Glu He Lys Asp Lys He Asp
165 170 175
Lys Gin Leu His Asn Asn Asp His Leu Phe Glu Gly Leu Phe Gly Glu
180 185 190
Lys Glu Asp Leu Lys Lys Leu Val Ser Met Phe Gly Gin Leu Arg Phe 195 200 205
Gin Lys Arg Trp Ser Gin Thr Pro Arg Val Pro Gin Thr Ser Val Leu
210 215 220
Gly His Thr Leu Cys Val Ala He Met Gly Tyr Leu Leu Ser Phe Asp 225 230 235 240 Leu Lys Ala Cys Lys Ser Met Arg He Asn His Phe Leu Gly Gly Leu
245 250 255
Phe His Asp Leu Pro Glu He Leu Thr Arg Asp He He Thr Pro He
260 265 270
Lys Gin Ser Val Ala Gly Leu Asp His Cys He Lys Glu He Glu Lys 275 280 285
Lys Glu Met Gin Asn Lys Val Tyr Ser Phe Val Ser Leu Gly Val Gin
290 295 300
Glu Asp Leu Lys Tyr Phe Thr Glu Asn Glu Phe Lys Asn Arg Tyr Lys 305 310 315 320 Asp Lys Ser His Gin He Val Phe Thr Lys Asp Ala Glu Glu Leu Phe
325 330 335
Thr Leu Tyr Asn Ser Asp Glu Tyr Leu Gly Val Cys Gly Glu Leu Leu
340 345 350
Lys Val Cys Asp His Leu Ser Ala Phe Leu Glu Ala Gin He Ser Leu 355 360 365
Ser His Gly He Ser Ser Tyr Asp Leu He Gin Gly Ala Lys Asn Leu
370 375 380
Leu Glu Leu Arg Ser Gin Thr Glu Leu Leu Asp Leu Asp Leu Gly Lys 385 390 395 400 Leu Phe Arg Asp Phe Lys
405
(2) INFORMATION FOR SEQ ID NO: 101: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 335 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori ( ix) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION 1...335
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:
Val Leu Trp Val Leu Tyr Phe Leu Thr Ser Leu Phe He Cys Ser Leu 1 5 10 15 He Val Leu Trp Ser Lys Lys Ser Met Leu Phe Val Asp Asn Ala Asn 20 25 30
Lys He Gin Gly Phe His His Ala Arg Thr Pro Arg Ala Gly Gly Leu
35 40 45
Gly He Phe Leu Ser Phe Ala Leu Ala Cys Tyr Leu Glu Pro Phe Glu 50 55 60
Met Pro Phe Lys Gly Pro Phe Val Phe Leu Gly Leu Ser Leu Val Phe 65 70 75 80
Leu Ser Gly Phe Leu Glu Asp He Asn Leu Ser Leu Ser Pro Lys He 85 90 95 Arg Leu He Leu Gin Ala Val Gly Val Val Cys He He Ser Ser Thr 100 105 110
Pro Leu Val Val Ser Asp Phe Ser Pro Leu Phe Ser Leu Pro Tyr Phe
115 120 125
He Ala Phe Leu Phe Ala He Phe Met Leu Val Gly He Ser Asn Ala 130 135 140
He Asn He He Asp Gly Phe Asn Gly Leu Ala Ser Gly He Cys Ala
145 150 155 160
He Ala Leu Leu Val He His Tyr He Asp Pro Ser Ser Leu Ser Cys
165 170 175 Leu Leu Ala Tyr Met Val Leu Gly Phe Met Val Leu Asn Phe Pro Ser
180 185 190
Gly Lys He Phe Leu Gly Asp Gly Gly Ala Tyr Phe Leu Gly Leu Val
195 200 205
Cys Gly He Ser Leu Leu His Leu Ser Leu Glu Gin Lys He Ser Val 210 215 220
Phe Phe Gly Leu Asn Leu Met Leu Tyr Pro Val He Glu Val Leu Phe
225 230 235 240
Ser He Leu Arg Arg Lys He Lys Arg Gin Lys Ala Thr Met Pro Asp
245 250 255 Asn Leu His Leu His Thr Leu Leu Phe Lys Phe Leu Gin Gin Arg Ser
260 265 270
Phe Asn Tyr Pro Asn Pro Leu Cys Ala Phe He Leu He Leu Cys Asn
275 280 285
Leu Pro Phe He Leu He Ser Val Leu Phe Arg Leu Asp Ala Tyr Ala 290 295 300
Leu He Val He Ser Leu Val Phe He Ala Cys Tyr Leu He Gly Tyr 305 310 315 320
Ala Tyr Leu Asn Arg Gin Val Cys Ala Leu Glu Lys Arg Ala Phe 325 330 335
(2) INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...96
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:
Met Lys Lys Val He Val Ala Leu Gly Val Leu Ala Phe Ala Asn Val 1 5 10 15
Leu Met Ala Thr Asp Val Lys Ala Leu Val Lys Gly Cys Ala Ala Cys 20 25 30 His Gly Val Lys Phe Glu Lys Lys Ala Leu Gly Lys Ser Lys He Val 35 40 45
Asn Met Met Ser Glu Lys Glu He Glu Glu Asp Leu Met Ala Phe Lys
50 55 60
Ser Gly Ala Asn Lys Asn Pro Val Met Thr Ala Gin Ala Lys Lys Leu 65 70 75 80
Ser Asp Glu Asp He Lys Ala Leu Ala Lys Tyr He Pro Thr Leu Lys 85 90 95
(2) INFORMATION FOR SEQ ID NO:103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 156 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...156
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:
Met Arg Asp Phe Asn Asn He Gin He Thr Arg Leu Lys Val Arg Gin 1 5 10 15
Asn Ala Val Phe Glu Lys Leu Asp Leu Glu Phe Lys Asp Gly Leu Ser
20 25 30
Ala He Ser Gly Ala Ser Gly Val Gly Lys Ser Val Leu He Ala Ser 35 40 45 Leu Leu Gly Ala Phe Gly Leu Lys Glu Ser Asn Ala Ser Asn He Glu 50 55 60
Val Glu Leu He Ala Pro Phe Leu Asp Thr Glu Glu Tyr Gly He Phe 65 70 75 80
Arg Glu Asp Glu His Glu Pro Leu Val He Ser Val He Lys Lys Glu 85 90 95
Lys Thr Arg Tyr Phe Leu Asn Gin Thr Ser Leu Ser Lys Asn Thr Leu
100 105 110
Lys Ala Leu Leu Lys Gly Leu He Lys Arg Leu Ser Asn Asp Arg Phe 115 120 125 Ser Gin Asn Glu Leu Asn Asp He Leu Met Leu Ser Leu Leu Asp Gly 130 135 140
Tyr He Gin Asn Lys Asn Arg Arg Leu Ala Pro Phe 145 150 155 (2) INFORMATION FOR SEQ ID NO: 104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...118
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104: Val Met Leu Met Ala He Phe Thr Pro Tyr He Leu He Leu Lys Met 1 5 10 15
Met Lys Lys Ser Met Ser Leu Phe Ala Asn Met Gly Leu Glu Gin He
20 25 30
Phe Cys Asn Arg Asp He Lys Asp Leu Asn Asp Phe Val Phe Gly He 35 40 45
Glu Val Gly Leu Asp Ser Asn Ala Arg Lys Asn Arg Ser Arg Lys Ala
50 55 60
Met Glu Asn His Leu He Gly Leu Phe Val Gin Ala Gin Leu Asn Phe 65 70 75 80 Lys Glu Gin Val Asp He Arg Glu Phe Glu Asp Leu Arg Gin Ala Phe
85 90 95
Gly Asn Asp Thr Lys Lys Phe Asp Phe Val He Phe Ser Lys Glu Lys
100 105 110
Thr Tyr Phe His Arg Ser 115
(2) INFORMATION FOR SEQ ID NO: 105:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 355 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...355 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:
Met Asn He Lys He Leu Lys He Leu Val Gly Gly Leu Phe Phe Leu 1 5 10 15
Ser Leu Asn Ala His Leu Trp Gly Lys Gin Asp Asn Ser Phe Leu Gly 20 25 30
He Gly Glu Arg Ala Tyr Lys Ser Gly Asn Tyr Ser Lys Ala Ala Ser
35 40 45
Tyr Phe Lys Lys Ala Cys Asn Asp Gly Val Ser Glu Gly Cys Thr Gin 50 55 60 Leu Gly He He Tyr Glu Asn Gly Gin Gly Thr Arg He Asp Tyr Lys 65 70 75 80
Lys Ala Leu Glu Tyr Tyr Lys Thr Ala Cys Gin Ala Asp Asp Arg Glu
85 90 95
Gly Cys Phe Gly Leu Gly Gly Leu Tyr Asp Glu Gly Leu Gly Thr Ala 100 105 110
Gin Asn Tyr Gin Glu Ala He Asp Ala Tyr Ala Lys Ala Cys Val Leu
115 120 125
Lys His Pro Glu Ser Cys Tyr Asn Leu Gly He He Tyr Asp Arg Lys
130 135 140 He Lys Gly Asn Ala Ala Gin Ala Val Thr Tyr Tyr Gin Lys Ser Cys
145 150 155 160
Asn Phe Asp Met Ala Lys Gly Cys Tyr He Leu Gly Thr Ala Tyr Glu
165 170 175
Lys Gly Phe Leu Glu Val Lys Gin Ser Asn His Lys Ala Val He Tyr 180 185 190
Tyr Leu Lys Ala Cys Arg Leu Asn Glu Gly Gin Ala Cys Arg Ala Leu
195 200 205
Gly Ser Leu Phe Glu Asn Gly Asp Ala Gly Leu Asp Glu Asp Phe Glu
210 215 220 Val Ala Phe Asp Tyr Leu Gin Lys Ala Cys Ala Leu Asn Asn Ser Gly
225 230 235 240
Gly Cys Ala Ser Leu Gly Ser Met Tyr Met Leu Gly Arg Tyr Val Lys
245 250 255
Lys Asp Pro Gin Lys Ala Phe Asn Tyr Phe Lys Gin Ala Cys Asp Met 260 265 270
Gly Ser Ala Val Ser Cys Ser Arg Met Gly Phe Met Tyr Ser Gin Gly
275 280 285
Asp Thr Val Ser Lys Asp Leu Arg Lys Ala Leu Asp Asn Tyr Glu Arg 290 295 300 Gly Cys Asp Met Gly Asp Glu Val Gly Cys Phe Ala Leu Ala Gly Met 305 310 315 320
Tyr Tyr Asn Met Lys Asp Lys Glu Asn Ala He Met He Tyr Asp Lys
325 330 335
Gly Cys Lys Leu Gly Met Lys Gin Ala Cys Glu Asn Leu Thr Lys Leu
340 345 350
Arg Gly Tyr 355
(2) INFORMATION FOR SEQ ID NO: 106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 193 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...193
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:
Met Lys Glu Lys Asn Phe Trp Pro Leu Gly He Met Ser Val Leu He 1 5 10 15
Phe Gly Leu Gly He Val Val Phe Leu Val Val Phe Ala Leu Lys Asn
20 25 30
Ser Pro Lys Asn Asp Leu Val Tyr Phe Lys Gly His Asn Glu Val Asp 35 40 45 Leu Asn Phe Asn Ala Met Leu Lys Thr Tyr Glu Asn Phe Lys Ser Asn 50 55 60
Tyr Arg Phe Ser Val Gly Leu Lys Pro Leu Thr Glu Ser Pro Lys Thr 65 70 75 80
Pro He Leu Pro Tyr Phe Ser Lys Gly Thr His Gly Asp Lys Lys He 85 90 95
Gin Glu Asn Leu Leu Asn Asn Ala Leu He Leu Glu Lys Ser Asn Thr
100 105 110
Leu Tyr Ala Gin Leu Gin Pro Leu Lys Pro Ala Leu Asp Ser Pro Asn 115 120 125 He Gin Val Tyr Leu Ala Phe Tyr Pro Ser Gin Ser Gin Pro Arg Leu 130 135 140
Leu Gly Thr Leu Asp Cys Lys Asn Ala Cys Glu Pro Leu Lys Phe Asp 145 150 155 160
Leu Leu Glu Gly Asp Lys Val Gly Arg Tyr Lys He Leu Phe Lys Phe 165 170 175
Val Phe Lys Asn Lys Glu Glu Leu He Leu Glu Gin Leu Ala Phe Phe
180 185 190
Lys (2) INFORMATION FOR SEQ ID NO: 107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 289 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...289
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:
Leu Gly He Asn Met Cys Ser Lys Lys He Arg Asn Leu He Leu Cys 1 5 10 15
Phe Gly Phe He Leu Ser Leu Cys Ala Glu Glu Asn He Thr Lys Glu 20 25 30 Asn Met Thr Glu Thr Asn Thr Thr Glu Glu Asn Thr Pro Lys Asp Ala 35 40 45
Pro He Leu Leu Glu Glu Lys Arg Ala Gin Thr Leu Glu Leu Lys Glu
50 55 60
Glu Asn Glu Val Ala Lys Lys He Asp Glu Lys Ser Leu Leu Glu Glu 65 70 75 80
He His Lys Lys Lys Arg Gin Leu Tyr Met Leu Lys Gly Glu Leu His
85 90 95
Glu Lys Asn Glu Ser He Leu Phe Gin Gin Met Ala Lys Asn Lys Ser 100 105 110 Gly Phe Phe He Gly Val He Leu Gly Asp He Gly He Asn Ala Asn 115 120 125
Pro Tyr Glu Lys Phe Glu Leu Leu Ser Asn He Gin Ala Ser Pro Leu
130 135 140
Leu Tyr Gly Leu Arg Ser Gly Tyr Gin Lys Tyr Phe Ala Asn Gly He 145 150 155 160
Ser Ala Leu Arg Phe Tyr Gly Glu Tyr Leu Gly Gly Ala Met Lys Gly
165 170 175
Phe Lys Ser Asp Ser Leu Ala Ser Tyr Gin Thr Ala Ser Leu Asn He 180 185 190 Asp Leu Leu Met Asp Lys Pro He Asp Lys Glu Lys Arg Phe Ala Leu 195 200 205
Gly He Phe Gly Gly Val Gly Val Gly Trp Asn Gly Met Tyr Gin Asn
210 215 220
Leu Lys Glu He Arg Gly Tyr Ser Gin Pro Asn Ala Phe Gly Leu Val 225 230 235 240
Leu Asn Leu Gly Val Ser Met Thr Leu Asn Leu Lys His Arg Phe Glu
245 250 255
Leu Ala Leu Lys Met Pro Pro Leu Lys Glu Thr Ser Gin Thr Phe Leu 260 265 270 Tyr Tyr Phe Lys Ser Thr Asn He Tyr Tyr He Ser Tyr Asn Tyr Leu 275 280 285
Leu
(2) INFORMATION FOR SEQ ID NO: 108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 668 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...668
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108: Met Arg Lys Leu Phe He Pro Leu Leu Leu Phe Ser Ala Leu Glu Ala 1 5 10 15
Asn Glu Lys Asn Gly Phe Phe He Glu Ala Gly Phe Glu Thr Gly Leu
20 25 30
Leu Glu Gly Thr Gin Thr Gin Glu Lys Arg His Thr Thr Thr Lys Asn 35 40 45
Thr Tyr Ala Thr Tyr Asn Tyr Leu Pro Thr Asp Thr He Leu Lys Arg
50 55 60
Ala Ala Asn Leu Phe Thr Asn Ala Glu Ala He Ser Lys Leu Lys Phe 65 70 75 80 Ser Ser Leu Ser Pro Val Arg Val Leu Tyr Met Tyr Asn Gly Gin Leu
85 90 95
Thr He Glu Asn Phe Leu Pro Tyr Asn Leu Asn Asn Val Lys Leu Ser
100 105 110
Phe Thr Asp Ala Gin Gly Asn Thr He Asp Leu Gly Val He Glu Thr 115 120 125
He Pro Lys His Ser Lys He Val Leu Pro Gly Glu Ala Phe Asp Ser
130 135 140
Leu Lys Glu Ala Phe Asp Lys He Asp Pro Tyr Thr Leu Phe Leu Pro 145 150 155 160 Lys Phe Glu Ala Thr Ser Thr Ser He Ser Asp Thr Asn Thr Gin Arg
165 170 175
Val Phe Glu Thr Leu Asn Asn He Lys Thr Asn Leu He Met Lys Tyr
180 185 190
Ser Asn Glu Asn Pro Asn Asn Phe Asn Thr Cys Pro Tyr Asn Asn Asn 195 200 205
Gly Asn Thr Lys Asn Asp Cys Trp Gin Asn Phe Thr Pro Gin Thr Ala
210 215 220
Glu Glu Phe Thr Asn Leu Met Leu Asn Met He Ala Val Leu Asp Ser 225 230 235 240 Gin Ser Trp Gly Asp Ala He Leu Asn Ala Pro Phe Glu Phe Thr Asn 245 250 255
Ser Ser Thr Asp Cys Asp Ser Asp Pro Ser Lys Cys Val Asn Pro Gly
260 265 270
Val Asn Gly Arg Val Asp Thr Lys Val Asp Gin Gin Tyr He Leu Asn 275 280 285
Lys Gin Gly He He Asn Asn Phe Arg Lys Lys He Glu He Asp Ala
290 295 300
Val Val Leu Lys Asn Ser Gly Val Val Gly Leu Ala Asn Gly Tyr Gly 305 310 315 320 Asn Asp Gly Glu Tyr Gly Thr Leu Gly Val Glu Ala Tyr Ala Leu Asp
325 330 335
Pro Lys Lys Leu Phe Gly Asn Asp Leu Lys Thr He Asn Leu Glu Asp
340 345 350
Leu Arg Thr He Leu His Glu Phe Ser His Thr Lys Gly Tyr Gly His 355 360 365
Asn Gly Asn Met Thr Tyr Gin Arg Val Pro Val Thr Lys Asp Gly Gin
370 375 380
Val Glu Lys Asp Ser Asn Gly Lys Pro Lys Asp Ser Asp Gly Leu Pro 385 390 395 400 Tyr Asn Val Cys Ser Leu Tyr Gly Gly Ser Asn Gin Pro Ala Phe Pro
405 410 415
Ser Asn Tyr Pro Asn Ser He Tyr His Asn Cys Ala Asp Val Pro Ala
420 425 430
Gly Phe Leu Gly Val Thr Ala Ala Val Trp Gin Gin Leu He Asn Gin 435 440 445
Asn Ala Leu Pro He Asn Tyr Ala Asn Leu Gly Ser Gin Thr Asn Tyr
450 455 460
Asn Leu Asn Ala Ser Leu Asn Thr Gin Asp Leu Ala Asn Ser Met Leu 465 470 475 480 Ser Thr He Gin Lys Thr Phe Val Thr Ser Ser Val Thr Asn His His
485 490 495
Phe Ser Asn Ala Ser Gin Ser Phe Arg Ser Pro He Leu Gly Val Asn
500 505 510
Ala Lys He Gly Tyr Gin Asn Tyr Phe Asn Asp Phe He Gly Leu Ala 515 520 525
Tyr Tyr Gly He He Lys Tyr Asn Tyr Ala Lys Ala Val Asn Gin Lys
530 535 540
Val Gin Gin Leu Ser Tyr Gly Gly Gly He Asp Leu Leu Leu Asp Phe 545 550 555 560 He Thr Thr Tyr Ser Asn Lys Asn Ser Pro Thr Gly He Gin Thr Lys
565 570 575
Arg Asn Phe Ser Ser Ser Phe Gly He Phe Gly Gly Leu Arg Gly Leu
580 585 590
Tyr Asn Ser Tyr Tyr Val Leu Asn Lys Val Lys Gly Ser Gly Asn Leu 595 600 605
Asp Val Ala Thr Gly Leu Asn Tyr Arg Tyr Lys His Ser Lys Tyr Ser
610 615 620
Val Gly He Ser He Pro Leu He Gin Arg Lys Ala Ser Val Val Ser 625 630 635 640 Ser Gly Gly Asp Tyr Thr Asn Ser Phe Val Phe Asn Glu Gly Ala Ser
645 650 655
His Phe Lys Val Phe Phe Asn Tyr Gly Trp Val Phe 660 665 (2) INFORMATION FOR SEQ ID NO: 109: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...63
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109: Met Asn Thr Glu He Leu Thr He Met Leu Val Val Ser Val Leu Met 1 5 10 15
Gly Leu Val Gly Leu He Ala Phe Leu Trp Gly Val Lys Ser Gly Gin
20 25 30
Phe Asp Asp Glu Lys Arg Met Leu Glu Ser Val Leu Tyr Asp Ser Ala 35 40 45
Ser Asp Leu Asn Glu Ala He Leu Gin Glu Lys Arg Gin Lys Asn 50 55 60
(2) INFORMATION FOR SEQ ID NO: 110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 406 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...406
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:
Met Val Phe Phe His Lys Lys He He Leu Asn Phe He Tyr Ser Leu 1 5 10 15
Met Val Ala Phe Leu Phe His Leu Ser Tyr Gly Val Leu Leu Lys Ala
20 25 30
Asp Gly Met Ala Lys Lys Gin Thr Leu Leu Val Gly Glu Arg Leu Val 35 40 45 Trp Asp Lys Leu Thr Leu Leu Gly Phe Leu Glu Lys Asn His He Pro 50 55 60
Gin Lys Leu Tyr Tyr Asn Leu Ser Ser Gin Asp Lys Glu Leu Ser Ala 65 70 75 80
Glu He Gin Ser Asn Val Thr Tyr Tyr Thr Leu Arg Asp Ala Asn Asn 85 90 95
Thr Leu He Gin Ala Leu He Pro He Ser Gin Asp Leu Gin He His
100 105 110
He Tyr Lys Lys Gly Glu Asp Tyr Phe Leu Asp Phe He Pro He Val 115 120 125 Phe Thr Arg Lys Glu Arg Thr Leu Leu Leu Ser Leu Gin Thr Ser Pro 130 135 140
Tyr Gin Asp He Val Lys Ala Thr Asn Asp Pro Leu Leu Ala Asn Gin 145 150 155 160
Leu Met Asn Ala Tyr Lys Lys Ser Val Pro Phe Lys Arg Leu Val Lys 165 170 175
Asn Asp Lys He Ala He Val Tyr Thr Arg Asp Tyr Arg Val Gly Gin
180 185 190
Ala Phe Gly Gin Pro Thr He Lys Met Ala Met Val Ser Ser Arg Leu 195 200 205 His Gin Tyr Tyr Leu Phe Ser His Ser Asn Gly Arg Tyr Tyr Asp Ser 210 215 220
Lys Ala Gin Glu Val Ala Gly Phe Leu Leu Glu Thr Pro Val Lys Tyr 225 230 235 240
Thr Arg He Ser Ser Pro Phe Ser Tyr Gly Arg Phe His Pro Val Leu 245 250 255
Lys Val Lys Arg Pro His Tyr Gly Val Asp Tyr Ala Ala Lys His Gly
260 265 270
Ser Leu He His Ser Ala Ser Asp Gly Arg Val Gly Phe He Gly Val 275 280 285 Lys Ala Gly Tyr Gly Lys Val Val Glu He His Leu Asn Glu Leu Arg 290 295 300
Leu Val Tyr Ala His Met Ser Ala Phe Ala Asn Gly Leu Lys Lys Gly 305 310 315 320
Ser Phe Val Lys Lys Gly Gin He He Gly Arg Val Gly Ser Thr Gly 325 330 335
Leu Ser Thr Gly Pro His Leu His Phe Gly Val Tyr Lys Asn Ser Arg
340 345 350
Pro He Asn Pro Leu Gly Tyr He Arg Thr Ala Lys Ser Lys Leu His 355 360 365 Gly Lys Gin Arg Glu Val Phe Leu Glu Lys Ala Gin Tyr Ser Lys Gin 370 375 380
Lys Leu Glu Glu Leu Phe Lys Thr His Ser Phe Glu Lys Asn Ser Phe 385 390 395 400
Tyr Leu Leu Glu Gly Phe 405
(2) INFORMATION FOR SEQ ID NO: 111:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 296 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...296 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111:
Leu Phe Leu Val Lys Lys He Gly Val Val He Met He Leu Val Cys 1 5 10 15
Phe Leu Ala Cys Ser Gin Glu Ser Phe He Lys Met Gin Lys Lys Ala 20 25 30
Gin Glu Gin Glu Asn Asp Gly Ser Lys Arg Pro Ser Tyr Val Asp Ser
35 40 45
Asp Tyr Glu Val Phe Ser Glu Thr He Phe Leu Gin Asn Met Val Tyr 50 55 60 Gin Pro He Glu Glu Arg Asn Ala Phe Phe Gin Leu Thr Lys Asp Glu 65 70 75 80
Asp Asn Ser Phe Asn Pro Glu Asn Ser Val He Leu Leu Asn Glu Pro
85 90 95
Ser Asp Asn Ser Glu Lys Asn Leu Leu Ser Tyr Pro Asn Asp Pro Asn 100 105 110
Asn Asn Glu Asp Asn Ala Asn Asn Ser Gin Lys Asn Pro Phe Leu Tyr
115 120 125
Lys Pro Lys Arg Lys Thr Lys Asn Pro Lys Leu He Glu Tyr Ser Gin
130 135 140 Gin Asp Phe Tyr Pro Leu Lys Asn Gly Asp He He Met Ser Lys Glu
145 150 155 160
Gly Asp Gin Trp Leu He Glu He Gin Ser Lys Ala Leu Lys Arg Phe
165 170 175
Leu Lys Asp Gin Asn Asp Lys Asp Arg Gin He Gin Thr Phe Thr Phe 180 185 190
Asn Asp Thr Lys Thr Gin He Ala Gin He Lys Gly Lys He Ser Ser
195 200 205
Tyr Val Tyr Thr Thr Asn Asn Gly Ser Leu Ser Leu Arg Pro Phe Tyr
210 215 220 Glu Ser Phe Leu Leu Glu Lys Lys Ser Asp Asn Val Tyr Thr He Glu
225 230 235 240
Asn Lys Ala Leu Asp Thr Met Glu He Ser Lys Cys Gin Met Val Leu
245 250 255
Lys Lys His Ser Thr Asp Lys Leu Asp Ser Gin His Lys Ala He Ser 260 265 270
He Asp Leu Asp Phe Lys Lys Glu Arg Phe Lys Ser Asp Thr Glu Leu
275 280 285
Phe Leu Glu Cys Leu Lys Glu Ser 290 295
(2) INFORMATION FOR SEQ ID NO: 112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 248 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...248
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:»
Val Ser Tyr Asp Asn Thr Asp Asp Tyr Tyr Phe Pro Arg Asn Gly Val 1 5 10 15
He Phe Ser Ser Tyr Ala Thr Met Ser Gly Leu Pro Ser Ser Gly Thr 20 25 30 Leu Asn Ser Trp Asn Gly Leu Gly Gly Asn Val Arg Asn Thr Lys Val 35 40 45
Tyr Gly Lys Phe Ala Ala Tyr His His Leu Gin Lys Tyr Leu Leu He
50 55 60
Asp Leu He Ala Arg Phe Lys Thr Gin Gly Gly Tyr He Phe Arg Tyr 65 70 75 80
Asn Thr Asp Asp Tyr Leu Pro Leu Asn Ser Thr Phe Tyr Met Gly Gly
85 90 95
Val Thr Thr Val Arg Gly Phe Arg Asn Gly Ser He Thr Pro Lys Asp 100 105 110 Glu Phe Gly Leu Trp Leu Gly Gly Asp Gly He Phe Thr Ala Ser Thr 115 120 125
Glu Leu Ser Tyr Gly Val Leu Lys Ala Ala Lys Met Arg Leu Ala Trp
130 135 140
Phe Phe Asp Phe Gly Phe Leu Thr Phe Lys Thr Pro Thr Arg Gly Ser 145 150 155 160
Phe Phe Tyr Asn Ala Pro Thr Thr Thr Ala Asn Phe Lys Asp Tyr Gly
165 170 175
Val Val Gly Ala Gly Phe Glu Arg Ala Thr Trp Arg Ala Ser Thr Gly 180 185 190 Leu Gin He Glu Trp He Ser Pro Met Gly Pro Leu Val Leu He Phe 195 200 205
Pro He Ala Phe Phe Asn Gin Trp Gly Asp Gly Asn Gly Lys Lys Cys
210 215 220
Lys Gly Leu Cys Phe Asn Pro Asn Met Asn Asp Tyr Thr Gin His Phe 225 230 235 240
Glu Phe Ser Met Gly Thr Arg Phe 245
(2) INFORMATION FOR SEQ ID NO:113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 335 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...335
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:
Val Gin His Phe Asn Phe Leu Tyr Lys Asp Ser Leu Phe Ser He Ala 1 5 10 15
Leu Phe Thr Phe He He Ala Leu Val He Leu Leu Glu Gin Ala Arg
20 25 30
Ala Tyr Phe Thr Arg Lys Arg Asn Lys Lys Phe Leu Gin Lys Phe Ala 35 40 45 Gin Asn Gin Asn Ala Tyr Ala Ser Ser Glu Asn Leu Asp Glu Leu Leu 50 55 60
Lys His Ala Lys He Ser Ser Leu Met Phe Leu Ala Arg Ala Tyr Ser 65 70 75 80
Lys Ala Asp Val Glu Met Ser He Glu He Leu Lys Gly Leu Leu Asn 85 90 95
Arg Pro Leu Lys Asp Glu Glu Lys He Ala Val Leu Asp Leu Leu Ala
100 105 110
Lys Asn Tyr Phe Ser Val Gly Tyr Leu Gin Lys Thr Lys Asp Thr Val 115 120 125 Lys Glu He Leu Arg Phe Ser Pro Arg Asn Val Glu Ala Leu Leu Lys 130 135 140
Leu Leu His Ala Tyr Glu Leu Glu Lys Asp Tyr Ser Lys Ala Leu Glu 145 150 155 160
Thr Leu Glu Cys Leu Glu Glu Leu Glu Val Pro Lys He Glu Thr He 165 170 175
Lys Asn Tyr Leu Tyr Leu Met His Leu He Glu Asn Lys Glu Asp Ala
180 185 190
Ala Lys He Leu His Val Ser Lys Ala Ser Leu Asp Leu Lys Lys He 195 200 205 Ala Leu Asn His Leu Lys Ser His Asp Glu Asn Leu Phe Trp Gin Glu 210 215 220
He Asp Thr Thr Glu Arg Leu Glu Asn Val He Asp Leu Leu Trp Asp 225 230 235 240
Met Asn He Pro Ala Phe He Leu Glu Lys His Ala Leu Leu Gin Asp 245 250 255
He Ala Arg Ser Gin Gly Leu Leu Leu Asp His Lys Pro Cys Gin He
260 265 270
Phe Glu Leu Glu Val Leu Arg Ala Leu Leu His Ser Pro He Lys Ala 275 280 285 Ser Leu Thr Phe Glu Tyr Arg Cys Lys His Cys Lys Gin He Phe Pro 290 295 300
Phe Glu Ser His Arg Cys Pro Val Cys Tyr Gin Leu Ala Phe Met Asp 305 310 315 320
Met Val Leu Lys He Ser Lys Lys Thr His Ala Met Gly Val Asp 325 330 335 (2) INFORMATION FOR SEQ ID NO: 114:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 413 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...413 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114:
Met Arg Lys He Phe Ser Tyr He Ser Lys Val Leu Leu Phe He Gly 1 5 10 15
Val Val Tyr Ala Glu Pro Asp Ser Lys Val Glu Ala Leu Glu Gly Arg 20 25 30
Lys Gin Glu Ser Ser Leu Asp Lys Lys He Arg Gin Glu Leu Lys Ser
35 40 45
Lys Glu Leu Lys Asn Lys Glu Leu Lys Asn Lys Asp Leu Lys Asn Lys 50 55 60 Glu Glu Lys Lys Glu Thr Lys Ala Lys Arg Lys Pro Arg Ala Glu Val 65 70 75 80
His His Gly Asp Ala Lys Asn Pro Thr Pro Lys He Thr Pro Pro Lys
85 90 95
He Lys Gly Ser Ser Lys Gly Val Gin Asn Gin Gly Val Gin Asn Asn 100 105 110
Ala Pro Lys Pro Glu Glu Lys Asp Thr Thr Pro Gin Ala Thr Glu Lys
115 120 125
Asn Lys Glu Thr Ser Pro Ser Ser Gin Phe Asn Ser He Phe Gly Asn
130 135 140 Pro Asn Asn Ala Thr Asn Asn Thr Leu Glu Asp Lys Val Val Gly Gly
145 150 155 160
He Ser Leu Leu Val Asn Gly Ser Pro He Thr Leu Tyr Gin He Gin
165 170 175
Glu Glu Gin Glu Lys Ser Lys Val Ser Lys Ala Gin Ala Arg Asp Arg 180 185 190
Leu He Ala Glu Arg He Lys Asn Gin Glu He Glu Arg Leu Lys He
195 200 205
His Val Asp Asp Asp Lys Leu Asp Gin Glu Met Ala Met Met Ala Gin
210 215 220 Gin Gin Gly Met Asp Leu Asp His Phe Lys Gin Met Leu Met Ala Glu
225 230 235 240
Gly His Tyr Lys Leu Tyr Arg Asp Gin Leu Lys Glu His Leu Glu Met
245 250 255
Gin Glu Leu Leu Arg Asn He Leu Leu Thr Asn Val Asp Thr Ser Ser 260 265 270 Glu Thr Lys Met Arg Glu Tyr Tyr Asn Lys His Lys Glu Gin Phe Ser
275 280 285
He Pro Thr Glu He Glu Thr Val Arg Tyr Thr Ser Thr Asn Gin Glu
290 295 300 Asp Leu Glu Arg Ala Met Ala Asp Pro Asn Leu Glu Val Pro Gly Val
305 310 315 320
Ser Lys Ala Asn Glu Lys He Glu Met Lys Thr Leu Asn Pro Gin He
325 330 335
Ala Gin Val Phe He Ser His Glu Gin Gly Ser Phe Thr Pro Val Met 340 345 350
Asn Gly Gly Gly Gly Gin Phe He Thr Phe Tyr He Lys Glu Lys Arg
355 360 365
Gly Lys Asn Glu Val Ser Phe Ser Gin Ala Lys Gin Phe He Ala Gin 370 375 380 Lys Leu Val Glu Glu Ser Lys Asp Lys He Leu Glu Glu His Phe Glu 385 390 395 400
Lys Leu Arg Val Lys Ser Arg He Val Met He Arg Glu 405 410 (2) INFORMATION FOR SEQ ID NO: 115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 186 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...186
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115: Met He Lys Arg He Ala Cys He Leu Ser Leu Ser Ala Ser Leu Ala 1 5 10 15
Leu Ala Gly Glu Val Asn Gly Phe Phe Met Gly Ala Gly Tyr Gin Gin
20 25 30
Gly Arg Tyr Gly Pro Tyr Asn Ser Asn Tyr Ser Asp Trp Arg His Gly 35 40 45
Asn Asp Leu Tyr Gly Leu Asn Phe Lys Leu Gly Phe Val Gly Phe Ala
50 55 60
Asn Lys Trp Phe Gly Ala Arg Val Tyr Gly Phe Leu Asp Trp Phe Asn 65 70 75 80 Thr Ser Gly Thr Glu His Thr Lys Thr Asn Leu Leu Thr Tyr Gly Gly
85 90 95
Gly Gly Asp Leu He Val Asn Leu He Pro Leu Asp Lys Phe Ala Leu
100 105 110
Gly Leu He Gly Gly Val Gin Leu Ala Gly Asn Thr Trp Met Phe Pro 115 120 125 Tyr Asp Val Asn Gin Thr Arg Phe Gin Phe Leu Trp Asn Leu Gly Gly
130 135 140
Arg Met Arg Val Gly Asp Arg Ser Ala Phe Glu Ala Gly Val Lys Phe 145 150 155 160 Pro Met Val Asn Gin Gly Ser Lys Asp Val Gly Leu He Arg Tyr Tyr
165 170 175
Ser Trp Tyr Val Asp Tyr Val Phe Thr Phe 180 185 (2) INFORMATION FOR SEQ ID NO: 116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...242
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: Met Lys Lys Phe Phe Ser Gin Ser Leu Leu Ala Leu He He Ser Met 1 5 10 15
Asn Ala Val Ser Gly Met Asp Gly Asn Gly Val Phe Leu Gly Ala Gly
20 25 30
Tyr Leu Gin Gly Gin Ala Gin Met His Ala Asp He Asn Ser Gin Lys 35 40 45
Gin Ala Thr Asn Ala Thr He Lys Gly Phe Asp Ala Leu Leu Gly Tyr
50 55 60
Gin Phe Phe Phe Glu Lys His Phe Gly Leu Arg Leu Tyr Gly Phe Phe 65 70 75 80 Asp Tyr Ala His Ala Asn Ser He Lys Leu Lys Asn Pro Asn Tyr Asn
85 90 95
Ser Glu Ala Ala Gin Val Ala Ser Gin He Leu Gly Lys Gin Glu He
100 105 110
Asn Arg Leu Thr Asn He Ala Asp Pro Arg Thr Phe Glu Pro Asn Met 115 120 125
Leu Thr Tyr Gly Gly Ala Met Asp Val Met Val Asn Val He Asn Asn
130 135 140
Gly He Met Ser Leu Gly Ala Phe Gly Gly He Gin Leu Ala Gly Asn 145 150 155 160 Ser Trp Leu Met Ala Thr Pro Ser Phe Glu Gly He Leu Val Glu Gin
165 170 175
Ala Leu Val Ser Lys Lys Ala Thr Ser Phe Gin Phe Leu Phe Asn Val
180 185 190
Gly Ala Arg Leu Arg He Leu Lys His Ser Ser He Glu Ala Gly Val 195 200 205 Lys Phe Pro Met Leu Lys Lys Asn Pro Tyr He Thr Ala Lys Asn Leu
210 215 220
Asp He Gly Phe Arg Arg Val Tyr Ser Trp Tyr Val Asn Tyr Val Phe 225 230 235 240 Thr Phe
(2) INFORMATION FOR SEQ ID NO: 117: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 256 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...256
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117:
Met Gly Tyr Ala Ser Lys Leu Ala Leu Lys He Cys Leu Val Gly Leu 1 5 10 15 Cys Leu Phe Ser Thr Leu Gly Ala Glu His Leu Glu Gin Lys Gly Asn 20 25 30
Tyr He Tyr Lys Gly Glu Glu Ala Tyr Asn Asn Lys Glu Tyr Glu Arg
35 40 45
Ala Ala Ser Phe Tyr Lys Ser Ala He Lys Asn Gly Glu Ser Leu Ala 50 55 60
Tyr He Leu Leu Gly He Met Tyr Glu Asn Gly Arg Gly Val Pro Lys 65 70 75 80
Asp Tyr Lys Lys Ala Val Glu Tyr Phe Gin Lys Ala Val Asp Asn Asp 85 90 95 He Pro Arg Gly Tyr Asn Asn Leu Gly Val Met Tyr Lys Glu Gly Lys 100 105 110
Gly Val Pro Lys Asp Glu Lys Lys Ala Val Glu Tyr Phe Arg He Ala
115 120 125
Thr Glu Lys Gly Tyr Thr Asn Ala Tyr He Asn Leu Gly He Met Tyr 130 135 140
Met Glu Gly Arg Gly Val Pro Ser Asn Tyr Ala Lys Ala Thr Glu Cys
145 150 155 160
Phe Arg Lys Ala Met His Lys Gly Asn Val Glu Ala Tyr He Leu Leu
165 170 175 Gly Asp He Tyr Tyr Ser Gly Asn Asp Gin Leu Gly He Glu Pro Asp
180 185 190
Lys Asp Lys Ala Val Val Tyr Tyr Lys Met Ala Ala Asp Val Ser Ser
195 200 205
Ser Arg Ala Tyr Glu Gly Leu Ser Glu Ser Tyr Arg Tyr Gly Leu Gly 210 215 220 Val Glu Lys Asp Lys Lys Lys Ala Glu Glu Tyr Met Gin Lys Ala Cys 225 230 235 240
Asp Phe Asp He Asp Lys Asn Cys Lys Lys Lys Asn Thr Ser Ser Arg 245 250 255
(2) INFORMATION FOR SEQ ID NO: 118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 657 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...657
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118:
Met Arg Lys Leu Phe He Pro Leu Leu Leu Phe Ser Ala Leu Glu Ala 1 5 10 15
Asn Glu Lys Asn Gly Phe Phe He Glu Ala Gly Phe Glu Thr Gly Leu 20 25 30 Leu Glu Gly Thr Gin Thr Gin Glu Lys Arg His Thr Thr Thr Lys Asn 35 40 45
Thr Tyr Ala Thr Tyr Asn Tyr Leu Pro Thr Asp Thr He Leu Lys Arg
50 55 60
Ala Ala Asn Leu Phe Thr Asn Ala Glu Ala He Ser Lys Leu Lys Phe 65 70 75 80
Ser Ser Leu Ser Pro Val Arg Val Leu Tyr Met Tyr Asn Gly Gin Leu
85 90 95
Thr He Glu Asn Phe Leu Pro Tyr Asn Leu Asn Asn Val Lys Leu Ser 100 105 110 Phe Thr Asp Ala Gin Gly Asn Val He Asp Leu Gly Val He Glu Thr 115 120 125
He Pro Lys His Ser Lys He Val Leu Pro Gly Glu Ala Phe Asp Ser
130 135 140
Leu Lys He Asp Pro Tyr Thr Leu Phe Leu Pro Lys He Glu Ala Thr 145 150 155 160
Ser Thr Ser He Ser Asp Ala Asn Thr Gin Arg Val Phe Glu Thr Leu
165 170 175
Asn Lys He Lys Thr Asn Leu Val Val Asn Tyr Arg Asn Glu Asn Lys 180 185 190 Phe Lys Asp His Glu Asn His Trp Glu Ala Phe Thr Pro Gin Thr Ala 195 200 205
Glu Glu Phe Thr Asn Leu Met Leu Asn Met He Ala Val Leu Asp Ser
210 215 220
Gin Ser Trp Gly Asp Ala He Leu Asn Ala Pro Phe Glu Phe Thr Asn 225 230 235 240 Ser Pro Thr Asp Cys Asp Asn Asp Pro Ser Lys Cys Val Asn Pro Gly
245 250 255
Thr Asn Gly Leu Val Asn Ser Lys Val Asp Gin Lys Tyr Val Leu Asn 260 265 270 Lys Gin Asp He Val Asn Lys Phe Lys Asn Lys Ala Asp Leu Asp Val 275 280 285
He Val Leu Lys Asp Ser Gly Val Val Gly Leu Gly Ser Asp He Thr
290 295 300
Pro Ser Asn Asn Asp Asp Gly Lys His Tyr Gly Gin Leu Gly Val Val 305 310 315 320
Ala Ser Ala Leu Asp Pro Lys Lys Leu Phe Gly Asp Asn Leu Lys Thr
325 330 335
He Asn Leu Glu Asp Leu Arg Thr He Leu His Glu Phe Ser His Thr 340 345 350 Lys Gly Tyr Gly His Asn Gly Asn Met Thr Tyr Gin Arg Val Pro Val 355 360 365
Thr Lys Asp Gly Gin Val Glu Lys Asp Ser Asn Gly Lys Pro Lys Asp
370 375 380
Ser Asp Gly Leu Pro Tyr Asn Val Cys Ser Leu Tyr Gly Gly Ser Asn 385 390 395 400
Gin Pro Ala Phe Pro Ser Asn Tyr Pro Asn Ser He Tyr His Asn Cys
405 410 415
Ala Asp Val Pro Ala Gly Phe Leu Gly Val Thr Ala Ala Val Trp Gin 420 425 430 Gin Leu He Asn Gin Asn Ala Leu Pro He Asn Tyr Ala Asn Leu Gly 435 440 445
Ser Gin Thr Asn Tyr Asn Leu Asn Ala Ser Leu Asn Thr Gin Asp Leu
450 455 460
Ala Asn Ser Met Leu Ser Thr He Gin Lys Thr Phe Val Thr Ser Ser 465 470 475 480
Val Thr Asn His His Phe Ser Asn Ala Ser Gin Ser Phe Arg Ser Pro
485 490 495
He Leu Gly Val Asn Ala Lys He Gly Tyr Gin Asn Tyr Phe Asn Asp 500 505 510 Phe He Gly Leu Ala Tyr Tyr Gly He He Lys Tyr Asn Tyr Ala Lys 515 520 525
Ala Val Asn Gin Lys Val Gin Gin Leu Ser Tyr Gly Gly Gly He Asp
530 535 540
Leu Leu Leu Asp Phe He Thr Thr Tyr Ser Asn Lys Asn Ser Pro Thr 545 550 555 560
Gly He Gin Thr Lys Arg Asn Phe Ser Ser Ser Phe Gly He Phe Gly
565 570 575
Gly Leu Arg Gly Leu Tyr Asn Ser Tyr Tyr Val Leu Asn Lys Val Lys 580 585 590 Gly Ser Gly Asn Leu Asp Val Ala Thr Gly Leu Asn Tyr Arg Tyr Lys 595 600 605
His Ser Lys Tyr Ser Val Gly He Ser He Pro Leu He Gin Arg Lys
610 615 620
Ala Ser Val Val Ser Ser Gly Gly Asp Tyr Thr Asn Ser Phe Val Phe 625 630 635 640
Asn Glu Gly Ala Ser His Phe Lys Val Phe Phe Asn Tyr Gly Trp Val
645 650 655
Phe (2) INFORMATION FOR SEQ ID NO: 119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 167 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...167
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119:
Met Lys Leu Val Ser Leu He Val Ala Leu Val Phe Cys Cys Phe Leu 1 5 10 15
Gly Ala Val Glu Leu Pro Gly Val Tyr Gin Thr Gin Glu Phe Leu Tyr 20 25 30 Met Lys Ser Ser Phe Val Glu Phe Phe Glu His Asn Gly Lys Phe Tyr 35 40 45
Ala Tyr Gly He Ser Asp Val Asp Gly Ser Lys Ala Lys Lys Asp Lys
50 55 60
Leu Asn Pro Asn Pro Lys Leu Arg Asn Arg Ser Asp Lys Gly Val Val 65 70 75 80
Phe Leu Ser Asp Leu He Lys Val Gly Glu Gin Ser Tyr Lys Gly Gly
85 90 95
Lys Ala Tyr Asn Phe Tyr Asp Gly Lys Thr Tyr His Val Arg Val Thr 100 105 110 Gin Asn Ser Asn Gly Asp Leu Glu Phe Thr Ser Ser Tyr Asp Lys Trp 115 120 125
Gly Tyr Val Gly Lys Thr Phe Thr Trp Lys Arg Leu Ser Asp Glu Glu
130 135 140
He Lys Asn Leu Lys Leu Lys Arg Phe Asn Leu Asp Glu Val Leu Lys 145 150 155 160
Thr Leu Lys Asp Ser Pro He 165
(2) INFORMATION FOR SEQ ID NO: 120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 294 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...294
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:
Met Ser Asn Gin Ala Ser His Leu Asp Asn Phe Met Asn Ala Lys Asn 1 5 10 15
Pro Lys Ser Phe Phe Asp Asn Lys Gly Asn Thr Lys Phe He Ala He
20 25 30
Thr Ser Gly Lys Gly Gly Val Gly Lys Ser Asn He Ser Ala Asn Leu 35 40 45 Ala Tyr Ser Leu Tyr Lys Lys Gly Tyr Lys Val Gly Val Phe Asp Ala 50 55 60
Asp He Gly Leu Ala Asn Leu Asp Val He Phe Gly Val Lys Thr His 65 70 75 80
Lys Asn He Leu His Ala Leu Lys Gly Glu Ala Lys Leu Gin Glu He 85 90 95
He Cys Glu He Glu Pro Gly Leu Cys Leu He Pro Gly Asp Ser Gly
100 105 110
Glu Glu He Leu Lys Tyr He Ser Gly Ala Glu Ala Leu Asp Arg Phe 115 120 125 Val Asp Glu Glu Gly Val Leu Ser Ser Leu Asp Tyr He Val He Asp 130 135 140
Thr Gly Ala Gly He Gly Ala Thr Thr Gin Ala Phe Leu Asn Ala Ser 145 150 155 160
Asp Cys Val Val He Val Thr Thr Pro Asp Pro Ser Ala He Thr Asp 165 170 175
Ala Tyr Ala Cys He Lys He Asn Ser Lys Asn Lys Asp Glu Leu Phe
180 185 190
Leu He Ala Asn Met Val Ala Gin Pro Lys Glu Gly Arg Ala Thr Tyr 195 200 205 Glu Arg Leu Phe Lys Val Ala Lys Asn Asn He Ala Ser Leu Glu Leu 210 215 220
His Tyr Leu Gly Ala He Glu Asn Ser Ser Leu Leu Lys Arg Tyr Val 225 230 235 240
Arg Glu Arg Lys He Leu Arg Lys He Ala Pro Asn Asp Leu Phe Ser 245 250 255
Gin Ser He Asp Gin He Ala Ser Leu Leu Val Ser Lys Leu Glu Thr
260 265 270
Gly Thr Leu Glu He Pro Lys Glu Gly Leu Lys Ser Phe Phe Lys Arg 275 280 285 Leu Leu Lys Tyr Leu Gly . 290
(2) INFORMATION FOR SEQ ID NO: 121: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 372 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...372
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121:
Leu Glu Pro Ser Arg Asn Arg Leu Lys His Ala Ala Phe Phe Val Gly 1 5 10 15 Leu Phe He Val Leu Phe Leu He He Met Lys His Gin Thr Ser Pro 20 25 30
Tyr Ala Phe Thr His Asn Gin Ala Leu Val Thr Gin Thr Pro Pro Tyr
35 40 45
Phe Thr Gin Leu Thr He Pro Lys Pro Asn Asp Ala Leu Ser Ala His 50 55 60
Ala Ser Ser Leu He Ser Leu Pro Asn Asp Asn Leu Leu Ser Ala Tyr 65 70 75 80
Phe Ser Gly Thr Lys Glu Gly Ala Arg Asp Val Lys He Ser Ala Asn 85 90 95 Leu Phe Asp Ser Lys Thr Asn Arg Trp Ser Glu Ala Phe He Leu Leu 100 105 110
Thr Lys Glu Glu Leu Ser His His Ser His Glu Tyr He Lys Lys Leu
115 120 125
Gly Asn Pro Leu Leu Phe Leu His Asp Asn Lys He Leu Leu Phe Val 130 135 140
Val Gly Val Ser Met Gly Gly Trp Ala Thr Ser Lys He Tyr Gin Phe
145 150 155 160
Glu Ser Ala Leu Glu Pro He His Phe Lys Phe Ala Arg Lys Leu Ser
165 170 175 Leu Ser Pro Phe Leu Asn Leu Ser His Leu Val Arg Asn Lys Pro Leu
180 185 190
Asn Thr Thr Asp Gly Gly Phe Met Leu Pro Leu Tyr His Glu Leu Ala
195 200 205
Thr Gin Tyr Pro Leu Leu Leu Lys Phe Asp Gin Gin Asn Asn Pro Arg 210 215 220
Glu Leu Leu Arg Pro Asn Thr Leu Asn His Gin Leu Gin Pro Ser Leu
225 230 235 240
Thr Pro Phe Lys Asp Cys Ala Val Met Ala Phe Arg Asn His Ser Phe
245 250 255 Lys Asp Ser Leu Met Leu Glu Thr Cys Lys Thr Pro Thr Asp Trp Gin
260 265 270
Lys Pro He Ser Thr Asn Leu Lys Asn Leu Asp Asp Ser Leu Asn Leu
275 280 285
Leu Asn Leu Asn Gly He Leu Tyr Leu He His Asn Pro Ser Asp Leu 290 295 300
Ser Leu Arg Arg Lys Glu Leu Trp Leu Ser Lys Leu Glu Asn Ser Asn
305 310 315 320
Ser Phe Lys Thr Leu Lys Val Leu Asp Lys Ala Asn Glu Val Ser Tyr
325 330 335 Pro Ser Tyr Ser Leu Asn Pro His Phe He Asp He Val Tyr Thr Tyr 340 345 350
Asn Arg Ser His He Lys His He Arg Phe Asn Met Ala Tyr Leu Asn
355 360 365
Ser Leu Leu Lys 370
(2) INFORMATION FOR SEQ ID NO:122:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 978 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...978 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122:
Met Lys Lys Arg Lys His Val Ser Lys Lys Val Phe Asn Val He He 1 5 10 15
Leu Phe Val Ala Val Phe Thr Leu Leu Val Val He His Lys Thr Leu 20 25 30
Ser Asn Gly He His He Gin Asn Leu Lys He Gly Lys Leu Gly He
35 40 45
Ser Glu Leu Tyr Leu Lys Leu Asn Asn Lys Leu Ser Leu Glu Val Glu 50 55 60 Arg Val Asp Leu Ser Ser Phe Phe His Gin Lys Pro Thr Lys Lys Arg 65 70 75 80
Leu Glu Val Ser Asp Leu He Lys Asn He Arg Tyr Gly He Trp Ala
85 90 95
Val Ser Tyr Phe Glu Lys Leu Lys Val Lys Glu He He Leu Asp Asp 100 105 110
Lys Asn Lys Ala Asn He Phe Phe Asp Gly Asn Lys Tyr Glu Leu Glu
115 120 125
Phe Pro Gly He Lys Gly Glu Phe Ser Leu Glu Asp Asp Lys Asn He
130 135 140 Lys Leu Lys He He Asn Leu Leu Phe Lys Asp Val Lys Val Gin Val
145 150 155 160
Asp Gly Asn Ala His Tyr Ser Pro Lys Ala Arg Lys Met Ala Phe Asn
165 170 175
Leu He Val Lys Pro Leu Val Glu Pro Ser Ala Ala He Tyr Leu Gin 180 185 190
Gly Leu Thr Asp Leu Lys Thr He Glu Leu Lys He Asn Thr Ser Pro
195 200 205
Met Lys Ser Leu Ala Phe Leu Lys Pro Leu Phe Gin Arg Gin Ser Gin 210 215 220 Lys Asn Leu Lys Thr Trp He Phe Asp Lys He Gin Phe Ala Ser Phe 225 230 235 240
Lys He Asp Asn Ala Leu He Lys Ala Asn Phe Thr Pro Ser Glu Phe
245 250 255
He Pro Ser Leu Leu Glu Asn Ser Val Val Lys Ala Thr Leu He Lys 260 265 270
Pro Ser Val Val Phe Asn Asp Gly Leu Ser Pro He Lys Met Asp Lys
275 280 285
Thr Glu Leu He Phe Lys Asn Lys Gin Leu Leu He Gin Pro Gin Lys
290 295 300 He Thr Tyr Glu Thr Met Glu Leu Thr Gly Ser Tyr Ala Thr Phe Ser
305 310 315 320
Asn Leu Leu Glu Ala Pro Lys Leu Glu Val Phe Leu Lys Thr Thr Pro
325 330 335
Asn Tyr Tyr Gly Asp Ser He Lys Asp Leu Leu Ser Ala Tyr Lys Val 340 345 350
Val Leu Pro Leu Asp Lys He Ser Met Pro Ser Ser Ala Asp Leu Lys
355 360 365
Leu Thr Leu Gin Phe Leu Lys Asn Thr Ala Pro Leu Phe Ser Val Gin
370 375 380 Gly Ser Val Asn Leu Gin Glu Gly Thr Phe Ser Leu Tyr Asn He Pro
385 390 395 400
Leu Tyr Thr Gin Ser Ala Gin He Asn Leu Asp He Ala Gin Glu Tyr
405 410 415
Gin Tyr He Tyr He Asp Thr He His Thr Arg Tyr Ala Asn Met Leu 420 425 430
Asp Leu Asp Ala Lys He Ala Leu Asp Leu Gly Gin Lys Asn Leu Ser
435 440 445
Leu Asp Ser Leu Val His Lys He Gin Val Asn Thr Asn Asn Asn He
450 455 460 Asn Met Arg Ser Tyr Asp Pro Asn Asn Thr Gin Glu Asp Pro Gin Thr
465 470 475 480
Asn Phe Thr Leu Asp Leu Lys Ser Leu His Ser He He Gin Glu Gly
485 490 495
Glu Asn Ser Glu Val Phe Arg Arg Lys He He Asp Thr He Lys Ala 500 505 510
Gin Ser Glu Asp Lys Phe Thr Lys Asp Val Phe Tyr Ala Thr Gly Asp
515 520 525
Thr Leu Lys Ser Leu Ser Leu Ser Phe Asp Phe Ser Asn Pro Asp His
530 535 540 He Gin Trp Ser Val Pro Gin Leu Leu Leu Glu Gly Glu Phe Lys Asp
545 550 555 560
Asn Ala Tyr Thr Phe Lys He Lys Asp Leu Lys Lys He Lys Pro Tyr
565 570 575
Ser Pro He Met Asp Tyr He Ala Leu Lys Asp Gly Ser Leu Glu Val 580 585 590
Ser Thr Ser Asp Phe Val Asn He Asp Phe Phe Ala Lys Asp Leu Lys
595 600 605
He Asn Leu Pro He Tyr Arg Ser Asp Gly Ser His Phe Asp Ser Phe
610 615 620 Ser Leu Phe Gly Ser He Asn Lys Asp Glu He Ser Val Tyr Thr Pro
625 630 635 640
Ser Lys Ser He Ser He Lys Val Lys Gly Asp Gin Lys Asp He Thr
645 650 655
Leu Asn Asn He Asp Leu Ser He Asp Asp Phe Leu Asp Ser Lys Met 660 665 670 Pro Ala He Ala Gly Leu Phe Ser Lys Glu Arg Lys Glu Lys Pro Ser
675 680 685
Ser Lys Glu He Gin Asp Glu Asp Val Phe He Ser Ala Lys Gin Arg
690 695 700 Tyr Glu Lys Ala His Lys He He Pro He Ser Thr Arg He His Ala
705 710 715 720
Lys Asp Val Val Leu He Tyr Lys Lys Met Pro Phe Pro Leu Glu Asn
725 730 735
Leu Asp He Val Ala Gin Asp Asp Arg Val Lys He Asp Gly Asn Tyr 740 745 750
Lys Asn Ala Met He Met Ala Asp Leu Val His Gly Ala Leu Tyr Leu
755 760 765
Lys Ala His Asn Phe Ser Gly Asp Tyr He Asn Thr He Leu Gin Lys
770 775 780 Asp Phe Val Glu Gly Gly Leu Phe Thr Leu He Gly Ala Leu Glu Asp
785 790 795 800
Gin Val Phe Asn Gly Glu Leu Lys Phe Gin Asn Thr Ser Leu Lys Asn
805 810 815
Phe Ala Leu Met Gin Asn Met Val Asn Leu He Asn Thr He Pro Ser 820 825 830
Leu He Val Phe Arg Asn Pro His Leu Gly Ala Asn Gly Tyr Gin He
835 840 845
Lys Thr Gly Ser Val Val Phe Gly He Thr Lys Glu Tyr Leu Gly Leu
850 855 860 Glu Lys He Asp Leu Val Gly Lys Thr Leu Asp He Ala Gly Asn Gly
865 870 875 880
He He Glu Leu Asp Lys Asn Lys Leu Asp Leu Asn Leu Glu Val Ser
885 890 895
Thr He Lys Ala Leu Ser Asn Val Leu Asn Lys He Pro He Val Gly 900 905 910
Tyr Leu Val Leu Gly Lys Gly Gly Lys He Thr Thr Asn Val Asn Val
915 920 925
Lys Gly Thr Leu Asp Lys Pro Lys Thr Gin Val Thr Leu Ala Ser Asp
930 935 940 He He Gin Ala Pro Phe Lys He Leu Arg Arg He Phe Thr Pro He
945 950 955 960
Asp He He Val Asp Glu Val Lys Lys Asn He Asp Ser Lys Arg Lys
965 970 975
Leu Lys
(2) INFORMATION FOR SEQ ID NO: 123
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 477 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori ( ix) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION 1...477 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:
Met Asn Thr He He Arg Tyr Ala Ser Leu Trp Gly Leu Cys He Thr 1 5 10 15
Leu Thr Leu Ala Gin Thr Pro Ser Lys Thr Pro Asp Glu He Lys Gin 20 25 30
He Leu Asn Asn Tyr Ser His Lys Asn Leu Lys Leu He Asp Pro Pro
35 40 45
Thr Ser Ser Leu Glu Ala Thr Pro Gly Phe Leu Pro Ser Pro Lys Glu 50 55 60 Thr Ala Thr Thr He Asn Gin Glu He Ala Lys Tyr His Glu Lys Ser 65 70 75 80
Asp Lys Ala Ala Leu Gly Leu Tyr Glu Leu Leu Lys Gly Ala Thr Thr
85 90 95
Asn Leu Ser Leu Gin Ala Gin Glu Leu Ser Val Lys Gin Ala Met Lys 100 105 110
Asn His Thr He Ala Lys Ala Met Phe Leu Pro Thr Leu Asn Ala Ser
115 120 125
Tyr Asn Phe Lys Asn Glu Ala Arg Asp Thr Pro Glu Tyr Lys His Tyr
130 135 140 Asn Thr Gin Gin Leu Gin Ala Gin Val Thr Leu Asn Val Phe Asn Gly
145 150 155 160
Phe Ser Asn Val Asn Asn Val Lys Glu Lys Ser Ala Thr Tyr Arg Ser
165 170 175
Thr Val Ala Asn Leu Glu Tyr Ser Arg Gin Ser Val Tyr Leu Gin Val 180 185 190
Val Gin Gin Tyr Tyr Glu Tyr Phe Asn Asn Leu Ala Arg Met He Ala
195 200 205
Leu Gin Lys Lys Leu Glu Gin He Gin Thr Asp He Lys Arg Val Thr
210 215 220 Lys Leu Tyr Asp Lys Gly Leu Thr Thr He Asp Asp Leu Gin Ser Leu
225 230 235 240
Lys Ala Gin Gly Asn Leu Ser Glu Tyr Asp He Leu Asp Met Gin Phe
245 250 255
Ala Leu Glu Gin Asn Arg Leu Thr Leu Glu Tyr Leu Thr Asn Leu Ser 260 265 270
Val Lys Asn Leu Lys Lys Thr Thr He Asp Ala Pro Asn Leu Gin Leu
275 280 285
Arg Glu Arg Gin Asp Leu Val Ser Leu Arg Glu Gin He Ser Ala Leu
290 295 300 Arg Tyr Gin Asn Lys Gin Leu Asn Tyr Tyr Pro Lys He Asp Val Phe
305 310 315 320
Asp Ser Trp Leu Phe Trp He Gin Lys Pro Ala Tyr Ala Thr Gly Arg
325 330 335
Phe Gly Asn Phe Tyr Pro Gly Gin Gin Asn Thr Ala Gly Val Thr Ala 340 345 350
Thr Leu Asn He Phe Asp Asp He Gly Leu Ser Leu Gin Lys Gin Ser
355 360 365
He Met Leu Gly Gin Leu Ala Asn Glu Lys Asn Leu Ala Tyr Lys Lys 370 375 380 Leu Glu Gin Glu Lys Asp Glu Gin Leu Tyr Arg Lys Ser Leu Asp He 385 390 395 400
Ala Arg Ala Lys He Glu Ser Ser Lys Ala Ser Leu Asp Ala Ala Asn
405 410 415
Leu Ser Phe Ala Asn He Lys Arg Lys Tyr Asp Ala Asn Leu Val Asp 420 425 430
Phe Thr Thr Tyr Leu Arg Gly Leu Thr Thr Arg Phe Asp Ala Glu Val
435 440 445
Ala Tyr Asn Leu Ala Leu Asn Asn Tyr Glu Val Gin Lys Ala Asn Tyr 450 455 460 He Phe Asn Ser Gly His Lys He Asp Asp Tyr Val His 465 470 475
(2) INFORMATION FOR SEQ ID NO: 124: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 412 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...412
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124:
Met Leu Ser Phe He Ser Ala Phe Asp Lys Arg Gly Val Ser He Arg 1 5 10 15 Leu Leu Thr Ala Leu Leu Leu Leu Phe Ser Leu Gly Leu Ala Lys Asp 20 25 30
Leu Glu He Gin Thr Phe Val Ala Lys Tyr Leu Ser Lys Asn Gin Lys
35 40 45
He Gin Ala Leu Gin Glu Gin He Asp Ala Leu Asp Ser Gin Glu Lys 50 55 60
Val Val Ser Lys Trp Asp Asn Pro He Leu Tyr Leu Gly Tyr Asn Asn 65 70 75 80
Ala Asn Val Ser Asp Phe Phe Arg Leu Asp Ser Thr Leu Met Gin Asn 85 90 95 Met Ser Leu Gly Leu Ser Gin Lys Val Asp Leu Asn Gly Lys Lys Leu 100 105 110
Thr Gin Ser Lys Met He Asn Leu Glu Lys Gin Lys Lys He Leu Glu
115 120 125
Leu Lys Lys Thr Lys Gin Gin Leu Val He Asn Leu Met He Asn Gly 130 135 140
He Glu Asn Tyr Lys Asn Gin Gin Glu He Glu Leu Leu Asn Thr Ala
145 150 155 160
He Lys Asn Leu Glu Asn Thr Leu Tyr Gin Ala Asn His Ser Ser Ser
165 170 175 Pro Asp Leu He Ala He Ala Lys Leu Glu He Leu Lys Ser Leu Leu 180 185 190
Glu He Gin Lys Asn Asp Leu Glu Val Ala Leu Ser Ser Ser His Tyr
195 200 205
Ser Met Gly Glu Leu Thr Phe Lys Glu Asn Glu He Leu Ser He Ala 210 215 220
Pro Lys Asn Phe Glu Phe Asn Asn Glu Gin Glu Leu His Asn He Ser
225 230 235 240
Ala Thr Asn Tyr Asp He Ala He Ala Arg Leu Asp Glu Glu Lys Ala
245 250 255 Gin Lys Asp He Thr Leu Ala Lys Lys Ser Phe Leu Glu Asp He Asn
260 265 270
Val Thr Gly Val Tyr Tyr Phe Arg Ser Lys Gin Tyr Tyr Asn Tyr Asp
275 280 285
Met Phe Ser Val Ala Leu Ser He Pro Leu Pro Leu Tyr Gly Lys Gin 290 295 300
Ala Lys Leu Val Glu Gin Lys Lys Lys Glu Ser Leu Ala Phe Lys Ser
305 310 315 320
Glu Val Glu Asn Ala Lys Asn Lys Thr Arg His Leu Ala Leu Lys Leu
325 330 335 Leu Lys Lys Leu Glu Thr Leu Gin Lys Asn Leu Glu Ser He Asn Lys
340 345 350
He He Lys Gin Asn Glu Lys He Ala Gin He Tyr Ala Leu Asp Leu
355 360 365
Lys Thr Asn Gly Asp Tyr Asn Ala Tyr Tyr Asn Ala Leu Asn Asp Lys 370 375 380
He Thr He Gin He Thr Gin Leu Glu Thr Leu Ser Ala Leu Asn Ser 385 390 395 400
Ala Tyr Leu Ser Leu Gin Asn Leu Lys Gly Leu Glu 405 410
(2) INFORMATION FOR SEQ ID NO: 125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 137 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...137
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125:
Met Arg He Val Arg Asn Leu Phe Leu Val Ser Phe Val Ala Tyr Ser 1 5 10 15
Ser Ala Phe Ala Ala Asp Leu Glu Thr Gly Thr Lys Asn Asp Lys Lys 20 25 30 Ser Gly Lys Lys Phe Tyr Lys Leu His Lys Asn His Gly Ser Glu Thr 35 40 45
Glu Thr Lys Asn Asp Lys Lys Leu Tyr Asp Phe Thr Lys Asn Ser Gly
50 55 60
Leu Glu Gly Val Asp Leu Glu Lys Ser Pro Asn Leu Lys Ser His Lys 65 70 75 80
Lys Ser Asp Lys Lys Phe Tyr Lys Gin Leu Ala Lys Asn Asn He Ala
85 90 95
Glu Gly Val Ser Met Pro He Val Asn Phe Asn Lys Ala Leu Ser Phe 100 105 110 Gly Pro Tyr Phe Glu Arg Thr Lys Ser Lys Lys Thr Gin Tyr Met Asp 115 120 125
Gly Gly Leu Met Met His He Arg Phe 130 135 (2) INFORMATION FOR SEQ ID NO: 126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...309
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126: Leu Met Pro Gin Asn Gin Leu Val He Thr He He Asp Glu Ser Gly 1 5 10 15
Ser Lys Gin Leu Lys Phe Ser Lys Asn Leu Lys Arg Asn Leu He He
20 25 30
Ser Val Val He Leu Leu Leu He Val Gly Leu Gly Val Gly Phe Leu 35 40 45
Lys Phe Leu He Ala Lys Met Asp Thr Met Thr Ser Glu Arg Asn Ala
50 55 60
Val Leu Arg Asp Phe Arg Gly Leu Tyr Gin Lys Asn Tyr Ala Leu Ala 65 70 75 80 Lys Glu He Lys Asn Lys Arg Glu Glu Leu Phe He Val Gly Gin Lys
85 90 95
He Arg Gly Leu Glu Ser Leu He Glu He Lys Lys Gly Ala Asn Gly
100 105 110
Gly Gly His Leu Tyr Asp Glu Val Asp Leu Glu Asn Leu Ser Leu Asn 115 120 125
Gin Lys His Leu Ala Leu Met Leu He Pro Asn Gly Met Pro Leu Lys
130 135 140
Thr Tyr Ser Ala He Lys Pro Thr Lys Glu Arg Asn His Pro He Lys 145 150 155 160 Lys He Lys Gly Val Glu Ser Gly He Asp Phe He Ala Pro Leu Asn 165 170 175
Thr Pro Val Tyr Ala Ser Ala Asp Gly He Val Asp Phe Val Lys Thr
180 185 190
Arg Ser Asn Ala Gly Tyr Gly Asn Leu Val Arg He Glu His Ala Phe 195 200 205
Gly Phe Ser Ser He Tyr Thr His Leu Asp His Val Asn Val Gin Pro
210 215 220
Lys Ser Phe He Gin Lys Gly Gin Leu He Gly Tyr Ser Gly Lys Ser 225 230 235 240 Gly Asn Ser Gly Gly Glu Lys Leu His Tyr Glu Val Arg Phe Leu Gly
245 250 255
Lys He Leu Asp Ala Glu Lys Phe Leu Ala Trp Asp Leu Asp His Phe
260 265 270
Gin Ser Ala Leu Glu Glu Asn Lys Phe He Glu Trp Lys Asn Leu Phe 275 280 285
Trp Val Leu Glu Asp He Val Gin Leu Gin Glu His Val Asp Lys Asp
290 295 300
Thr Leu Lys Gly Gin 305
(2) INFORMATION FOR SEQ ID NO: 127
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 332 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...332
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127:
Val Leu Tyr Phe Leu Thr Ser Leu Phe He Cys Ser Leu He Val Leu 1 5 10 15
Trp Ser Lys Lys Ser Met Leu Phe Val Asp Asn Ala Asn Lys He Gin 20 25 30 Gly Phe His His Ala Arg Thr Pro Arg Ala Gly Gly Leu Gly He Phe 35 40 45
Leu Ser Phe Ala Leu Ala Cys Tyr Leu Glu Pro Phe Glu Met Pro Phe
50 55 60
Lys Gly Pro Phe Val Phe Leu Gly Leu Ser Leu Val Phe Leu Ser Gly 65 70 75 80
Phe Leu Glu Asp He Asn Leu Ser Leu Ser Pro Lys He Arg Leu He
85 90 95
Leu Gin Ala Val Gly Val Val Cys He He Ser Ser Thr Pro Leu Val 100 105 110 Val Ser Asp Phe Ser Pro Leu Phe Ser Leu Pro Tyr Phe He Ala Phe 115 120 125
Leu Phe Ala He Phe Met Leu Val Gly He Ser Asn Ala He Asn He
130 135 140
He Asp Gly Phe Asn Gly Leu Ala Ser Gly He Cys Ala He Ala Leu 145 150 155 160
Leu Val He His Tyr He Asp Pro Ser Ser Leu Ser Cys Leu Leu Ala
165 170 175
Tyr Met Val Leu Gly Phe Met Val Leu Asn Phe Pro Ser Gly Lys He 180 185 190 Phe Leu Gly Asp Gly Gly Ala Tyr Phe Leu Gly Leu Val Cys Gly He 195 200 205
Ser Leu Leu His Leu Ser Leu Glu Gin Lys He Ser Val Phe Phe Gly
210 215 220
Leu Asn Leu Met Leu Tyr Pro Val He Glu Val Leu Phe Ser He Leu 225 230 235 240
Arg Arg Lys He Lys Arg Gin Lys Ala Thr Met Pro Asp Asn Leu His
245 250 255
Leu His Thr Leu Leu Phe Lys Phe Leu Gin Gin Arg Ser Phe Asn Tyr 260 265 270 Pro Asn Pro Leu Cys Ala Phe He Leu He Leu Cys Asn Leu Pro Phe 275 280 285
He Leu He Ser Val Leu Phe Arg Leu Asp Ala Tyr Ala Leu He Val
290 295 300
He Ser Leu Val Phe He Ala Cys Tyr Leu He Gly Tyr Ala Tyr Leu 305 310 315 320
Asn Arg Gin Val Cys Ala Leu Glu Lys Arg Ala Phe 325 330
(2) INFORMATION FOR SEQ ID NO: 128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...271
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 128:
Met Asn He Phe Lys Arg He He Cys Val Thr Ala He Val Leu Gly 1 5 10 15
Phe Phe Asn Leu Leu Asp Ala Lys His His Lys Glu Lys Lys Glu Asp
20 25 30
His Lys He Thr Arg Glu Leu Lys Val Gly Ala Asn Pro Val Pro His 35 40 45 Ala Gin He Leu Gin Ser Val Val Asp Asp Leu Lys Glu Lys Gly He 50 55 60
Lys Leu Val He Val Ser Phe Thr Asp Tyr Val Leu Pro Asn Leu Ala 65 70 75 80
Leu Asn Asp Gly Ser Leu Asp Ala Asn Tyr Phe Gin His Arg Pro Tyr 85 90 95
Leu Asp Arg Phe Asn Leu Asp Arg Lys Met His Leu Val Gly Leu Ala
100 105 110
Asn He His Val Glu Pro Leu Arg Phe Tyr Ser Gin Lys He Thr Asp 115 120 125 He Lys Asn Leu Lys Lys Gly Ser Val He Ala Val Pro Asn Asp Pro 130 135 140
Ala Asn Gin Gly Arg Ala Leu He Leu Leu His Lys Gin Gly Leu He 145 150 155 160
Ala Leu Lys Asp Pro Ser Asn Leu Tyr Ala Thr Glu Phe Asp He Val 165 170 175
Lys Asn Pro Tyr Asn He Lys He Lys Pro Leu Glu Ala Ala Leu Leu
180 185 190
Pro Lys Val Leu Gly Asp Val Asp Gly Ala He He Thr Gly Asn Tyr 195 200 205 Ala Leu Gin Ala Lys Leu Thr Gly Ala Leu Phe Ser Glu Asp Lys Asp 210 215 220
Ser Pro Tyr Ala Asn Leu Val Ala Ser Arg Glu Asp Asn Ala Gin Asp 225 230 235 240
Glu Ala He Lys Ala Leu He Glu Ala Leu Gin Ser Glu Lys Thr Arg 245 250 255
Lys Phe He Leu Asp Thr Tyr Lys Gly Ala He He Pro Ala Phe 260 265 270
(2) INFORMATION FOR SEQ ID NO: 129:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 316 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...316
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129:
Met Gin Glu Phe Ser Leu Trp Cys Asp Phe He Glu Arg Asp Phe Leu 1 5 10 15
Glu Asn Asp Phe Leu Lys Leu He Asn Lys Gly Ala He Cys Gly Ala
20 25 30
Thr Ser Asn Pro Ser Leu Phe Cys Glu Ala He Thr Lys Ser Ala Phe
35 40 45 Tyr Gin Asp Glu He Ala Lys Leu Lys Gly Lys Lys Ala Lys Glu He 50 55 60
Tyr Glu Thr Leu Ala Leu Lys Asp He Leu Gin Ala Ser Ser Ala Leu 65 70 75 80
Met Pro Leu Tyr Glu Lys Asp Pro Asn Asn Gly Tyr He Ser Leu Glu 85 90 95
He Asp Pro Phe Leu Glu Asp Asp Ala He Lys Ser He Asp Glu Ala
100 105 110
Lys Arg Leu Phe Lys Thr Leu Asn Arg Pro Asn Val Met He Lys Val 115 120 125 Pro Ala Ser Glu Ser Ala Phe Glu Val He Ser Ala Leu Ala Gin Ala 130 135 140
Ser He Pro He Asn Val Thr Leu Val Phe Ser Pro Lys He Ala Gly 145 150 155 160
Glu He Ala Gin He Leu Ala Lys Glu Ala Arg Lys Arg Ala Val He 165 170 175
Ser Val Phe Val Ser Arg Phe Asp Lys Glu He Asp Pro Leu Val Pro
180 185 190
Gin Asn Leu Gin Ala Gin Ser Gly He Met Asn Ala Thr Glu Cys Tyr 195 200 205 Tyr Gin He Asn Gin His Ala Asn Lys Leu He Ser Thr Leu Phe Ala 210 215 220
Ser Thr Gly Val Lys Ser Asn Ser Leu Ala Lys Asp Tyr Tyr He Lys 225 230 235 240
Ala Leu Cys Phe Lys Asn Ser He Asn Thr Ala Pro Leu Asp Ala Leu 245 250 255
Asn Ala Tyr Leu Leu Asp Pro Asn Thr Glu Cys Gin Thr Pro Leu Lys
260 265 270
He Thr Glu He Glu Ala Phe Lys Lys Glu Leu Lys Thr His Asn He 275 280 285 Asp Leu Glu Asn Thr Ala Gin Lys Leu Leu Lys Glu Gly Leu He Ala 290 295 300
Phe Lys Gin Ser Phe Glu Lys Leu Leu Ser Ser Phe 305 310 315 (2) INFORMATION FOR SEQ ID NO: 130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...260
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130: Met Lys Thr Asn Gly His Phe Lys Asp Phe Ala Trp Lys Lys Cys Phe 1 5 10 15
Leu Gly Ala Ser Val Val Ala Leu Leu Val Gly Cys Ser Pro His He
20 25 30
He Glu Thr Asn Glu Val Ala Leu Lys Leu Asn Tyr His Pro Ala Ser 35 40 45
Glu Lys Val Gin Ala Leu Asp Glu Lys He Leu Leu Leu Arg Pro Ala
50 55 60
Phe Gin Tyr Ser Asp Asn He Ala Lys Glu Tyr Glu Asn Lys Phe Lys 65 70 75 80 Asn Gin Thr Thr Leu Lys Val Glu Glu He Leu Gin Asn Gin Gly Tyr
85 90 95
Lys Val He Asn Val Asp Ser Ser Asp Lys Asp Asp Phe Ser Phe Ala
100 105 110
Gin Lys Lys Glu Gly Tyr Leu Ala Val Ala Met Asn Gly Glu He Val 115 120 125
Leu Arg Pro Asp Pro Lys Arg Thr He Gin Lys Lys Ser Glu Pro Gly
130 135 140
Leu Leu Phe Ser Thr Gly Leu Asp Lys Met Glu Arg Val Leu He Pro 145 150 155 160 Ala Gly Phe Val Lys Val Thr He Leu Glu Pro Met Ser Gly Glu Ser
165 170 175
Leu Asp Ser Phe Thr Met Asp Leu Ser Glu Leu Asp He Gin Glu Lys
180 185 190
Phe Leu Lys Thr Thr His Ser Ser His Ser Gly Gly Leu Val Ser Thr 195 200 205
Met Val Lys Gly Thr Asp Asn Ser Asn Asp Ala He Lys Ser Ala Leu
210 215 220
Asn Lys He Phe Ala Ser He Met Gin Glu Met Asp Lys Lys Leu Thr 225 230 235 240 Gin Arg Asn Leu Glu Ser Tyr Gin Lys Asp Ala Lys Glu Leu Lys Asn
245 250 255
Lys Arg Asn Arg 260 (2) INFORMATION FOR SEQ ID NO: 131:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1382 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...1382
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131: Leu Asn Phe Asn Asn Leu Thr Ala Asn Gly Ala Leu Asn Phe Asn Gly 1 5 10 15
Tyr Ala Pro Ser Leu Thr Lys Ala Leu Met Asn Val Ser Gly Gin Phe
20 25 30
Val Leu Gly Asn Asn Gly Asp He Asn Leu Ser Asp He Asn He Phe 35 40 45
Asp Asn He Thr Lys Ser Val Thr Tyr Asn He Leu Asn Ala Gin Lys
50 55 60
Gly He Thr Gly He Ser Gly Ala Asn Gly Tyr Glu Lys He Leu Phe 65 70 75 80 Tyr Gly Met Lys He Gin Asn Ala Thr Tyr Ser Asp Asn Asn Asn He
85 90 95
Gin Thr Trp Ser Phe He Asn Pro Leu Asn Ser Ser Gin He He Gin
100 105 110
Glu Ser He Lys Asn Gly Asp Leu Thr He Glu Val Leu Asn Asn Pro 115 120 125
Asn Ser Ala Ser Asn Thr He Phe Asn He Ala Pro Glu Leu Tyr Asn
130 135 140
Tyr Gin Asp Ser Lys Gin Asn Pro Thr Gly Tyr Ser Tyr Asp Tyr Ser 145 150 155 160 Asp Asn Gin Ala Gly Thr Tyr Tyr Leu Thr Ser Asn He Lys Gly Leu
165 170 175
Phe Thr Pro Lys Gly Ser Gin Thr Pro Gin Thr Pro Gly Thr Tyr Ser
180 185 190
Pro Phe Asn Gin Pro Leu Asn Ser Leu Asn He Tyr Asn Lys Gly Phe 195 200 205
Ser Ser Glu Asn Leu Lys Thr Leu Leu Gly He Leu Ser Gin Asn Ser
210 215 220
Ala Thr Leu Lys Glu Met He Glu Ser Asn Gin Leu Asp Asn He Thr 225 230 235 240 Asn He Asn Glu Val Leu Gin Leu Leu Asp Lys He Lys He Thr Gin
245 250 255
Ala Gin Lys Gin Ala Leu Leu Glu Thr He Asn His Leu Thr Asp Asn
260 265 270
He Asn Gin Thr Phe Asn Asn Gly Asn Leu Val He Gly Ala Thr Gin 275 280 285
Asp Asn Val Thr Asn Ser Thr Ser Ser He Trp Phe Gly Gly Asn Gly
290 295 300
Tyr Ser Ser Pro Cys Ala Leu Asp Ser Ala Thr Cys Ser Ser Phe Arg 305 310 315 320 Asn Thr Tyr Leu Gly Gin Leu Leu Gly Ser Thr Ser Pro Tyr Leu Gly
325 330 335
Tyr He Asn Ala Asp Phe Lys Ala Lys Ser He Tyr He Thr Gly Thr
340 345 350
He Gly Ser Ser Asn Ala Phe Glu Ser Gly Gly Ser Ala Asp Val Thr 355 360 365
Phe Gin Ser Ala Asn Asn Leu Val Leu Asn Lys Ala Asn He Glu Ala
370 375 380
Gin Ala Thr Asp Asn He Phe Asn Leu Leu Gly Gin Glu Gly He Asp 385 390 395 400 Lys He Phe Asn Gin Gly Asn Leu Ala Asn Val Leu Ser Gin Met Ala
405 410 415
Met Glu Lys He Lys Gin Ala Gly Gly Leu Gly Asn Phe He Glu Asn
420 425 430
Ala Leu Ser Pro Leu Ser Lys Glu Leu Pro Ala Ser Leu Gin Asp Glu 435 440 445 Thr Leu Gly Gin Leu He Gly Gin Asn Asn Leu Asp Asp Leu Leu Asn
450 455 460
Asn Ser Gly Val Met Asn Glu He Gin Asn He He Ser Gin Lys Leu 465 470 475 480 Ser He Phe Gly Asn Phe Val Thr Pro Ser He He Glu Asn Tyr Leu
485 490 495
Ala Lys Gin Ser Leu Lys Ser Met Leu Asp Asp Lys Gly Leu Leu Asn
500 505 510
Phe He Gly Gly Tyr He Asp Ala Ser Glu Leu Ser Ser He Leu Gly 515 520 525
Val He Leu Lys Asp He Thr Asn Pro Pro Thr Ser Leu Gin Lys Asp
530 535 540
He Gly Val Val Ala Asn Asp Leu Leu Asn Glu Phe Leu Gly Gin Asp 545 550 555 560 Val Val Lys Lys Leu Glu Ser Gin Gly Leu Val Ser Asn He He Asn
565 570 575
Asn Val He Ser Gin Gly Gly Leu Ser Gly Val Tyr Asn Gin Gly Leu
580 585 590
Gly Ser Val Leu Pro Pro Ser Leu Gin Asn Ala Leu Lys Glu Asn Asp 595 600 605
Leu Gly Thr Leu Leu Ser Pro Arg Gly Leu His Asp Phe Trp Gin Lys
610 615 620
Gly Tyr Phe Asn Phe Leu Ser Asn Gly Tyr Val Phe Val Asn Asn Ser 625 630 635 640 Ser Phe Ser Asn Ala Thr Gly Gly Ser Leu Asn Phe Val Ala Asn Lys
645 650 655
Ser He He Phe Asn Gly Asp Asn Thr He Asp Phe Ser Lys Tyr Gin
660 665 670
Gly Ala Leu He Phe Ala Ser Asn Gly Val Ser Asn He Asn He Thr 675 680 685
Thr Leu Asn Ala Thr Asn Gly Leu Ser Leu Asn Ala Gly Leu Asn Asn
690 695 700
Val Ser Val Gin Lys Gly Glu He Cys He Asn Leu Ala Asn Cys Pro 705 710 715 720 Thr Thr Lys Asn Ser Ser Pro Ala Asn Ser Ser Val Thr Pro Thr Asn
725 730 735
Glu Ser Leu Ser Val His Ala Asn Asn Phe Thr Phe Leu Gly Thr He
740 745 750
He Ser Asn Gly Ala He Asp Leu Ser Gin Val Thr Asn Asn Ser Val 755 760 765
He Gly Thr Leu Asn Leu Asn Glu Asn Ala Thr Leu Gin Ala Asn Asn
770 775 780
Leu Thr He Thr Asn Ala Phe Asn Asn Ala Ser Asn Ser Thr Ala Asn 785 790 795 800 He Asp Gly Asn Phe Thr Leu Asn Gin Gin Ala Thr Leu Ser Thr Asn
805 810 815
Ala Ser Gly Leu Asn Val Met Gly Asn Phe Asn Ser Tyr Gly Asp Leu
820 825 830
Val Phe Asn Leu Ser His Ser Val Ser His Ala He He Asn Thr Gin 835 840 845
Gly Thr Ala Thr He Met Ala Asn Asn Asn Pro Leu He Gin Phe Asn
850 855 860
Ala Ser Ser Lys Glu Val Gly Thr Tyr Thr Leu He Asp Ser Ala Lys 865 870 875 880 Ala He Tyr Tyr Gly Tyr Asn Asn Gin He Thr Gly Gly Ser Ser Leu 885 890 895
Asp Asn Tyr Leu Lys Leu Tyr Ala Leu He Asp He Asn Gly Lys His
900 905 910
Met Val Met Thr Asp Asn Gly Leu Thr Tyr Asn Gly Gin Ala Val Ser 915 920 925
Val Lys Asp Gly Gly Leu Val Val Gly Phe Lys Asp Ser Gin Asn Gin
930 935 940
Tyr He Tyr Thr Ser He Leu Tyr Asn Lys Val Lys He Ala Val Ser 945 950 955 960 Asn Asp Pro He Asn Asn Pro Gin Ala Pro Thr Leu Lys Gin Tyr He
965 970 975
Ala Gin He Gin Gly Val Gin Ser Val Asp Ser He Asp Gin Ala Gly
980 985 990
Gly Asn Gin Ala He Asn Trp Leu Asn Lys He Phe Glu Thr Lys Gly 995 1000 1005
Ser Pro Leu Phe Ala Pro Tyr Tyr Leu Glu Ser His Ser Thr Lys Asp
1010 1015 1020
Leu Thr Thr He Ala Gly Asp He Ala Asn Thr Leu Glu Val He Ala 1025 1030 1035 1040 Asn Pro Asn Phe Lys Asn Asp Ala Thr Asn He Leu Gin He Asn Thr
1045 1050 1055
Tyr Thr Gin Gin Met Ser Arg Leu Ala Lys Leu Ser Asp Thr Ser Thr
1060 1065 1070
Phe Ala Arg Ser Asp Phe Leu Glu Arg Leu Glu Ala Leu Lys Asn Lys 1075 1080 1085
Arg Phe Ala Asp Ala He Pro Asn Ala Met Asp Val He Leu Lys Tyr
1090 1095 1100
Ser Gin Arg Asn Arg Val Lys Asn Asn Val Trp Ala Thr Gly Val Gly 1105 1110 1115 1120 Gly Ala Ser Phe He Ser Gly Gly Thr Gly Thr Leu Tyr Gly He Asn
1125 1130 1135
Val Gly Tyr Asp Arg Phe He Lys Gly Val He Val Gly Gly Tyr Ala
1140 1145 1150
Ala Tyr Gly Tyr Ser Gly Phe His Ala Asn He Thr Gin Ser Gly Ser 1155 1160 1165
Ser Asn Val Asn Val Gly Val Tyr Ser Arg Ala Phe He Lys Arg Ser
1170 1175 1180
Glu Leu Thr Met Ser Leu Asn Glu Thr Trp Gly Tyr Asn Lys Thr Phe 1185 1190 1195 1200 He Asn Ser Tyr Asp Pro Leu Leu Ser He He Asn Gin Ser Tyr Arg
1205 1210 1215
Tyr Asp Thr Trp Thr Thr Asp Ala Lys He Asn Tyr Gly Tyr Asp Phe
1220 1225 1230
Met Phe Lys Asp Lys Ser Val He Phe Lys Pro Gin Val Gly Leu Ser 1235 1240 1245
Tyr Tyr Tyr He Gly Leu Ser Gly Leu Arg Gly He Met Asp Asp Pro
1250 1255 1260
He Tyr Asn Gin Phe Arg Ala Asn Ala Asp Pro Asn Lys Lys Ser Val 1265 1270 1275 1280 Leu Thr He Asn Phe Ala Leu Glu Ser Arg His Tyr Phe Asn Lys Asn
1285 1290 1295
Ser Tyr Tyr Phe Val He Ala Asp Val Gly Arg Asp Leu Phe He Asn
1300 1305 1310
Ser Met Gly Asp Lys Met Val Arg Phe He Gly Asn Asn Thr Leu Ser 1315 1320 1325 Tyr Arg Asp Gly Gly Arg Tyr Asn Thr Phe Ala Ser He He Thr Gly
1330 1335 1340
Gly Glu He Arg Leu Phe Lys Thr Phe Tyr Val Asn Ala Gly He Gly 1345 1350 1355 1360 Ala Arg Phe Gly Leu Asp Tyr Lys Asp He Asn He Thr Gly Asn He
1365 1370 1375
Gly Met Arg Tyr Ala Phe 1380 (2) INFORMATION FOR SEQ ID NO: 132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...262
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 132: Met Lys Lys He Gly Leu Ser Leu Cys Leu Val Leu Ser Leu Gly Phe 1 5 10 15
Leu Lys Ala His Glu Val Ser Ala Glu Glu He Ala Asp He Phe Tyr
20 25 30
Lys Leu Asn Ala Lys Glu Pro Lys Met Lys He Asn His Thr Lys Gly 35 40 45
Phe Cys Ala Lys Gly Val Phe Leu Pro Asn Pro Gin Ala Arg Glu Asp
50 55 60
Leu Glu Val Pro Leu Leu Asn Glu Lys Glu He Pro Ala Ser Val Arg 65 70 75 80 Tyr Ser Leu Gly Gly Val Ala Met Asp Asp Lys Ser Lys Val Arg Gly
85 90 95
Met Ala Leu Lys Leu Glu Asn Gin Asn Ala Ser Trp Thr Met Val Met
100 105 110
Leu Asn Thr Glu He Asn Phe Ala Lys Asn Pro Glu Glu Phe Ala Gin 115 120 125
Phe Phe Glu Met Arg Leu Pro Lys Asn Gly Lys Val Asp Glu Ala Arg
130 135 140
He Lys Lys Leu Tyr Glu Glu Val Pro Ser Tyr Arg Asn Phe Ala Ala 145 150 155 160 Tyr Met Lys Thr He Gly He Ser Ser Ser Val Ala Asn Thr Pro Tyr
165 170 175
Tyr Ser Val His Ala Phe Lys Phe Lys Asp Lys Lys Glu Lys Leu Leu
180 185 190
Pro Ala Arg Trp Lys Phe Val Pro Lys Glu Gly Val Lys Tyr Leu Asn 195 200 205 Pro Gin Glu Leu Lys Gin Lys Asp Ser Asn Tyr Leu Leu Ser Ser Phe
210 215 220
Gin Gin His Leu Lys Asn Lys Pro He Glu Tyr Gin Met Tyr Leu Val 225 230 235 240 Phe Ala Asn Gin Asn Asp Ala Thr Asn Asp Thr Thr Ala Leu Trp Lys
245 250 255
Gly Ser He Arg Asn Tyr 260 (2) INFORMATION FOR SEQ ID NO: 133:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 246 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...246
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133: Met Lys Gin Phe Lys Lys Lys Pro Lys Lys He Lys Arg Ser His Gin 1 5 10 15
Asn Gin Lys Thr He Leu Lys Arg Pro Leu Trp Leu Met Pro Leu Leu
20 25 30
He Gly Gly Phe Ala Ser Gly Val Tyr Ala Asp Gly Thr Asp He Leu 35 40 45
Gly Leu Ser Trp Gly Glu Lys Ser Gin Lys Val Cys Val His Arg Pro
50 55 60
Trp Tyr Ala He Trp Ser Cys Asp Lys Trp Glu Glu Lys Thr Gin Gin 65 70 75 80 Phe Thr Gly Asn Gin Leu He Thr Lys Thr Trp Ala Gly Gly Asn Ala
85 90 95
Ala Asn Tyr Tyr His Ser Gin Asn Asn Gin Asp He Thr Ala Asn Leu
100 105 110
Lys Asn Asp Asn Gly Thr Tyr Phe Leu Ser Gly Leu Tyr Asn Tyr Thr 115 120 125
Gly Gly Glu Tyr Asn Gly Gly Asn Leu Asp He Glu Leu Gly Ser Asn
130 135 140
Ala Thr Phe Asn Leu Gly Ala Ser Ser Gly Asn Ser Phe Thr Ser Trp 145 150 155 160 Tyr Pro Asn Gly His Thr Asp Val Thr Phe Ser Ala Gly Thr He Asn
165 170 175
Val Asn Asn Ser Val Glu Val Gly Asn Arg Val Gly Ser Gly Ala Gly
180 185 190
Thr His Thr Gly Thr Ala Thr Leu Asn Leu Asn Ala Asn Lys Val Thr 195 200 205 Ile Asn Ser Asn He Ser Ala Tyr Lys Thr Ser Gin Val Asn Val Gly
210 215 220
Asn Ala Asn Ser Val He Thr He Asn Ser Val Ser Leu Asn Gly Glu
225 230 235 240 Tyr Leu Gin Phe Phe Ser
245
(2) INFORMATION FOR SEQ ID NO: 134: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 245 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...245
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134:
Met He Lys Lys Thr Leu Ala Ser Val Leu Leu Gly Leu Ser Leu Met 1 5 10 15 Ser Val Leu Asn Ala Lys Glu Cys Val Ser Pro He Thr Arg Ser Val 20 25 30
Lys Tyr His Gin Gin Ser Ala Glu He Arg Ala Leu Gin Leu Gin Ser
35 40 45
Tyr Lys Met Ala Lys Met Ala Leu Asp Asn Asn Leu Lys Leu Val Lys 50 55 60
Asp Lys Lys Pro Ala Val He Leu Asp Leu Asp Glu Thr Val Leu Asn 65 70 75 80
Thr Phe Asp Tyr Ala Gly Tyr Leu Val Lys Asn Cys He Lys Tyr Thr 85 90 95 Pro Glu Thr Trp Asp Lys Phe Glu Lys Glu Gly Ser Leu Thr Leu He 100 105 110
Pro Gly Ala Leu Asp Phe Leu Glu Tyr Ala Asn Ser Lys Gly Val Lys
115 120 125
He Phe Tyr He Ser Asn Arg Thr Gin Lys Asn Lys Ala Phe Thr Leu 130 135 140
Lys Thr Leu Lys Ser Phe Lys Leu Pro Gin Val Ser Glu Glu Ser Val
145 150 155 160
Leu Leu Lys Glu Lys Gly Lys Pro Lys Ala Val Arg Arg Glu Leu Val
165 170 175 Ala Lys Asp Tyr Ala He Val Leu Gin Val Gly Asp Thr Leu His Asp
180 185 190
Phe Asp Ala He Phe Ala Lys Asp Ala Lys Asn Ser Gin Glu Gin Gin
195 200 205
Ala Lys Val Leu Gin Asn Ala Gin Lys Phe Gly Thr Glu Trp He He 210 215 220 Leu Pro Asn Ser Leu Tyr Gly Thr Trp Glu Asp Gly Pro He Lys Ala 225 230 235 240
Trp Gin Asn Lys Lys 245
(2) INFORMATION FOR SEQ ID NO:135:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 288 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...288
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135:
Leu Trp Cys Leu Lys Thr Pro He He Gly His Gly Met Lys Lys Lys 1 5 10 15
Ala Lys Val Phe Trp Cys Cys Phe Lys Met He Arg Trp Leu Tyr Leu 20 25 30 Ala Val Phe Phe Leu Leu Ser Val Ser Asp Ala Lys Glu He Ala Met 35 40 45
Gin Arg Phe Asp Lys Gin Asn His Lys He Phe Glu He Leu Ala Asp
50 55 60
Lys Val Ser Ala Lys Asp Asn Val He Thr Ala Ser Gly Asn Ala He 65 70 75 80
Leu Leu Asn Tyr Asp Val Tyr He Leu Ala Asp Lys Val Arg Tyr Asp
85 90 95
Thr Lys Thr Lys Glu Ala Leu Leu Glu Gly Asn He Lys Val Tyr Arg 100 105 110 Gly Glu Gly Leu Leu Val Lys Thr Asp Tyr Val Lys Leu Ser Leu Asn 115 120 125
Glu Lys Tyr Glu He He Phe Pro Phe Tyr Val Gin Asp Ser Val Ser
130 135 140
Gly He Trp Val Ser Ala Asp He Ala Ser Gly Lys Asp Gin Lys Tyr 145 150 . 155 160
Lys He Lys Asn Met Ser Ala Ser Gly Cys Ser He Asp Asn Pro He
165 170 175
Trp His Val Asn Ala Thr Ser Gly Ser Phe Asn Met Gin Lys Ser His 180 185 190 Leu Ser Met Trp Asn Pro Lys He Tyr Val Gly Asp He Pro Val Leu 195 200 205
Tyr Leu Pro Tyr He Phe Met Ser Thr Ser Asn Lys Arg Thr Thr Gly
210 215 220
Phe Leu Tyr Pro Glu Phe Gly Thr Ser Asn Leu Asp Gly Phe He Tyr 225 230 235 240 Leu Gin Pro Phe Tyr Leu Ala Pro Lys Asn Ser Trp Asp Met Thr Phe
245 250 255
Thr Pro Gin He Arg Tyr Lys Arg Gly Phe Gly Leu Asn Phe Glu Ala 260 265 270 Arg Tyr He Asn Ser Lys Thr Gin Val Phe He Gin Cys Ala Leu Phe 275 280 285
(2) INFORMATION FOR SEQ ID NO:136: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 128 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...128
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136:
Leu Met Phe Lys Lys Met Cys Leu Ser Leu Leu Met He Ser Gly Val 1 5 10 15 Cys Val Gly Ala Lys Asp Leu Asp Phe Lys Leu Asp Tyr Arg Ala Thr 20 25 30
Gly Gly Lys Phe Met Gly Lys Met Thr Asp Ser Ser Leu Leu Ser He
35 40 45
Thr Ser Met Asn Asp Glu Pro Val Val He Lys Asn Leu He Val Asn 50 55 60
Arg Gly Asn Ser Cys Glu Ala Thr Lys Lys Val Glu Pro Lys Phe Gly 65 70 75 80
Asp Lys Phe Lys Lys Glu Lys Leu Phe Asp His Glu Leu Lys Tyr Ser 85 90 95 Gin Gin He Phe Tyr Arg Leu Asp Cys Lys Pro Asn Gin Leu Leu Glu 100 105 110
Val Lys He He Thr Asp Lys Gly Glu Tyr Tyr His Lys Phe Ser Lys 115 120 125 (2) INFORMATION FOR SEQ ID NO: 137:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 169 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...169
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137: Met Gin Ala Leu Lys Ser Leu Leu Glu Val He Thr Lys Leu Gin Asn 1 5 10 15
Leu Gly Gly Tyr Leu Met His He Ala He Phe He He Phe He Trp
20 25 30
He Gly Gly Leu Lys Phe Val Pro Tyr Glu Ala Glu Gly He Ala Pro 35 40 45
Phe Val Ala Asn Ser Pro Phe Phe Ser Phe Met Tyr Lys Phe Glu Lys
50 55 60
Pro Ala Tyr Lys Gin His Lys Met Ser Glu Ser Gin Ser Met Gin Glu 65 70 75 80 Glu Met Gin Asp Asn Pro Lys He Val Glu Asn Lys Glu Trp His Lys
85 90 95
Glu Asn Arg Thr Tyr Leu Val Ala Glu Gly Leu Gly He Thr He Met
100 105 110
He Leu Gly He Leu Val Leu Leu Gly Leu Trp Met Pro Leu Met Gly 115 120 125
Val Val Gly Gly Leu Leu Val Ala Gly Met Thr He Thr Thr Leu Phe
130 135 140
Phe Phe He His Asn Ala Arg Ser Val Cys Gin Ser Ala Phe Pro Met 145 150 155 160 Ala Phe Trp Gly Trp Lys Ala Ser Gly
165
(2) INFORMATION FOR SEQ ID NO: 138: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 487 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...487
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 138 :
Met He Glu Trp Met Gin Asn His Arg Lys Tyr Leu Val Val Thr He 1 5 10 15 Trp He Ser Thr He Ala Phe He Ala Ala Gly Met He Gly Trp Gly 20 25 30
Gin Tyr Ser Phe Ser Leu Asp Ser Asp Ser Ala Ala Lys Val Gly Gin
35 40 45
He Lys He Ser Gin Glu Glu Leu Ala Gin Glu Tyr Arg Arg Leu Lys 50 55 60
Asp Ala Tyr Ala Glu Ser He Pro Asp Phe Lys Glu Leu Thr Glu Asp 65 70 75 80
Gin He Lys Ala Met His Leu Glu Lys Ser Ala Leu Asp Ser Leu He 85 90 95 Asn Gin Ala Leu Leu Arg Asn Phe Ala Leu Asp Leu Gly Leu Gly Ala 100 105 110
Thr Lys Gin Glu Val Ala Lys Glu He Arg Lys Thr Asn Val Phe Gin
115 120 125
Lys Asp Gly Val Phe Asp Glu Glu Leu Tyr Lys Asn He Leu Lys Gin 130 135 140
Ser His Tyr Arg Pro Lys His Phe Glu Glu Ser Val Glu Arg Leu Leu
145 150 155 160
He Leu Gin Lys He Ser Ala Leu Phe Pro Lys Thr Thr Thr Pro Leu
165 170 175 Glu Gin Ser Ser Leu Ser Leu Trp Ala Lys Leu Gin Asp Lys Leu Asp
180 185 190
He Leu He Leu Asn Pro Asn Asp Val Lys He Ser Leu Asn Glu Glu
195 200 205
Glu Met Lys Lys Tyr Tyr Glu Asn His Arg Lys Asp Phe Lys Lys Pro 210 215 220
Thr Ser Phe Lys Thr Arg Ser Leu Tyr Phe Asp Ala Ser Leu Glu Lys
225 230 235 240
Thr Asp Leu Lys Glu Leu Glu Glu Tyr Tyr His Lys Asn Lys Val Ser
245 250 255 Tyr Leu Asp Lys Glu Gly Lys Leu Gin Asp Phe Lys Ser Val Gin Glu
260 265 270
Gin Val Lys His Asp Leu Asn Met Gin Lys Ala Asn Glu Lys Ala Leu
275 280 285
Arg Ser Tyr He Ala Leu Lys Lys Gly Asn Ala Gin Asn Tyr Thr Thr 290 295 300
Gin Asp Phe Glu Lys Asn Asn Ser Pro Tyr Thr Ala Glu He Thr Gin
305 310 315 320
Lys Leu Thr Ala Leu Lys Pro Leu Glu Val Leu Lys Pro Glu Pro Phe
325 330 335 Lys Asp Gly Phe He Val Val Gin Leu Val Ser Gin He Lys Asp Glu
340 345 350
Leu Gin Asn Phe Asp Glu Ala Lys Ser Ala Leu Lys Thr Arg Leu Thr
355 360 365
Gin Glu Lys Thr Leu Met Ala Leu Gin Thr Leu Ala Lys Glu Lys Leu 370 375 380
Lys Asp Phe Lys Gly Lys Ser Val Gly Tyr Val Ser Pro Asn Phe Gly
385 390 395 400
Gly Thr He Ser Glu Leu Asn Gin Glu Glu Ser Ala Lys Phe He Asn
405 410 415 Thr Leu Phe Asn Arg Gin Glu Lys Lys Gly Phe Val Thr He Gly Asn
420 425 430
Lys Val Val Leu Tyr Gin He Thr Glu Gin Asn Phe Asn His Pro Phe
435 440 445
Ser Ala Glu Glu Asn Gin Tyr Met Gin Arg Leu Val Asn Asn Thr Lys 450 455 460 Thr Asp Phe Phe Asp Lys Ala Leu He Glu Glu Leu Lys Lys Arg Tyr
465 470 475 480
Lys He Val Lys Tyr He Gin 485
(2) INFORMATION FOR SEQ ID NO: 139:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 142 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori (ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...142
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139:
Met Lys Thr Asn Phe Tyr Lys He Lys Leu Leu Phe Ala Trp Cys Leu 1 5 10 15
He He Gly Met Phe Asn Ala Pro Leu Asn Ala Asp Gin Asn Thr Asp 20 25 30 He Lys Asp He Ser Pro Glu Asp Met Ala Leu Asn Ser Val Gly Leu 35 40 45
Val Ser Arg Asp Gin Leu Lys He Glu He Pro Lys Glu Thr Leu Glu
50 55 60
Gin Lys Val Ala He Leu Asn Asp Tyr Asn Asp Lys Asn Val Asn He 65 70 75 80
Lys Phe Asp Asp He Ser Leu Gly Ser Phe Gin Pro Asn Asp Asn Leu
85 90 95
Gly He Asn Ala Met Trp Gly He Gin Asn Leu Leu Met Ser Gin Met 100 105 110 Met Ser Asn Tyr Gly Pro Asn Asn Ser Phe Met Tyr Gly Tyr Ala Pro 115 120 125
Thr Tyr Ser Asp Ser Ser Phe Leu Pro Pro He Leu Gly Tyr 130 135 140 (2) INFORMATION FOR SEQ ID NO: 140:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...208
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140: Leu He Asn Asn Asn Asn Asn Asn Lys Lys Leu Arg Gly Phe Phe Leu 1 5 10 15
Lys Val Leu Leu Ser Leu Val Val Phe Ser Ser Tyr Gly Ser Ala Asn
20 25 30
Asp Asp Lys Glu Ala Lys Lys Glu Ala Leu Glu Lys Glu Lys Asn Thr 35 40 45
Pro Asn Gly Leu Val Tyr Thr Asn Leu Asp Phe Asp Ser Phe Lys Ala
50 55 60
Thr He Lys Asn Leu Lys Asp Lys Lys Val Thr Phe Lys Glu Val Asn 65 70 75 80 Pro Asp He He Lys Asp Glu Val Phe Asp Phe Val He Val Asn Arg
85 90 95
Val Leu Lys Lys He Lys Asp Leu Lys His Tyr Asp Pro Val He Glu
100 105 110
Lys He Phe Asp Glu Lys Gly Lys Glu Met Gly Leu Asn Val Glu Leu 115 120 125
Gin He Asn Pro Glu Val Lys Asp Phe Phe Thr Phe Lys Ser He Ser
130 135 140
Thr Thr Asn Lys Gin Arg Cys Phe Leu Ser Leu His Gly Glu Thr Arg 145 150 155 160 Glu He Leu Cys Asp Asp Lys Leu Tyr Asn Val Leu Leu Ala Val Phe
165 170 175
Asn Ser Tyr Asp Pro Asn Asp Leu Leu Lys His He Ser Thr He Glu
180 185 190
Ser Leu Lys Lys He Phe Tyr Thr He Thr Cys Glu Ala Val Tyr Leu 195 200 205
(2) INFORMATION FOR SEQ ID NO: 141:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 245 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...245 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141: Met Ala Gly Thr Gin Ala He Tyr Glu Ser Ser Ser Ala Gly Phe Leu 1 5 10 15
Ser Gin Val Ser Ser He He Ser Ser Thr Ser Gly Val Ala Gly Pro 20 25 30
Phe Ala Gly He Val Ala Gly Ala Met Thr Ala Ala He He Pro He
35 40 45
Val Val Gly Phe Thr Asn Pro Gin Met Thr Ala He Met Thr Gin Tyr 50 55 60 Asn Gin Ser He Ala Glu Ala Val Ser Val Pro Met Lys Ala Ala Asn 65 70 75 80
Gin Gin Tyr Asn Gin Leu Tyr Gin Gly Phe Asn Asp Gin Ser Met Ala
85 90 95
Val Gly Asn Asn He Leu Asn He Ser Lys Leu Thr Gly Glu Phe Asn 100 105 110
Ala Gin Gly Asn Thr Gin Ser Ala Gin He Ser Ala Val Asn Ser Gin
115 120 125
He Ala Ser He Leu Ala Ser Asn Thr Thr Pro Lys Asn Pro Ser Ala
130 135 140 He Glu Ala Tyr Ala Thr Asn Gin He Ala Val Pro Ser Val Pro Thr
145 150 155 160
Thr Val Glu Met Met Ser Gly He Leu Gly Asn He Thr Ser Ala Ala
165 170 175
Pro Lys Tyr Ala Leu Ala Leu Gin Glu Gin Leu Arg Ser Gin Ala Ser 180 185 190
Asn Ser Ser Met Asn Asp Thr Ala Asp Ser Leu Asp Ser Cys Thr Ala
195 200 205
Leu Gly Ala Leu Val Gly Ser Ser Lys Val Phe Phe Ser Cys Met Gin 210 215 220 He Ser Met Thr Pro Met Ser Val Ser Met Pro Thr Val Met Pro Asn 225 230 235 240
Thr Ser Gly Cys His 245 (2) INFORMATION FOR SEQ ID NO: 142:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 367 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...367
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 142 : Met He Lys Ser Val Glu He Glu Asn Tyr Lys Asn Phe Glu His Leu 1 5 10 15
Lys Met Glu Asn Phe Lys Leu He Asn Phe Phe Thr Gly Gin Asn Asp
20 25 30
Ala Gly Lys Thr Asn Leu Leu Glu Ala Leu Tyr Thr Asn Thr Gly Leu 35 40 45
Cys Asp Pro Thr Ala Asn Gin Val Ser Leu Pro Pro Glu His Ala Val
50 55 60
Asn He Ser Glu Phe Arg Lys He Lys Leu Asp Ala Asp Asn Leu Lys 65 70 75 80 Thr Phe Phe Tyr Gin Gly Asn Thr Ala Asn Pro He Ser He Arg Thr
85 90 95
Glu Phe Glu His Ala Thr He Pro Leu Thr He Gin Tyr Pro Thr Gin
100 105 110
Thr Ser Tyr Ser Lys Asp He Asn Leu Asn Ser Asp Asp Ala His Met 115 120 125
Thr Asn Leu He Asn Thr Thr He Thr Lys Pro Gin Leu Gin Phe Ser
130 135 140
Tyr Asn Pro Ser Leu Ser Pro Met Thr Met Thr Tyr Glu Phe Glu Arg 145 150 155 160 Gin Asn Leu Gly Leu He His Ser Asn Leu Asp Lys He Ala Gin Thr
165 170 175
Tyr Lys Glu Asn Ala Met Phe He Pro He Glu Leu Ser He Val Asn
180 185 190
Ser Leu Lys Ala Leu Glu Asn Leu Gin Leu Ala Ser Lys Glu Lys Glu 195 200 205
Leu He Glu He Leu Gin Cys Phe Asn Pro Asn He Leu Asn Ala Asn
210 215 220
Thr He Arg Lys Ser Val Tyr He Gin He Lys Asp Glu Asn Thr Pro 225 230 235 240 Leu Glu Glu Ser Pro Lys Arg Leu Leu Asn Leu Phe Gly Trp Gly Phe
245 250 255
He Lys Phe Phe He Met Val Ser He Leu He Asp Asn Arg Val Lys
260 265 270
Tyr Leu Phe He Asp Glu He Glu Ser Gly Leu His His Thr Lys Met 275 280 285
Gin Glu Phe Leu Lys Ala Leu Phe Lys Leu Ala Gin Lys Leu Gin He
290 295 300
Gin He Phe Ala Thr Thr His Asn Lys Glu Phe Leu Leu Asn Ala He 305 310 315 320 Asn Thr He Ser Asp Asn Glu Thr Gly Val Phe Lys Asp He Ala Leu
325 330 335
Phe Glu Leu Glu Lys Glu Ser Ala Ser Gly Phe He Arg His Ser Tyr
340 345 350
Ser Met Leu Glu Lys Ala Leu Tyr Arg Gly Met Glu Val Arg Gly 355 360 365
(2) INFORMATION FOR SEQ ID NO: 143:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 409 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...409 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143:
Met Ser Leu He Arg Val Asn Gly Glu Ala Phe Lys Leu Ser Leu Glu 1 5 10 15
Ser Leu Glu Glu Asp Pro Phe Glu Thr Lys Glu Thr Leu Glu Thr Leu 20 25 30
Glu Thr Leu He Lys Gin Thr Ser Val Val Leu Leu Ala Ala Gly Glu
35 40 45
Ser Lys Arg Phe Ser Arg Ala He Lys Lys Gin Trp Leu Arg Ser His 50 55 60 His Thr Pro Leu Trp Leu Ser Val Tyr Glu Ser Phe Lys Glu Ala Leu 65 70 75 80
Asp Phe Lys Glu Val He Leu Val Val Ser Glu Leu Asp Tyr Val Tyr
85 90 95
He Gin Arg His Tyr Pro Lys He Lys Leu Val Lys Gly Gly Ala Ser 100 105 110
Arg Gin Glu Ser Val Arg Asn Ala Leu Lys Val He Asp Ser Thr Tyr
115 120 125
Thr He Thr Ser Asp Val Ala Arg Gly Leu Ala Asn Met Glu Ala Leu
130 135 140 Lys Ser Leu Phe Leu Thr Leu Gin Gin Thr Ser His Tyr Cys He Ala
145 150 155 160
Pro Tyr Leu Pro Cys Tyr Asp Thr Ala He Tyr Tyr Asn Glu Ala Leu
165 170 175
Asp Arg Glu Ala He Lys Leu He Gin Thr Pro Gin Leu Ser His Thr 180 185 190
Lys Thr Leu Gin Ser Ala Leu Asn Gin Gly Gly Phe Lys Asp Glu Ser
195 200 205
Ser Ala He Leu Gin Ala Phe Pro Asn Ser Val Ser Tyr He Glu Gly
210 215 220 Ser Lys Asp Leu His Lys Leu Thr Thr Ser Gly Asp Leu Lys Phe Phe
225 230 235 240
Thr Pro Phe Phe Asn Pro Ala Lys Asp Thr Phe He Gly Met Gly Phe
245 250 255
Asp Thr His Ala Phe He Lys Asp Lys Pro Met Val Leu Gly Gly Val 260 265 270
Val Leu Asp Cys Glu Phe Gly Leu Lys Ala His Ser Asp Gly Asp Ala
275 280 285
Leu Leu His Ala Val He Asp Ala He Leu Gly Ala He Lys Gly Gly
290 295 300 Asp He Gly Glu Trp Phe Pro Asp Asn Asp Pro Lys Tyr Lys Asn Ala
305 310 315 320
Ser Ser Lys Glu Leu Leu Lys He Val Leu Asp Phe Ser Gin Ser He
325 330 335
Gly Phe Glu Leu Leu Glu Met Gly Ala Thr He Phe Ser Glu He Pro 340 345 350 Lys He Thr Pro Tyr Lys Pro Ala He Leu Glu Asn Leu Ser Gin Leu
355 360 365
Leu Gly Leu Glu Lys Ser Gin He Ser Leu Lys Ala Thr Thr Met Glu 370 375 380 Lys Met Gly Phe He Gly Lys Gin Glu Gly Leu Leu Val Gin Ala His 385 390 395 400
Val Ser Met Arg Tyr Lys Gin Lys Leu 405 (2) INFORMATION FOR SEQ ID NO: 144:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 270 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...270
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:144: Met Lys Lys Phe Val Ala Leu Gly Leu Leu Ser Ala Val Leu Ser Ser 1 5 10 15
Ser Leu Leu Ala Glu Gly Asp Gly Val Tyr He Gly Thr Asn Tyr Gin
20 25 30
Leu Gly Gin Ala Arg Leu Asn Ser Asn He Tyr Asn Thr Gly Asp Cys 35 40 45
Thr Gly Ser Val Val Gly Cys Pro Pro Gly Leu Thr Ala Asn Lys His
50 55 60
Asn Pro Gly Gly Thr Asn He Asn Trp His Ser Lys Tyr Ala Asn Gly 65 70 75 80 Ala Leu Asn Gly Phe Gly Leu Asn Val Gly Tyr Lys Lys Phe Phe Gin
85 90 95
Phe Lys Ser Leu Asp Met Thr Ser Lys Trp Phe Gly Phe Arg Val Tyr
100 105 110
Gly Leu Phe Asp Tyr Gly His Ala Asp Leu Gly Lys Gin Val Tyr Ala 115 120 125
Pro Asn Lys He Gin Leu Asp Met Val Ser Trp Gly Val Gly Ser Asp
130 135 140
Leu Leu Ala Asp He He Asp Lys Asp Asn Ala Ser Phe Gly He Phe 145 150 155 160 Gly Gly Val Ala He Gly Gly Asn Thr Trp Lys Ser Ser Ala Ala Asn
165 170 175
Tyr Trp Lys Glu Gin He He Glu Ala Lys Gly Pro Asp Val Cys Thr
180 185 190
Pro Thr Tyr Cys Asn Pro Asn Ala Pro Tyr Ser Thr Asn Thr Ser Thr 195 200 205 Val Ala Phe Gin Val Trp Leu Asn Phe Gly Val Arg Ala Asn He Tyr
210 215 220
Lys His Asn Gly Val Glu Phe Gly Val Arg Val Pro Leu Leu He Asn 225 230 235 240 Lys Phe Leu Ser Ala Gly Pro Asn Ala Thr Asn Leu Tyr Tyr His Leu
245 250 255
Lys Arg Asp Tyr Ser Leu Tyr Leu Gly Tyr Asn Tyr Thr Phe 260 265 270 (2) INFORMATION FOR SEQ ID NO: 145:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 438 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE: (A) NAME/KEY: misc_feature
(B) LOCATION 1...438
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145: Met Ala Tyr Lys Pro Asn Lys Lys Lys Leu Lys Glu Leu Arg Glu Gin 1 5 10 15
Pro Asn Leu Phe Ser He Leu Asp Lys Gly Asp Val Ala Thr Asn Asn
20 25 30
Pro Val Glu Glu Ser Asp Lys Ala Asn Lys He Gin Glu Pro Leu Pro 35 40 45
Tyr Val Val Lys Thr Gin He Asn Lys Ala Ser Met He Ser Arg Asp
50 55 60
Pro He Glu Trp Ala Lys Tyr Leu Ser Phe Glu Lys Arg Val Tyr Lys 65 70 75 80 Asp Asn Ser Lys Glu Asp Val Asn Phe Phe Ala Asn Gly Glu He Lys
85 90 95
Glu Ser Ser Arg Val Tyr Glu Ala Asn Lys Glu Gly Phe Glu Arg Arg
100 105 110
He Thr Lys Arg Tyr Asp Leu He Asp Arg Asn He Asp Arg Asn Arg 115 120 125
Glu Phe Phe He Lys Glu He Glu He Leu Thr His Thr Asn Ser Leu
130 135 140
Lys Glu Leu Lys Glu Gin Gly Leu Glu He Gin Leu Thr His His Asn 145 150 155 160 Glu Thr His Lys Lys Ala Leu Glu Asn Gly Asn Glu He Val Lys Glu
165 170 175
Tyr Asp His Leu Lys Asp He Tyr Gin Glu Val Glu Arg Thr Lys Asp
180 185 190
Gly Gly Leu Val Arg Glu He He Pro Ser He Ser Ser Ala Glu Tyr 195 200 205 Phe Lys Leu Tyr Asn Lys Leu Pro Phe Glu Ser He Asn Asn Glu Asn
210 215 220
Thr Lys Leu Asn Thr Asn Asp Asn Glu Glu Val Lys Lys Leu Glu Phe 225 230 235 240 Glu Leu Ala Lys Glu Val His He Leu He Leu Glu Gin Gin Leu Leu
245 250 255
Ser Ala Thr Asn Tyr Tyr Ser Trp He Asp Lys Asp Asp Asn Ala Asn
260 265 270
Phe Ala Trp Lys Met His Arg Leu He Asn Glu Asn Lys Leu Lys Glu 275 280 285
Asn His Leu Ser Ala Asn Asn Ala Asn Lys He Lys Gin Phe Phe Phe
290 295 300
Asn Asn Gly Ser He Leu Gly Trp Thr Lys Glu Glu Gin Ser Ala He 305 310 315 320 Gin Glu Asn Arg Asp Tyr Ser Leu Arg Ser Ala Leu Leu Ser Leu Glu
325 330 335
Glu He Ala Gin Ala Lys He Glu Leu Gin Lys Tyr Tyr Glu Ser Val
340 345 350
Tyr Val Asn Gly Asp Gly Asn Lys Arg Glu He Lys Pro Phe Lys Glu 355 360 365
He Leu Arg Asp Thr Asn Asn Phe Glu Lys Ala Tyr Lys Glu Arg Tyr
370 375 380
Asp Lys Leu Val Ser Leu Ser Ala Ala He He Gin Ala Lys Glu Gly 385 390 395 400 Gly Asn Glu Arg Pro Asn Ser Ser Ala Asn Asn Asn Asn Pro He Lys
405 410 415
Asn Thr He Glu Thr Asn Thr Ser Asn Asn He He Gin Asn Asn Asp
420 425 430
Asn He He He Gin He 435
(2) INFORMATION FOR SEQ ID NO: 146:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 215 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...215 (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 146 :
Met Gin Ala Leu Lys Ser Leu Leu Glu Val He Thr Lys Leu Gin Asn 1 5 10 15
Leu Gly Gly Tyr Leu Met His He Ala He Phe He He Phe He Trp 20 25 30 He Gly Gly Leu Lys Phe Val Pro Tyr Glu Ala Glu Gly He Ala Pro
35 40 45
Phe Val Ala Asn Ser Pro Phe Phe Ser Phe Met Tyr Lys Phe Glu Lys 50 55 60 Pro Ala Tyr Lys Gin His Lys Met Ser Glu Ser Gin Ser Met Gin Glu 65 70 75 80
Glu Met Gin Asp Asn Pro Lys He Val Glu Asn Lys Glu Trp His Lys
85 90 95
Glu Asn Arg Thr Tyr Leu Val Ala Glu Gly Leu Gly He Thr He Met 100 105 110
He Leu Gly He Leu Val Leu Leu Gly Leu Trp Met Pro Leu Met Gly
115 120 125
Val Val Gly Gly Leu Leu Val Ala Gly Met Thr He Thr Thr Leu Ser
130 135 140 Phe Leu Phe Thr Thr Pro Glu Val Phe Val Asn Gin His Phe Pro Trp
145 150 155 160
Leu Ser Gly Ala Gly Arg Leu Val Val Lys Asp Leu Ala Leu Phe Ala
165 170 175
Gly Gly Leu Phe Val Ala Gly Phe Asp Ala Lys Arg Tyr Leu Glu Gly 180 185 190
Lys Gly Phe Cys Leu Met Asp Arg Ser Ser Val Gly He Lys Thr Lys
195 200 205
Cys Ser Ser Gly Cys Cys Ser 210 215
(2) INFORMATION FOR SEQ ID NO: 147:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147:
TATACCATGG TGGGCGCTAA 20
(2) INFORMATION FOR SEQ ID NO: 148:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...23
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148: ATGAATTCGA GTAAGGATTT TTG 23 (2) INFORMATION FOR SEQ ID NO: 149:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 149: TTAACCATGG TGAAAAGCGA TA 22 (2) INFORMATION FOR SEQ ID NO: 150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...23
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150:
TAGAATTCGC ATAACGATCA ATC 23
(2) INFORMATION FOR SEQ ID NO: 151:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 151:
ATATCCATGG TGAGTTTGAT GA 22
(2) INFORMATION FOR SEQ ID NO: 152:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 152: ATGAATTCAA TTTTTTATTT TGCCA 25 (2) INFORMATION FOR SEQ ID NO: 153:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...21
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 153: AATTCCATGG TGGGGGCTAT G 21 (2) INFORMATION FOR SEQ ID NO: 154:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...23 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154: ATGAATTCTC GATAGCCAAA ATC 23 (2) INFORMATION FOR SEQ ID NO: 155:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155: AATTCCATGG TGCATAACTT CCATT 25 (2) INFORMATION FOR SEQ ID NO: 156:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 156: AAGAATTCTC TAGCATCCAA ATGGA 25 (2) INFORMATION FOR SEQ ID NO: 157:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...24
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 157: ATTTCCATGG TCATGTCTCA TATT 24 (2) INFORMATION FOR SEQ ID NO: 158:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...23
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 158: ATGAATTCCA TCTTTTATTC CAC 23 (2) INFORMATION FOR SEQ ID NO: 159:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...27
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159: AACCATGGTG ATTTTAAGCA TTGAAAG 27 (2) INFORMATION FOR SEQ ID NO : 160:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...28
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160: AAGAATTCCA CTCAAAATTT TTTAACAG 28 (2) INFORMATION FOR SEQ ID NO: 161:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 161:
GATCATCCAT ATGTTATCTT CTAAT 25
(2) INFORMATION FOR SEQ ID NO: 162:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...23
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 162:
TGAATTCAAC CATTTTAACC CTG 23
(2) INFORMATION FOR SEQ ID NO:163:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...27
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 163: TATACCATGG TGAAATTTTT TCTTTTA 27 (2) INFORMATION FOR SEQ ID NO : 164 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 164: AGAATTCAAT TGCGTCTTGT AAAAG 25 (2) INFORMATION FOR SEQ ID NO: 165:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165:
TATACCATGG TGATGGACAA ACTC 24
(2) INFORMATION FOR SEQ ID NO: 166:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...23
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 166:
ATGAATTCCC ACTTGGGGCG ATA 23
(2) INFORMATION FOR SEQ ID NO: 167:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 167:
TTATGGATCC AAACCAATTA AAACT 25 (2) INFORMATION FOR SEQ ID NO: 168:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...23
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 168:
TATCTCGAGT TATAGAGAAG GGC 23
(2) INFORMATION FOR SEQ ID NO: 169:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 169:
TTAACCATGG TGAAAAGCGA TA 22
(2) INFORMATION FOR SEQ ID NO: 170:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...24
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 170: TAGAATTCGC CTCTAAAACT TTAG 24 (2) INFORMATION FOR SEQ ID NO: 171:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 171: TTAACCATGG TGAAAAGCGA TA 22 (2) INFORMATION FOR SEQ ID NO : 172:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...23
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 172:
TAGAATTCGC ATAACGATCA ATC 23
(2) INFORMATION FOR SEQ ID NO:173:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:173
ATATCCATGG TGAGTTTGAT GA 22
(2) INFORMATION FOR SEQ ID NO: 174:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 174: ATGAATTCAA TTTTTTATTT TGCCA 25 (2) INFORMATION FOR SEQ ID NO: 175:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...23
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 175: AATTCCATGG CTATCCAAAT CCG 23 (2) INFORMATION FOR SEQ ID NO: 176:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 176:
ATGAATTCGC CAAAATCGTA GTATT 25
(2) INFORMATION FOR SEQ ID NO: 177:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...24
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 177:
GATACCATGG AATTTATGAA AAAG 24
(2) INFORMATION FOR SEQ ID NO: 178:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...25
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 178:
TGAATTCGAA AAAGTGTAGT TATAC 25 (2) INFORMATION FOR SEQ ID NO: 179:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...19
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 179:
CCCTTCATTT TAGAAATCG 19
(2) INFORMATION FOR SEQ ID NO: 180:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 180:
ATTTCAACCA ATTCAATGCG 20
(2) INFORMATION FOR SEQ ID NO: 181:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...20
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 181: GCCCCTTTTG ATTTGAAGCT 20 (2) INFORMATION FOR SEQ ID NO: 182:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 182: TCGCTCCAAG ATACCAAGAA GT 22 (2) INFORMATION FOR SEQ ID NO: 183:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 183
CTTGAATTAG GGGCAAAGAT CG 22
(2) INFORMATION FOR SEQ ID NO:184:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 184
ATGCGTTTTT ACCCAAAGAA GT 22
(2) INFORMATION FOR SEQ ID NO: 185:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 185: ATAACGCCAC TTCCTTATTG GT 22 (2) INFORMATION FOR SEQ ID NO: 186:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...19
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 186: CTTTGGGTAA AAACGCATC 19 (2) INFORMATION FOR SEQ ID NO: 187:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 187: CGATCTTTGA TCCTAATTCA 20
(2) INFORMATION FOR SEQ ID NO: 188:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...19
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 188:
ATCAAGTTGC CTATGCTGA 19
(2) INFORMATION FOR SEQ ID NO: 189:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 189:
TTGAACACTT TTGATTATGC GG 22 (2) INFORMATION FOR SEQ ID NO: 190:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...23
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 190: GGATTATGCG ATTGTTTTAC AAG 23 (2) INFORMATION FOR SEQ ID NO: 191:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...21
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 191: GTCTTTAGCA AAAATGGCGT C 21 (2) INFORMATION FOR SEQ ID NO: 192:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...21
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 192: AATGAGCGTA AGAGAGCCTT C 21 (2) INFORMATION FOR SEQ ID NO: 193:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...18
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:193 CTTATGGGGG TATTGTCA 18 (2) INFORMATION FOR SEQ ID NO: 194:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...18
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:194:
AGCATGTGGG TATCCAGC 18
(2) INFORMATION FOR SEQ ID NO: 195:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...19
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:195:
AGGTTGTTGC CTAAAGACT 19
(2) INFORMATION FOR SEQ ID NO: 196:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...18
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 196:
CTGCCTCCAC CTTTGATC 18
(2) INFORMATION FOR SEQ ID NO: 197:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...19
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:197:
ACCAATATCA ATTGGCACT 19
(2) INFORMATION FOR SEQ ID NO: 198:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...18 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 198: ACTTGGAAAA GCTCTGCA 18 (2) INFORMATION FOR SEQ ID NO: 199:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...19
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 199: CTTGCTTGTC ATATCTAGC 19 (2) INFORMATION FOR SEQ ID NO: 200:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...18
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:200: GTTGAAGTGT TGGTGCTA 18 (2) INFORMATION FOR SEQ ID NO: 201:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 201: CAAGCAAGTG GTTTGGTTTT AG 22 (2) INFORMATION FOR SEQ ID NO: 202:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...22
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 202: TGGAAAGAGC AAATCATTGA AG 22 (2) INFORMATION FOR SEQ ID NO: 203:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...21
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:203: GCCCATAATC AAAAAGCCCA T 21 (2) INFORMATION FOR SEQ ID NO: 204:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...24
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 204: CTAAAACCAA ACCACTTGCT TGTC 24 (2) INFORMATION FOR SEQ ID NO: 205:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...16
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 205:
GTAAAACGAC GGCCAG 16
(2) INFORMATION FOR SEQ ID NO:206:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...17
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:206:
CAGGAAACAG CTATGAC 17
(2) INFORMATION FOR SEQ ID NO: 207:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION 1...21
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 207: ATCTTACCTA TCACCTCAAA T 21
(2) INFORMATION FOR SEQ ID NO: 208:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: Helicobacter pylori
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION 1...21
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 208:
AGACAGCAAC ATCTTTGTGA A 21

Claims

1. An isolated nucleic acid comprising a nucleotide sequence encoding an H pylori polypeptide at least about 60% homologous to an amino acid sequence selected from the group consisting of SEQ ID NO: 74-SEQ ID NO: 146.
2. An isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori polypeptide selected from the group consisting of SEQ ID NO: 74-SEQ ID NO: 146.
3. An isolated nucleic acid which encodes an H pylori polypeptide, comprising a nucleotide sequence at least about 60% homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 73, or a complement thereof.
4. The isolated nucleic acid of claim 1, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 73, or a complement thereof.
5. An isolated nucleic acid molecule encoding an H. pylori polypeptide, comprising a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 73, or a complement thereof.
6. An isolated nucleic acid comprising a nucleotide sequence of at least 8 nucleotides in length, wherein the sequence hybridizes under stringent hybridization conditions to a nucleic acid having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 73, or a complement thereof.
7. An isolated nucleic acid comprising a nucleotide sequence encoding an
H. pylori cell envelope polypeptide or a fragment thereof, said nucleic acid selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 48, SEQ ID NO: 16, SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 7, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 11, SEQ ID NO: 71, SEQ ID NO: 17, SEQ ID NO: 57, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 21, or a complement thereof.
8. The isolated nucleic acid of claim 7, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 25, and SEQ ID NO: 48, or a complement thereof.
9. The isolated nucleic acid of claim 7, wherein said H pylori cell envelope polypeptide or a fragment thereof is an H pylori outer membrane polypeptide or a fragment thereof encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 7, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 1 1, and SEQ ID NO: 71, or a complement thereof.
10. The isolated nucleic acid of claim 9, wherein said H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 11 and SEQ ID NO:71, or a complement thereof.
11. The isolated nucleic acid of claim 9, wherein said H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 45, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 7, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, or a complement thereof.
12. An isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori cell envelope polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 98, SEQ ID NO: 121, SEQ ID NO: 89, SEQ ID NO: 83, SEQ ID NO: 118, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 80, SEQ ID NO: 1 12, SEQ ID NO: 128, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID
NO: 131, SEQ ID NO: 74, SEQ ID NO: 115, SEQ ID NO: 87, SEQ ID NO: 116, SEQ
ID NO: 84, SEQ ID NO: 144, SEQ ID NO: 90, SEQ ID NO: 130. SEQ ID NO: 78, SEQ
ID NO: 79, SEQ ID NO: 81, and SEQ ID NO: 94.
13. The isolated nucleic acid of claim 12, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 98, and SEQ ID NO: 121.
14. The isolated nucleic acid of claim 12, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 83, SEQ ID NO: 118, SEQ ID NO: 108, SEQ ID NO: 1 10, SEQ ID NO: 80, SEQ ID NO: 112, SEQ ID NO: 128, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 74, SEQ ID NO: 115, SEQ ID NO: 87, SEQ ID NO: 116, SEQ ID NO: 84, SEQ ID NO: 144, SEQ ID NO: 90, and SEQ ID NO: 130.
15. The isolated nucleic acid of claim 14, wherein said H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof selected from the group consisting of SEQ ID NO: 74, SEQ ID NO: 1 15, SEQ ID NO: 87, SEQ ID NO: 1 16, and SEQ ID NO: 84 and SEQ ID NO: 144.
16. The isolated nucleic acid of claim 14, wherein said H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 1 18, SEQ ID NO: 108, SEQ ID NO: 1 10, SEQ ID NO: 80, SEQ ID NO: 112, SEQ ID NO: 128, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, and SEQ ID NO: 131.
17. An isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori secreted polypeptide or a fragment thereof, said nucleic acid selected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 32, SEQ ID NO: 51, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 22. SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41. SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, or a complement thereof.
18. An isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori secreted polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 145, SEQ ID NO: 105, SEQ ID NO: 124, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 95, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 1 11, SEQ ID NO: 1 13, SEQ ID NO: 114, SEQ ID NO: 1 17, SEQ ID NO: 1 19, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141.
19. An isolated nucleic acid comprising a nucleotide sequence encoding an H. pylori cellular polypeptide or a fragment thereof, said nucleic acid selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 73, or a complement thereof.
20. An isolated nucleic acid comprising a nucleotide sequence encoding an H pylori cellular polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 93, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 120, SEQ ID NO: 123, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 146.
21. A probe comprising a nucleotide sequence consisting of at least 8 nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 73, or a complement thereof.
22. A recombinant expression vector comprising the nucleic acid of any of claims 1, 2, 3, 4, 5, 6, 7, 12, 17, 18, 19 or 20 operably linked to a transcription regulatory element.
23. A cell comprising a recombinant expression vector of claim 22.
24. A method for producing an H. pylori polypeptide comprising culturing a cell of claim 23 under conditions that permit expression of the polypeptide.
25. The method of claim 24, further comprising purifying the polypeptide from the cell.
26. A method for detecting the presence of a Helicobacter nucleic acid in a sample comprising:
(a) contacting a sample with a nucleic acid of any of claims 6 or 21 so that a hybrid can form between the probe and a Helicobacter nucleic acid in the sample; and
(b) detecting the hybrid formed in step (a), wherein detection of a hybrid indicates the presence of a Helicobacter nucleic acid in the sample.
27. An isolated H pylori polypeptide comprising an amino acid sequence at least about 60% homologous to an H. pylori polypeptide selected from the group consisting of SEQ ID NO: 74-SEQ ID NO: 146.
28. An isolated H. pylori polypeptide which is encoded by a nucleic acid comprising a nucleotide sequence at least about 60% homologous to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 73.
29. The isolated H. pylori polypeptide of claim 28, wherein said polypeptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 73.
30. An isolated H. pylori polypeptide which is encoded by a nucleic acid which hybridizes under stringent hybridization conditions to a nucleic acid selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 73, or a complement thereof.
31. An isolated H. pylori polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 74-SEQ ID NO: 146.
32. An isolated H. pylori cell envelope polypeptide or a fragment thereof, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 98, SEQ ID NO: 121, SEQ ID NO: 89, SEQ ID NO: 83, SEQ ID NO: 118, SEQ ID NO: 108, SEQ ID NO: 1 10, SEQ ID NO: 80, SEQ ID NO: 112, SEQ ID NO: 128, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 74, SEQ ID NO: 1 15, SEQ ID NO: 87, SEQ ID NO: 116, SEQ ID NO: 84, SEQ ID NO: 144, SEQ ID NO: 90, SEQ ID NO: 130, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 81, and SEQ ID NO: 94.
33. The isolated polypeptide of claim 32, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H pylori inner membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 98, and SEQ ID NO: 121.
34. The isolated polypeptide of claim 32, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 83, SEQ ID NO: 118, SEQ ID NO: 108, SEQ ID NO: 1 10, SEQ ID NO: 80, SEQ ID NO: 112, SEQ ID NO: 128, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 74, SEQ ID NO: 115, SEQ ID NO: 87, SEQ ID NO: 1 16, SEQ ID NO: 84, SEQ ID NO: 144, SEQ ID NO: 90, and SEQ ID NO: 130.
35. The isolated polypeptide of claim 34, wherein said H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof selected from the group consisting of SEQ ID NO: 74, SEQ ID NO: 1 15, SEQ ID NO: 87, SEQ ID NO: 1 16, and SEQ ID NO: 84 and SEQ ID NO: 144.
36. The isolated polypeptide of claim 34, wherein said H. pylori outer membrane polypeptide or a fragment thereof is an H. pylori polypeptide having a terminal phenylalanine residue or a fragment thereof selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 118, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 80, SEQ ID NO: 112, SEQ ID NO: 128, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, and SEQ ID NO: 131.
37. An isolated H. pylori cell envelope polypeptide or a fragment thereof, wherein said polypeptide is encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 48, SEQ ID NO: 16, SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 7, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 11, SEQ ID NO: 71, SEQ ID NO: 17, SEQ ID NO: 57, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 21.
38. The isolated polypeptide of claim 37, wherein said H pylori cell envelope polypeptide or a fragment thereof is an H. pylori inner membrane polypeptide or a fragment thereof encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 25, and SEQ ID NO: 48.
39. The isolated polypeptide of claim 37, wherein said H. pylori cell envelope polypeptide or a fragment thereof is an H. pylori outer membrane polypeptide or a fragment thereof encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 7, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 11, and SEQ ID NO: 71.
40. The isolated polypeptide of claim 39, wherein said H pylori outer membrane polypeptide or a fragment thereof is an H pylori polypeptide having a terminal phenylalanine residue and a C-terminal tyrosine cluster or a fragment thereof encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 42, SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 11 and SEQ ID NO:71.
41. The isolated polypeptide of claim 39, wherein said H. pylori outer membrane polypeptide or a fragment thereof is an H pylori polypeptide having a terminal phenylalanine residue or a fragment thereof encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 45, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 7, SEQ ID NO: 39, SEQ ID NO: 55, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58.
42. An isolated H. pylori cellular polypeptide or a fragment thereof, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 93, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 120, SEQ ID NO: 123, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 146.
43. An isolated H. pylori cellular polypeptide or a fragment thereof, wherein said polypeptide is encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 73.
44. An isolated H. pylori secreted polypeptide or a fragment thereof, wherein said polypeptide is selected from the group consisting of SEQ ID NO: 145, SEQ ID NO:
105, SEQ ID NO: 124, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 95, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 117, SEQ ID NO: 1 19, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141.
45. An isolated H. pylori secreted polypeptide or a fragment thereof, wherein said polypeptide is encoded by a nucleic acid selected from the group consisting of SEQ ID NO: 72, SEQ ID NO: 32, SEQ ID NO: 51, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68.
46. A fusion protein comprising an H pylori polypeptide which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 74-SEQ ID NO: 146 operatively linked to a non-H. pylori polypeptide.
47. A vaccine formulation for prophylaxis or treatment of an H. pylori infection comprising an effective amount of at least one isolated nucleic acid of any of claims 1, 2, 3, 4, 5, 6, 7, 12, 17, 18, 19, or 20.
48. A vaccine formulation for prophylaxis or treatment of an H. pylori infection comprising an effective amount of at least one H pylon polypeptide or a fragment thereof of any of claims 26, 27, 28, 29, 30, 31 , 32, 37, 42, 43, 44 or 45.
49. A vaccine formulation of claim 47, further comprising a pharmaceutically acceptable carrier.
50. A vaccine formulation of claim 48, further comprising a pharmaceutically acceptable carrier.
51. A vaccine formulation of claim 49, wherein the pharmaceutically acceptable carrier comprises an adjuvant.
52. A vaccine formulation of claim 50, wherein the pharmaceutically acceptable carrier comprises an adjuvant.
53. A vaccine formulation of claim 49, wherein the pharmaceutically acceptable carrier comprises a delivery system.
54. A vaccine formulation of claim 50, wherein the pharmaceutically acceptable carrier comprises a delivery system.
55. A vaccine formulation of claim 53, wherein the delivery system comprises a live vector.
56. A vaccine formulation of claim 54, wherein the delivery system comprises a live vector.
57. A vaccine formulation of claim 55, wherein the live vector is a bacteria or a virus.
58. A vaccine formulation of claim 56, wherein the live vector is a bacteria or a virus.
59. A vaccine formulation of claim 53, wherein the pharmaceutically acceptable carrier further comprises an adjuvant.
60. A vaccine formulation of claim 54, wherein the pharmaceutically acceptable carrier further comprises an adjuvant.
61. A method of treating or reducing a risk of H pylori infection in a subject comprising administering to a subject a vaccine formulation of claim 47, such that treatment or reduction of risk of H pylori infection occurs.
62. A method of treating or reducing a risk of H pylori infection in a subject comprising administering to a subject a vaccine formulation of claim 48, such that treatment or reduction of risk of H. pylori infection occurs.
63. A method of producing a vaccine formulation comprising: combining at least one isolated H. pylori polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 74-SEQ ID NO: 146 with a pharmaceutically acceptable carrier to thereby form a vaccine formulation.
64. A method of producing a vaccine formulation comprising:
(a) providing at least one isolated H. pylori polypeptide or a fragment thereof selected from the group consisting of SEQ ID NO: 74-SEQ ID NO: 146; and
(b) combining at least one said isolated H pylori polypeptide or a fragment thereof with a pharmaceutically acceptable carrier to thereby form a vaccine formulation.
65. A method of producing a vaccine formulation comprising:
(a) culturing a cell under condition that permit expression of an H. pylori polypeptide or a fragment thereof selected from the group consisting of SEQ ID
NO: 74-SEQ ID NO: 146;
(b) isolating said H. pylori polypetide from said cell; and
(c) combining at least one said isolated H pylori polypeptide or a fragment thereof with a pharmaceutically acceptable carrier to thereby form a vaccine formulation.
EP97913847A 1996-10-28 1997-10-28 Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof Withdrawn EP0973394A4 (en)

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US75973996A 1996-12-06 1996-12-06
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PCT/US1997/019575 WO1998018323A1 (en) 1996-10-28 1997-10-28 Nucleic acid and amino acid sequences relating to helicobacter pylori and vaccine compositions thereof
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