DE19639601A1 - Parapox viruses that contain foreign DNA, their production and their use in vaccines - Google Patents

Abstract

The invention concerns recombinantly produced parapoxviruses which carry in their genome deletions or insertions in the form of foreign hereditary information and contain other hereditary information. The invention also concerns the production of such constructs and their use in vaccines.

Description

The present invention relates to recombinant parapox viruses, their Manufacture and vaccines that contain them.

The genetically modified parapox viruses according to the invention carry in their Genome deletions and / or insertions. The deletion of genomic sections of the Parapox viruses and / or the insertion of foreign DNA leads, if necessary, to reduce or lose their pathogenicity (attenuation). By insertion cations become genetic information that represent pathogens or for other biolo encode genetically active substances into the genome of the parapox viruses builds. This foreign genetic information is used for expression, for example in Cell cultures, tissues or in intact organisms.

The recombinant parapox viruses produced according to the invention are called Vaccine used. The insertions into the genome of the parapox viruses resolve in Vaccinated a defense reaction against the pathogen caused by the foreign heir information are represented. In addition, the general immune system of the vaccine increased. (In the following, the term parapox viruses is used PPV abbreviated.)

Parapox viruses themselves can have an immunomodulatory effect because they are non-pathogens stimulate specific immune reactions in the organism. That's how preparations are made from parapox viruses, for example in veterinary medicine to increase the general successfully used my defenses. Vaccines based on recom Binary parapox viruses can be produced as a biological pro products for improved control of infectious diseases because they both build long-lasting, pathogen-specific immunity as also a very quickly occurring pathogen-specific protection in the organism induce.

For example, a preparation of parapoxvirus ovis (Baypamun®, Bayer AG, Leverkusen) successfully in veterinary medicine to increase all common defenses against infectious diseases. This is based on the PPV strain D1701 hereinafter abbreviated to D1701. Investigations The effectiveness of Baypamun showed that different fractions of the body  own immune system can be stimulated on a cellular and humoral level. Infection experiments with different target animals verified the protective effect this immunomodulator against various infectious diseases. It could shown that the protective effect occurs very quickly after treatment, but only for a relatively short time, i.e. H. usually 1 week, lasts.

Vaccines that have a pathogen-specific effect, however, require depending on the Antigens for the establishment of protection from several days to weeks, but offer then a long period of protection lasting from months to years.

The combination of the immunomodulatory principle of parapox viruses with Antigens that induce pathogen-specific protection are new and allowed thus the manufacture of products that are both fast-moving, provide broad non-pathogen protection against infection as well provide long-lasting, pathogen-specific infection protection.

The Vertebrate Smallpox (Chordopoxvirinae) family is divided into independent genera divided. The present invention relates to Genus of parapox viruses, which are both structurally and genetically distinguish the other smallpox viruses. The parapox viruses are in three different species classified (Lit. # 1):

• - Parapoxvirus ovis (also Ecthyma Contagiosum Virus, Virus der Contagious pustular dermatitis or Orf virus), which is the prototype of the Gender applies
• - Parapoxvirus bovis 1 (also Bovine Papular Stomatitis Virus or Stoma titis papulosa virus) and
• - Parapoxvirus bovis 2 (also udderpox virus, Paravaccinia virus, pseudo cowpox virus or dairy knot virus called).

Parapox virus representatives are also described, which consist of camels, red deer, chamois, seals, seals, sea lions and squirrels the. Whether this is an independent species within the Para genus poxviruses or isolates of the species described above is still not finally clarified.  

Parapox virus infections can cause animal and animal diseases Cause people (zoonotic agents). Lit. # 1 gives an overview of the clinical pictures described so far. The fight against disease is possible through the use of prophylactic measures, such as Vaccines. The vaccines available so far, which are based exclusively on Parapopxvirus ovis were developed, however, show only an unsatisfactory Effectiveness (Lit. # 2).

Knowledge of the organization of the genome of parapox viruses are currently limited to determining the size of the genome Guanine and cytosine content of the nucleic acid, comparative restriction enzyme Analyzes, the cloning of individual genome fragments, sequence analyzes of Sub-areas and resulting limited description of individual Virus genes (for overview, ref. # 1, ref. # 6). Among others, Lyttle et al. (Lit. # 6) described a VEGF gene, the exact function of which is not known.

Recombinant parapox viruses or parts of these viruses, which are recombinant Nucleic acids expressed in biological systems offer the possibility for the development of improved parapoxvirus vaccines. The present The invention therefore relates to improved parapox virus vaccines.

The invention also relates to the parapox virus as a carrier (vector) of foreign genetic information that is expressed about the virus use. Vectors based on parapox viruses were theoretically in the Literature discussed, but not yet due to technical difficulties realized. For example, it is not yet known in which parts of the genome of parapox viruses an insertion of foreign DNA and its expression is possible. The experiments, like the orthopox virus, the gene for thymidine kinase Using it as an insertion site for parapox viruses did not produce any results. Mazur and co-workers (ref. # 3) describe the identification of a genome section of parapox viruses that are similar to the thymidine kinase gene of vaccinia virus (an orthopox virus) is said to have its own extensive sub However, the existence of such a gene in parapox could be searched do not confirm viruses. Other authors (ref. # 1) were also able to use a thymidine kinase Cannot find gene in parapox viruses. Robinson and Lyttle say in 1992 that they find alternative insertion sites on the genome of parapox viruses could (Lit. # 1), but a description of these places was not given. To  no successful use of Para has been described today smallpox viruses as vectors.

Smallpox viruses based on Avipox, Capripox, Swinepox and Raccoonpox viruses or vaccinia virus are vectors for the transmission of foreign genetic Information already described. The knowledge gained cannot can be transmitted to parapox viruses. Like comparative studies always have shown again there are morphological, structural and genetic Differences between the individual genes of smallpox viruses. So you can for example the parapox viruses with serological methods from the others Genera of smallpox viruses differ, suggesting different protein patterns and related to different genetic information. For example, some smallpox viruses have the ability to Agglutination of erythrocytes. This activity is via a surface protein mediates, the so-called hemagglutinin (HA). The gene for HA is called Insertion site for foreign DNA in vaccinia virus, which belongs to the genus Orthopox viruses heard, used. Parapox viruses do not have this activity. Furthermore, in the genera Avipox and Orthopox, the gene preferred which codes for thymidine kinase (TK) as an insertion site for foreign DNA used. Own investigations and experiments by other authors (Ref. # 1) showed that this gene does not exist at all with poxpox viruses, and therefore not as Insertion point can be used. The wealth of knowledge that others have Smallpox virus is available, so it cannot affect the parapox virus be transmitted.

The present invention relates to

• 1. Recombinantly produced parapox viruses with insertions and / or dele ions.
• 2. Recombinantly produced parapox viruses with insertions and / or dele in genome sections that are not necessary for virus replication are.
• 3. Recombinantly produced parapox viruses with insertions and / or dele genome sections that are necessary for virus replication.  
• 4. Recombinantly produced parapox viruses, the insertions and / or dele ions in the areas of Hind III fragment I from D1701 contain the cannot be expressed.
• 5. Recombinantly produced parapox viruses, the insertions and / or dele ions in the areas of Hind III fragment I from D1701 contain the be expressed.
• 6. Recombinantly produced parapox viruses according to 1 to 5, insert tions and / or deletions in Hind III fragment I of D1701 or in DNA corresponding to this fragment from other parapox viruses loka are listed.
• 7. Recombinantly produced parapox viruses, the insertions and / or dele ions in the region of the VEGF gene or adjacent to it.
• 8. Recombinantly produced parapox viruses, the insertions and / or dele ions in the region of the PK gene or adjacent to it.
• 9. Recombinantly produced parapox viruses, the insertions and / or dele tion in the area of the ITR section or adjacent to it hold.
• 10. Recombinantly produced parapox viruses, the insertions and / or dele ions in the region or adjacent to the gene which is responsible for the 10 KDa protein encoded, included.
• 11. Recombinantly produced parapox viruses, the insertions and / or dele ions in the region of the gene HD1R or adjacent to it.
• 12. Recombinantly produced parapox viruses, the insertions and / or dele ions in the region of the F9L gene or adjacent to it.
• 13. Plasmid containing Hind III fragment I from D1701 or this fragment corresponding DNA from other parapox viruses.  
• 14. Plasmid containing Hind III fragment I from D1701, which in this Fragment contains deletions and / or insertions in areas for which Virus replication is necessary.
• 15. Plasmid containing Hind III fragment I from D1701, which in this Fragment deletions and / or insertions in areas not for Virus replication is necessary.
• 16. Plasmid containing Hind III fragment I from D1701, which in this Contains fragment deletions and / or insertions in the areas that are not necessary for virus replication and are in areas that are not expressed.
• 17. Plasmid containing Hind III fragment I from D1701, which in this Contains fragment deletions and / or insertions in the areas that are not necessary for virus replication and those on areas lying, which are expressed.
• 18. Plasmid containing Hind III fragment I from D1701 which is in or adjacent to contains deletions and / or insertions of the VEGF gene of this fragment.
• 19. Plasmid containing Hind III fragment I from D1701 which is in or adjacent to contains deletions and / or insertions of the PK gene of this fragment.
• 20. Plasmid containing Hind III fragment I from D1701 which is in or adjacent to to the ITR section of this fragment deletions and / or insertions contains.
• 21. Plasmid containing Hind III fragment I from D1701 which is in or adjacent to ent to deletions and / or insertions to the HD1R gene and / or F9L gene holds.
• 22. Plasmid containing DNA fragment from D1701, which is in or adjacent to the gene coding for the 10 KDa protein, deletions and / or Contains insertions.  
• 23. Plasmid-containing part of Hind III fragment I from D1701, in which Deletions and / or insertions according to 13 to 22 are included.
• 24. Plasmid according to 13 to 23, wherein the DNA fragment from D1701 by a DNA from other parapox viruses corresponding to this fragment was replaced is.
• 25. Plasmid according to 13 to 24, wherein the Hind III fragment I completely or only partially available.
• 26. Genome fragment Hind III fragment I of D1701 or parts thereof or the fragments from other parapox viruses corresponding to this fragment with the sequence according to sequence protocol ID8.
• 27. DNA section or parts thereof from Hind III fragment I of D1701 or the section corresponding to this section or parts thereof other parapox viruses that encode VEGF protein according to sequence Protocol ID1.
• 28. DNA section or parts thereof from Hind III fragment I of D1701 or the section corresponding to this section or parts thereof other parapox viruses that encode PK protein according to sequence Protocol ID2.
• 29. DNA section or parts thereof from parapox viruses for gene HD1R the sequence according to sequence protocol ID3.
• 30. DNA section or parts thereof of parapox viruses for F9L with the Sequence according to sequence protocol ID5.
• 31. DNA section or parts thereof from parapox viruses for the ITR area with the sequence according to sequence protocol ID4.
• 32. Gene products made based on the sequences of the DNA segments according to 26 to 31.  
• 33. Recombinantly produced parapox viruses according to 1 to 12, which as Inserts contain foreign DNA, which are used for immunogenic components code from other pathogens.
• 34. Recombinantly produced parapox viruses according to 1 to 12 and 33, which as Inserts contain foreign DNA that encode immune mediators.
• 35. A method for producing the viruses according to 1 to 12, 33 and 34, thereby characterized in that the plasmids according to 13 to 25 in itself recombined in a known manner with parapox viruses in cells and on the selected viruses.
• 36. Process for the preparation of the plasmids according to 22, characterized in that
  • 1. selects a suitable parapox virus strain,
  • 2. cleanses his genome,
  • 3. treated the purified genome with restriction enzymes,
  • 4. the fragments obtained are inserted into plasmids and
  • 5. selected for the plasmids which contain the gene coding for the 10 KDa protein, and
  • 6. optionally inserts and / or deletions into the gene coding for the 10 KDa protein.
• 37. Process for the preparation of the plasmids according to 13 to 21 and 23 to 25, characterized in that
  • 1. selects a suitable parapox virus strain,
  • 2. cleanses his genome,
  • 3. treated the purified genome with restriction enzymes,
  • 4. the fragments obtained are inserted into plasmids and
  • 5. selected for the plasmids which contain the Hind III fragment I or fragments or components thereof corresponding thereto,
  • 6. and optionally inserts and / or deletions into these fragments in the plasmids obtained.
• 38. Process for the preparation of the Hind III fragment I or the DNA section which codes for the 10 KDa protein, from D1701 or the area corresponding to this fragment or section from other parapox viruses or parts thereof, characterized in that
  • 1. selects a suitable parapox virus strain,
  • 2. cleanses his genome,
  • 3. treated the purified genome with restriction enzymes,
  • 4. and selected the desired fragments or sections or
  • 5. If necessary, the fragments of the genome obtained are first inserted into plasmids, the plasmids with the desired fragments are isolated, these plasmids are multiplied and the desired fragments are isolated therefrom.
• 39. A process for producing the gene products according to 32, thereby characterized in that the fragments obtainable according to 38 are suitable Expression systems transferred and by means of these systems the genes be expressed.
• 40. Use of the recombinantly produced parapox viruses according to 1 to 12 in vaccines.
• 41. Use of the recombinantly produced parapox viruses according to 1 to 12 in products that immunize as well as the non-pathogen stimulate specific immune defense.  
• 42. Use of the recombinantly produced parapoxviruses in immune modulators that stimulate the non-pathogen-specific immune defense.
• 43. Use of the recombinantly produced parapox viruses for the heterologous expression of foreign DNA.
• 44. Use of the recombinantly produced parapox viruses as vectors for foreign DNA.
• 45. Use of the plasmids according to 13 to 25 for expression parapockenspe specific genome sections.
• 46. Use of the plasmids according to 13 to 25 for the preparation of dia Gnostic means.
• 47. Use of the genome fragments according to 26 to 31 for the production of diagnostic means.
• 48. DNA section according to sequence protocol ID6 (promoter of the VEGF gene).
• 49. Use of the DNA section according to 48 as a promoter for the expresses sion of DNA with parapox viruses.

The genome fragments of parapoxviruses described in Plas miden or viruses can be inserted or as free DNA sections may include the specified DNA sequences and their variants and homologues.

The terms listed above have the following meanings:

• - attenuation is a
  Process in which the parapox viruses have been reduced by changes in their genome or have become non-pathogenic or virulent for animals or humans.
• - are deletions
  missing DNA fragments from the genome of parapox viruses.
• - are deletion plasmids
  Plasmids carrying parapox virus genome sections in addition to the plasmid DNA, from which sections have been removed.
• - Essential (essential) genome sections for virus replication
  Parts of the entire genome of parapox viruses that are indispensable for the multiplication of parapox viruses, ie for the formation of infectious virus progeny.
• - (Non-essential) genome sections are not necessary for virus replication
  Parts of the entire genome of parapoxviruses that are unnecessary for the multiplication of parapoxviruses, ie for the formation of infectious virus progeny.
• - Foreign DNA elements (foreign DNA) are
  DNA fragments, e.g. B. foreign genes or nucleotide sequences that are not originally present in the parapox virus used according to the invention.
  Foreign DNA is inserted into the parapox virus for the following reasons: 1. for expression of the foreign DNA
  2. to inactivate functions of DNA sections of the para pockenvirus
  3. to mark the parapox virus. Depending on these reasons, different foreign DNA is inserted. If foreign DNA is to be expressed in accordance with (1), the foreign DNA inserted will carry at least one open reading frame which codes for one or more desired foreign protein (s). The foreign DNA may also contain its own or third-party regulatory sequences. The length of the inserted foreign DNA depends on the fact that the desired function of the expressed protein is ensured. In general, the length is between 1 nucleotide and 20 kbp, preferably between 0.5 and 15 kbp.
  Examples include genes for immunogens from viruses, such as in example
  Herpes virus suid 1
  Equine herpes virus 1 to 4
  Bovine herpes virus 1 to 5
  Foot and mouth disease virus, classical swine fever virus
  Bovine Respiratory Syncytial Virus
  Bovine parainfluenza virus 3
  Bovine Virus Diarrhea Virus
  Influenza virus
  or from bacteria, such as
  Pasteurella spec.
  Salmonella spec.
  Actinobacillus spec.
  Chlamydia spec.
  or from parasites such as
  Toxoplasma
  Dirofilaria
  Echinococcus.
  If, according to (2), foreign DNA is to be inserted, it is in principle sufficient to insert a suitable foreign nucleotide to interrupt the vector virus DNA sequence. The maximum length of the foreign DNA inserted for inactivation depends on the absorption capacity of the vector virus for foreign DNA. In general, the length of the foreign DNA is between 1 nucleotide and 20 kbp, preferably between 0.1 and 15 kbp.
  If, according to (3), DNA sequences are to be inserted for labeling, their length depends on the detection method for identifying the marked virus. In general, the length of the foreign DNA is between 1 nucleotide and 20 kbp, preferably between 20 nucleotides and 15 kbp.
• - gene bank
  is the entirety of fragments of a genome contained in reproducible vectors. It is by fragmentation of the genome and insertion of all, individual or a large part of the fragments in ver reproducible vectors, such as. B. Plasmids obtained.
• - Genome fragment is
  a portion of a genome that can be isolated or inserted into a vector that is capable of reproduction.
• - Inactivation by insertion means
  that the inserted foreign DNA prevents the expression or function of parapox virus-specific genome sequences.
• - are insertions
  DNA fragments that were additionally built into the genome of parapox virus. Depending on the reason for the insertion, the length of the DNA sections can be between 1 nucleotide and several thousand nucleotides (see also definition for "foreign DNA").
• - are insertion plasmids
  Plasmids, in particular bacterial plasmids, which contain the foreign DNA to be inserted, flanked by DNA sequences of the parapox virus.
• - (Suitable) insertion sites are
  Loci in a virus genome that are suitable for the uptake of foreign DNA.
• - cloning means
  that the genomic DNA from parapox viruses is isolated and fragmented. The fragments or a selection of the fragments are inserted into common DNA vectors (bacterial plasmids or phage vectors or eukaryotic vectors).
  "Molecular Cloning" 2nd edition, 1989, ed. J. Sambrook, EF Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press gives a selection of methods for the production and cloning of DNA fragments. The DNA vectors with the parapox virus DNA fragments serve as inserts for. B. the production of identical copies of the originally isolated DNA fragments of parapox viruses.
• - marking by insertion means
  that the inserted foreign DNA enables the later identification of the modified parapox virus.
• - "Open reading frame" is a nucleotide sequence (open reading frame, ORF), which is not interrupted by a stop codon. Usually becomes based on the DNA or the RNA sequence checked whether an open reading grid is present. Crick, F.H.C., L. Barnett, S. Brenner and R.J. Watts Tobin, 1961. "General Nature of the Genetic Code for Proteins" Nature 192: 1227-1232.
• - are regulatory sequences
  DNA sequences that influence the expression of genes. Such are known from "Molecular Biology of the Gene", Watson, JD; Hopkins, NH; Roberts, JW; Steitz, JA and Weiner, AM 1987), The Benja min / Cummings Publishing Company, Menlo Park. The VEGF promoter as described in sequence protocol ID6 is preferred.
• - are recombinant smallpox viruses
  Parapox viruses with insertions and / or deletions in their genome. The insertions and deletions were made using molecular biological methods.
• - Repetitive (DNA) sequences are
  Sequences of nucleotide building blocks that occur repeatedly in succession or scattered across the genome of the parapox virus.
• - vector virus is
  a parapox virus that is suitable for inserting foreign DNA and can transport the inserted foreign DNA in its genome into infected cells or organisms and thus, where appropriate, enables expression of the foreign DNA there.

The production of the parapox viruses according to the invention according to 1 to 12 (above) is described as follows:

• 1. Selection of a suitable parapox virus strain
• 2. Identification of genome sections with insertion sites in the Genome of the parapox virus
• 2.a Identification of parapox virus genome sections with insert sites in genes that are not essential for virus replication are,
• 2.b Identification of parapox virus genome sections with insert sites in genes that are essential for virus replication,
• 2.c Identification of genome sections with insertion sites in Be range outside genes in the genome of the parapox virus and / or in gene duplications
• 2.d Additional methods for the identification of genome sections with Insertion points
• 2.e requirements for an insertion point
• 2.1 Identification of insertion points
• 2.1.1 Purification of the genome from parapox viruses  
• 2.1.2 Cloning of the genome fragments, establishment of a gene bank
• 2.1.3 Sequencing for the Identification of Genes or Genome Sections outside of genes
• 2.1.4 Selection of the clones with parapox virus genome fragments for Further processing
• 2.2 ITR region, VEGF, PK gene, gene coding for the 10 KDa- Protein and the area between the PK and HDIR genes as Insertion points
• 2.2.1 Cloning of the VEGF gene
• 2.2.2 Cloning of the protein kinase gene
• 2.2.3 Cloning the area with the gene for the 10 KDa protein encoded
• 2.2.4 Cloning the inverted terminal repeat area or the Genome section that between the PK gene and the HDIR gene lies
• 3. Construction of insertion plasmids or of deletion plasmids, which contain the foreign DNA to be inserted,
• 3.1 Identification or production of unique, d. H. unique restriction enzyme recognition sites ("unique restric tion sites ") in the cloned genome fragments and insertion of Foreign DNA
• 3.2 Deletion of genome sequences in the cloned genome fragments and insertion of foreign DNA
• 3.3 A combination of # 3.1 and # 3.2
• 4. Construction of a recombinant parapox virus according to 1 to 12 (above).

1. Selection of a suitable parapox virus strain

Basically, all parapox virus types or strains are for the Implementation of the present invention suitable. Preferred strains are those that have titers <10⁵ PFU (Plaque Forming Unit) / ml in let a tissue culture multiply and from the medium of the infected Show cells as extracellular, infectious virus. Before from the genus of parapox viruses the species Orfvirus is mentioned.  

Parapox virus strain D1701 may be mentioned as particularly preferred, deposited according to the Budapest contract on April 28th, 1988 with Pasteur Institute, C.N.C.M. under reg.no. CNC′M I-751, as well as its variants and Mutants.

The viruses multiply in the usual way in tissue cultures animal cells such as mammalian cells, e.g. B. in sheep cells, cattle cells, preferred in bovine cells such as the permanent bovine kidney cell BK- Kl-3A (or its descendants) or monkey cells like the permanent one Monkey kidney cells MA104 or Vero (or their descendants).

The propagation takes place in a conventional manner in stationary, roller or carrier cultures in the form of closed cell groups or in Suspension cultures. The cells are used as propagation media for the cells uses all known cell culture media e.g. B. described in the product Flow Laboratories GmbH catalog PO Box 1249, Meckenheim, Germany, in particular the Minimal Essential Medium (MEM), the as essential components amino acids, vitamins, salts and coals contains hydrates, completed with buffer substances such as B. Sodium Bicar bonate or hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and opp also animal sera, such as. B. sera from cattle, horses or their Fetus. The use of fetal calf serum is particularly preferred or serum replacement BMS in a concentration of 1-30% by volume preferably 2-10% by volume.

The cells and cell turf used to multiply the viruses are in usually almost to confluence or up to optimal cell density increased. Cellular growth is preferred before infection with viruses Removed medium and the cells preferentially with virus propagation washed medium. Virus propagation media are used, all cell culture media known per se, in particular the one mentioned above MEM. Then it is infected with a virus suspension. In the virussus the virus is so diluted in the virus propagation medium that with an MOI (= multiplicity of infection, corresponds to infectious virus particles per cell) of 0.01-50, preferably 0.10-10.  

The viruses are propagated with or without the addition of animal sera. For the case that serum is used, this becomes the propagation medium in a concentration of 1-30 vol .-%, preferably 1-10 vol .-% added give.

Infection and virus replication occurs at temperatures between rooms temperature and 40 ° C, preferably between 32 and 39 ° C, especially be preferably at 37 ° C for several days, preferably until complete Destruction of the infected cells. The virus harvest can still be cell-stained denes virus additionally released mechanically or by ultrasound will.

The virus-containing medium of the infected cells can then continue to be worked, e.g. B. by removing the cell debris by filtration with pore sizes of z. B. 0.2-0.45 microns and / or low-speed centri fugation.

Filtrate or centrifugation supernatant can be used for virus enrichment and - cleaning can be used. For this purpose, filtrate or supernatant are one high-speed centrifugation until sedimentation of the virus particles under throw. If necessary, further cleaning steps by e.g. B. Zen trifugation in a density gradient.

2. Identification of genome sections with insertion sites on the genome of the parapox virus

Different areas of the genome of the parapox virus can be inserted serve for insertion of foreign DNA. Foreign DNA can be used be cured

• a. in genes that are not essential for virus replication in vitro and / or are in vivo,
• b. in genes that are essential for virus replication and / or
• c. in areas that do not act as genes, i.e. outside of genes lie and / or in gene duplications.

2.a Identification of genome sections with insertion sites in genes that are not essential for virus replication, on the genome of the Parapox virus definition

Viral genes that are not essential for virus replication can be disturbed for example by deletions or insertions without this blocks the reproduction of the virus. You can thus as Sites for insertion of foreign DNA in the construction of vectors serve.

identification

i. Such genes are found for example by comparative Investigations between different strains of parapox virus. Genes that are not in one or more strains of a virus occur but are found in other tribes are not essential for virus replication.

ii. Genes that are not essential for virus replication can can also be identified in an alternative way. After Sequencing of genome fragments from parapox viruses, the for example, can be present as cloned fragments these DNA sequences for possible "open reading frames" ("Open Reading Frames ", ORF). If you find an ORF, its function as a gene via the detection of a transcription and / or translation verified. To determine if the found Gene is not essential for virus replication, the gene by molecular genetic methods from the genome of the para pox virus removed, partially destroyed or by imported Mutation (s) interrupted, and the resulting virus on propagation capability examined. Can the virus exist without existence replicate the manipulated gene, it is a non-essential gene.  

Examples of identified insertion sites

i. As an example of a non-essential gene from parapox viruses here called the VEGF gene of the parapoxvirus ovis. This gene comes from the investigated field strains of parapoxvirus ovis (New-Zealand-2, New-Zealand-7 and D1701 before (Ref. # 6). Some Parapox viruses, for example strains of the Parapoxvirus bovis 1 do not contain this VEGF gene. So the VEGF gene is for Parapox viruses are non-essential for virus replication and can serve as an insertion site for foreign DNA. The identification of the Area with the VEGF gene on the genome of parapox viruses can be identified using the sequence shown in the protocol ID1 DNA sequence done. About the usual methods of molecular the gene can be biologically analyzed using hybridization experiments, Genome sequence analysis and / or polymerase chain reactions can be found on the genome of a parapox virus.

ii. As another example of a non-essential gene from Para pockenviren is the gene for the 10 KDa (kilodalton) protein from Called parapox viruses. This gene comes from field strains of Parapoxvirus ovis (New Zealand-2, New Zealand-7 and D1701). The gene for the 10 KDa protein from parapox viruses is not essential for virus replication and can be used as an insertion point for Serve foreign DNA. Identification of the area with the gene for the 10 KDa protein on the genome of parapox viruses using conventional methods of molecular biology such as with the help of polymerase chain reactions (PCR) on the genome of a parapox virus can be found. Lit. # 8 shows the DNA sequence of the 10 KDa protein gene. The ones that can be used for a PCR Primers are mentioned for example in Ref. # 8. The DNA sequence shows a 10 KD-specific PCR product of the ORF strain Sequence listing ID11.

The non-essential gene can be used in parts for the insertion of foreign DNA or completely removed from the genome of the parapox virus. It it is also possible to insert the foreign DNA into the non-essential gene insert without removing areas of the parapox virus gene.  

Restriction enzyme recognition, for example, can be used as insertion sites to serve.

2.b Identification of genome sections with insertion sites in genes that are essential for virus replication, on the genome of parapox virus definition

Disruption of genes that are essential for virus replication leads a blockade in virus replication. One removes for example Parts of one of these genes, or the entire gene, breaks down virus replication at a specific point in the virus's reproductive cycle. Infectious cations or treatments with such mutants do not lead to one Release of infectious offspring from the animal. If you replace parts of a or an entire essential gene (s) by foreign DNA or inserted Foreign DNA into essential genes, vector vaccines can be constructed by anyone those who are not able to reproduce in the vaccinated and therefore not as infectious pathogens are excreted.

Principle of identification

i. Such genes are found for example by comparative Investigations (genome analyzes and / or expression studies) between different strains of parapoxvirus. Genes in all Virus strains and isolates are very true Probably also essential for PPV propagation.

ii. Genes that are essential for virus replication are also revealed identified an alternative way. After sequencing viral genome fragments, for example as a cloned frag elements are present, these DNA sequences are possible "Open Reading Frames" ("ORF) examined will. If you find an ORF, its function as a gene is over verified evidence of transcription and / or translation will. To determine whether the gene found is essential for the  Virus propagation is the gene through molecular genetic Methods in the genome of the parapox virus destroyed, for example by removing part or all of the gene or by Insertion of foreign DNA, and the resulting virus on Ver ability to multiply examined. May the virus mutant obtained not replicate, this is an essential gene.

Examples of insertion sites

One example is the PK gene from parapox viruses. The PK gene is expressed late in the virus propagation cycle. The identification of the PK gene on the genome of parapox viruses can be Sequence protocol ID2 shown DNA sequence take place. About the usual methods of molecular biology can be the area with the gene for example by hybridization experiments, genome sequence analyze and / or polymerase chain reactions on the genome of a Parapox virus can be found.

The essential gene can be used in parts or for the insertion of foreign DNA completely removed from the genome of the parapox virus. It is but also possible to insert the foreign DNA into the essential gene, without removing areas from the parapox virus gene.

2.c Identification of genome sections with insertion sites in areas outside of genes on the genome of the parapox virus and / or in Gene duplications definition

Genome sections that do not code for functional gene products, and have no essential regulatory functions (so-called intergenic Sections) are in principle suitable as insertion sites for foreign DNA. Areas with repetitive sequences are particularly suitable since changes Compensation in some areas by remaining sequence repetitions  can be settled. This also includes genes that are in two or multiple copies occur, so-called gene duplications.

Genes in the ITR region or in duplicate sections of the PPV genome exist in two copies in the virus genome, so they are diploid. After Ent remove or damage a copy of such a gene and insert it Foreign DNA can be obtained from stable PPV recombinants, too if the damaged gene is important for virus replication. A second, undamaged gene copy could be used for gene functionality suffice.

identification

i. The identification of genome sequences that are not for gene products encode, is carried out via sequence analysis of the genome of the parapox viruses. Genome areas in those in such sequence analyzes for example, neither an ORF nor virus-specific transcription is found and there is no evidence of regulatory Function, represent potential insertion points. Especially the Represent restriction enzyme recognition sites in these areas animals potential insertion sites. To check whether a suitable Nete insertion site is known about molecular biology methods of foreign DNA into the potential insertion site be serated and the resulting virus mutant on life ability to be examined. If the mutant virus in the possible insertion site carries foreign DNA, is capable of reproduction, the investigated body is a suitable one Insertion point.

ii. The identification of repetitive sequences as well as gene duplicates For example, genome hybridization experiments are used elements and / or via sequence analyzes. With the hybridisie Development experiments become cloned or isolated genome fragments a parapox virus (from a parapox virus gene bank) as Probes for hybridizations with restriction enzyme digested Parapox virus total genome used. The genome fragments of the parapox virus, which contains more than one fragment of the  Whole genome of the parapox virus hybridize, contain one or several repetitive sequences. To the repetitive sequence or duplicate genome areas on the genome fragment exactly localize, the nucleotide sequence is determined. Identical se sequences on the fragments are repetitive. Become homologous Areas of DNA at two relatively distant locations in the genome found, it is gene duplication that is related to a second Site of the PPV genome was translocated. To see if a potential insertion point a suitable insertion point in the Whole genome of the parapox virus is foreign DNA in one repetitive sequence or inserted into a copy of the gene duplication and the PPV genome fragment with the insert in the Virus genome can be incorporated. Then the foreign DNA containing recombinant virus tested for reproductive ability. The recombinant virus containing foreign DNA multiplies the recognition site identified above is suitable as an insertion site.

Examples of insertion sites

i. The genome may be mentioned as examples of intergenic areas section between the gene for the protein kinase and the HD1R- Gene (sequence protocol ID7).

ii. The ITR region is an example of a repeat sequence (Inverted Terminal Repeat Region) called (sequence protocol ID4).

iii. This is an example of diploid genes in PPV strain D1701 potential gene "ORF 3" (in the ITR region) and the VEGF gene called. Hybridization studies proved that in the present Strain D1701 duplicated an area containing the VEGF gene and has been translocated to the other end of the virus genome so that two VEGF gene copies are available.

Using the sequences in the sequence protocol ID4 (ITR sequence with Gene "ORF3") and ID7 (area between PK and HDIR gene)  the corresponding genome areas in other parapox viruses using the usual methods of molecular biology for example hybridization experiments, genome sequence analysis sen and / or polymerase chain reactions can be found.

2.d Other methods of identifying insertion sites

In general, possible insertion sites on the genome of Para pockenviren also found over modifications of the viral genome sequences the. Genome sites at which nucleotide exchanges, deletions and / or Insertions or combinations of these, but not virus multiplication block, represent possible insertion points. To check whether a potential insertion point represents a suitable insertion point using known molecular biological methods foreign DNA in the potential insertion site and the resulting virus mutant Viability examined. If the virus recombinant is capable of reproduction the examined place is a suitable one Insertion point.

2.e requirements for the insertion points

The insertion sites have to be used to record foreign DNA sequences be suitable for a length that varies according to their above tasks judges.

Insertion sites into which foreign DNA with a length of 1 are preferred Nucleotide up to 15 kbp can be inserted.

The capacity to take up foreign DNA can be increased by setting Deletions in the genome of the virus are enlarged. The deletions prove gene are in a size of 1 nucleotide to 20 kbp, preferably of 1-10 kB.

2.1 Identification of insertion points 2.1.1 Purification of the genome from parapox viruses

For the molecular genetic cloning of insertion sites of para pockenviren the genome of the parapockenviruses is first cleaned. Starting from the virus produced according to FIG. 1 (above), the genome isolated and cleaned. Native viral DNA is extracted preferred over the treatment of the purified virions with aqueous solutions detergents and proteases.

Anionic, cationic, amphoteric, non- ionic detergents. Ionic detergents are preferably used. Sodium dodecyl sulfate and sodium lauryl sulfate are particularly preferred.

All proteases in the presence of Deter are mentioned as proteases gens work such. B. Proteinase K and Pronase. Preferred is mentioned Proteinase K.

Detergents are used in concentrations of 0.1-10% by volume. 0.5-3% by volume are preferred.

Proteases are in concentrations of 0.01-10 mg / ml virus lysate set, 0.05-0.5 mg / ml virus lysate are preferred.

It is preferred in aqueous buffered solution in the presence of DNase inhibitors worked. The following may be mentioned as buffer substances: salts weak acids with strong bases such as B. Tris (hydroxymethylamino methane), salts of strong acids with weak bases such as B. primary phos phate or mixtures thereof.

The following buffer system is preferred: Tris (hydroxymethyl aminomethane).

The buffer substances or buffer systems are incorporated in concentrations set that ensure pH values at which the DNA does not denature. PH values of 5-9, particularly preferably 6-8.5, all are preferred particularly preferred 7-8, especially work in the neutral area called.  

DNase inhibitors are e.g. B. ethylenediaminetetraacetic acid in concentration of 0.1-10 mmoles, preferably about 1 mmole.

The lipophilic components of the virus lysate are then extracted. Solvents such as phenol, chloroform and iso serve as extracting agents amyl alcohol or mixtures thereof. A mixture is preferred first from phenol and chloroform / isoamyl alcohol used, the Extrak tion takes place in one or more stages.

In the last stage of the extraction, chloroform / isoamyl is preferred alcohol used. Alternatively, phenol first and then Chloroform / isoamyl alcohol can be used.

Other methods for isolating the virus DNA are e.g. B. Centrifugation a virus lysate in a CsCl density gradient or in the gel electro phosphorus (Sharp et al. Biochem. 1973 (12) pp. 3055-3063).

The extraction of nucleic acids is described in "Molecular Cloning", A Labora tory Manual, 2nd Edition 1989, ed J. Sambrook, E.F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press.

The DNA extracted in this way is preferably extracted from the aqueous solution with e.g. B. Alcohol, preferably with ethanol or isopropanol and with the addition of monovalent salts such as B. alkali chlorides or acetates, preferred Lithium chloride, sodium chloride or sodium acetate, potassium acetate falls.

The concentration of alcohol is between 40 and 100% by volume, preferably between 60 and 80 vol.%, particularly preferably at approx. 70 vol%.

The chloride or acetate concentration is between 0.01 and 1 molar, preferably between 0.1 and 0.8 molar. If LiCl is used, it lies Concentration between 0.1 and 1 molar, preferably between 0.4 and 0.8 molar.  

Methods for the precipitation of nucleic acids are in "Molecular Cloning" loc. cit. described in detail. The precipitated DNA is z. B. Centrifugation isolated from the aqueous suspension, preferably with Alcohol, e.g. B. 70 vol .-% ethanol, washed and finally in aqueous Buffer solution resolubilized.

Tris (hydroxymethylaminomethane) in Kon concentrations of 1-100 mM, 10-50 mM is preferred. Preferred pH Values are 6-8.5, particularly preferably 7-8.

Other additives may be mentioned e.g. B. EDTA (ethylenediaminotetraacetic acid) in concentrations of 0.1-10 mM, preferably 1-10 mM.

Alternatively, the precipitated DNA can also be in 0.01 or 0.1 × SSC buffer (Molecular Cloning) loc. cit. or resolu in ammonium carbonate buffer be accounted for.

2.1.2 Cloning of the genome fragments, establishment of a gene bank

The viral DNA purified in this way is followed by restriction endonuclease Regulation of the manufacturers treated. Suitable restriction endonucleases are those that have at least one interface specific to them on the Detect virus genome. Restriction endonucleases (restriction enzymes) are z. B. EcoRI, BamHI, SalI, HindIII, PstI, XbaI, AflIII or BspMII. The resulting DNA fragments can be determined according to the "Molecular Cloning", A Laboratory Manual, 2nd Edition 1989, ed. J. Sambrook, E.F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press into the corresponding recognition sequences of bacterial plasmid Vectors, phages or cosmids are molecularly cloned. Especially are preferred for low molecular weight DNA fragments (up to approx. 15 kbp) Plasmid vectors, for DNA fragments from approx. 15 kbp up to approx. 45 kbp Cosmide.

2.1.3 Sequencing to identify genes or sections of genomes outside of genes

The nucleotide sequences of the cloned virus DNA prepared above Fragments or their subclones are made according to common methods such as for example in Virological Working Methods, Volume III: Biochemical and biophysical methods, Dernick et al., 1989, VEB Gustav Fischer Publisher are described. From the determined Nucleotide sequences can then be "open reading frames" (Open Reading Frames, ORFs) that signal possible genes. The existence the genes can then be transcribed or translated with mole verify biological methods. To see if that was found whose gene is not essential for virus replication, the gene can molecular genetic methods can be destroyed by looking at it removed in part or entirely from the PPV genome and that resulting virus are examined for reproductive ability. Can the Replicate virus without the existence of the removed gene, it is is a non-essential gene.

Sequencing also allows repetition to be identified sequences, so-called repetitive sequences.

For further identification are clones with fragments of the para pockenvirus genome interesting, the genes, parts of genes or areas outside of genes, for example repetitive sequences and / or Gene duplications included.

2.1.4 Selection of the clones with parapox virus genome fragments for further processing

Which of the clones obtained above with genome fragments of parapox Virus processed depends on whether the virus to be manufactured recombinant parapox viruses (i) capable of replication or (ii) replication should be defective.

i. Recombinant parapox viruses are to be produced that despite the insertion and / or deletion the ability to form in have not lost fectious offspring, i.e. capable of replication cloned viral genome fragments are processed further  Contain genes or genomic regions outside of genes that are not essential for virus replication or gene duplication contain.

Checking whether it is the present gene or genome area is a non-essential region of the virus genome or a gene duplication, which is due to the compensation tion by the identical gene, can be destroyed via virus mutants are determined. For this, the gene or the genome area examined in the parapox virus by moleku larbiological methods inactivated, for example by partially or complete deletion of the area in question, and Ver The viral mutant's ability to reproduce was examined. Can the virus mutant despite the inactivation of the gene in question or Increase genome range, it is the examined Gene or genome region around a non-essential region.

Cloned genome fragments of the parapox virus are preferred, that intact the non-essential genes, d. H. completely included. There In addition, the genes or genome should be on both ends areas also contain the flanking viral genome areas be. The length of the flanking areas should be more than 100 Base pairs. If such genome clones are not available, they can they from existing genome clones with molecular biological me methods are produced. Contain the cloned genome fragments additional genome areas necessary for the present manufacture are not required, these can be done by further cloning be removed.

ii. Recombinant parapox viruses are to be produced that are based on due to the insertion and / or deletion the ability to form have lost infectious offspring, that is replication defective cloned viral genome fragments are processed further Contain genes or genomic regions outside of genes that are essential for virus replication.  

Checking whether it is the present gene or genome area is an essential region of the virus genome can be determined via virus mutants. For this, the examined Gene or the genome area examined in the parapox virus molecular biological methods inactivated, for example by partial or complete deletion of the area in question, and investigated the ability of the virus mutant to reproduce. Can the Virus mutant itself due to the inactivation of the gene in question or no longer increase genome range, it is about the gene or genome region under investigation by an essential Region.

Cloned genome fragments of the parapox virus are preferred, that keep the essential genes intact, d. H. completely included. About that addition, both ends of the genes or genome areas should the flanking viral genome areas are also included. The length of the flanking areas should be more than 100 bases pairs. If there are no such genome clones, they can from existing genome clones using molecular biological methods getting produced. Contain the cloned genome fragments additionally genome areas that are not for the present production can be removed by further cloning will.

2.2 ITR region, VEGF gene, PK gene, gene coding for the 10 KDa protein and the area between the PK gene and the HDIR gene as an insertion put

Should the ITR region, the VEGF gene, the PK gene, the gene for 10 encoded KDa protein or the intergenic area between the PK gene and the HDIR gene as an insertion site in a parapox virus the corresponding areas of the parapox virus Genomes containing the insertion sites are isolated. About who preferred the corresponding areas of the parapox virus genome cloned.  

2.2.1 Cloning of the VEGF gene

The gene encoding VEGF is on the genome of the parapox virus localized and then in part or in its entirety with his flanking genome sections isolated. For this, it is preferred that Parapox virus according to FIG. 1 and, also preferred, the genome cleaned according to # 2.1.1.

• a. The VEGF gene is, for example, via a polymerase chain Reaction (PCR) amplified. The ones necessary for this reaction Start sequences (primers) are derived from the sequence ID1 in the protocol DNA sequence of the VEGF gene shown. That he held amplificate is then preferably molecularly cloned.
• b. The region which contains the VEGF gene and its flanking genome sections is preferably obtained by fragmentation of the para pox virus genome and isolation and cloning of the corresponding genome fragments. For this, the purified genome of the virus is treated with restriction enzyme, particularly preferably with the enzyme HindIII, as described in # 2.1.2. In order to identify the genome fragment or fragments which carry the VEGF gene and its flanking genome sections, the genome fragments obtained after enzyme digestion are preferably separated.
  Electrophoretic and chromatographic methods can be used as methods for separating the DNA fragments. In the chromatographic process, gel filtration should be mentioned. Agarose or polyacrylamide may be mentioned as carriers in the electrophoretic processes. As electrophoresis buffers may be mentioned, for. B. ethylenediaminetetraacetic acid phosphate buffer (EPP) or tris (hydroxymethyl) aminomethane-borate-ethylenediaminetetraacetic acid buffer (TBE).
  A detailed list and description of electrophoresis buffers can be found in
  • - Current Protocols in Molecular Biology 1987-1988, Verlag Wiley-Interscience, 1987
  • - A Practical Guide to Molecular Cloning, Perbal, 2 ^nd edition, Wiley Interscience, 1988
  • - Molecular cloning, loc. cit.
  • - Virological Working Methods, Volume III, Gustav Fischer Verlag, 1989

described.
The implementation of the method is described, for example, in "Molecular Cloning" loc. cit., or in Virolog. Working methods, Volume III, loc. cit.
The genome fragments which carry the VEGF gene and its flanking sequences are identified, for example, by hybridization with defined nucleic acid probes. For this purpose, the separated genome fragments are transferred to filters and hybridized with VEGF-specific and labeled nucleic acid probes according to the Southern blot method. The method of transferring the genome fragments and hybridization can be carried out according to standard protocols, as described under "Southern blotting" in Molecular cloning loc. cit. are described. The oligo nucleotides or nucleic acid fragments which can be used as probes can be derived from the sequence listing Seq ID No 1. For example, the TaqI subfragment (366 bp), as can be seen from Seq ID No 1, is used as a hybridization probe.
The genome fragments, which have been shown to contain parts or preferably all of the VEGF gene and the flanking genomic sections, are isolated and cloned. The isolation of the corresponding genome fragments is carried out, for example, from the electrophoresis gel via electroelution from the corresponding gel area. Alternatively, for example, using a "low-melting agarose" method, the genome fragment (s) can be isolated from the electrophoresis gel by conventional methods.

To clone the VEGF gene, the genome Fragments inserted into bacterial or eukaryotic vectors. Especially bacterial plasmid or phage vectors are preferred first. For Insertion of the genome fragment will be double-stranded plasmid or Phage vector DNA molecules treated with restriction enzymes so that suitable ends for the insertion arise.

Known plasmids are plasmids, e.g. B. pSPT18 / 19, pAT153, pACYC184, pUC18 / 19, pBR322, pSP64 / 65.

As phage vectors z. B. the well-known Lambda phoenix variants such as e.g. B. -ZAP, -gt10 / 11 or Phage M13mp18 / 19.

The restriction enzymes that can be used are known per se, for. B. from genes Volume 92 (1989) Elsevier Science Publishers BV Amsterdam.

The plasmid treated with the restriction enzyme or the phage vector becomes with an excess of the DNA fragment to be inserted z. B. about in Ratio 5 to 1, mixed and treated with DNA ligases to make up the Covalently bind DNA fragment end-to-end in the vector. Ligases are enzymes that are capable of two DNA molecules via 3′-OH-5′- Connect leftovers. The ligation approach is used to increase the Plasmids or phages in pro- or eukaryotic cells, preferably in Bacteria, introduced and multiplied. Z serve as bacteria. B. Escherichia coli strain K-12 and its derivatives e.g. B. K 12-600 (Molecular Cloning loc. cit.). The preparation of the ligation approach and the Bacterial culture takes place in a manner known per se as in Molecular Cloning loc. cit. described. The bacteria, the plasmids with inserted Containing foreign DNA or phages with foreign DNA are selected.

The identity of the foreign DNA is preferred via hybridization riments and particularly preferably verified by sequence analysis. If necessary. subcloning is carried out in order to include intact VEGF genes to obtain flanking genome sections as nucleic acid clones.  

2.2.2 Cloning of the protein kinase gene

The gene that encodes the protein kinase is located on the genome of the PPV localized and then in parts or entirely with its flanking Genome sections isolated.

How the cloning of the VEGF gene is described in detail (above) the pox virus preferentially multiplied, its genome purified and the area on the genome that codes for the PK gene via a polymerase chain Reaction and preferably obtained by subsequent cloning.

Alternatively, this area can be used for cloning the VEGF gene described about the fragmentation of the PPV genome, preferably with subsequent cloning of the fragments and selection of the fragments or clones, the parts of the PK gene or preferably the entire PK gene containing flanking DNA sequences of the PPV genome will. DNA molecules that act as primers for a PCR or as probes for a Hybridization that can be used are in sequence protocol ID2 or ID9 recognizable.

If necessary, subclonings are made to keep them intact PK genes including their flanking genome sections on the genome of the Get PPV.

2.2.3 Cloning of the gene coding for the 10 KDa protein

The gene that codes for the 10 KDa protein is corresponding to that Methods as detailed for the VEGF gene and the PK gene was described (above), located on the genome of the PPV and in Share or preferably entirely with its flanking PPV genome sequences isolated.

The area of the PPV genome that contains the gene for the 10 KDa- Contains protein or parts thereof, preferably via PCR and / or cloning and identify and select the appropriate clones.  

DNA molecules that act as primers for a PCR or as probes for a Hybridization can be used, see Ref. # 8 will. If necessary, subclonings are made to keep them intact Genes coding for the 10 KDa protein including its flanking To obtain PPV genome sections.

2.2.4 Cloning the inverted terminal repeat area or the genome cut that lies between the PK gene and the HDIR gene

The procedure corresponds to that for the cloning of the VEGF gene has been described in detail (above). The DNA molecules that make up Iso the corresponding area as a primer of a PCR or as a probe can be used for hybridization are in the sequence Protocol ID4 (ITR region) and 7 (area between PK gene and HDIR gene) evident.

3. Construction of insertion plasmids that contain the foreign DNA to be inserted contain, or of deletion plasmids

Based on the genome identified, located and cloned in # 2 Fragments of parapox viruses are known as insertion plasmids asked to insert foreign DNA into the genome of the para smallpox viruses can be used later. Carrying the insertion plasmids then the foreign DNA to be inserted into the parapox virus, flanked by Genome sections of the parapox virus. There are different possibilities Possibilities of producing the insertion plasmids: Here are examples thinks:

• 3.1 Identification or production of unique occurrences, i.e. H. sin gular restriction enzyme recognition sites ("unique restriction sites") in the cloned genome fragments and insertion of foreign DNA,  
• 3.2 Deletion of genome sequences in the cloned genome fragments and Insertion of foreign DNA
• 3.3 A combination of # 3.1 and # 3.2

3.1 Identification or production of unique restriction enzymes "unique restriction sites" according to 2.1.4 or 2.2 cloned genome fragments and insertion of foreign DNA

The nucleotide sequences of the Genome fragments of the parapox viruses provide direct information about sin gular restriction enzyme recognition sites at which the fragment after Treatment with the corresponding enzyme opened in only one place becomes. If necessary. can by insertion of, for example, synthetic herge provided oligo nucleotides, for example so-called "polylinkers", singu Restriction enzyme recognition sites are incorporated. For this, the Parapox virus genome fragment with the insertion site with a restriction tion enzyme that only opens the fragment at one point. That so opened fragment is incubated with a polylinker and ligase to to specifically target other restriction enzyme recognition sites.

Polylinkers are DNA sequences that contain at least two restriction enzymes wear identification points connected in series.

As a ligase z. B. called T4 DNA ligase.

The plasmids obtained are in pro- or eukaryotic cells, Bacteria, propagated and selected.

Alternatively, you can also use the "PCR Mutagenesis" method as described by Jacobs et al. (Arch. Virol. 131, 251-264), new singular Restriction enzyme recognition sites in the parapox virus genome gmente be installed.  

(Jacobs, L., H.-J. Rziha, T.G. Kimman, A.L.J. Gielkens and J.T. van Oirschot. 1993, Arch. Virol. 131, 251-264, LIT)

In the singular restrictions identified and / or produced here Zymerk recognitionstellen can by the known methods of moles kularbiologie the foreign DNA to be inserted into the parapox virus be cured. The parapox virus genome fragment with the insertion site is treated with one or more restriction enzymes, which on the Separate the insertion point or in the inserted polylinker. The Foreign DNA is, for example, according to the methods known per se "sticky-end" or "blunt-end" ligation, using ligases in the recognition place advertised. After the ligase reaction, the incubation or eukaryotic cells, preferably bacteria, and the plas mide increased and selected.

The above-mentioned methods used to produce the insertion plasmids are applied in detail in "Molecular Cloning", A Laboratory Manual, 2nd Edition 1989, ec. J Sambrook, E.F. Fritsch and T.Maniatis, Cold Spring Harbor Laboratory Press.

3.2 Deletion of genome sequences in the cloned genome fragments and Insertion of foreign DNA

The deletion of subfragments from the cloned parapox virus Genome fragments can be, for example, by treatment with restrictions enzymes. Restriction enzymes are one possible approach preferred that more than one, particularly preferably 2 recognition sites in recognize the genome fragment. After the enzyme treatment, the fragments obtained, as described above, for example electrophoresis table separated, isolated and the corresponding fragments with example wise ligase treatment put together again. The plasmids obtained are multiplied and the deleted plasmids are selected.

In an alternative approach, a unique restriction enzyme is used identification point on the parapox virus genome fragment as the starting point for a bidirectional degradation of the fragments with an endonuclease  for example, the enzyme Bal31. The reaction time determines here the length of the deletion, the extent of which is taken kinetically Samples can be followed in electrophoresis. After the reaction the newly created fragment ends can be supplemented with oligonucleotides to, for example, new unique restriction enzyme recognition places to install. The incorporation of foreign DNA can then, as above described.

4. Construction of a recombinant parapox virus according to 1 to 12

Insertion of foreign DNA into the parapox virus genome:
The following procedures are used to incorporate the foreign genetic element into the vector virus:

• a. Simultaneous transfection of the insertion or deletion plasmid DNA and infection with parapox virus in suitable host cells,
• b. Transfection of the insertion or deletion plasmid and DNA closing infection with the parapox virus in appropriate Host cells,
• c. Parapox virus infection and subsequent transfection with the insertion or deletion plasmid DNA in suitable hosts cells.

The methods of the methods suitable here are described in "Methods in Virology Vol. VI "(1977), ed. K. Maramorosch, H. Koprowski, Academic Press New York, San Francisco, London. The is preferred Transfection the method of the so-called calcium phosphate technique ("Methods in Virology Vol. VI" (1977), ed. K. Maramorosch, H. Koprowski, Academic Press New York, San Francisco, London).

Method #c (above) is preferably used. Here are the following Steps necessary:

• 1. Parapox virus infection:
  For the production of the parapox viruses with foreign DNA, cell cultures are preferred which allow good virus multiplication and efficient transfection, for example the permanent bovine kidney cell BK-Kl-3A. A method is also preferred in which the cells are first infected with the virus and then trans fected in suspension with purified plasmid DNA.
• 2. Preparation of the insertion or deletion plasmid DNA:
  The transformed cells obtained by the methods described above, for example bacteria which contain insertion or deletion plasmids, are propagated and the plasmids are isolated from the cells in a manner known per se and further purified. The cleaning is done e.g. B. by an isopycnic centrifugation in the density gradient of z. B. CsCl or by affinity purification on commercially available silicate particles (z. B. Qiagen).
• 3. Transfection:
  The plasmid DNA is used circularly or linearized for transfection.
• 4. Cell cultivation:
  The cells are cultivated according to the methods described above (selection of a suitable parapox virus strain). If a cytopathogenic effect occurs, the culture medium is removed, cell debris is removed, if necessary, by centrifugation or filtration and, if appropriate, is stored, and viruses are worked up using the conventional methods of individual plaque cleaning.

The following method is very particularly preferred during production recombinant parapox viruses:

• 1. BK-KL3A cells are counted and in suspension with plaque purified parapoxvirus (PPV) with multiplicity of the infection of 1 (MOI = 1) infected and 2 to 4 hours with gentle stirring  incubated at 37 ° C. The transfection then takes place with purified insertion or deletion plasmid DNA with pelleted Cells. In parallel, non-infected cells are in the same meadow transfected.
• 2. After transfection using the calcium phosphate method (Ca / P method) (van der Eb, 1973) and glycerol shock become the infected and transfected cells in a 1:10 ratio with only transfected (or untreated) cells mixed in order to Further cultivation the direct virotoxic effect of smallpox to avoid viruses. The addition of transfected cells increases the Likelihood that virus will advance from cell to cell infection again strikes plasmid DNA and a re combination can occur. The cell mixture is then in 6-hole plates or other culture vessels stationary at 37 ° C cul tiviert until microscopically typical virus destruction in the form of plaques is visible.

Recombinant viruses containing foreign DNA are selected depending on the inserted foreign DNA z. B. by:

• a. Expression of the foreign DNA with the aid of the recombinant viruses,
• b. Detection of the presence of the foreign DNA, e.g. B. by DNA / DNA hybridizations,
• c. The amplification of the foreign DNA by means of the PCR.

to a.

The expression of the foreign DNA at the protein level can be demonstrated are by z. B. Infection of cells with the virus and subsequent Immunofluorescence analysis with specific antibodies against that of the Foreign DNA encoded protein or by immunoprecipitation or Western Blotting with antibodies against the protein encoded by the foreign DNA from the lysates of infected cells.

The expression of the foreign DNA at the RNA level can be demonstrated are generated by hybridizing the RNA from cells infected with virus  DNA that is at least partially identical to the inserted foreign DNA.

to b.

For this, the DNA is obtained from the virus in question and with Nucleic acid, at least in part with the inserted foreign DNA is identical, hybridized.

The single plaque cleaned and identified as recombinants Vek Tor viruses are preferred again for presence and / or express sion of the foreign DNA checked. Recombinant vector viruses that Containing and / or expressing DNA stably stands for the others Application available.

Examples

The present invention is particularly preferred in a range of para poxvirus genome, which is suitable for the insertion and expression of homologous and hetero suitable genes or parts thereof. This genomic fragment is generated by a 5516 base pair HindIII DNA fragment of the parapoxvirus ovis strain orf D1701 (and its respective derivatives) (sequence Protocol ID8).

1. Cloning of HindIII fragment I

After cleaving purified viral DNA with the restriction enzyme HindIII, the resulting DNA fragments were separated by agarose gel electrophoresis, the approximately 5.6 kbp fragment I was cut out and isolated by Qiaex purification (Qiagen). This DNA fragment was cloned into the vector plasmid pSPT18 (Boehringer, Mannheim), which had been digested with HindIII and treated with CIP (calf intestinal phosphatase) using standard techniques (Maniatis et al.). The resulting recombinant plasmids pORF-1 and pORF-2 differ only in the orientation of the insert. The creation of a restriction map enabled further subcloning, as shown in Fig. 1. All recombinant plasmid DNAs were tested for restriction-digested viral or plasmid DNA by Southern blot hybridization to check their identity and viral origin.

2. DNA sequencing

DNA sequencing was carried out using double stranded DNA various recombinant plasmids and SP6 and T7 specific primers, that bind to both ends of the cloning site of the vector plasmid pSPT18, accomplished. The Sanger dideoxy chain termination method was developed after the recommendations of the manufacturer (Pharmacia - LKB) in the presence of ³⁵S- [α] - dATP and T7 DNA polymerase performed. According to the received DNA sequence, a variety of oligonucleotides were synthesized and used for Sequencing both strands of the fully inserted HindIII fragment I used. To sequencing artifacts or band compression due to the was able to resolve the relatively high G + C content of the viral DNA insert (64.78%)  7-Desaza-GTP was used and the sequencing products were added if necessary, separated in formamide-containing denaturing polyacrylamide gels.

3. Identification of potential genes

Computer-aided analysis of the DNA sequence obtained (5516 nucleotides) revealed several possible open reading frames (ORF). The amino acid sequence derived from this ORF was used for gene homology searches (LIT, GCG). As a result, significant amino acid homologies with the following genes were detected (see also Fig. 1).

• 3.1 An ORF with an amino acid homology (36.1 to 38.3% identity; 52.8 58.6% similarity) to the vascular endothelial growth factor (VEGF) of various mammalian species (e.g. mouse, rat, guinea pig, Cattle, humans) and also to the VEGF gene that was recently introduced in the Parapoxvirus strains NZ-2 and NZ-7 have been described (Lit. # 6) found. A corresponding gene homolog is with other smallpox viruses, like various orthopox viruses, not known. This ORF that as VEGF is comprised of 399 nucleotides and encodes a polypeptide with 132 amino acids with a calculated molecular weight of 14.77 kDa. Transcription analysis using Northern blot Hybridization, RNA protection tests and primer extension tests with whole or oligo (dT) -selected RNA coming from infected cells was isolated, demonstrated the expression of VEGF as an early gene from about 2 hours after infection (p.i.). It was found that the specific mRNA covers 422 to 425 bases, starting immediately downstream of one Sequence that is 100% homology with the critical area of a consen susmotivs shows that vaccinia genes typical of early promoters Virus is. The 3'-end of the VEGF mRNA could be in a consensus sequence be mapped, the early transcripts of e.g. B. Vaccinia virus genes is common. The size of this mRNA was determined by Northern blotting estimated about 500 bases, indicating a poly (A) section with a Length of about 100 bases indicates.
• 3.2 Another ORF was found, that for a potential protein kinase (PK) with homology to a corresponding gene encoded in several other orthopox viruses (e.g. vaccinia, Variola or Shope fibroma virus)  occurs and is known as F10L. This gene homolog is used in Infection cycle of orf D1701 transcribed late (from 12 to 16 hours pi.). The transcription start point is located briefly downstream of one Area that has good homology to known promoters later genes of the vaccinia virus shows.
• 3.3 Further possible ORFs have been found that do not show any striking homologies to any known gene sequence. Of particular interest could be a potential gene called the HDIR gene ( Fig. 1). Analyzes such as those described above showed the transcription of a specific early mRNA with a size of 1.6 kb.
• 3.4 Finally, an ORF was found using a computer that overlaps the 3′-end of F10L and the 5′-end of VEGF (F9L, Fig. 1). A comparison with the described genes showed a low (dispersed) homology with the F9L gene of the vaccinia virus.
• 3.5 Compared to known DNA sequences from the Orf strains NZ2 and NZ7, the start of the so-called ITR region in the genome of D1701 at nucleotide position 1611 of the HindIII fragment I could be determined. The ITR region is a sequence region occurring at the end of the pox virus genome, which is also present in the opposite orientation at the other genome end and is therefore referred to as an "inverted repeat region" (ITR). The result of the sequence comparison coincides with attempts to locate the genome of the HindIII fragment I of D1701 cloned in pORF-1. The mapping of this fragment and the presumably identical HindIII fragment H is shown in Fig. 2. From this it can be concluded that the ITR of the D1701 genome comprises approximately 2.6 kbp.
  Attempts to determine the 3'end of the VEGF mRNA of D1701 revealed that between about 40 and 220 bp after the transition to ITR, at least one other virus-specific RNA starts in the ITR. The corresponding gene was named ORF3 due to the amino acid homology to NZ2 ( Fig. 1). Before the putative 5'-end of the ORF3 mRNA is a consensus sequence, which is typical for an early poxvirus promoter. No homology to other known genes has been found so far.

4. Introduction of DNA sequences into HindIII fragment I

The possibility of using the described HindIII DNA fragment (in the plasmids pORF-1 and pORF-2 cloned) for the introduction of homologous or heterologous DNA sequences were developed. In the following three ver different strategies are used to achieve this goal.

4.1 Insertion in intergenic, non-coding areas

Identify or create new singular restriction sites that are located in an intergenic, non-coding part. These sites are then used to insert foreign DNA sequences encoding the functional and detectable gene products (e.g. the lacZ gene, from E. coli) or parts thereof. The plasmid pGSRZ was constructed for the following examples containing the E. coli functional lacZ gene under the control of the 11K promoter of the vaccinia virus. For this purpose, the relevant DNA parts of the plasmid pUCIILZ (Sutter et al., 1992) were isolated and cloned into the plasmid pSPT18. This functional 11K-lacZ gene combination (hereinafter referred to as lacZ cassette) can be obtained by isolating a 3.2 kbp SmaI-SalI fragment from pGSRZ ( FIG. 3).

Example 4.1.1

The plasmid pORF-PB ( Fig. 1) contains a single NruI site, which is located between the protein kinase (F10L) and the HDIR gene. After linearization of pORF-PB with this restriction enzyme, the lacZ cassette (after filling reaction) was ligated "blunt end". Recombinant plasmids containing the functional lacZ gene were cleaved after e.g. B. selected with BglI, and the correct insertion by Southern blot hybridization with a lacZ-specific probe and by partial DNA sequencing of the transition of the lacZ and Orf DNA was demonstrated.

Example 4.1.2

The same procedure as described in the example above was used shortly downstream of the VEGF gene (cleavage at the BstEII site, Fig. 1) and in the potential ORF 3 gene in the ITR region (partial degradation with XbaI in order to cleave the XbaI Site of the multiple pSPT18 cloning site).

Example 4.1.3

In order to introduce a new simple restriction site at any desired site, the technique of PCR mutagenesis as outlined in Fig. 10 and recently described (Jacobs, L., H.-J. Rziha, TG Kimman , ALJ Gielkens and JT van Oirschot. 1993. Arch. Virol. 131, 251-264, LIT). For this purpose, two PCR reactions are carried out separately, the primer pair E1 + EV2 (PCR A) or EV1 + XB (PCR B) being used. All primers cover 25 nucleotides that are identical to the Orf DNA sequence at the indicated sequence positions. While primer XB the authentic sequence z. B. around the XbaI site ( Fig. 1), a new EcoRI site was introduced in primer E1 at the 5′-end and in primers EV1 and EV2 (which are complementary to each other) a new EcoRV site. The EcoRV site, which is not originally present in the entire sequence of pORF-1 or -2, has been moved to the site chosen for the introduction of the lacZ cassette. The PCR products obtained from reactions A and B are cleaned, denatured into single strands and mixed together under reassociation conditions; then they are used for the final PCR C reaction. E1 and XB are now used as primers in order to extend the left 793 bp of pORF-1 in this example. After gel isolation and purification, the resulting PCR product from reaction C is cleaved with EcoRI and XbaI and then ligated with the plasmit pORF-XB cleaved by EcoRI and XbaI. As a result, the plasmid pORF-1EV ( Fig. 10) contains the EcoRV restriction site at the desired position; it can then be used for linearization and ligation (with blunt ends) with the lacZ cassette.

In addition, mismatches can occur for the method described in this example tion primers are used, the defined base changes or a dele tion of a single base included, e.g. B. translation stop codons or  Amino acid deletions at any desired location in the viral DNA sequence produce.

4.2 Intragonal insertion without deletion of Orf sequences

In the coding sequences of one of the ORFs described can Cleavage at restriction sites that occur only once in the selected gene, new or additional sequences are introduced.

Example 4.2.1

The unique XcmI site located in the right part of the gene homolog F10L was used to linearize the plasmid pORF-1. Both the lacZ cassette and the cleaved pORF-1 DNA were blunt-ended by T4 or Klenow DNA polymerase, ligated and competent E. coli bacteria (DH5αF ′) were used for the transformation. The resulting bacterial colonies were tested for positive recombinant plasmids by colony filter hybridization using a lacZ-specific probe, and the corresponding plasmid DNAs were then subjected to restriction cleavage. Fig. 8 shows schematically the insertion of the lacZ cassette into the XcmI site of the pORF-1. The plasmids pCE9 and pCE10 contain the 3.2 kbp LacZ cassette inserted into the XcmI site in pORF-1 ( Fig. 9).

Example 4.2.2

The VEGF coding region contains a single StyI site that is as above was used to insert the lacZ cassette.

4.3 Deletion of viral sequences

The following examples use both coding (intra genetic deletions) as well as non-coding (intergenic deletions) area che described:  

Example 4.3.1

Using restriction enzymes, defined parts of the HindIII DNA fragment are removed to replace the deleted viral sequences by inserting foreign genes or parts thereof. This was done by cleaving the plasmid pORF-PA with the restriction enzyme NruI (at a location in the ITR region, FIG. 1), a 396 bp fragment being removed. After a replenishment reaction, the lacZ cassette was ligated as described above. Fig. 4 shows schematically the deletion of the 396 bp fragment from pORF-PA and the insertion of the lacZ cassette. Figures 5 and 6 show the deletion / insertion plasmids pCE4 and pCE5 thus constructed, derived from pORF-PA. Fig. 7 shows the DNA fragment pattern 14172 00070 552 001000280000000200012000285911406100040 0002019639601 00004 14053r of the plasmids pCE4 and pCE5 after restriction enzyme digestion.

Example 4.3.2

Individual restriction sites in the HindIII DNA fragment are used as starting points in order to bring about a bidirectional deletion of sequences by the action of the endonuclease Bal31, as is schematically outlined in FIG. 11. For restriction digestion, the enzyme StyI was used for the plasmid pORF-PA and the enzyme XcmI for the plasmid pORF-1 or pORF-XB in order to open the genes which code for VEGF or protein kinase F10L ( FIGS. 1 and 11). After addition of the exonuclease Bal31, equal parts of the reaction were removed every 2 minutes and the reaction was stopped. Splitting the current values z. B. with the restriction enzyme BglI and subsequent gel electrophoresis allowed the calculation of the size of the DNA segment deleted by Bal31. Mixtures from the appropriate times were then used to flatten the DNA ends by a fill reaction. Two complementary oligonucleotides, which are new unique Smal, SalI and EcoRV restriction sites, were hybridized and the resulting double-stranded primer molecules (referred to as "EcoRV" linkers) ligated to the blunt-ended Bal31 products. After bacterial transformation, plasmid DNA was isolated and digested with EcoRV, which contains no recognition site in the DNA sequence of the Orf-HindIII fragment. Therefore, each plasmid DNA with an EcoRV site contained the inserted linker sequence and was then used to ligate the blunt-ended lacZ cassette into the new EcoRV site. The exact size of the DNA deletion generated in each resulting recombinant plasmid DNA could be determined by sequencing with the single-stranded EcoRV linkers and with suitable LacZ gene-specific primers.

5. Detection and identification of the VEGF gene in others Parapox viruses

Knowledge of the DNA region encoding VEGF in D1701 allows for this Manufacture of specific DNA probes and PCR primers to this gene in others Parapoxvirus strains identify extremely different restrictions can have profiles. To this end, the following options were used tested: (i) isolation of a TaqI subfragment (366 bp in size) from pORF-PA as Hybridization probe that represents the central portion of the D1701 VEGF gene; (ii) Amplification of the complete VEGF-ORF using suitable synthetic Primer and subsequent plasmid cloning of the PCR product; (iii) primers that different parts of the VEGF gene were covered for PCR in the presence of PPV DNAs used as templates as well as specific hybridization probes.

After radioactive labeling, these probes were successfully used for Southern as well as dot / spot blot hybridization with the genomic DNA of various Strains of Parapox ovis, Parapox bovis 1 (Bovine papular stomatitis; BPS) and Parapox bovis 2 (milker knot). After cleaving the DNA of the different PPV strains with restriction enzymes were hybridized with defined DNA fragments found for further detailed Mapping the location of the VEGF gene in the different PPV genomes ver can be applied. Consequently, cloning and subcloning enables corresponding fragments the production of recombinant PPV, similar to it is described in Chapter 4 for the Orf virus D1701.

In addition, the same probes can be used for comparative RNA analysis as Northern blood hybridization, used to express potential Test VEGF genes in other PPV strains.  

6. Generation of D1701 recombinants

After the preparation and analysis of insertion / deletion plasmids that the contained lacZ cassette, 20 µg of purified plasmid DNA were used for transfection from cells infected with D1701 for approximately 3 hours. For this purpose the cell line BK KL-3A can be used has proven that it is efficient z. B. with the calcium phosphate technology trans can be infected and is permissive for D1701 and for other PPV strains. On modified method for transfecting cells in suspension established. If there were clear cytopathogenic effects, the cells were included Overlayed agarose, which contained X-gal as a substrate for β-galactosidase, resulting in leads to a dark blue staining of cells, the lacZ-positive recombinant Replicate D1701. Then blue plaques were picked and at least three times in BK-KL-3A cells multiplied and plaque-cleaned until stable lacZ virus recombinant was obtained.

7. VEGF promoter

As outlined in Chapter 3.1, the D1701 VEGF gene is an early gene The specific mRNA is found in D1701 virus-infected cells of two to four Hours after infection at large until later times of infection Quantities transcribed. Therefore the identified promoter region of D1701- VEGF is generally very useful for controlling the expression of foreign genes or parts thereof in recombinant PPV viruses. The sequence that the VEGF promoter (35 to 40 nucleotides; sequence protocol ID6), can be isolated are by (i) PCR using the appropriate primers that match the Flank the promoter region, (ii) subcloning the corresponding DNA fragment or (iii) synthesizing the promoter sequence as an oligonucleotide. After the ver linking the VEGF promoter to any gene or one of interest DNA sequence can be the resulting gene cassette for the production of recombinant PPV by any procedure as described in the previous chapter is used.

bibliography

1. Robinson AJ, Lyttle DJ
Recombinant Poxviruses ed. By MM Binns, born Smith CRC Press Boca Raton 1992 p 285-327
2. A. Mayr, M. Buttner
Chapter 7 (Ecthyma (Orf) Virus) in: Virus Infections of Vertebrates, Vol. 3: Virus Infections of Ruminants, 1990, Elsevier Science Publishers BV, The Netherlands
3. Mazur, C .; Rangel-Filho, FB; Galler, R
Molecular analysis of contagious pustular dermatitis virus: a simplified method for viral DNA extraction from scab material (1991) J. Virol. Methods 35, 265-272
4. AA Mercer et. al.
10th Int. Conf. on Poxvimses and Iridoviruses April 30th-5th May 1994 Proceedings
5. Fleming, SB et al.
Genomic analysis of a transposition-deletion variant of orf virus reveals a 3.3 kbp region of non-essential DNA (1995)
J. Gen. Virol 76, 2969-2978
6. Lyttle, DJ, Fraser, KM, Fleming, SB, Mercer, AA, Robinson, AJ
Homologs of Vascular Endothedial Growth Factor are encoded by the poxvirus Orf virus. (1994) J. Virol. 68, 84-92
7. Sutter, G., Moss, B Nonreplicating vaccinia vector efficiently expresses recombinant genes. (1992) Proc. Natl. Acad. Sci. USA 89, 10847-10851
18. Naase, M .; Nicholson, BH; Fsaser, KM; Mesces, AA; Robinsin, AJ
An orf virius sequence showing homology to the 14K "fusion" protein of vaccinia virus (1991) J.gen. Virol. 72, 1177-1181.

In the sequences ID No 1-11 described in the sequence listing will follow Information given:

ID NO 1

Sequence ID1 of the application shows that on the HindIII / I fragment of the stem with ORF D1701 localized VEGF gene.

It corresponds to the nucleotide positions 3361 to 3900 of the HindIII / I fragment (ID8).
Additional Information:
Early promoter: nucleotides 48 to 65
5'-end mRNA: nucleotides 79 and 80, respectively
3'-end mRNA: nucleotides 498 to 500
Start codon: nucleotides 92 to 94
Stop codon: nucleotides 488 to 490

ID NO 2

Sequence ID2 of the application shows that on the HindIII / I fragment of the stem with ORF D1701 localized protein kinase gene.

It corresponds to nucleotide position 1180 to 2921 of the HindIII / I fragment (ID8).
Additional Information:
Late promoter core-motif: nucleotides 48 to 66
RNA start signal: nucleotides 72 to 80
5'-end mRNA: nucleotide 88
Start codon: nucleotides 94 to 96
Stop codon: nucleotides 1738 to 1740

ID NO 3

Sequence ID3 of the application shows that on the HindIII / I fragment of the stem With ORF D1701 localized HD1R gene segment.

It corresponds to nucleotide position 1 to 1080 of the HindIII / I fragment (ID8).
Additional Information:
Stop codon: nucleotides 1075 to 1077

ID NO 4

Sequence ID4 of the application shows that on the HindIII / I fragment of the stem With ORF D1701 localized ITR region and the one located in this region ORF3 gene.

It corresponds to nucleotide positions 3901 to 5515 of the HindIII / I fragment (ID8).
Additional Information:
Start of ITR region: nucleotide 5
Early promoter: nucleotides 19 to 30
Start codon ORF3: nucleotides 111 to 113
Stop codon ORF3: nucleotides 562 to 564

ID NO 5

Sequence ID5 of the application shows that on the HindIII / I fragment of the stem with ORF D1701 localized F9L gene.

It corresponds to nucleotide positions 2682 to 3580 of the HindIII / I fragment (ID8).
Additional Information:
Start codon: nucleotides 48 to 50
Stop codon: nucleotides 861 to 863

ID NO 6

Sequence ID6 of the application shows that on the HindIII / I fragment of the stem with ORF D1701 localized VBGF promoter region.

It corresponds to nucleotide positions 3387 to 3478 of the HindIII / I fragment (ID8).
Additional Information:
Early promotor consensus: nucleotides 24 to 41
Starting point of transcription: nucleotides 53 to 55
Start codon: nucleotides 68 to 70

ID NO 7

Sequence ID7 of the application shows that on the HindIII / I fragment of the stem With ORF D1701 located between the HD1R and PKF10L genes located intergenic region.

It corresponds to the nucleotide positions 1051 to 1300 of the HindIII / I fragment (ID8).
Stop codon HD1R: nucleotides 24 to 26
Start codon PKF10L: nucleotides 223 to 225

ID NO 8

Sequence ID8 of the application shows the complete nucleotide sequence of the HindIII / I fragment of ORF strain D1701.

ID NO 9

Sequence ID9 of the application shows a variant of that on the HindIII / I question ment of the ORF D1701 strain localized protein kinase gene.

It corresponds to the nucleotide positions 1180 to 2799 of the HindIII / I fragment (ID8).
Additional Information:
Late promoter core-motif: nucleotides 48 to 66
RNA start signal: nucleotides 72 to 80
5'-end mRNA: nucleotide 88
Start codon: nucleotides 94 to 96
Stop codon: nucleotides 1583 to 1585

ID NO 10

Sequence ID10 of the application shows a variant of the on the HindIII / I- Fragment of strain ORF D1701 localized F9L gene.

It corresponds to nucleotide positions 2680 to 3459 of the HindIII / I fragment (ID8).
Additional Information:
Start codon: nucleotides 48 to 50
Stop codon: nucleotides 722 to 724

ID NO 11

Sequence ID9 of the application shows one on the EcoRI / E fragment of the ORF D1701 strain located 10 kD gene fragment.

List of pictures

Fig. 1 shows the physical map of the HindIII-I fragment from Orf D1701 in the plasmids pORF-1 / -2.

Fig. 2 shows the physical map of the HindIII restriction. Enzyme recognition sites on the D1701 genome.

Fig. 3 shows the LacZ cassette with the LacZ gene coupled to the 11K promoter.

Fig. 4 shows plasmid pORF-PA, in which the 3.2 Kbp LacZ cassette was inserted after a deletion of 395 bp.

Fig. 5 shows plasmid pCE4 as a construct from pORF-PA and the LacZ cassette from pGSRZ, the orientation of which is analogous to the VEGF gene.

Fig. 6 shows plasmid pCE5 as a construct from pORF-PA and the LacZ cassette, the orientation of which runs counter to the VEGF gene.

Fig. 7 shows DNA fragment patterns of the treatment of pCE4 and pCE5 with restriction enzymes PVuII and SacI separated in agarose gel electrophoresis.

Fig. 8 shows plasmid pORF1, into which the 3.2 Kbp LacZ cassette was inserted after linearization by X cm I.

Fig. 9 shows plasmid pCE9 / 10, which consists of the construct of pORF1 and the LacZ cassette. The insert shows a course analogous to the PKF 10 gene.

Fig. 10 shows schematically the PCR mutagenesis.

Fig. 11 shows schematically the Bal 31 mediated deletion of DNA fragments.

Claims (49)

1. Recombinantly produced parapox viruses (PPV) with insertions and / or Deletions. 2. Recombinantly produced parapox viruses with insertions and / or dele in genome sections that are not necessary for virus replication are. 3. Recombinantly produced parapox viruses with insertions and / or dele genome sections that are necessary for virus replication. 4. Recombinantly produced parapox viruses, the insertions and / or dele tions in the areas of Hind III fragment I from PPV strain D1701 as well as its variants and homologs that are not expressed will. 5. Recombinantly produced parapox viruses, the insertions and / or dele tions in the areas of Hind III fragment I from PPV strain D1701 as well as its variants and homologs that are expressed. 6. recombinantly produced parapox viruses according to claims 1 to 5, insertions and / or deletions in Hind III fragment I of the PPV Strain D1701 and its variants and homologues or the latter Fragment corresponding DNA localized from other parapox viruses are. 7. Recombinantly produced parapox viruses, the insertions and / or dele ions in the region of the VEGF gene or adjacent to it. 8. Recombinantly produced parapox viruses, the insertions and / or dele ions in the region of the PK gene or adjacent to it. 9. Recombinantly produced parapox viruses, the insertions and / or dele tions in the area of the ITR section or adjacent to it hold.   10. Recombinantly produced parapox viruses, the insertions and / or dele ions in the region or adjacent to the gene which is responsible for the 10 KDa protein encoded, included. 11. Recombinantly produced parapox viruses, the insertions and / or dele ions in the region of the HD1R gene or adjacent to it. 12. Recombinantly produced parapox viruses, the insertions and / or dele ions contained in or adjacent to the F9L gene. 13. Plasmid containing Hind III fragment I from PPV strain D1701 and corresponding to its variants and homologues or to this fragment DNA from other parapox viruses. 14. Plasmid containing Hind III fragment I from PPV strain D1701 and its variants and homologues, the deletions in this fragment and / or insertions included in areas responsible for virus replication are necessary. 15. Plasmid containing Hind III fragment I from PPV strain D1701 and its variants and homologues, the deletions in this fragment and / or insertions in areas that are not necessary for virus replication are contains. 16. Plasmid containing Hind III fragment I from PPV strain D1701 and its variants and homologues, the deletions in this fragment and / or contains insertions in areas not for the Virus replication is necessary and is in areas that are not be expressed. 17. Plasmid containing Hind III fragment I from PPV strain D1701 and its variants and homologues, the deletions in this fragment and / or contains insertions in areas that are not for the virus multiplication are necessary and are located on areas that are expressed will.   18. Plasmid containing Hind III fragment I from PPV strain D1701 and its variants and homologues that are in or adjacent to the VEGF gene this fragment contains deletions and / or insertions. 19. Plasmid containing Hind III fragment I from PPV strain D1701 and its variants and homologues that are in or adjacent to the PK gene this fragment contains deletions and / or insertions. 20. Plasmid containing Hind III fragment I from PPV strain D1701 and its variants and homologues that are in or adjacent to ITR section of this fragment contains deletions and / or insertions. 21. Plasmid containing Hind III fragment I from PPV strain D1701 and its variants and homologues that are in or adjacent to the HDIR gene and / or F9L gene contains deletions and / or insertions. 22. Plasmid containing DNA fragment from D1701 and its variants and Homologs that are in or adjacent to the gene for the 10 KDa protein encodes deletions and / or insertions. 23. Plasmid-containing part of Hind III fragment I from PPV strain D1701 as well as its variants and homologues, in which deletions and / or Inserts according to claims 13 to 22 are included. 24. A plasmid according to claims 13 to 23, wherein the Hind III fragment I from PPV strain D1701 and its variants and homologues through a DNA from other parapox viruses corresponding to this fragment was replaced is. 25. Plasmid according to claims 13 to 24, wherein the Hind III fragment I as well as its variants and homologues completely or only partially is present. 26. Genome fragment Hind III fragment I from PPV strain D1701 and its Variants and homologues or parts thereof or of this fragment corresponding fragments from other parapox viruses with the sequence according to sequence listing ID No 8.   27. DNA section or parts thereof from Hind III fragment I of PPV Strain D1701 and its variants and homologues or the latter Section or parts thereof corresponding section from other Para pockenviren, which codes for VEGF protein according to sequence protocol ID1. 28. DNA section from Hind III fragment I of PPV strain D1701 as well as its variants and homologues, which codes for PK protein according to Sequence listing ID NO2 or ID NO9 (gene variant). 29. DNA section or parts thereof from parapox viruses coding for gene HD1R with the sequence according to sequence protocol ID NO3 and its Variants and homologues. 30. DNA section or parts thereof of parapox viruses for F9L with the Sequence according to sequence protocol ID NO5 (F9L gene) and its Variants (ID NO10) and homologues. 31. DNA section or parts thereof from parapox viruses for the ITR area with the sequence according to sequence protocol ID4 (ITR area) and its Variants and homologues. 32. Gene products made based on the sequences of the DNA segments according to claims 26 to 31. 33. Recombinantly produced parapoxviruses according to claims 1 to 12, the contain foreign DNA as inserts, which are used for immunogenic components code from other pathogens. 34. Recombinantly produced parapox viruses according to claims 1 to 12 and 33, which contain foreign DNA as inserts, which are used for immune mediators encode. 35. A method for producing the viruses according to claims 1 to 12, 33 and 34, characterized in that the plasmids according to claims 13 to 25 recombined in a manner known per se with parapox viruses in cells and selected for the desired viruses.   36. A method for producing the plasmids according to claim 22, characterized in that

• 1. selects a suitable parapox virus strain,
• 2. cleanses his genome,
• 3. treated the purified genome with restriction enzymes,
• 4. the fragments obtained are inserted into plasmids and
• 5. selected for the plasmids which contain the gene which codes for the 10 KDA protein and its variants and homologues, and
• 6. optionally inserts and / or deletions into the gene coding for the 10 KDa protein.

37. Process for the preparation of the plasmids according to Claims 13 to 21 and 23 to 25, characterized in that

• 1. selects a suitable parapox virus strain,
• 2. cleanses his genome,
• 3. treated the purified genome with restriction enzymes,
• 4. the fragments obtained are inserted into plasmids and
• 5. selected for the plasmids which contain the HindIIIi fragment and variants and homologues or fragments or components thereof corresponding thereto, and
• 6. optionally insertions and / or deletions in these fragments in the plasmids obtained.

38. Process for the preparation of the Hind IIIi fragment or the DNA section which codes for the 10 KDa protein, from PPV strain D1701 or the region corresponding to this fragment or section from other parapox viruses or parts thereof, characterized in that one

• 1. selects a suitable parapox virus strain,
• 2. cleanses his genome,
• 3. treated the purified genome with restriction enzymes,
• 4. and selected the desired fragments or sections or
• 5. If necessary, the fragments of the genome obtained are first inserted into plasmids, the plasmids with the desired fragments are isolated, these plasmids are multiplied and the desired fragments are isolated therefrom.

39. A method for producing the gene products according to claim 32, characterized characterized in that the fragments obtainable according to claim 38 in transferred suitable expression systems and by means of these systems the Genes expressed. 40. Use of the recombinantly produced parapox viruses according to An say 1 to 12 in vaccines. 41. Use of the recombinantly produced parapox viruses according to An say 1 to 12 in products that immunize as well Stimulate non-pathogen-specific immune defense. 42. Use of the recombinantly produced parapoxviruses in immune modulators that stimulate the non-pathogen-specific immune defense. 43. Use of the recombinantly produced parapox viruses for the heterologous expression of foreign DNA.   44. Use of the recombinantly produced parapox viruses as vectors for foreign DNA. 45. Use of the plasmids according to claims 13 to 25 for expression parapox-specific genome sections. 46. Use of the plasmids according to claims 13 to 25 for the preparation of diagnostic agents. 47. Use of the genome fragments according to claims 26 to 31 for Manufacture of diagnostic agents. 48th DNA section as well as variants and homologues according to the sequence listing ID NO6 (promoter of the VEGF gene). 49. Use of the DNA section as well as variants and homologues according to Claim 48 as a promoter for the expression of DNA with para smallpox viruses.