WO2001036587A2 - Gene for encoding merozoite surface protein of plasmodium vivax and use thereof - Google Patents
Gene for encoding merozoite surface protein of plasmodium vivax and use thereof Download PDFInfo
- Publication number
- WO2001036587A2 WO2001036587A2 PCT/KR2000/001302 KR0001302W WO0136587A2 WO 2001036587 A2 WO2001036587 A2 WO 2001036587A2 KR 0001302 W KR0001302 W KR 0001302W WO 0136587 A2 WO0136587 A2 WO 0136587A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gene
- sequence
- protein
- plasmodium
- recombinant protein
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 42
- 210000003936 merozoite Anatomy 0.000 title claims abstract description 19
- 241000223810 Plasmodium vivax Species 0.000 title claims description 22
- 108010052285 Membrane Proteins Proteins 0.000 title abstract description 15
- 102000018697 Membrane Proteins Human genes 0.000 title abstract description 15
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 34
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 34
- 238000002965 ELISA Methods 0.000 claims abstract description 13
- 239000013604 expression vector Substances 0.000 claims abstract description 5
- 201000004792 malaria Diseases 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 238000010367 cloning Methods 0.000 claims description 8
- 238000009007 Diagnostic Kit Methods 0.000 claims description 6
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 6
- 108091008146 restriction endonucleases Proteins 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims 1
- 102000036639 antigens Human genes 0.000 abstract description 30
- 108091007433 antigens Proteins 0.000 abstract description 30
- 241000224016 Plasmodium Species 0.000 abstract description 22
- 208000015181 infectious disease Diseases 0.000 abstract description 18
- 238000003745 diagnosis Methods 0.000 abstract description 10
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 241000588724 Escherichia coli Species 0.000 abstract description 4
- 210000004899 c-terminal region Anatomy 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 37
- 108020004414 DNA Proteins 0.000 description 22
- 239000000427 antigen Substances 0.000 description 19
- 210000002966 serum Anatomy 0.000 description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 210000004369 blood Anatomy 0.000 description 14
- 239000008280 blood Substances 0.000 description 14
- 238000002835 absorbance Methods 0.000 description 13
- 239000013642 negative control Substances 0.000 description 13
- 239000000872 buffer Substances 0.000 description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 10
- 201000010099 disease Diseases 0.000 description 10
- 239000012634 fragment Substances 0.000 description 10
- 239000000758 substrate Substances 0.000 description 10
- 210000003743 erythrocyte Anatomy 0.000 description 8
- 239000013641 positive control Substances 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 238000001962 electrophoresis Methods 0.000 description 7
- 208000024891 symptom Diseases 0.000 description 7
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 6
- 206010035503 Plasmodium vivax infection Diseases 0.000 description 6
- 201000009976 Plasmodium vivax malaria Diseases 0.000 description 6
- 208000005469 Vivax Malaria Diseases 0.000 description 6
- 239000002131 composite material Substances 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 241000282414 Homo sapiens Species 0.000 description 5
- 108010057081 Merozoite Surface Protein 1 Proteins 0.000 description 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 241000255925 Diptera Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 208000007502 anemia Diseases 0.000 description 3
- 210000001772 blood platelet Anatomy 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000002405 diagnostic procedure Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 210000003046 sporozoite Anatomy 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical class OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 206010061192 Haemorrhagic fever Diseases 0.000 description 2
- 206010019233 Headaches Diseases 0.000 description 2
- 241000711549 Hepacivirus C Species 0.000 description 2
- 241000700721 Hepatitis B virus Species 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000012807 PCR reagent Substances 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 206010037660 Pyrexia Diseases 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- -1 al. Chemical compound 0.000 description 2
- 230000003460 anti-nuclear Effects 0.000 description 2
- 230000011681 asexual reproduction Effects 0.000 description 2
- 238000013465 asexual reproduction Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 231100000869 headache Toxicity 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 206010039766 scrub typhus Diseases 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000000405 serological effect Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 1
- HQWQVBJUIIJTRE-LKRNKTNVSA-N 4-amino-n-(5,6-dimethoxypyrimidin-4-yl)benzenesulfonamide;(s)-[2,8-bis(trifluoromethyl)quinolin-4-yl]-[(2r)-piperidin-2-yl]methanol;5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine;hydron;chloride Chemical compound Cl.CCC1=NC(N)=NC(N)=C1C1=CC=C(Cl)C=C1.COC1=NC=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=C1OC.C([C@@H]1[C@@H](O)C=2C3=CC=CC(=C3N=C(C=2)C(F)(F)F)C(F)(F)F)CCCN1 HQWQVBJUIIJTRE-LKRNKTNVSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 101000705294 Arabidopsis thaliana Oxygen-evolving enhancer protein 1-2, chloroplastic Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 208000002091 Febrile Seizures Diseases 0.000 description 1
- 101000636168 Grapevine leafroll-associated virus 3 (isolate United States/NY1) Movement protein p5 Proteins 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 101710085938 Matrix protein Proteins 0.000 description 1
- 101710127721 Membrane protein Proteins 0.000 description 1
- 101100151229 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) msp-4 gene Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000223960 Plasmodium falciparum Species 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 208000001871 Tachycardia Diseases 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- MJOQJPYNENPSSS-XQHKEYJVSA-N [(3r,4s,5r,6s)-4,5,6-triacetyloxyoxan-3-yl] acetate Chemical compound CC(=O)O[C@@H]1CO[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@H]1OC(C)=O MJOQJPYNENPSSS-XQHKEYJVSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000011841 epidemiological investigation Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 206010016284 febrile convulsion Diseases 0.000 description 1
- 230000027950 fever generation Effects 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 201000004108 hypersplenism Diseases 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000000055 macrogametocyte Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000000052 microgametocyte Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000013102 re-test Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000014639 sexual reproduction Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000011120 smear test Methods 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 201000009225 splenic sequestration Diseases 0.000 description 1
- 210000004215 spore Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000006794 tachycardia Effects 0.000 description 1
- 208000008203 tachypnea Diseases 0.000 description 1
- 206010043089 tachypnoea Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 210000004916 vomit Anatomy 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
- C07K14/445—Plasmodium
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates a gene encoding merozoite surface protein of Plasmodium vivax, a method of use thereof in preparing recombinant antigen protein, the resulted recombinant antigen protein and a diagnosis reagent comprising the same protein
- this invention is useful for the diagnosis of malarial infection by detecting antigen-antibody composites obtained from reaction the antigen with a test sample in question BACKGROUND ART
- Malaria is one of generally known fever diseases worldwide and an increasing health problem throughout the world including Korea More than a billion people live in malaria-prevalent area, at least a hundred million patients suffer from each year and more than one million cases among them die from that disease Therefore, this disease is considered as one of the most important six diseases that the Health World Organ ⁇ sat ⁇ on ⁇ (WHO) designates Plasmodium vivax parasitize on human bodies and destroys erythrocytes to cause various symptoms such as periodic febrile seizure, anemia, splenectasis and so forth
- Malarial infection is known to progress under the following steps Malarial sporozoites introduced from the mouth of mosquitoes into human body propagate within hepatocytes for several days(8-9days) and go into erythrocytes carrying out the asexual reproduction After 48 hours, the sphtted merozoites destroy the erythrocytes and flow along blood vessel and move into another erythrocytes During repeatedly such asexual reproduction, some of merozoites are transformed into macrogametocytes and/or microgametocytes These cytes perform sexual reproduction, as introduced into body of mosquito These cytes become spores through zygote-ookimete-oocyst steps(sporgony), and are present in salivary gland until they transfers to another object As above mentioned in life cycle, the clinical property for mala ⁇ a is that sporozoites of Plasmodium parasitize in erythrocytes after dividing and increasing steps within hepatic cells then producing a number of cyptomerozo
- malaria models having typical symptom and clinical progress it has been shown that a cold stage to feel a chill, have a headache and/or vomit is firstly appeared for a few minutes to one or two hours, a second hot stage of representing heated and dried skin, tachycardia, tachypnea is progressed for 3-6 hours followed by a wet stage of perspiring
- the pyrogenetic stage of malarial disease depends on species of Plasmodium, for example 48 hours for Plasmodium vivax.
- anemia symptom is caused by the destroy of erythrocytes so called hemocytocatheresis, the splenectasis is due to the deposition of the broken erythrocytes and hemoglobin and the decrease of blood platelet may be derived from antibody-mediated splenic sequestration of blood platelet coated with Plasmodium.
- malarial disease can be determined and/or diagnosed by carefully observing its clinical symptom. If it has a regular and typical pyrogenetic cycle, the species of Plasmodium may be identified. Thick smear test is a method performed by smearing and drying large volume of blood, complete hemolysis of erythrocytes followed by the examination of the remained Plasmodium and leukocytes with a microscope so that it can conveniently determine whether a subject is at the malaria-positive or negative conditions. However, this test needs a staining procedure of blood samples, therefore taking several hours to observe the blood film with a microscope.
- the present inventor has found that C-termmal regions of merozoite surface protein of Plasmodium vivax has unexpectedly high antigemcity Therefore, it is still another object of the present invention to provide a diagnostic reagent containing recombinant protein obtained by the expression of the gene encoding such regions in host vector after cloning such gene, as well as its use in determining whether any subject m question is under malarial infection condition.
- Mala ⁇ al infection has been generally known to be caused from four types of Plasmodium; P vivax, P. falciparum, P malariae and P ovale.
- P vivax both of P vivax and P malariae cause mostly the malarial infection.
- P vivax is distributed over the most extensive area and is the major plasmodium of malarial infection discovered m Korea, as described by Sho, jn-tak, "Human parasitology" which is incorporated herewith for reference.
- MSP-1, MSP-2 and MSP-4 of merozoite surface proteins of Plasmodium are coupled to GPI-hpids m outer membrane of the merozoite of Plasmodium to represent antigemcity against host (Camila I et al., Vaccine 17, 2959- 2968, 1999).
- merozoite surface protein of Plasmodium vivax has a ammo acid sequence denoted as Sequence ID No. 2, together with DNA sequence denoted as Sequence ID No.l . These both sequences have been previously deposited as AF200953 on November 3, 1999 with GenBank. Cloning such DNA sequence and expressing the gene in E. coli vector according to gene recombination technique result in 27 5 kDa recombinant antigen protein.
- the present inventor noticed that it is possible to use a particular MSP-1 recombinant protein of Plasmodium vivax reacting with serum of a Plasmodium-infected patient m determining and/or detecting malarial infection. Therefore, He performed the cloning process for C-terminal region of gene encoding MSP-1 from Plasmodium vivax Repeatedly It has been found that about 27 kDa recombinant protein produced from the partial gene (noted as Sequence ID No. 3) of cloned merozoite surface protein has very high antigemcity against host.
- the present invention provides novel recombinant protein obtained from the gene encoding merozoite surface protein of cloned Plasmodium and an Enzyme-Lmked ImmunoSorbent Assay(ELISA) type diagnostic kit comprising such protein
- ELISA Enzyme-Lmked ImmunoSorbent Assay
- 27.5 kDa and 27 kDa recombinant proteins of Plasmodium can be used as specific antigens, and also in preparation of DNA probe, monoclonal antibody or vaccine.
- the present invention further provides an improved method for quickly and conveniently diagnosing and/or determining whether a subject is infected with Plasmodium, and concurrently having specificity and sensitivity only to patient infected with
- Plasmodium especially with Plasmodium vivax other than patients for example having hepatitis B or
- the process to determine gene sequence encoding MSP-1 from P. vivax comprises a plurality of steps;
- the first step is to design p ⁇ mer by using ⁇ -cyanoethyl phosphormidite method in order to isolate MSP-related gene.
- the primer 1 (5'-GAATTCAAAATCGAGAGCATGATCG-3') is designed to have a specificity cleaved with restriction enzyme EcoR I whilst another p ⁇ mer 2 (5'- GTCGACGACATTGAATAGGAGGTC-3') is designed to have a specificity cleaved with rest ⁇ ction enzyme Hind III
- DNA segments related to MSP are isolated from Plasmodium vivax by reacting genomic DNA with such primers obtained from the first step.
- the third step is to determine the base sequence of said DNA segment.
- recombinant antigen protein is produced by the expression of such sequential regions on E.coh vector pET21a(Novagen).
- the fifth step comprises the investigation of antigemcity of the recombinant antigen protein prepared through above speps.
- the diagnostic kit for ELISA containing such prepared recombinant protein is manufactured in the last sixth step
- the inventor has carried out the cloning process of gene (Sequence ID No.3) encoding C- terminal of merozoite surface proteins of Plasmodium vivax and prepared recombinant antigen protein from the gene.
- Sequence ID No. 3 The sequence represented by Sequence ID No. 3 is characterized in that:
- Type (of sequence) nucleic acid in) Type (of molecules) : genomic DNA
- 27 kDa recombinant protein is generated in the same procedure as 27.5 kDa protein mentioned above, except that in the first step, p ⁇ mer l(5'-GTGGAATTCGAGTACGAGTCC-3') is designed to have a specificity cleaved with restriction enzyme EcoR I whilst another primer 2 (5'-
- TCTGTCGACAAGCTCCATGCA-3' is designed to have a specificity cleaved with restriction enzyme Sal I.
- the present invention comprises detecting antigen-antibody composites formed by reacting malarial infected blood (particularly, blood infected with vivax malaria) with said recombinant protein.
- the diagnostic method comprises detecting the presence of an antibody against malarial antigen in blood according to the principle of known Enzyme Linked Immunosorbent Assay(ELISA). More particularly, the diagnostic method comprises adsorbing malarial antigens to each well of a 96-wells microplate as a solid supporting body and reacting malarial antigen adsorbed to the well with an antibody existed in blood of the subject to be test according to the following steps:
- rabbit anti-human lmmunoglobuhn antibody, anti-canine immunoglobulin antibody and rabbit anti-felme immunoglobulin all of them being conjugated with horseradish peroxidase (HRP) are added to such well to induce its specific coupling to antibody region of the above formed antigen-antibody composite.
- HRP conjugated second antibody as a chromopho ⁇ c enzyme is removed m a washing step.
- TMB solut ⁇ on[(3,3',5,5'-Tetramethyl benzidme, Merck] as a substrate is added to the material obtained from the second step. If the antigen-antibody composite from the second step was coupled with the second antibody, because of chromopho ⁇ c reaction it appears yellow color
- the chromaticity of final chromopho ⁇ c reactant is determined by means of the microwell reader. With this, whether malarial antibody exists in blood can be determined. According to the principle of three-steps diagnosis illustrated above, polystyrene beads, nitrocellulose strips and so forth can be used as solid supporting bodies for adhering antigen.
- a diagnostic kit consisting of essential reagents to detect malarial antibody in blood depending on the diagnostic principle desc ⁇ bed above, in addition to malarial antigen adsorbed to the solid supporting body.
- the malarial antigen used in the present invention is 27 kDa or 27.5 kDa recombinant protein
- Such recombinant protein is obtained form a clone having high reactivity with malaria-infected serum, and is produced by generally known methods to produce recombinant protein
- FIG 1 shows an electrophoretic results of the recombinant malarial antigen protein
- M denotes standard molecular weight protein
- S is an isolated malarial antigen appearing at a location corresponding 27.5 kDa of molecular weight.
- C means crude protein without pu ⁇ fication
- FIG. 2 shows a result of Western blotting for the 27.5 kDa recombinant protein
- ANA means anti-nuclear antibody
- EHF is serum of hemorrhagic fever case
- HBV is serum of hepatitis B virus infected case
- HCV denotes serum of hepatitis C virus infected case
- aRt is serum of Richettsia Tsutsugamushi-mfected case
- RA is serum of Rheumatoid arthritis patient
- VDRL means serum of venereal disease case
- aPv is serum of vivax malaria case and nor is serum of normal case.
- FIG 4 shows an electrophoretogram of 27kDa recombinant malarial antigen.
- M denotes standard molecular weight protein.
- (1) in this figure shows uncoupled protein fragments, (2) means purified protein fragment 1 while, and (3) means purified protein fragment 2.
- FIG 5 illustrates another measurement result of the antibody against 27kDa recombinant protein in blood.
- (-) shown in this figure is negative standard and (+) is positive standard, respectively, and malaria means serum of patient suffering from malaria while normal being serum of normal case.
- (-) shown in this figure is negative standard and (+) is positive standard, respectively, and malaria means serum of patient suffering from malaria while normal being serum of normal case.
- 0.5g(wet weight) of Plasmodium vivax was frozen under liquid nitrogen and the resulted solids were ground by means of mortar.
- To the ground powders was added with 1.2ml of digestion buffer consisted of lOOmM NaCl, lOmM t ⁇ s-HCl with pH8.0, 25mM EDTA, 0.5% SDS and 0.12mg of proteinase K, per lOOmg of Plasmodium and was shaken at 50 ° C for 18 hours to dissolve cell membranes.
- the obtained sample was added with an equivalent amount of mixture of phenol/chloroform/isoamyl alcohol at a ratio of 25:24: 1 and thoroughly admixed together, following by cent ⁇ fugation by means of swinging bucket rotor(Sorval RC-5, USA) at 20,000 g for 10 minutes at room temperature. Thereafter, supernatant was transferred into another tube and 7.5M ammonium acetate (m the amount equal to 1/2 of supernatant) and 100% ethanol (in the amount equal to 2 times supernatant) were added into the tube, respectively, and then the sample was cent ⁇ fuged at 10,000g for 2 minutes. After this, the obtained pellets were washed with 70% ethanol and dried.
- DNA was thoroughly dissolved with TE buffer comprising lOmM t ⁇ s-HCl and ImM EDTA with pH8.0 for 3 hours at 65 "C To remove the remaining RNA, The resulted sample was treated with 0.1% SDS and lug/ul DNase-free RNase and allowed to place at 37 ° C for lhour following by organic extraction and ethanol precipitation.
- lOul of the reactant solution was added with 2ul of a sample buffer(6X, 0.25% Bromophenol blue, 0.25% Xylene cyano FF, 40% sucrose) and then was electrophoresed on 1% agarose gels.
- the amplified DNA fragment was cleaved and purified by using the Geneclean II k ⁇ t(B ⁇ o
- the purified DNA was cloned on pT7Blue T-vector(Novagen).
- the resultant cloned plasmid DNA was purified by alkahsis method (Birnboim HC et al., Ish-Horowicz 0 and Barke JF, 1981 ) and then by the Geneclean II kit.
- DNA purified above was added to T7 promotor primer and T3 reverse primer (ABI cloning system) and PCR amplified, following by the electrophoresis treatment at 2,500V and 30mA for 12 hours on 4.75% polyacrylamide gel containing 8M urea.
- the result of this electrophoresis was analyzed by the Microgene software program (manufactured by ABI Corp.) The result is proposed as Sequence ID No. 1.
- Sequence ID No.l is the sequence for gene encoding MSP of P. vivax which was deposited with GenBank as AF200953 (November 3, 1999). (but, not published by November 2, 2002)
- the cloned gene above was introduced into E. coli expression vector pET21 a(Novagen Corp ) and treated with heat shock to produce pET21-Pv, and then incubated on with 50ug/ml of Ampicillin-contaming LB plate (0.5% yeast extracts, 1% t ⁇ pton, 1% sodium chloride and 1.5% agar) at 37 °C to select the recombinant clone.
- the selected clone was introduced 10ml of LB amp+ and incubated within a shaking incubator at 200 rpm for 16 hours. In order to express recombinant protein, incubated cells were added to 1-.
- LB amp+ medium incubated to reach 0.6-0.8 at OD600 and then cultured with the addition of 4ml of 1M IPTG(B ⁇ oneer Corp.) for 3 hours. After the cent ⁇ fugation, the collected cells were subjected to a washing step with 100ml of 20mM T ⁇ s- Cl(pH8.0) following by another cent ⁇ fugation to collect the washed cells, and then was sonificated with 50ml Buffer A (20mM T ⁇ s-Cl(pH8.0), 150mM NaCl, 8M urea).
- N ⁇ 2+-NTA agarose beads were washed with a buffer B(5mM Imidazole, 20mM T ⁇ s-Cl(pH8.0), 150mM NaCl) and then poured into and passed through a CIO column (Parmacia Biotech Inc.) to eluate the protein depending on Imidazole concentration. About 27.5kDa of recombinant protein was resulted from the above experiment (see FIG. 1).
- Western blotting method was carried out. About 0.5ug of plasma protein was electrophoresed on 10% polyacrylamide gels under the presence of SDS based on Laemmh's method(Laemmh, Nature 227, 680(1970)) and transferred the protein isolated from the electophresis gels to the nitrocellulose filter membrane (manufactured by Milhpore Corp., pore size 0.45um) according to Towbm's method (Towbin et al., Proc, Natl.
- the filter was reacted with a solution containing 0.5% bovme-serum albumin (BSA) to prevent nonspecific binding
- BSA bovme-serum albumin
- the test serum from vivax malaria patient was added to PBS buffer containing 0.05% Tween 20 (lOmM sodium phosphate, 0.15M NaCl, pH7.0) at the volume of 10,000 and then the filter was washed twice each for 15 minutes in said PBS buffer containing 0.05% Tween 20.
- the filter was added with the second antibody (that is, goat anti-rabbit immunoglobulin G diffled with horseradish peroxidase) to react together and then was washed with said PBS buffer.
- TMB solution was applied to the filter to be chromophored. The result of this experiment is shown in Figure 2 accompanied heremabove.
- Antigen adsorption plate The diluted recombinant malarial antigen protein with 0.05M bicarbonates buffer(S ⁇ gma Corp., capsule) was adsorbed to 96 wells at a volume of lOOul each well.
- Serum 1 containing malarial antibody is diluted by the addition of said specimen diluent solution 100.
- Negative control solution Serum 1 of normal case is diluted by the addition of said specimen diluent solution 100.
- Substrate concentration solution TMB(3,3',5,5'- Tetramethylbenzidme) as a substrate was diluted at a ratio of 1 : 100 with substrate buffer.
- Substrate buffer Citric acid phosphate buffer(pH5.0) was added with 0.035% hydrogen peroxide.
- such recombinant antigen represented a single band for each of vivax malaria-infected blood and serum whilst no reaction was found out for normal case (not shown in Figure)
- the present inventive antigen may specifically couple to antibodies of vivax malaria-infected patients
- the diagnostic sensitivity was observed to 97% (29 of 30 cases) and the diagnostic specificity being 100% (34 of 34 cases) From the result of anti-nuclear patients (representing a diagnostic specificity of 100%, 0 of 12 cases), hemorrhagic fever cases (a diagnostic specificity of 89%, 1 of 9 cases), hepatitis B virus infected cases (a diagnostic specificity of 92 5%, 3 of 40 cases), hepatitis C virus infected cases (a diagnostic specificity of 87 5%, 3 of 24 cases), Richettsia Tsutsugamushi infected cases (a diagnostic specificity of 94 4%, 0 of 8 cases), Rheumatoid arthritis patients (a diagnostic specificity of 94 4%, 1 of 18 cases) and VDRL cases (a diagnostic specificity of 100%, 0 of 40 cases), it has been found that 27 5 kDa recomb
- Example 2 Materials and Methods The same procedure as in Example 1 was repeated to isolate DNA from Plasmodium lOul of the obtained genomic DNA, lOul of primer 1 (5'-GTGGAATTCGAGTACGAGTCC- 3') having a specificity cleaved with restriction enzyme EcoR I and lOul of primer 2 (5 - TCTGTCGACAAGCTCCATGCA-3') having a specificity cleaved with rest ⁇ ction enzyme Sal I were added to GeneAmp PCR reagent kit system with AmpliTaq DNA Polymerase (Perkin-Elmer Cetus Corp ) The resulted PCR reactant solution was subject to PCR amplification in the same manner as described in Example 1, to generate about 618bp DNA fragments after electrophoresis
- Example 2 Furthermore, by the same procedure of Example 1 the purification and cloning treatments were carried out To obtained cloned gene was expressed in pET21a (Novagen Corp.), E. coli expression vector to create about 27 kDa recombinant protein according to the same manner as illustrated m Example 1. (see FIG. 4)
- the average absorbance of negative control solution must be withm 0.000 to 0.100 If any one of such three absorbencies is out of the above range, the average value should be calculated from only the remained both absorbencies. However, in case of where at least two absorbencies are out of the above range it is necessary to perform the re-exammation and/or re-test the absorbance for the negative control solution.
- the average absorbance of positive control solution must be at least 0.500 and the difference between both absorbencies must be less than 0.100. If such average absorbance is out of the above range, it will be expected to have problems in test procedure or the reagent. So, after finding out and eliminating the problems, the re-examination of the absorbance for the positive control solution should be performed. (3) Calculation of Cutoff value
- the present invention can clone a specific gene encoding merozoite surface protein of Plasmodium vivax, and use it in prepa ⁇ ng recombinant antigen protein capable of identifying and determining whether any one has the malarial infection, by means of Enzyme-Linked ImmunoSorbent Assay (ELISA) method.
- ELISA Enzyme-Linked ImmunoSorbent Assay
Abstract
The present invention provides a gene encoding merozoite surface protein of Plasmodium and a method for preparing 27.5 kDa recombinant antigen protein by expressing the above gene in E. coli expression vector. The invention also provides a method of using the gene encoding C-terminal of prior known merozoite surface protein of Plasmodium in determining and/or diagnosing the malarial infection, comprising to clone the gene and to produce about 27 kDa recombinant protein from such gene. More particularly, the invention provides the more simple and convenient method for diagnosing the malarial infection having improvements in higher sensitivity and specificity, compared with conventional diagnosis methods, by applying the enzyme-linked immunosorbent assay(ELISA) method.
Description
GENE FOR ENCODING MEROZOITE SURFACE PROTEIN OF PLASMODIUM VIVAX
AND USE THEREOF
TECHNICAL FIELD The present invention relates a gene encoding merozoite surface protein of Plasmodium vivax, a method of use thereof in preparing recombinant antigen protein, the resulted recombinant antigen protein and a diagnosis reagent comprising the same protein Particularly, this invention is useful for the diagnosis of malarial infection by detecting antigen-antibody composites obtained from reaction the antigen with a test sample in question BACKGROUND ART
Malaria is one of generally known fever diseases worldwide and an increasing health problem throughout the world including Korea More than a billion people live in malaria-prevalent area, at least a hundred million patients suffer from each year and more than one million cases among them die from that disease Therefore, this disease is considered as one of the most important six diseases that the Health World OrganιsatιonΙ(WHO) designates Plasmodium vivax parasitize on human bodies and destroys erythrocytes to cause various symptoms such as periodic febrile seizure, anemia, splenectasis and so forth
Recently, owing to the increase of overseas trips, a imported malaria which has been infected on hιs(her) travels and shows the symptoms after hιs(her) returning home is an important problem even in the advanced countries, not limited to Korea
Malarial infection is known to progress under the following steps Malarial sporozoites introduced from the mouth of mosquitoes into human body propagate within hepatocytes for several days(8-9days) and go into erythrocytes carrying out the asexual reproduction After 48 hours, the sphtted merozoites destroy the erythrocytes and flow along blood vessel and move into another erythrocytes During repeatedly such asexual reproduction, some of merozoites are transformed into macrogametocytes and/or microgametocytes These cytes perform sexual reproduction, as introduced into body of mosquito These cytes become spores through zygote-ookimete-oocyst steps(sporgony), and are present in salivary gland until they transfers to another object
As above mentioned in life cycle, the clinical property for malaπa is that sporozoites of Plasmodium parasitize in erythrocytes after dividing and increasing steps within hepatic cells then producing a number of cyptomerozoites. The peπod of time after one has been infected with Plasmodium from a mosquito until the parasitic stage of sporozoite mentioned above, is latent period of the malarial disease, which is variable and may be ranged from a few weeks to several months Accordingly, even the winter season without mosquito there may be cases of malaria because the disease has the long ιncubatιon(latent) period of several months.
In malaria models having typical symptom and clinical progress, it has been shown that a cold stage to feel a chill, have a headache and/or vomit is firstly appeared for a few minutes to one or two hours, a second hot stage of representing heated and dried skin, tachycardia, tachypnea is progressed for 3-6 hours followed by a wet stage of perspiring The pyrogenetic stage of malarial disease depends on species of Plasmodium, for example 48 hours for Plasmodium vivax.
In addition to the pyrexia symptom, patients appear the conditions of illness such as anemia, headache, splenectasis, decrease of blood platelet and so forth. The anemia symptom is caused by the destroy of erythrocytes so called hemocytocatheresis, the splenectasis is due to the deposition of the broken erythrocytes and hemoglobin and the decrease of blood platelet may be derived from antibody-mediated splenic sequestration of blood platelet coated with Plasmodium.
It will be appreciated that malarial disease can be determined and/or diagnosed by carefully observing its clinical symptom. If it has a regular and typical pyrogenetic cycle, the species of Plasmodium may be identified. Thick smear test is a method performed by smearing and drying large volume of blood, complete hemolysis of erythrocytes followed by the examination of the remained Plasmodium and leukocytes with a microscope so that it can conveniently determine whether a subject is at the malaria-positive or negative conditions. However, this test needs a staining procedure of blood samples, therefore taking several hours to observe the blood film with a microscope.
Also, it takes more than one hour to observe the sample, because the viewer skilled in the art needs to observe the results moving at least 100 fields under the microscope after dripping immersion oil Accordingly, in order to diagnose malaria, a number of users skilled for the microscope are
required together with long time and labor in large quantities. Due to lack of such skilled man in this technical fields and labor m malaria-incident areas, diagnosis and treatment of such diseases are carried out by only observing clinical symptoms thereof. Such works without confirmation test performed by any laboratory method are unsuitable and may increase tolerance to drug and incidence of side effects
In view of the foregoing, it is recently necessary to research and develop serological diagnostic methods to quickly and efficiently determine malarial infection
At present, it has been found that, in order to diagnose vivax malaria, fusion proteins can be added to C-termmal regions of merozoite surface protein to produce 19kDa recombinant protein which has a strong antigemcity and can be available for the diagnosis malarial infection and preparation of a vaccine against malaria. However, as a result of epidemiological investigation, it has been reported that the diagnostic accuracy is only about 60 to 80%. (Infection and Immunity, May 1997, p.1606- 16141, Southeast Asian J Trop Med Public Health Vol 29, No.4, December 1998; Am J Trop Med Hyg 60(3), 1999 pp357-363; Vaccine 17, 1999, 2959-2968). DISCLOSURE OF INVENTION
From the fact merozoite surface protein of Plasmodium falcψarum is shown to have antigemcity in malarial diseases caused by such Plasmodium, as previously disclosed by Derek Wakehn, Immunity to parasite, 44-54, 1996 which is incorporated herewith for reference, the present inventor intended to apply it to diagnosis of malarial infection Accordingly, it is an object of the present invention to provide novel gene sequence encoding merozoite surface protein of Plasmodium vivax, which is available for serological diagnosis of malaria
It is a further object of the present invention to provide a method for preparing a recombinant protein from such cloned gene and its use m determining malarial infection, as well as diagnosis reagent including it
Also, the present inventor has found that C-termmal regions of merozoite surface protein of Plasmodium vivax has unexpectedly high antigemcity Therefore, it is still another object of the present invention to provide a diagnostic reagent containing recombinant protein obtained by the
expression of the gene encoding such regions in host vector after cloning such gene, as well as its use in determining whether any subject m question is under malarial infection condition.
Malaπal infection has been generally known to be caused from four types of Plasmodium; P vivax, P. falciparum, P malariae and P ovale. Among these Plasmodium, both of P vivax and P malariae cause mostly the malarial infection. Particularly, P vivax is distributed over the most extensive area and is the major plasmodium of malarial infection discovered m Korea, as described by Sho, jn-tak, "Human parasitology" which is incorporated herewith for reference. MSP-1, MSP-2 and MSP-4 of merozoite surface proteins of Plasmodium, are coupled to GPI-hpids m outer membrane of the merozoite of Plasmodium to represent antigemcity against host (Camila I et al., Vaccine 17, 2959- 2968, 1999).
According to the present invention, merozoite surface protein of Plasmodium vivax has a ammo acid sequence denoted as Sequence ID No. 2, together with DNA sequence denoted as Sequence ID No.l . These both sequences have been previously deposited as AF200953 on November 3, 1999 with GenBank. Cloning such DNA sequence and expressing the gene in E. coli vector according to gene recombination technique result in 27 5 kDa recombinant antigen protein.
The present inventor noticed that it is possible to use a particular MSP-1 recombinant protein of Plasmodium vivax reacting with serum of a Plasmodium-infected patient m determining and/or detecting malarial infection. Therefore, He performed the cloning process for C-terminal region of gene encoding MSP-1 from Plasmodium vivax Repeatedly It has been found that about 27 kDa recombinant protein produced from the partial gene (noted as Sequence ID No. 3) of cloned merozoite surface protein has very high antigemcity against host.
In particular, the present invention provides novel recombinant protein obtained from the gene encoding merozoite surface protein of cloned Plasmodium and an Enzyme-Lmked ImmunoSorbent Assay(ELISA) type diagnostic kit comprising such protein According to the present invention, 27.5 kDa and 27 kDa recombinant proteins of Plasmodium can be used as specific antigens, and also in preparation of DNA probe, monoclonal antibody or vaccine. By utilizing such diagnostic kit comprising the recombinant protein according to this invention, it has advantages that it
is possible to more conveniently and easily diagnose malarial infection and to detect and/or evaluate the treatment effect against such infection.
In addition to the above, the present invention further provides an improved method for quickly and conveniently diagnosing and/or determining whether a subject is infected with Plasmodium, and concurrently having specificity and sensitivity only to patient infected with
Plasmodium, especially with Plasmodium vivax other than patients for example having hepatitis B or
C, venereal dιsease(VDRL), autoimmune dιsease(Rheumatoιd arthritis, ANA) and so forth.
The invention is further illustrated by the following description.
Determination of gene sequence encoding MSP-1 of Plasmodium vivax and preparation of 27.5 kDa recombinant antigen protein
The process to determine gene sequence encoding MSP-1 from P. vivax comprises a plurality of steps; The first step is to design pπmer by using β -cyanoethyl phosphormidite method in order to isolate MSP-related gene.
The primer 1 (5'-GAATTCAAAATCGAGAGCATGATCG-3') is designed to have a specificity cleaved with restriction enzyme EcoR I whilst another pπmer 2 (5'- GTCGACGACATTGAATAGGAGGTC-3') is designed to have a specificity cleaved with restπction enzyme Hind III
In a next step, DNA segments related to MSP are isolated from Plasmodium vivax by reacting genomic DNA with such primers obtained from the first step. The third step is to determine the base sequence of said DNA segment.
In the fourth step, recombinant antigen protein is produced by the expression of such sequential regions on E.coh vector pET21a(Novagen).
The fifth step comprises the investigation of antigemcity of the recombinant antigen protein prepared through above speps. In addition, the diagnostic kit for ELISA containing such prepared recombinant protein is manufactured in the last sixth step
Preparation of about 27 kDa recombinant protein
The inventor has carried out the cloning process of gene (Sequence ID No.3) encoding C-
terminal of merozoite surface proteins of Plasmodium vivax and prepared recombinant antigen protein from the gene.
The sequence represented by Sequence ID No. 3 is characterized in that:
1) Length (of sequence) : 618 n) Type (of sequence) : nucleic acid in) Type (of molecules) : genomic DNA
IV) Presumption of sequence : No v) Anti sense : No vi) Title : Plasmodium vivax (isolated from Homo Sapiens in Korea) vu) Name/Key (identifying feature) : CDS vm) Location : 4557
IX) Identification method : S
27 kDa recombinant protein is generated in the same procedure as 27.5 kDa protein mentioned above, except that in the first step, pπmer l(5'-GTGGAATTCGAGTACGAGTCC-3') is designed to have a specificity cleaved with restriction enzyme EcoR I whilst another primer 2 (5'-
TCTGTCGACAAGCTCCATGCA-3') is designed to have a specificity cleaved with restriction enzyme Sal I.
Diagnosis of malarial infection
In order to achieve the purpose mentioned above, the present invention comprises detecting antigen-antibody composites formed by reacting malarial infected blood (particularly, blood infected with vivax malaria) with said recombinant protein.
That is, in an aspect of the present invention, the diagnostic method comprises detecting the presence of an antibody against malarial antigen in blood according to the principle of known Enzyme Linked Immunosorbent Assay(ELISA). More particularly, the diagnostic method comprises adsorbing malarial antigens to each well of a 96-wells microplate as a solid supporting body and reacting malarial antigen adsorbed to the well with an antibody existed in blood of the subject to be test according to the following steps:
Firstly, serum or blood as the sample is reacted within the well adsorbed with antigen at a
proper temperature for desired time. In case where antibody against malaria is m blood, it will be appreciated that antigen-antibody composite is formed
In a second step, rabbit anti-human lmmunoglobuhn antibody, anti-canine immunoglobulin antibody and rabbit anti-felme immunoglobulin all of them being conjugated with horseradish peroxidase (HRP) are added to such well to induce its specific coupling to antibody region of the above formed antigen-antibody composite. In this case, if there was no composite it will be understood that no coupling process was generated so that HRP conjugated second antibody as a chromophoπc enzyme is removed m a washing step.
In a third step, TMB solutιon[(3,3',5,5'-Tetramethyl benzidme, Merck] as a substrate is added to the material obtained from the second step. If the antigen-antibody composite from the second step was coupled with the second antibody, because of chromophoπc reaction it appears yellow color Next, after completing the reaction with the addition of diluted sulfuπc acid, the chromaticity of final chromophoπc reactant is determined by means of the microwell reader. With this, whether malarial antibody exists in blood can be determined. According to the principle of three-steps diagnosis illustrated above, polystyrene beads, nitrocellulose strips and so forth can be used as solid supporting bodies for adhering antigen.
In another feature of the present invention, provided is a diagnostic kit consisting of essential reagents to detect malarial antibody in blood depending on the diagnostic principle descπbed above, in addition to malarial antigen adsorbed to the solid supporting body. The malarial antigen used in the present invention is 27 kDa or 27.5 kDa recombinant protein
Such recombinant protein is obtained form a clone having high reactivity with malaria-infected serum, and is produced by generally known methods to produce recombinant protein
The following examples are for illustration only and are not intended to limit the invention in any way BRIEF DESCRIPTION OF DRAWINGS
FIG 1 shows an electrophoretic results of the recombinant malarial antigen protein M denotes standard molecular weight protein, S is an isolated malarial antigen appearing at a location corresponding 27.5 kDa of molecular weight. C means crude protein without puπfication,
FIG. 2 shows a result of Western blotting for the 27.5 kDa recombinant protein; FIG. 3 shows a measurement result of the antibody against such 27.5 kDa recombinant protein in blood; ANA means anti-nuclear antibody, EHF is serum of hemorrhagic fever case, HBV is serum of hepatitis B virus infected case, HCV denotes serum of hepatitis C virus infected case, aRt is serum of Richettsia Tsutsugamushi-mfected case, RA is serum of Rheumatoid arthritis patient, VDRL means serum of venereal disease case, aPv is serum of vivax malaria case and nor is serum of normal case.
FIG 4 shows an electrophoretogram of 27kDa recombinant malarial antigen. M denotes standard molecular weight protein. (1) in this figure shows uncoupled protein fragments, (2) means purified protein fragment 1 while, and (3) means purified protein fragment 2.
FIG 5 illustrates another measurement result of the antibody against 27kDa recombinant protein in blood. (-) shown in this figure is negative standard and (+) is positive standard, respectively, and malaria means serum of patient suffering from malaria while normal being serum of normal case. EXAMPLES
EXAMPLE 1
Materials and Methods
(1) Isolation of DNA from Plasmodium
0.5g(wet weight) of Plasmodium vivax was frozen under liquid nitrogen and the resulted solids were ground by means of mortar. To the ground powders was added with 1.2ml of digestion buffer consisted of lOOmM NaCl, lOmM tπs-HCl with pH8.0, 25mM EDTA, 0.5% SDS and 0.12mg of proteinase K, per lOOmg of Plasmodium and was shaken at 50 °C for 18 hours to dissolve cell membranes. The obtained sample was added with an equivalent amount of mixture of phenol/chloroform/isoamyl alcohol at a ratio of 25:24: 1 and thoroughly admixed together, following by centπfugation by means of swinging bucket rotor(Sorval RC-5, USA) at 20,000 g for 10 minutes at room temperature. Thereafter, supernatant was transferred into another tube and 7.5M ammonium acetate (m the amount equal to 1/2 of supernatant) and 100% ethanol (in the amount equal to 2 times supernatant) were added into the tube, respectively, and then the sample was centπfuged at 10,000g
for 2 minutes. After this, the obtained pellets were washed with 70% ethanol and dried.
DNA was thoroughly dissolved with TE buffer comprising lOmM tπs-HCl and ImM EDTA with pH8.0 for 3 hours at 65 "C To remove the remaining RNA, The resulted sample was treated with 0.1% SDS and lug/ul DNase-free RNase and allowed to place at 37°C for lhour following by organic extraction and ethanol precipitation.
(2) Isolation of DNA fragment having about 489 bp lOul of genomic DNA from the above process, lOul of pnmer 1 (5'- GAATTCAAAATCGAGAGCATGATCG-3') having a specificity cleaved with restπction enzyme EcoR I and lOul of pπmer 2 (5'-GTCGACGACATTGAATAGGAGGTC-3') having a specificity cleaved with Hind III were added to GeneAmp PCR reagent kit system with AmpliTaq DNA Polymerase (Perkin-Elmer Cetus Corp.). 99.5ul of the resulted PCR reactant solution was introduced into a DNA Thermal Cycler (Perkin-Elmer Cetus Corp. model=9600) to carry out PCT amplification PCR amplification was achieved by repeatedly performing total 45 cycles the following steps of, denaturing DNA at 94 °C for 1.5 minutes; annealing DNA at 25 °C for 1.5 minutes; and synthesizing at 72 °C for 3 minutes. In order to identify amplified DNA fragments, lOul of the reactant solution was added with 2ul of a sample buffer(6X, 0.25% Bromophenol blue, 0.25% Xylene cyano FF, 40% sucrose) and then was electrophoresed on 1% agarose gels. At this, the electrophoresis treatment was performed with tπs-acetate/efhylenediame tetraacetate(TAE) electrophoresis buffer at 100V for 30 minutes to result m the isolation of about 489 bp DNA fragments. (3) Purification and Cloning of DNA fragment
After the electrophoresis of amplified DNA fragment obtained from the above process on 1% agarose gels, the amplified DNA fragment was cleaved and purified by using the Geneclean II kιt(Bιo
101 Inc.) The purified DNA was cloned on pT7Blue T-vector(Novagen). The resultant cloned plasmid DNA was purified by alkahsis method (Birnboim HC et al., Ish-Horowicz 0 and Barke JF, 1981 ) and then by the Geneclean II kit.
(4) Sequencing
DNA purified above was added to T7 promotor primer and T3 reverse primer (ABI cloning system) and PCR amplified, following by the electrophoresis treatment at 2,500V and 30mA for 12
hours on 4.75% polyacrylamide gel containing 8M urea. The result of this electrophoresis was analyzed by the Microgene software program (manufactured by ABI Corp.) The result is proposed as Sequence ID No. 1.
Sequence ID No.l is the sequence for gene encoding MSP of P. vivax which was deposited with GenBank as AF200953 (November 3, 1999). (but, not published by November 2, 2002)
(5) Preparation of about 27.5kDa recombinant protein
The cloned gene above was introduced into E. coli expression vector pET21 a(Novagen Corp ) and treated with heat shock to produce pET21-Pv, and then incubated on with 50ug/ml of Ampicillin-contaming LB plate (0.5% yeast extracts, 1% tπpton, 1% sodium chloride and 1.5% agar) at 37 °C to select the recombinant clone. The selected clone was introduced 10ml of LB amp+ and incubated within a shaking incubator at 200 rpm for 16 hours. In order to express recombinant protein, incubated cells were added to 1-. LB amp+ medium, incubated to reach 0.6-0.8 at OD600 and then cultured with the addition of 4ml of 1M IPTG(Bιoneer Corp.) for 3 hours. After the centπfugation, the collected cells were subjected to a washing step with 100ml of 20mM Tπs- Cl(pH8.0) following by another centπfugation to collect the washed cells, and then was sonificated with 50ml Buffer A (20mM Tπs-Cl(pH8.0), 150mM NaCl, 8M urea). By centπfugmg, only supernants were isolated and Nι2+-NTA agarose beads were washed with a buffer B(5mM Imidazole, 20mM Tπs-Cl(pH8.0), 150mM NaCl) and then poured into and passed through a CIO column (Parmacia Biotech Inc.) to eluate the protein depending on Imidazole concentration. About 27.5kDa of recombinant protein was resulted from the above experiment (see FIG. 1).
(6) Western Blotting with 27.5kDa recombinant protein
In order to detect and identify the specificity of antigen protein obtained above, Western blotting method was carried out. About 0.5ug of plasma protein was electrophoresed on 10% polyacrylamide gels under the presence of SDS based on Laemmh's method(Laemmh, Nature 227, 680(1970)) and transferred the protein isolated from the electophresis gels to the nitrocellulose filter membrane (manufactured by Milhpore Corp., pore size 0.45um) according to Towbm's method (Towbin et al., Proc, Natl. Acad, Sci, USA 76, 4350(1979)) In this case, the filter was reacted with a solution containing 0.5% bovme-serum albumin (BSA) to prevent nonspecific binding Thereafter,
the test serum from vivax malaria patient was added to PBS buffer containing 0.05% Tween 20 (lOmM sodium phosphate, 0.15M NaCl, pH7.0) at the volume of 10,000 and then the filter was washed twice each for 15 minutes in said PBS buffer containing 0.05% Tween 20. The filter was added with the second antibody (that is, goat anti-rabbit immunoglobulin G rebelled with horseradish peroxidase) to react together and then was washed with said PBS buffer. Finally, as a substrate TMB solution was applied to the filter to be chromophored. The result of this experiment is shown in Figure 2 accompanied heremabove.
(7) ELISA method for the determination of lmmuno-specificity and sensitivity of antigen
* Antigen adsorption plate: The diluted recombinant malarial antigen protein with 0.05M bicarbonates buffer(Sιgma Corp., capsule) was adsorbed to 96 wells at a volume of lOOul each well.
* Specimen diluent solution: To 0.15M PBS(0.2M phosphate buffer, 0.13M NaCl, pH 7.2) were added both of 0.05% Tween 20 and 0.5% casem.
* Positive control solution: Serum 1 containing malarial antibody is diluted by the addition of said specimen diluent solution 100. * Negative control solution: Serum 1 of normal case is diluted by the addition of said specimen diluent solution 100.
* Concentrated washing solution: 10 times concentrated PBS phosphate buffer (0.13M NaCl, pH7.2) was added with 0 5% Tween 20 and diluted 10 times with distilled water.
* Concentrated enzyme marker solution: Rabbit anti-human immunoglobulin (Serotec) conjugated with horseradish peroxidase (HRP) was diluted m the conjugate diluent solution -prepared by adding 0.05% Tween 20 to 0.15M PBS(0.2M phosphate buffer, 0.13M NaCl, pH7.2) - at a ratio of 1 :40 and then diluted by 1000 times with said specimen diluent solution )
* Substrate concentration solution: TMB(3,3',5,5'- Tetramethylbenzidme) as a substrate was diluted at a ratio of 1 : 100 with substrate buffer. * Substrate buffer: Citric acid phosphate buffer(pH5.0) was added with 0.035% hydrogen peroxide.
* Reaction stopping solution: 2M sulfuπc acid
(8) Adhesion of 27.5kDa recombinant protein to solid supporting body
After diluting 27.5kDa recombinant protein to concentration of l-5ug/ml with 0 05M bicarbonates buffer capsules(Sιgma), the dilute solution was allotted to polystyrene ELISA plate(Immunoplate, Nunc) at a volume of lOOul/well and allowed to adsorb antigen at 4°C for 18 hours. The plate was lyophilized and stored in vinyl res s bags including a drying agent at about 4°C The plate was observed to be stable under the above condition more than one month.
(9) Use of recombinant protein
(l) The ELISA plate was opened under room temperature.
(n) The number of specimen diluent solutions (contained in the analysis kit) equal to total number of three negative control solutions, two positive control solutions and the specimens to be test were allotted into wells at a volume of 200ul/well (except a substrate blank)
(in) Each of negative controls, positive controls and specimen samples was introduced in said wells containing diluted solution at a volume of 2ul/well, was hit carefully for about 5 to 10 seconds and then sealed by a plate sealing tape.
(IV) The sealed plate was subjected to reaction at 37 °C ± 1 for 60 minutes. (v) After completing the reaction, the reacted material was washed five times after removing the seal tape. The well was thoroughly filled with washing solution and left for 15-30 seconds The remained solution was discarded.
(vi) 50ul of enzyme marker solution was allotted into each well and then sealed by a plate sealing tape (vn) The sealed plate was subjected to reaction at 37 °C ± 1 for 60 minutes.
(vin) Concentrated washing solution was used to rinse the sealed plate.
(IX) Pre-prepared substrate buffers were allotted into all of wells including substrate blank at a volume of 1 OOul/well
(x) The prepared wells were subjected to reaction m a dark space at 15-30°C (xi) Reaction stopping solutions (contained in the diagnostic kit) were allotted into all of wells including substrate blank at a volume of 1 OOul/well
(xπ) The resulted product was analyzed by determining its absorbance(OD) at 450nm In case of where double wavelength spectrophotometer is used, reference wavelength ranges from 620-650
nm
RESULT
As shown in A of FIG 2, such recombinant antigen represented a single band for each of vivax malaria-infected blood and serum whilst no reaction was found out for normal case (not shown in Figure) Based on the above result, it will be understood that the present inventive antigen may specifically couple to antibodies of vivax malaria-infected patients
Referring to FIG 3, in case of where the 27 5 kDa recombinant protein was used as antigen, the diagnostic sensitivity was observed to 97% (29 of 30 cases) and the diagnostic specificity being 100% (34 of 34 cases) From the result of anti-nuclear patients (representing a diagnostic specificity of 100%, 0 of 12 cases), hemorrhagic fever cases (a diagnostic specificity of 89%, 1 of 9 cases), hepatitis B virus infected cases (a diagnostic specificity of 92 5%, 3 of 40 cases), hepatitis C virus infected cases (a diagnostic specificity of 87 5%, 3 of 24 cases), Richettsia Tsutsugamushi infected cases (a diagnostic specificity of 94 4%, 0 of 8 cases), Rheumatoid arthritis patients (a diagnostic specificity of 94 4%, 1 of 18 cases) and VDRL cases (a diagnostic specificity of 100%, 0 of 40 cases), it has been found that 27 5 kDa recombinant protein according to this invention has remarkably surprising diagnostic specificity and sesitivity EXAMPLE 2
Materials and Methods The same procedure as in Example 1 was repeated to isolate DNA from Plasmodium lOul of the obtained genomic DNA, lOul of primer 1 (5'-GTGGAATTCGAGTACGAGTCC- 3') having a specificity cleaved with restriction enzyme EcoR I and lOul of primer 2 (5 - TCTGTCGACAAGCTCCATGCA-3') having a specificity cleaved with restπction enzyme Sal I were added to GeneAmp PCR reagent kit system with AmpliTaq DNA Polymerase (Perkin-Elmer Cetus Corp ) The resulted PCR reactant solution was subject to PCR amplification in the same manner as described in Example 1, to generate about 618bp DNA fragments after electrophoresis
Furthermore, by the same procedure of Example 1 the purification and cloning treatments were carried out
To obtained cloned gene was expressed in pET21a (Novagen Corp.), E. coli expression vector to create about 27 kDa recombinant protein according to the same manner as illustrated m Example 1. (see FIG. 4)
According to the same manner as in Example 1, the antigemcity and the sensitivity of obtained 27 kDa recombinant protein were determined.
RESULT
As illustrated in FIG. 5, in case of where the present inventive 27 kDa recombinant protein was used as antigen the protein has been observed to have remarkably significant diagnostic sensitivity and specificity of 100% (16 of 16 cases) and (72 of 72 cases) respectively.
JUDGEMENT OF RESULT
(1) Calculation of average absorbance of negative control (NC) solution
According to the method of use descπbed above, absorbance of each negative control(NC) solution was determined and then its average being calculated based on the following three values with reference of Table 1 below: [Table 1]
Average absorbance for negative control solution NC= (0.024+0.028+ 0.031)/3 = 0.028 The average absorbance of negative control solution must be withm 0.000 to 0.100 If any one of such three absorbencies is out of the above range, the average value should be calculated from only the remained both absorbencies. However, in case of where at least two absorbencies are out of the above range it is necessary to perform the re-exammation and/or re-test the absorbance for the negative control solution.
(2) Calculation of average absorbance of positive control(PC) solution
According to the method of use described above, absorbance of each positive control(PC)
solution was determined and then its average being calculated based on the following two values with reference of Table 2 below: [Table 2]
Average absorbance for positive control solution PC= (0.768+0.156)12 = 0.762 The average absorbance of positive control solution must be at least 0.500 and the difference between both absorbencies must be less than 0.100. If such average absorbance is out of the above range, it will be expected to have problems in test procedure or the reagent. So, after finding out and eliminating the problems, the re-examination of the absorbance for the positive control solution should be performed. (3) Calculation of Cutoff value
Cutoff value may be calculated by adding 0.260 to average absorbance value of negative control solution. For the instance mentioned above, the cutoff value is NC + 0.260 = 0.028 + 0.260 = 0.228.
INDUSTRIAL APPLICABILITY As illustrated above, the present invention can clone a specific gene encoding merozoite surface protein of Plasmodium vivax, and use it in prepaπng recombinant antigen protein capable of identifying and determining whether any one has the malarial infection, by means of Enzyme-Linked ImmunoSorbent Assay (ELISA) method.
Claims
Claims
1 A gene encoding merozoite surface proteιn(MSP) of Plasmodium vivax, designated as Sequence ID No 1
2 A method for determining the gene sequence designated as Sequence ID No 1 from genomic DNA of Plasmodium vivax using a conservative region of MSP, primer 1 (5'-
GAATTCAAAATCGAGAGCATGATCG-3') having a specificity cleaved with restriction enzyme EcoR I and primer 2 (5'-GTCGACGACATTGAATAGGAGGTC-3') having a specificity cleaved
3 About 27 5 kDa recombinant protein including the ammo acid sequence designated as Sequence ID No 2, prepared by the expression of gene of claim 1 in E coli expression vector pET21a
4 A method for cloning gene designated as Sequence ID No 3 from genomic DNA of Plasmodium vivax using a conservative region of MSP, primer 1 (5'- GTGGAATTCGAGTACGAGTCC-3') having a specificity cleaved with restnction enzyme EcoR I and primer 2 (5'-TCTGTCGACAAGCTCCATGCA-3') having a specificity cleaved with restnction enzyme Sal I
5 About 27 kDa recombinant protein including the amino acid designated as Sequence ID No 4, prepared by the expression of gene designated as Sequence ID No 3 in E coli expression vector pET21a
6 The protein of according to claim 3 or 5 wherein it has antigemcity against Malarial antibody 7 An enzyme-linked immunosorbent assay(ELISA) type diagnostic kit containing the recombinant protein according to claims 3 or 5 for determining whether a subject is infected with Malaria
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU14209/01A AU1420901A (en) | 1999-11-15 | 2000-11-15 | Gene for encoding merozoite surface protein of plasmodium vivax and use thereof |
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019990050616A KR20010046731A (en) | 1999-11-15 | 1999-11-15 | DNA sequence of merozoite surface protein of Plasmodium vivax |
| KR1999/50616 | 1999-11-15 | ||
| KR2000/22041 | 2000-04-25 | ||
| KR1020000022041A KR20010097729A (en) | 2000-04-25 | 2000-04-25 | Malaria antibody diagnostic kits and methods for the same |
| KR2000/27305 | 2000-05-20 | ||
| KR1020000027305A KR20010106041A (en) | 2000-05-20 | 2000-05-20 | Malaria antibody-diagnostic kits and methods for manufacturing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2001036587A2 true WO2001036587A2 (en) | 2001-05-25 |
| WO2001036587A3 WO2001036587A3 (en) | 2002-02-28 |
Family
ID=27350093
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2000/001302 WO2001036587A2 (en) | 1999-11-15 | 2000-11-15 | Gene for encoding merozoite surface protein of plasmodium vivax and use thereof |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU1420901A (en) |
| WO (1) | WO2001036587A2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2378949A (en) * | 2001-08-16 | 2003-02-26 | Morten Steen Hanefeld Dziegiel | Human antibodies against Plasmodium falciparum MSP-3 antigen |
| WO2009111599A3 (en) * | 2008-03-06 | 2012-02-02 | Abbott Laboratories | Plasmodium malariae and plasmodium ovale genes and uses thereof |
-
2000
- 2000-11-15 AU AU14209/01A patent/AU1420901A/en not_active Abandoned
- 2000-11-15 WO PCT/KR2000/001302 patent/WO2001036587A2/en active Application Filing
Non-Patent Citations (2)
| Title |
|---|
| MANCILLA LI ET AL.: 'Plasmodium vivax: dimorphic DNA sequences from the MSP-1 gene code for regions that are immunogenic in natural infections' EXP. PARASITO. vol. 79, no. 2, September 1994, pages 148 - 158 * |
| PAN W. ET AL.: 'Vaccine candidate MSP-1 from plasmodium falciparum: a redesigned 4917 bp polynucleotide enables synthesis and isolation of full-length protein from escherichia coli and mammalian cells' NUCLEIC ACIDS RES. vol. 27, no. 4, February 1999, pages 1094 - 1103 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2378949A (en) * | 2001-08-16 | 2003-02-26 | Morten Steen Hanefeld Dziegiel | Human antibodies against Plasmodium falciparum MSP-3 antigen |
| GB2378949B (en) * | 2001-08-16 | 2005-09-07 | Morten Steen Hanefeld Dziegiel | Recombinant anti-plasmodium falciparum antibodies |
| US7811569B2 (en) | 2001-08-16 | 2010-10-12 | Institut Pasteur | Recombinant anti-Plasmodium falciparum antibodies |
| WO2009111599A3 (en) * | 2008-03-06 | 2012-02-02 | Abbott Laboratories | Plasmodium malariae and plasmodium ovale genes and uses thereof |
| US8268981B2 (en) | 2008-03-06 | 2012-09-18 | Abbott Laboratories | Plasmodium malariae and plasmodium ovale genes and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2001036587A3 (en) | 2002-02-28 |
| AU1420901A (en) | 2001-05-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Van Gelder et al. | Serodiagnosis of toxoplasmosis by using a recombinant form of the 54-kilodalton rhoptry antigen expressed in Escherichia coli | |
| JP3245143B2 (en) | Toxoplasma gondii antigen, its preparation and its use | |
| KR20010012813A (en) | Compounds for diagnosis of tuberculosis and methods for their use | |
| JP2002530050A (en) | Compounds and methods for the diagnosis of tuberculosis | |
| JPH11502923A (en) | T. Compounds and methods for detecting CRUZI infection | |
| JP4234207B2 (en) | Diagnosis method of allergic bronchopulmonary aspergillosis | |
| JPH0347133A (en) | Antigen of langerhans island cell obtained by molecular cloning | |
| EP1255852B1 (en) | Immunoassay and diagnostic reagent for malaria | |
| JP2005506512A (en) | Latent human tuberculosis model, diagnostic antigen, and method of use | |
| US5254459A (en) | Nucleotide and amino acid sequences of protein MTP40 of M. tuberculosis and synthetic peptides derived therefrom | |
| AU2002324578A1 (en) | Early detection of mycobacterial disease using peptides | |
| EP1463526A2 (en) | Early detection of mycobacterial disease using peptides | |
| WO2001036587A2 (en) | Gene for encoding merozoite surface protein of plasmodium vivax and use thereof | |
| EP0516859A1 (en) | Non-a non-b hepatitis virus antigen protein | |
| US5171839A (en) | Nucleotide and amino acid sequences of protein mtp40 of m. tuberculosis and synthetic peptides derived therefrom | |
| EP1362910B1 (en) | Lectins for analyzing sugar chains and method of using the same | |
| US5169940A (en) | Nucleotide sequences of protein MTP40 of M. tuberculosis | |
| JP2002508506A (en) | T. Compounds and methods for detection and prevention of cruzi infection | |
| Kouguchi et al. | Characterization of various recombinant antigens from Echinococcus multilocularis for use in the immunodiagnosis | |
| JP3225248B2 (en) | Detection method | |
| KR101270662B1 (en) | Diagnostic kits of Brucella abortus using 26kDa recombinant antigenic protein and Lipopolysaccaride | |
| KR100380306B1 (en) | Reagent composition and method for detecting malaria antibodies | |
| Robles et al. | Isolation of the Taenia crassiceps antigens from a phage display cDNA library and evaluation of their use for diagnosis of neurocysticercosis | |
| AU645482B2 (en) | A malaria antigen | |
| CA2097912A1 (en) | Method of detecting methicillin-resistant staphylococcus aureus, novel peptides, and dnas encoding the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
| AK | Designated states |
Kind code of ref document: A3 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
| REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
| 122 | Ep: pct application non-entry in european phase |