CN1290579C - Recombinant foot-and-mouth disease virus VP1 confluent protein vaccine - Google Patents
Recombinant foot-and-mouth disease virus VP1 confluent protein vaccine Download PDFInfo
- Publication number
- CN1290579C CN1290579C CNB2003101001751A CN200310100175A CN1290579C CN 1290579 C CN1290579 C CN 1290579C CN B2003101001751 A CNB2003101001751 A CN B2003101001751A CN 200310100175 A CN200310100175 A CN 200310100175A CN 1290579 C CN1290579 C CN 1290579C
- Authority
- CN
- China
- Prior art keywords
- foot
- mouth disease
- disease virus
- fusion rotein
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 101900061472 Foot-and-mouth disease virus Capsid protein VP1 Proteins 0.000 title claims abstract description 75
- 229940023143 protein vaccine Drugs 0.000 title abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 94
- 241000710198 Foot-and-mouth disease virus Species 0.000 claims abstract description 73
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 18
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 17
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 16
- 239000002773 nucleotide Substances 0.000 claims abstract description 13
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 13
- 241000588724 Escherichia coli Species 0.000 claims abstract description 12
- 230000002265 prevention Effects 0.000 claims abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 72
- 230000004927 fusion Effects 0.000 claims description 68
- 230000000799 fusogenic effect Effects 0.000 claims description 57
- 241001465754 Metazoa Species 0.000 claims description 22
- 229960005486 vaccine Drugs 0.000 claims description 12
- 108010023958 glycyl-threonyl-alanyl-methionyl-arginyl-isoleucyl-leucyl-glycyl-glycyl-valyl-isoleucine Proteins 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000005215 recombination Methods 0.000 claims description 4
- 230000006798 recombination Effects 0.000 claims description 4
- 229920000232 polyglycine polymer Polymers 0.000 abstract description 9
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract 1
- 229920002704 polyhistidine Polymers 0.000 abstract 1
- 230000009385 viral infection Effects 0.000 abstract 1
- 101710132601 Capsid protein Proteins 0.000 description 25
- 101710197658 Capsid protein VP1 Proteins 0.000 description 23
- 101710118046 RNA-directed RNA polymerase Proteins 0.000 description 23
- 101710108545 Viral protein 1 Proteins 0.000 description 23
- 239000013612 plasmid Substances 0.000 description 22
- 241000700199 Cavia porcellus Species 0.000 description 19
- 239000007788 liquid Substances 0.000 description 17
- 235000018102 proteins Nutrition 0.000 description 17
- 102000004169 proteins and genes Human genes 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 230000003053 immunization Effects 0.000 description 16
- 238000002649 immunization Methods 0.000 description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 230000029087 digestion Effects 0.000 description 14
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 description 13
- 238000000034 method Methods 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 13
- 210000002966 serum Anatomy 0.000 description 12
- 239000000872 buffer Substances 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 238000005267 amalgamation Methods 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 10
- 239000012634 fragment Substances 0.000 description 10
- 238000006062 fragmentation reaction Methods 0.000 description 10
- 238000002156 mixing Methods 0.000 description 10
- 241000700605 Viruses Species 0.000 description 9
- 238000013467 fragmentation Methods 0.000 description 9
- 230000036039 immunity Effects 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 102220023256 rs387907547 Human genes 0.000 description 8
- 239000012153 distilled water Substances 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000007983 Tris buffer Substances 0.000 description 6
- 102220369447 c.1352G>A Human genes 0.000 description 6
- 150000002460 imidazoles Chemical class 0.000 description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 6
- 206010010254 Concussion Diseases 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 230000009514 concussion Effects 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000010353 genetic engineering Methods 0.000 description 5
- 235000013336 milk Nutrition 0.000 description 5
- 239000008267 milk Substances 0.000 description 5
- 210000004080 milk Anatomy 0.000 description 5
- 238000010369 molecular cloning Methods 0.000 description 5
- 230000003472 neutralizing effect Effects 0.000 description 5
- 230000001681 protective effect Effects 0.000 description 5
- 229940126583 recombinant protein vaccine Drugs 0.000 description 5
- 241000283690 Bos taurus Species 0.000 description 4
- 229910052782 aluminium Inorganic materials 0.000 description 4
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 229960000074 biopharmaceutical Drugs 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 235000013877 carbamide Nutrition 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 208000030194 mouth disease Diseases 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 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 3
- 241000282898 Sus scrofa Species 0.000 description 3
- 239000004411 aluminium Substances 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 102220369446 c.1274G>A Human genes 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010411 cooking Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000005498 polishing Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 102220023257 rs387907546 Human genes 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 101710094648 Coat protein Proteins 0.000 description 2
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 101710125418 Major capsid protein Proteins 0.000 description 2
- 101710141454 Nucleoprotein Proteins 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 241000709664 Picornaviridae Species 0.000 description 2
- 101710083689 Probable capsid protein Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 2
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- 230000009849 deactivation Effects 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 229940031551 inactivated vaccine Drugs 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 210000000214 mouth Anatomy 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N nickel Substances [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 210000005164 penile vein Anatomy 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001665 trituration Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 239000002435 venom Substances 0.000 description 2
- 210000001048 venom Anatomy 0.000 description 2
- 231100000611 venom Toxicity 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 101710145634 Antigen 1 Proteins 0.000 description 1
- 241000710189 Aphthovirus Species 0.000 description 1
- 240000004355 Borago officinalis Species 0.000 description 1
- 235000007689 Borago officinalis Nutrition 0.000 description 1
- 102100031974 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 4 Human genes 0.000 description 1
- 101800000594 Capsid protein 1 Proteins 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 241000282994 Cervidae Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 238000011238 DNA vaccination Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102100034274 Diamine acetyltransferase 1 Human genes 0.000 description 1
- 101100117236 Drosophila melanogaster speck gene Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101000703754 Homo sapiens CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 4 Proteins 0.000 description 1
- 101000641077 Homo sapiens Diamine acetyltransferase 1 Proteins 0.000 description 1
- 101000713305 Homo sapiens Sodium-coupled neutral amino acid transporter 1 Proteins 0.000 description 1
- 101000640813 Homo sapiens Sodium-coupled neutral amino acid transporter 2 Proteins 0.000 description 1
- 101000716973 Homo sapiens Thialysine N-epsilon-acetyltransferase Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000080590 Niso Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 102100020926 Thialysine N-epsilon-acetyltransferase Human genes 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229940103272 aluminum potassium sulfate Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000012501 chromatography medium Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 210000000003 hoof Anatomy 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- CTCHDPAJHVDPRN-UHFFFAOYSA-N integrin Chemical compound C=1C(OC)=CC(O)=C(C(C=2CC=C(C)C)=O)C=1OC=2C1=CC=C(O)C=C1O CTCHDPAJHVDPRN-UHFFFAOYSA-N 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
- 210000000088 lip Anatomy 0.000 description 1
- 229940023832 live vector-vaccine Drugs 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910001453 nickel ion Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 229940093429 polyethylene glycol 6000 Drugs 0.000 description 1
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 description 1
- 235000019353 potassium silicate Nutrition 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000008279 sol Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000002105 tongue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000036269 ulceration Effects 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 210000000605 viral structure Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical class OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 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
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a recombinant foot-and-mouth disease virus VP1 fusion protein vaccine, particularly a recombinant fusion protein produced in Escherichia coli by fusing a foot-and-mouth disease virus VP1 peptide segment, 2-polyglycine and a 6-polyhistidine coding gene. The present invention provides an amino acid sequence of a recombinant foot-and-mouth disease virus VP1 fusion protein and a nucleotide sequence thereof. The present invention also provides the action of foot-and-mouth disease virus infection prevention of the recombinant foot-and-mouth disease virus VP1 fusion protein.
Description
Invention field
The present invention relates to the genetic engineering field, be specifically related to the genetic engineering recombinant protein vaccine, particularly relate to a kind of recombined foot-and-mouth disease virus VP1 amalgamation protein vaccine (hereinafter being also referred to as recombined foot-and-mouth disease virus VP1 fusion rotein sometimes) that prevents the animal foot and mouth disease virus to infect.It is that foot and mouth disease virus VP1 peptide section, 2 polyglycines, 6 polyhistidyl encoding genes are merged, recombination fusion protein in escherichia coli production, after being applied to animal body, can bring out the neutrality antibody of body generation, have the biologic activity that prevention animal foot and mouth disease virus infects at foot and mouth disease virus.
Background of invention
(foot-and-mouthdisease FMD) is the deadly infectious disease that is caused by foot and mouth disease virus to foot and mouth disease.Pig, cattle, sheep all can infect foot and mouth disease virus and foot and mouth disease takes place, and pathological changes such as blister, ulceration and speck appear in the lighter at positions such as mouth, tongue, lip, hoof, breast, and death takes place weight person.The generation of foot and mouth disease and popular meeting cause huge direct and indirect economic loss [Jiang Pengfei, 1999].International Office of Epizootics (OIE) classifies foot and mouth disease as the category-A livestock contagious disease.Countries in the world are all attached great importance to prevention and control foot and mouth disease.Except that massacring the domestic animal that infects foot and mouth disease, vaccination is the major measure [M.Woolhouse, 2001] of prevention and control foot and mouth disease.Traditional foot-and-mouth disease vaccine refuels the inactivated vaccine that adjuvant emulsion makes by the foot and mouth disease virus of the preliminary purification of deactivation, and the susceptible domestic animal is had suitable protective effect, but exists deactivation insufficient and cause the possibility of foot and mouth disease.In addition, the great-hearted foot and mouth disease virus of employing in producing this vaccine process is a kind of very important potential source of infection.
Twentieth century is since the beginning of the eighties, and many countries have dropped into a large amount of human and material resources and financial resources are developed novel foot-and-mouth disease vaccine, as recombinant protein vaccine, and synthetic peptide vaccine, [Broekhuigsen M P, 1987 such as live vector vaccine and dna vaccination; MorganD O, 1990; Ward, G., 1997; Berinstein A, 2000].Some vaccines have shown effect preferably, but major part is still among breadboard exploration.
Foot and mouth disease virus (foot-and-mouth disease virus, FMDV) be the pathogen of foot and mouth disease, be the member of Picornaviridae (Picornaviridae) Hostis (Aphthovirus), 7 serotypes have been had now found that, promptly 0, A, C type (title Europe class), SAT1, SAT2, SAT3 type (South Africa 1,2,3 types also claim African type) and AsiaI type (the Asia I type claims Asian type).O type foot and mouth disease virus is a popular wider serotype in the world wide in recent years.Studies show that, at humoral immunization in the human body foot-and-mouth disease virus resistant infects, play a major role.Be in the carcass, the ability that the level of foot and mouth disease virus neutralizing antibody and its opposing foot and mouth disease virus are attacked is tangible positive correlation [Pay TW, 1987].Based on this observation, many laboratorys have been done a large amount of experiments, seek the target spot that can stimulate body to produce virucidin on foot and mouth disease virus, find to exist in the coat protein (VP) of foot and mouth disease virus structure [Crowther JR, 1993 of five candidates; Kitson JD, 1990], wherein three are arranged in coat protein 1 (Vp1).Experiment showed, that isolated Vp1 albumen has good immunogenicity [Bachrach HL, 1975:Kleid DG, 1981 from foot and mouth disease virus; Strohmaier K, 1982; Rodriguez A, 1994].
The neutrality antibody that produces with the proteic peptide section of Vp1 (141-160 and 200-213) immune guinea pig and cattle can watch for animals and avoid infecting foot and mouth disease virus [Bittle JL, 1982; Pfaff E, 1982; DiMarchi R, 1986]." ring " shape structure that VP1 140-160 aminoacid constitutes is exposed to virus surface, R-G-D sequence wherein is at the foot and mouth disease virus high conservative, be the key structure of foot and mouth disease virus, may mediate foot and mouth disease virus and enter infected cells [JacksonT, 2002 in conjunction with cell surface receptor; Almeida MR, 1998].Can induce the high-caliber neutralizing antibody of generation according to the synthetic peptide section of VP1 circulus.Also there are some researches show, use the Vp1 protein immune animal of separating from foot and mouth disease virus or gene engineering method obtains, can generating unit divide the protection effect.The albumen that Vp1 albumen and some is derived from other microorganisms by genetic engineering means merges and can improve the proteic immunogenicity of Vp1 [Clarke BE, 1987:Corchero JL, 1996].
List of references:
Jiang Pengfei, Zhao Qizu, Xie Qingge, foot and mouth disease progress Scientia Agricultura Sinica 1999,32 (6): 93～100Almeida MR, Rieder E, Chinsangaram J, Ward G, Beard C, Grubman MJ, Mason PW.Construction and evaluation of an attenuated vaccine for foot-and-mouth disease:difficulty adapting the leader proteinase-deleted strategy to the serotype 01 virus.Virus Res 1998 May; 55 (1): 49-60
Bachrach?HL，Moore?DM，McKercher?PD，Polatnick?J.Immune?and?antibody?responses?toan?isolated?capsid?protein?of?foot-and-mouth?disease?virus.J?Immunol.1975Dec；115(6)：1636-41.
Berinstein?A，Tami?C，Taboga?O，Smitsaart?E，Carrillo?E.Protective?immunity?againstfoot-and-mouth?disease?virus?induced?by?a?recombinant?vaccinia?virus.Vaccine.2000Apr?28；18(21)：2231-8.
Bittle?JL，Houghten?RA，Alexander?H，Shinnick?TM，Sutcliffe?JG，Lerner?RA，RowlandsDJ，Brown?F.Protection?against?foot-and-mouth?disease?by?immunization?with?achemically?synthesized?peptide?predicted?from?the?viral?nucleotide?sequence.Nature.1982?Jul?1；298(5869)：30-3.
Broekhuigsen?M?P，Blom?Ton，Rijn?et?al.fusion?proteins?with?multiple?copies?ofthe?major?antigenic?determinant?of?FMDV?protect?both?the?natural?host?and?laboratoryanimals.J.Gen.Virol，1987，(68)；3137-3143.
Clarke?BE，Newton?SE，Carroll?AR，Francis?MJ，Appleyard?G，Syred?AD，Highfield?PE，Rowlands?DJ，Brown?F.Improved?immunogenicity?of?a?peptide?epitope?after?fusion?tohepatitis?B?core?protein.Nature.1987?Nov?26-Dec?2；330(6146)：381-4.
Corchero?JL，Villaverde?A.Antigenicity?of?a?viral?peptide?displayed?onbeta-galactosidase?fusion?proteins?is?influenced?by?the?presence?of?the?homologouspartner?protein.FEMS?Microbiol?Lett.1996?Nov?15；145(1)：77-82.
Crowther?JR，Farias?S，Carpenter?WC，Samuel?AR.Identification?of?a?fifthneutralizable?site?on?type?O?foot-and-mouth?disease?virus?following?characterizationof?single?and?quintuple?monoclonal?antibody?escape?mutants.J?Gen?Virol.1993?Aug；74(Pt?8)：1547-53.
DiMarchi?R，Brooke?G，Gale?C，Cracknell?V，Doel?T，Mowat?N.Protection?of?cattleagainst?foot-and-mouth?disease?by?a?synthetic?peptide.Science.1986?May?2；232(4750)：639-41.
Jackson?T，Mould?AP，Sheppard?D，King?AM.Integrin?alphavbetal?is?a?receptor?forfoot-and-mouth?disease?virus.J?Virol?2002?Feb；76(3)：935-41
Kitson?JD，McCahon?D，Belsham?GJ.Sequence?analysis?of?monoclonal?antibody?resistantmutants?of?type?O?foot?and?mouth?disease?virus：evidence?for?the?involvement?of?thethree?surface?exposed?capsid?proteins?in?four?antigenic?sites.Virology.1990Nov；179(1)：26-34.
Kleid?DG，Yansura?D，Small?B，Dowbenko?D，Moore?DM，Grubman?MJ，McKercher?PD，MorganDO，Robertson?BH，Bachrach?HL.Cloned?viral?protein?vaccine?for?foot-and-mouthdisease：responses?in?cattle?and?swine.Science.1981?Dec?4；214(4525)：1125-9.
Lubroth?J，Grubman?MJ，Burrage?TG，Newman?JF，Brown?F.Absence?of?protein?2C?fromclarified?foot-and-mouth?disease?virus?vaccines?provides?the?basis?fordistinguishing?convalescent?from?vaccinated?animals.Vaccine.1996?Apr：14(5)：419-27.
M.Woolhouse?and?A.Donaldson，Managing?foot-and-mouth?disease：the?science?ofcontrolling?disease?outbreaks.Nature?410(2001)，pp.515-516.
Morgan?D?O，Moore，M.Protection?of?cattle?and?swine?against?FMD，using?biosyntheticpeptide?vaccines.Am.J.Vet.Res，1990，51(1)40-45.
Pay?TW，Hingley?PJ.Correlation?of?140S?antigen?dose?with?the?serum?neutralizingantibody?response?and?the?level?of?protection?induced?in?cattle?by?foot-and-mouthdisease?vaccines.Vaccine.1987?Mar；5(1)：60-4.
Pfaff?E，Mussgay?M，Bohm?HO，Schulz?GE，Schaller?H.Antibodies?against?a?preselectedpeptide?recognize?and?neutralize?foot?and?mouth?disease?virus.EMBO?J.1982；1(7)：869-74.
Ward，G.，Rieder，E.&?Mason，P.W.(1997).Plasmid?DNA?encoding?replicatingfoot-and-mouth?disease?virus?genomes?induces?antiviral?immune?responses?in?swine.Journal?of?Virology?71，7442-7447.
Rodriguez?A，Saiz?JC，Novella?IS，Andreu?D，Sobrino?F.Antigenic?specificity?ofporcine?T?cell?response?against?foot-and-mouth?disease?virus?structural?proteins：identification?of?T?helper?epitopes?in?VP1.Virology.1994?Nov?15；205(1)：24-33.Strohmaier?K，Franze?R，Adam?KH.Location?and?characterization?of?the?antigenicportion?of?the?FMDV?immunizing?protein.J?Gen?Virol.1982Apr；59(Pt?2)：295-306.
Summary of the invention
An aspect of of the present present invention provides a kind of recombined foot-and-mouth disease virus VP1 amalgamation protein vaccine, and it is that foot and mouth disease virus VP1 peptide section, 2 polyglycines and 6 polyhistidyl encoding genes are merged, at the recombination fusion protein of escherichia coli production.
Another aspect of the present invention provides the aminoacid sequence and the nucleotide sequence thereof of recombined foot-and-mouth disease virus VP1 fusion rotein.
Another aspect of the present invention provides the nucleotide sequence and the amino acid sequence coded thereof of foot and mouth disease virus VP1BT1 fusogenic peptide.
Another aspect of the present invention provides the nucleotide sequence and the amino acid sequence coded thereof of foot and mouth disease virus VP1BT2 fusogenic peptide.
Another aspect of the present invention provides the connected mode of foot and mouth disease virus VP1BT1 fusogenic peptide, comprises existence, connected mode and the position of 2 polyglycines.
Another aspect of the present invention provides the connected mode of foot and mouth disease virus VP1BT2 fusogenic peptide, comprises existence, connected mode and the position of 2 polyglycines.
The foot and mouth disease virus VP1 peptide section fusion rotein that another aspect of the present invention provides both sides that 6 polyhistidyls are arranged.
Another aspect of the present invention relates to recombined foot-and-mouth disease virus VP1 fusion rotein according to the described recombined foot-and-mouth disease virus VP1 of claim 1 fusion rotein, it can bring out the neutrality antibody of body generation at foot and mouth disease virus after being applied to animal body, have the biologic activity of the infection of prevention animal foot and mouth disease virus.
In addition, it is pointed out that on the basis of the application's contextual disclosure that aspect and other creative beneficial effect that other has substantive distinguishing features of the present invention are conspicuous to those skilled in the art.
Brief Description Of Drawings
Accompanying drawing 1: the recombined foot-and-mouth disease virus VP1 fusion rotein SDS-PAGE electrophoretogram of expression
Accompanying drawing 2: the recombined foot-and-mouth disease virus VP1 fusion rotein SDS-PAGE electrophoretogram of purification
Accompanying drawing 3: recombined foot-and-mouth disease virus VP1 fusion protein immunization Cavia porcellus (1) serum is to the protective effect of neonatal rat
Accompanying drawing 4: recombined foot-and-mouth disease virus VP1 fusion protein immunization Cavia porcellus (2) serum is to the protective effect of neonatal rat
Accompanying drawing 5: recombined foot-and-mouth disease virus VP1 fusion protein immunization Cavia porcellus (3) serum is to the protective effect of neonatal rat
The specific embodiment
In the context of the present invention, employed term generally has the implication of those of ordinary skill in the art's common sense unless otherwise indicated.Especially, following term has following implication:
Recombinant protein vaccine: be to utilize molecule clone technology, the gene clone that coding is had immunogenic albumen or polypeptide is on protokaryon or eukaryotic expression vector, have immunogenic protein or polypeptide with this carrier what antibacterial or eukaryotic cell (yeast cells or mammalian cell) were produced, this protein or polypeptide can stimulate body (human or animal) that specific humoral immunoresponse(HI) and/or cellullar immunologic response take place after being applied to body.If on protokaryon or eukaryotic expression vector, is recombinant protein vaccine with antiviral such as hoof-and-mouth disease toxic action in antibacterial or eukaryotic cell (yeast cells or mammalian cell) production by viral gene encoded protein matter or polypeptide with it with the gene clone with immunogenic albumen or polypeptide of virus as foot-and-mouth disease virus gene coding.This vaccine can induce body that specific humoral immunoresponse(HI) and/or cellullar immunologic response take place after being applied to animal, prevents the infection that virus causes as foot and mouth disease virus.
The foot and mouth disease virus of animal (FMDV) infects: be the contagious disease of being suffered from altogether by the cloven-hoofed animal that FMDV causes.FMDV is a kind of RNA viruses, and its genome comprises 7500-8000 base approximately.FMDV mainly propagates in the mode of saliva, and the speed of propagation is very fast, easily causes large-area popular.Cattle, pig, sheep, deer etc. are the infection animal of foot and mouth disease virus.
Foot and mouth disease virus VP1 antigen: be the part of FMD virus coat protein, it can induce body to produce the foot and mouth disease virus neutralizing antibody with protective effect.
Foot and mouth disease virus VP1 peptide section comprises foot and mouth disease virus VP1BT1 fusogenic peptide and foot and mouth disease virus VP1BT2 fusogenic peptide
Foot and mouth disease virus VP1BT1 fusogenic peptide is meant the polypeptide with aminoacid sequence shown in SEQ ID NO 2.
GGVSNVRGDLQVLAQKAERALPGGEENYGGETQVQRRQHTDISFILDRFVKVTP(SEQ?ID?NO?2)。
Foot and mouth disease virus VP1 BT2 fusogenic peptide is meant to have and is meant the polypeptide with aminoacid sequence shown in SEQ ID NO 4.
The peptide section of GGVSNVRGDLQVLAQKAKRALPGGPSDARHKQKIVAPAKQLLNFDL (SEQ ID NO4) aminoacid sequence.
2 peptides that 2 polyglycines are made up of 2 adjacent glycine.
Foot and mouth disease virus VP1 peptide section fusion rotein encoding gene is meant the DNA with the nucleotide sequence shown in SEQ ID NO5, and its encoded protein matter is foot and mouth disease virus VP1 peptide section fusion rotein.
Recombined foot-and-mouth disease virus VP1 amalgamation protein vaccine encoding gene has the nucleotide sequence shown in SEQ ID NO8, its encoded protein matter is the recombined foot-and-mouth disease virus VP1 amalgamation protein vaccine or the group foot and mouth disease virus VP1 fusion rotein of weighing, and it has the aminoacid sequence shown in SEQ ID NO 9.
The invention provides a kind of recombined foot-and-mouth disease virus VP1 amalgamation protein vaccine, it is that foot and mouth disease virus VP1 peptide section, 2 polyglycines, 6 polyhistidyl encoding genes are merged, at the recombination fusion protein of escherichia coli production.Recombined foot-and-mouth disease virus VP1 amalgamation protein vaccine of the present invention has the aminoacid sequence shown in nucleotide sequence shown in the SEQ ID NO8 and the SEQ ID NO9.
Foot and mouth disease virus VP1BT1 fusogenic peptide in the recombined foot-and-mouth disease virus VP1 fusion rotein provided by the invention has the aminoacid sequence shown in nucleotide sequence shown in the SEQ IDNO:1 and the SEQ ID NO2.
Foot and mouth disease virus VP1BT2 fusogenic peptide in the recombined foot-and-mouth disease virus VP1 fusion rotein provided by the invention has the aminoacid sequence shown in nucleotide sequence shown in the SEQ IDNO:3 and the SEQ ID NO 4.
Foot and mouth disease virus VP1 peptide section fusion rotein encoding gene in the recombined foot-and-mouth disease virus VP1 fusion rotein provided by the invention has the nucleotide sequence shown in SEQ ID NO 5, and its encoded protein matter is foot and mouth disease virus VP1 peptide section fusion rotein.
Foot and mouth disease virus VP1BT1 fusogenic peptide in the recombined foot-and-mouth disease virus VP1 fusion rotein provided by the invention has the connected mode shown in the SEQ IDNO:2, comprises existence, connected mode and the position of 2 polyglycines.
Foot and mouth disease virus VP1BT2 fusogenic peptide in the recombined foot-and-mouth disease virus VP1 fusion rotein provided by the invention has the connected mode shown in the SEQ IDNO:4, comprises existence, connected mode and the position of 2 polyglycines.
Foot and mouth disease virus VP1 peptide section fusion rotein in the recombined foot-and-mouth disease virus VP1 fusion rotein provided by the invention has the connected mode shown in SEQID NO:5.
There is the sequence of 6 polyhistidyls the both sides of the foot and mouth disease virus VP1 peptide section fusion rotein in the recombined foot-and-mouth disease virus VP1 fusion rotein provided by the invention.
Recombined foot-and-mouth disease virus VP1 fusion rotein provided by the invention adopts escherichia coli to produce.
Recombined foot-and-mouth disease virus VP1 fusion rotein provided by the invention can bring out the neutrality antibody of body generation at foot and mouth disease virus after being applied to animal, have the biologic activity of the infection of prevention animal foot and mouth disease virus.
The invention still further relates to this genetic engineering recombiant protein and be used for preventing purposes of vaccine product of infection of animal foot and mouth disease virus and the vaccine product that contains this genetic engineering recombiant protein in preparation.It will be appreciated by persons skilled in the art that these vaccine products can prepare with the known various conventional methods in this area.
Recombinant protein vaccine of the present invention can be given animal inoculation by hypodermic mode, and the dosage of inoculation is 100-5000 μ g.For stiffening effect, can carry out 1 time booster immunization in 14 or 21 days in the immunity back first time.
Below in conjunction with concrete preparation embodiment and biology effect embodiment, and the present invention is described in further detail with reference to accompanying drawing.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
Embodiment
In following embodiment, various processes of Xiang Ximiaoshuing and method are not conventional methods as known in the art, for example Molecular Cloning one book (J.Sambrook, Cold Spring Harbor Laboratory Press, Molecularcloning, 1989) described method.
Embodiment 1, synthetic VP1BT1 fusogenic peptide encoding gene
Adopt the synthetic VP1 BT1 fusogenic peptide encoding gene of PCR method.Designed and synthesized PCR primer with following sequence:
Primer 1: ' CTGCAAGTACTGGCTCAAAAAGCAGAACGTGCTCTGCCGGGTGGCGAAGAAAACTA CGGTGGT3 '
Primer 2: 5 ' TGAAGGAGATGTCAGTGTGCTGACGACGCTGAACCTGAGTTTCACCACCGTAGTTT TCTTCGCC3 '
Primer 3:5 ' GAATTCAGATCTGGCGGTGTATCTAACGTACGTGGTGACCTGCAAGTACTGGCTCA A3 '
Primer 4:5 ' AAGCTTCCCGGAGTAACTTTAACGAAACGGTCCAGGATGAAGGAGATGTCAGTGTG 3 '
Adopt primer 1,2 synthetic dna fragmentation (fragment 1,2) with following sequence:
CTGCAAGTAC?TGGCTCAAAA?AGCAGAACGT?GCTCTGCCGG?GTGGCGAAGA?AAACTACGGT?GGTGAAACTC
AGGTTCAGCG?TCGTCAGCAC?ACTGACATCT?CCTTCA
For touching plate, adopt the VP1 BT1 fusogenic peptide encoding gene at primer 3,4 synthetic band restricted enzyme point of contacts, both sides with fragment 1,2, (SEQ ID NO:1) is as follows for its sequence:
GAATTCAGAT?CTGGCGGTGT?ATCTAACGTA?CGTGGTGACC?TGCAAGTACT?GGCTCAAAAA?GCAGAACGTG
CTCTGCCGGG?TGGCGAAGAA?AACTACGGTG?GTGAAACTCA?GGTTCAGCGT?CGTCAGCACA?CTGACATCTC
CTTCATCCTG?GACCGTTTCG?TTAAAGTTAC?TCCGGGATCC
AAGCTT
The PCR operation sequence that adopts is:
In one 500 μ l microcentrifugal tubes, add following reagent:
10 * PCR buffer, 5 μ l
(500mmol/L?KCl，100mmol/L?Tris-Cl，
15mmol/L?MgCl
2)
dNTPs(10mmol/L) 1μl
5 ' end and 3 ' each 0.5 μ l of end primer (0.01mmol/L)
Taq archaeal dna polymerase (5u/ μ l) 0.25 μ l
Add deionized water to final volume 50 μ l
Mix the back and add 3 in mineral oil
Reaction condition: 94 ℃, 30 "; 55 ℃, 1 '; 72 ℃, behind 2 ', 30 cycle periods, 72 ℃ were extended 10 minutes.
The PCR product is done 2% agarose gel electrophoresis (1xTAE, 150-200mA, 0.5 hour).Adopt the DNA of Beijing ancient cooking vessel state company to reclaim the test kit recovery synthetic VP1 BT1 of PCR fusogenic peptide encoding gene dna fragmentation.Downcut the gel that contains dna fragmentation from agarose gel, put into a centrifuge tube.Add the sol solutions (Beijing ancient cooking vessel state company) of 3 times of volumes, 45-55 ℃ of water-bath 5-10min melts glue fully.Add 10ul glass milk (Beijing ancient cooking vessel state company), flick pipe end mixing, then at 45-55 ℃ of water-bath 5-10min, during every 2-3min mixing once.Centrifugal 60 seconds of 5000g abandons supernatant.Add the 400ul rinsing liquid, flick pipe end mixing glass milk, the same then centrifugal, abandon supernatant.Add the 400ul rinsing liquid again, flick pipe end mixing glass milk, the same then centrifugal supernatant of abandoning, and with the sample injector Ex-all rinsing liquid of trying one's best.Room temperature is dried glass milk then.Add 10-30 μ l aseptic double-distilled water glass milk suspended, 45-55 ℃ water-bath 5-10 minute.The centrifugal 2min of 10000g, it is standby to reclaim supernatant (containing VP1 BT1 fusogenic peptide encoding gene dna fragmentation).
VP1 BT1 fusogenic peptide encoding gene dna fragmentation is cloned into pMD18-T Vect plasmid (TakaRa company).
The coupled reaction system:
VP1 BT1 fusogenic peptide encoding gene dna fragmentation (sequence is referring to SEQ ID NO:1) 0.05-0.3pmol
PMD18-T Vect (TakaRa company) 0.5-1 μ l
Solution I (TakaRa company) 5 μ l
Distilled water adds together to 10 μ l
16 ℃ were reacted 1 hour
There is the pMD18-T Vect plasmid of VP1 BT1 fusogenic peptide encoding gene dna fragmentation to be transformed into e. coli jm109 (Novagen company) clone.
The preparation method of competence e. coli jm109 cell is: get an e. coli jm109 list bacterium colony in 2ml LB culture medium, 37 ℃, the concussion of 225rpm speed was cultivated 12-16 hour; Get the above-mentioned culture of 1ml and be inoculated in the 100ml LB culture medium, 37 ℃ of C, it is about 0.5 (about 3 hours) that the speed concussion of 225rpm is cultivated until the OD value; With bacterium liquid ice bath 2 hours, 2,500 * g then collected thalline in centrifugal 20 minutes for 4 ℃; The Trituration buffer that adding 100ml is ice-cold (100mmol/LCaCl2,70mmol/L MgCl2, the 40mmol/L sodium acetate, pH5.5), mixing was put 45 minutes on ice; 1,800 * g, 4 ℃ centrifugal 10 minutes, abandon supernatant, add the ice-cold Trituration buffer suspension cell of 10ml; By every part 200 μ l packing, can preserve 1-2 week for 4 ℃.If need long preservation, but glycerol adding to final concentration is 15%, put-70 ℃ standby.
The method that transforms is: 200 μ l competent cells are put on ice melted, add 3 μ l DMSO or beta-mercaptoethanols then, after the mixing, add 2 μ l coupled reaction liquid (containing recombiant plasmid), mixing was put 30 minutes on ice; 42 ℃ 45 seconds, put back to rapidly then in the ice 1-2 minute; Add 2ml LB culture fluid, 37 ℃, the speed of 225rpm is swayed and was cultivated 1 hour; 4,000 * g centrifugal 10 seconds, abandon supernatant, with the resuspended thalline of 200 μ l LB culture fluid; Bacterium liquid is laid on contains on the antibiotic LB agar culture plate, smoothen, room temperature was placed 20-30 minute, was inverted in 37 ℃ of incubators and cultivated 12-16 hour.
Select positive colony, extract plasmid (J.Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989).
Embodiment 2, synthetic VP1BT2 fusogenic peptide encoding gene
Adopt the synthetic VP1 BT1 fusogenic peptide encoding gene of PCR method.Designed and synthesized PCR primer with following sequence:
Primer 7:5 ' GTGACCTGCAAGTACTGGCTCAAAAAGCTAAACGTGCTCTGCCGGGTGGTCCGTCT 3 '
Primer 8:5 ' CGGAGCAACGATTTTCTGTTTGTGACGAGCGTCAGACGGACCACCCGGCA3 '
Primer 9:5 ' CCATGGAATTCAGATCTGGTGGTGTTTCTAACGTTCGTGGTGACCTGCAAGTACTG GC3 '
Primer 10:5 ' AAGCTTGGATCCCAGGTCGAAGTTCAGCAGCTGTTTAGCCGGAGCAACGATTTTCT G3 '
Adopt primer 7,8 synthetic dna fragmentations (fragment 7,8) with following sequence:
GTGACCTGCA?AGTACTGGCT?CAAAAAGCTA?AACGTGCTCT?GCCGGGTGGT?CCGTCTGACG?CTCGTCACAA
ACAGAAAATC?GTTGCTCCG
For touching plate, adopt the VP1 BT2 fusogenic peptide encoding gene at primer 9,10 synthetic band restricted enzyme point of contacts, both sides with fragment 7,8, have the following sequence shown in SEQ ID NO:3:
CCATGGAATT?CAGATCTGGT?GGTGTTTCTA?ACGTTCGTGG?TGACCTGCAA?GTACTGGCTC?AAAAAGCTAA
ACGTGCTCTG?CCGGGTGGTC?CGTCTGACGC?TCGTCACAAA?CAGAAAATCG?TTGCTCCGGC?TAAACAGCTG
CTGAACTTCG?ACCTGGGATC?CAAGCTT
PCR operation sequence that adopts and embodiment 1 are roughly the same.The method of recovery, clone VP1BT2 fusogenic peptide encoding gene and embodiment 1 are roughly the same.The pMD18-T Vect plasmid that will contain VP1BT2 fusogenic peptide encoding gene is transformed into competence e. coli jm109 cell (method is analogous to embodiment 1).Select positive colony, extract plasmid (J.Sambrook, Cold Spring HarborLaboratory Press, Molecular cloning, 1989).
Embodiment 3, structure VP1 peptide section fusion rotein encoding gene
With the VP1BT1 fusogenic peptide encoding gene on the BamHI digestion pMD18-T Vect plasmid, with the BT2 fusogenic peptide encoding gene on the Bg1II digestion pMD18-TVect plasmid, with the T4 ligase two digestion fragments connections are obtained BT1 fusogenic peptide encoding gene-BT2 fusogenic peptide encoding gene fusion gene (SEQ ID NO:5), its sequence is as follows:
GAATTCAGAT?CTGGCGGTGT?ATCTAACGTA?CGTGGTGACC?TGCAAGTACT?GGCTCAAAAA?GCAGAACGTG
CTCTGCCGGG?TGGCGAAGAA?AACTACGGTG?GTGAAACTCA?GGTTCAGCGT?CGTCAGCACA?CTGACATCTC
CTTCATCCTG?GACCGTTTCG?TTAAAGTTAC?TCCGGGATCT?GGTGGTGTTT?CTAACGTTCG?TGGTGACCTG
CAAGTACTGG?CTCAAAAAGC?TAAACGTGCT?CTGCCGGGTG?GTCCGTCTGA?CGCTCGTCAC?AAACAGAAAA
TCGTTGCTCC?GGCTAAACAG?CTGCTGAACT?TCGACCTGGG?ATCCAAGCTT
Digest VP1 BT1 fusogenic peptide encoding gene-VP1 BT2 fusogenic peptide encoding gene fusion gene respectively with BamHI and Bg1II, obtain the VP1 BT1 fusogenic peptide encoding gene-VP1 BT2 fusogenic peptide encoding gene fusion gene fragment of BamHI digestion and the VP1 BT1 fusogenic peptide encoding gene-VP1 BT2 fusogenic peptide encoding gene fusion gene fragment of Bg1II digestion, with the T4 ligase two digestion fragments are connected the 2 aggressiveness genes (SEQ ID NO:6) that obtain VP1 BT1 fusogenic peptide encoding gene-BT2 fusogenic peptide encoding gene fusion gene, its sequence is as follows:
GAATTCAGAT?CTGGCGGTGT?ATCTAACGTA?CGTGGTGACC?TGCAAGTACT?GGCTCAAAAA?GCAGAACGTG
CTCTGCCGGG?TGGCGAAGAA?AACTACGGTG?GTGAAACTCA?GGTTCAGCGT?CGTCAGCACA?CTGACATCTC
CTTCATCCTG?GACCGTTTCG?TTAAAGTTAC?TCCGGGATCT?GGTGGTGTTT?CTAACGTTCG?TGGTGACCTG
CAAGTACTGG?CTCAAAAAGC?TAAACGTGCT?CTGCCGGGTG?GTCCGTCTGA?CGCTCGTCAC?AAACAGAAAA
TCGTTGCTCC?GGCTAAACAG?CTGCTGAACT?TCGACCTGGG?ATCTGGCGGT?GTATCTAACG?TACGTGGTGA
CCTGCAAGTA?CTGGCTCAAA?AAGCAGAACG?TGCTCTGCCG?GGTGGCGAAG?AAAACTACGG?TGGTGAAACT
CAGGTTCAGC?GTCGTCAGCA?CACTGACATC?TCCTTCATCC?TGGACCGTTT?CGTTAAAGTT?ACTCCGGGAT
CTGGTGGTGT?TTCTAACGTT?CGTGGTGACC?TGCAAGTACT?GGCTCAAAAA?GCTAAACGTG?CTCTGCCGGG
TGGTCCGTCT?GACGCTCGTC?ACAAACAGAA?AATCGTTGCT?CCGGCTAAAC?AGCTGCTGAA?CTTCGACCTG
GGATCCAAGC?TT(SEQ?ID?NO：6)
2 aggressiveness genes with BamHI digestion VP1 BT1 fusogenic peptide encoding gene-VP1 BT2 fusogenic peptide encoding gene fusion gene, with Bg1II digestion VP1 BT1 fusogenic peptide encoding gene-VP1 BT2 fusogenic peptide encoding gene fusion gene, with the T4 ligase two digestion fragments are connected the 3 aggressiveness genes (SEQ ID NO:7) that obtain VP1 BT1 fusogenic peptide encoding gene-VP1 BT2 fusogenic peptide encoding gene fusion gene, its sequence is as follows:
GAATTCAGAT?CTGGCGGTGT?ATCTAACGTA?CGTGGTGACC?TGCAAGTACT?GGCTCAAAAA?GCAGAACGTG
CTCTGCCGGG?TGGCGAAGAA?AACTACGGTG?GTGAAACTCA?GGTTCAGCGT?CGTCAGCACA?CTGACATCTC
CTTCATCCTG?GACCGTTTCG?TTAAAGTTAC?TCCGGGATCT?GGTGGTGTTT?CTAACGTTCG?TGGTGACCTG
CAAGTACTGG?CTCAAAAAGC?TAAACGTGCT?CTGCCGGGTG?GTCCGTCTGA?CGCTCGTCAC?AAACAGAAAA
TCGTTGCTCC?GGCTAAACAG?CTGCTGAACT?TCGACCTGGG?ATCTGGCGGT?GTATCTAACG?TACGTGGTGA
CCTGCAAGTA?CTGGCTCAAA?AAGCAGAACG?TGCTCTGCCG?GGTGGCGAAG?AAAACTACGG?TGGTGAAACT
CAGGTTCAGC?GTCGTCAGCA?CACTGACATC?TCCTTCATCC?TGGACCGTTT?CGTTAAAGTT?ACTCCGGGAT
CTGGTGGTGT?TTCTAACGTT?CGTGGTGACC?TGCAAGTACT?GGCTCAAAAA?GCTAAACGTG?CTCTGCCGGG
TGGTCCGTCT?GACGCTCGTC?ACAAACAGAA?AATCGTTGCT?CCGGCTAAAC?AGCTGCTGAA?CTTCGACCTG
GGATCTGGCG?GTGTATCTAA?CGTACGTGGT?GACCTGCAAG?TACTGGCTCA?AAAAGCAGAA?CGTGCTCTGC
CGGGTGGCGA?AGAAAACTAC?GGTGGTGAAA?CTCAGGTTCA?GCGTCGTCAG?CACACTGACA?TCTCCTTCAT
CCTGGACCGT?TTCGTTAAAG?TTACTCCGGG?ATCTGGTGGT?GTTTCTAACG?TTCGTGGTGA?CCTGCAAGTA
CTGGCTCAAA?AAGCTAAACG?TGCTCTGCCG?GGTGGTCCGT?CTGACGCTCG?TCACAAACAG?AAAATCGTTG
CTCCGGCTAA?ACAGCTGCTG?AACTTCGACC?TGGGATCCAA?GCTT(SEQ?ID?NO：7)。
3 aggressiveness genes of VP1 BT1 fusogenic peptide encoding gene-VP1BT2 fusogenic peptide encoding gene fusion gene both had been VP1 peptide section fusion rotein encoding gene.
VP1 peptide section fusion rotein encoding gene is cloned into pMD18-T Vect plasmid, and the method for employing and embodiment 1 are roughly the same used.The pMD18-T Vect plasmid that will contain VP1 peptide section fusion rotein encoding gene is transformed into competence e. coli jm109 cell (method is analogous to embodiment 1).Select positive colony, extract plasmid (J.Sambrook, Cold Spring HarborLaboratory Press, Molecular cloning, 1989).
Embodiment 4, structure recombined foot-and-mouth disease virus VP1 fusion rotein encoding gene
The pMD18-T Vect plasmid and the pET28 plasmid that contain VP1 peptide section fusion rotein encoding gene (SEQ ID NO:5) with EcoRI and HindIII digestion.
The digestion reaction that contains the pMD18-T Vect plasmid DNA of VP1 peptide section fusion rotein encoding gene (SEQ ID NO:5):
Plasmid DNA 1 μ g
10 * buffer (10 * M buffer Lot:A1032, TaKaRa) 1 μ l
Restricted enzyme HindIII (10 units/μ l) 1 μ l
Restricted enzyme EcoRI (10 units/μ l) 1 μ l
With distilled water polishing to 10 μ l
Mix the back in 37 ℃ of incubation 30-120 minutes.
The digestion reaction of pET-28a (+) plasmid:
Plasmid DNA 1 μ g
10 * buffer (10 * K buffer Lot:A1018, TaKaRa) 1 μ l
Restricted enzyme EcoRI (10 units/μ l) 1 μ l
Restricted enzyme HindIII (10 units/μ l) 1 μ l
With distilled water polishing to 10 μ l
After the mixing, in 37 ℃ of incubation 30-120 minutes.
Will with the VP1 peptide section fusion rotein encoding gene dna fragmentation of EcoRI and HindIII digestion be connected with procaryotic cell expression carrier pET-28a (+) plasmid (U.S. Novagen company) of HindIII digestion through restricted enzyme EcoRI.
Coupled reaction:
PET-28a (+) plasmid (0.5 μ g/ μ l) 2 μ l
VP1 peptide section fusion rotein encoding gene dna fragmentation DNA (300ng/ μ l) 5 μ l
10 * connection buffer
(T4?Ligation?Solution?Lot：CA2901，TaKaRa) 1μl
T4DNA ligase (T4 Ligation Code No:D2011A, TaKaRa) 1 μ l
With distilled water polishing to 10 μ l
Mix rearmounted 14-16 ℃ water-bath 6-12 hour.
Reorganization pET-28a (+) plasmid that will contain foot and mouth disease virus VP1 peptide section fusion rotein encoding gene is transformed into escherichia coli expression bacterium BL21 (BL21 (DE3).
Insertion fragment in pET-28a (+) plasmid is carried out dna sequencing.Utilize the sequence of pET-28a (+) plasmid EcoRI and both sides, HindIII point of contact to merge and recombined foot-and-mouth disease virus VP1 fusion rotein encoding gene, (SEQ ID NO:8) is as follows for its sequence:
ATGGGCAGCA?GCCATCATCA?TCATCATCAC?AGCAGCGGCC?TGGTGCCGCG?CGGCAGCCAT?ATGGCTAGCA
TGACTGGTGG?ACAGCAAATG?GGTCGCGGAT?CCGAATTCAG?ATCTGGCGGT?GTATCTAACG?TACGTGGTGA
CCTGCAAGTA?CTGGCTCAAA?AAGCAGAACG?TGCTCTGCCG?GGTGGCGAAG?AAAACTACGG?TGGTGAAACT
CAGGTTCAGC?GTCGTCAGCA?CACTGACATC?TCCTTCATCC?TGGACCGTTT?CGTTAAAGTT?ACTCCGGGAT
CTGGTGGTGT?TTCTAACGTT?CGTGGTGACC?TGCAAGTACT?GGCTCAAAAA?GCTAAACGTG?CTCTGCCGGG
TGGTCCGTCT?GACGCTCGTC?ACAAACAGAA?AATCGTTGCT?CCGGCTAAAC?AGCTGCTGAA?CTTCGACCTG
GGATCTGGCG?GTGTATCTAA?CGTACGTGGT?GACCTGCAAG?TACTGGCTCA?AAAAGCAGAA?CGTGCTCTGC
CGGGTGGCGA?AGAAAACTAC?GGTGGTGAAA?CTCAGGTTCA?GCGTCGTCAG?CACACTGACA?TCTCCTTCAT
CCTGGACCGT?TTCGTTAAAG?TTACTCCGGG?ATCTGGTGGT?GTTTCTAACG?TTCGTGGTGA?CCTGCAAGTA
CTGGCTCAAA?AAGCTAAACG?TGCTCTGCCG?GGTGGTCCGT?CTGACGCTCG?TCACAAACAG?AAAATCGTTG
CTCCGGCTAA?ACAGCTGCTG?AACTTCGACC?TGGGATCTGG?CGGTGTATCT?AACGTACGTG?GTGACCTGCA
AGTACTGGCT?CAAAAAGCAG?AACGTGCTCT?GCCGGGTGGC?GAAGAAAACT?ACGGTGGTGA?AACTCAGGTT
CAGCGTCGTC?AGCACACTGA?CATCTCCTTC?ATCCTGGACC?GTTTCGTTAA?AGTTACTCCG?GGATCTGGTG
GTGTTTCTAA?CGTTCGTGGT?GACCTGCAAG?TACTGGCTCA?AAAAGCTAAA?CGTGCTCTGC?CGGGTGGTCC
GTCTGACGCT?CGTCACAAAC?AGAAAATCGT?TGCTCCGGCT?AAACAGCTGC?TGAACTTCGA?CCTGGGATCC
AAGCTTGCGG?CCGCACTCGA?GCACCACCAC?CACCACCACT?GA(SEQ?ID?NO：9)。The recombined foot-and-mouth disease virus VP1 fusion rotein that this gene (1092 bases) coding is made up of 363 amino acid residues, (SEQ ID NO:9) is as follows for the aminoacid sequence of this recombiant protein:
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFRSGGVSNVRGDLQVLAQKAERALPGGEENYGGETQVQRRQHTDI
SFILDRFVKVTPGSGGVSNVRGDLQVLAQKAKRALPGGPSDARHKQKIVAPAKQLLNFDLGSGGVSNVRGDLQVLAQKAE
RALPGGEENYGGETQVQRRQHTDISFILDRFVKVTPGSGGVSNVRGDLQVLAQKAKRALPGGPSDARHKQKIVAPAKQLL
NFDLGSGGVSNVRGDLQVLAQKAERALPGGEENYGGETQVQRRQHTDISFILDRFVKVTPGSGGVSNVRGDLQVLAQKAK
RALPGGPSDARHKQKIVAPAKQLLNFDLGSKLAAALEHHHHHH
The expression of embodiment 5, recombined foot-and-mouth disease virus VP1 amalgamation protein vaccine and split bacterium
In 100ml LB culture medium, in the 500ml conical flask, it is 0.6 that 37 ℃ of water-bath concussions are cultured to OD600 with the microbionation of single bacterium colony.It is 1mM that adding IPTG makes its final concentration, and 37 ℃ of water-baths concussions were cultivated 3 hours.With conical flask 5 minutes on ice, 4 ℃ centrifugal 5 minutes (5000 * g).Abandon supernatant, collect antibacterial, immediately use or frozen.Sampling is SDS-PAGE and is analyzed recombined foot-and-mouth disease virus VP1 Expression of Fusion Protein (accompanying drawing 1).
Antibacterial is resuspended in the cell pyrolysis liquid (the 5mM imidazoles is transferred pH to 7.9 for 20mM Tris, 0.5M NaCl), and the wet bacterium of 1 gram/3ml cell pyrolysis liquid adds DNA enzymic final concentration 2 mg/ml, adds 1mM Mg
2SO
4(10 μ l/ml) adds 50MmPMSF (0.5 μ l/ml), hatched on ice 30 fens, and 5,000rpm, centrifugal 15min abandons supernatant.
Precipitation is added in conjunction with liquid, and the precipitation that the wet bacterium of 1 gram produces adds 6ml in conjunction with liquid (20mM Tris, 0.5M NaCl, 5mM imidazoles, 6M carbamide, accent pH to 7.9).Ice bath jolts 2 hours.5,000rpm, centrifugal 30min receives supernatant, uses the supernatant upper prop.
Execute the purification of example 6, recombined foot-and-mouth disease virus VP1 amalgamation protein vaccine
(1), affinity chromatography
The dress post:
Adorn post (20mM phosphate, pH 7.2 1M NaCl) with phosphate buffer, chromatography media is Sepharose4B-Ni
2+(Pharmacia) and balance.
Distilled water with 5 bed volumes is washed post.
The 20mM metal ion solution (preparation of 100mM nickel ion solution: NiSO with 3-5 bed volume
46H
2O 13.1g, two water that heat up in a steamer are added to 1000ml) be added in the post.
Elution buffer (20mM Tris, pH 7.9,5M NaCl, 0.5M imidazoles, 4M carbamide) with 5 bed volumes is washed post.
Adsorption-buffering liquid (20mM Tris, pH7.9,0.5M NaCl, 5mM imidazoles) with 5 bed volumes is washed post.
In conjunction with:
Sample is added in the metalchelated post.
With the washing 1 buffer (20mM Tris, pH 7.9,0.5M NaCl, the 20mM imidazoles, 4M carbamide is washed post.
Eluting:
With eluting 2 buffer (20mM Tris, pH 7.9,0.5M NaCl, 1M imidazoles, 4M carbamide) eluting, collect the albumen of eluting., access the effluent of different periods with container, and carry out the SDS electrophoresis not only dropping to up to absorption value, judge purification effect, purity can reach more than 50%.
(2), desalination
With Sephadex G25 (Pharmacia) dress post.
Distilled water with 2 bed volumes is washed post.
With the 10mM PBS of 2 bed volumes, pH 7.2 washes post.
Last sample.
Use 10mM PBS, pH 7.2 eluting,
Collect the albumen of eluting, this albumen is recombined foot-and-mouth disease virus VP1 fusion rotein.
Recombined foot-and-mouth disease virus VP1 fusion rotein is preserved in lyophilizing.
The recombined foot-and-mouth disease virus VP1 fusion rotein of purification is identified that its purity reaches 95% (seeing accompanying drawing 2) with SDS-PAGE.
The immunity of embodiment 7, recombined foot-and-mouth disease virus VP1 fusion rotein
Immune programme for children:
Three immunity, 14 days at interval, initial immunity aluminum added adjuvant, and twice booster immunization do not add aluminium adjuvant.The dosage of each immunity be 50 micrograms/only/time.If recombined foot-and-mouth disease virus VP1 fusion protein immunization group, foot and mouth disease virus inactivated vaccine (Inner Mongol bio-pharmaceuticals factory group) group and PBS matched group, every group of 3 Cavia porcelluss.
The preparation aluminium adjuvant:
10%12 water aluminum potassium sulfate solutions move in 50 milliliters of conical flasks for 10 milliliters.Dropwise add 22.8 milliliters of 0.25N sodium hydroxide, concussion mixing while adding.
Incubated at room 10 minutes.Centrifugal 10 minutes of 1000g.Abandon supernatant.Add the resuspended sedimentary aluminium hydroxide of 50 ml distilled waters.
In the above-mentioned aluminium hydroxide of packing suspension to 20 centrifuge tube, 1 milliliter of every pipe.Centrifugal 10 minutes of 1000g. abandon supernatant.
200 microgram recombined foot-and-mouth disease virus VP1 fusion rotein are dissolved in the aluminium adjuvant that 400 microlitres prepare, piping and druming mixing, incubated at room 20 minutes.The lumbar injection immunity, 100 microlitres/every Cavia porcellus.
After the lumbar injection immunity the 21st day, do booster immunization.With PBS dissolving recombined foot-and-mouth disease virus VP1 fusion rotein, concentration is 50 micrograms/0.2 milliliter.Vein (penile vein) injection, 0.2 milliliter of every Cavia porcellus.0.2 milliliter of PBS of matched group Cavia porcellus injection.
After the lumbar injection immunity the 42nd day, do booster immunization for the second time.With PBS dissolving recombined foot-and-mouth disease virus VP1 fusion rotein, concentration is 50 micrograms/0.2 milliliter.Vein (penile vein) injection, 0.2 milliliter of every Cavia porcellus.0.2 milliliter of PBS of matched group Cavia porcellus injection.
After the lumbar injection immunity the 46th day.The aseptic guinea pig heart blood of getting.Be positioned in the test tube.37 ℃ 1 hour.Tilt to place for 4 ℃ and spend the night, treat that serum is fully separated out after, collect serum, aseptic subpackaged ,-20 ℃ of preservations.
The detection of embodiment 8, foot and mouth disease virus specific antibody (ELISA method)
The purification of foot and mouth disease virus carries out in Inner Mongol bio-pharmaceuticals factory.The BHK cell monolayer of learning from else's experience is cultivated the fresh O1 type hoof-and-mouth disease venom (Inner Mongol bio-pharmaceuticals factory) of amplification, 4000 rev/mins centrifugal 20 minutes, remove cell debris.Dropwise adding saturated polyethylene glycol 6000 to final concentration in supernatant is 7%, stirs 4 ℃ of static spending the night while dripping.5000 rev/mins centrifugal 30 minutes, collecting precipitation.To precipitate resuspendedly with the PB liquid of pH7.6, on the sucrose density gradient of 15%-45%, be further purified (centrifugal force is 35000 gram forces, and the time is 3 hours, and temperature is 4 ℃).The segmentation sampling is surveyed each section sample 260nm absorption value with the ultraviolet monitoring instrument, and the virion of 146s appears in the sucrose of 30%-35%.It with 5 times of PB liquid dilutions, is being contained on the PB liquid of 20% sucrose centrifugal 2 hours, and centrifugal force is 35000 gram forces, and temperature is 4 ℃, collecting precipitation.To precipitate and use PB liquid resuspended, measure the foot and mouth disease virus concentration of purification with the BCA method.
Foot-and-mouth disease virus antigen wrapper sheet with purification.Coating buffer is 0.05M, the carbonate buffer solution of pH value 9.6, and every hole 100 μ l contain foot-and-mouth disease virus antigen 1 μ g, and 4 ℃ of bags are spent the night.After washing plate 3 times with the PBS that contains 0.05% polysorbas20 (PBS-T) washing liquid, add confining liquid (PBS-T that contains 2%BSA) 200 μ l and placed 1 hour in 37 ℃ of incubators.Wash and to dilute (1: 2048) good serum to be checked 100 μ l behind the plate 3 times and add each hole, placed 1 hour in 37 ℃ of incubators.If 3 multiple holes.Wash plate 3 times, add enzyme labelled antibody (the goat-anti Cavia porcellus IgG of horseradish peroxidase-labeled) working solution, hatched 1 hour for 37 ℃, add tmb substrate solution and H
2O
2Placed 10-30 minute for 37 ℃, survey OD value (A492), with the average and the standard deviation ecbatic of each group OD value with microplate reader.
The result:
Blank: OD value: 0.005 ± 0.0005
Recombined foot-and-mouth disease virus VP1 fusion protein immunization Cavia porcellus 1:1.55 ± 0.013
Recombined foot-and-mouth disease virus VP1 fusion protein immunization Cavia porcellus 2:1.231 ± 0.021
Recombined foot-and-mouth disease virus VP1 fusion protein immunization Cavia porcellus 3:1.001 ± 0.013
3 PBS control group mice: 0.403 ± 0.014
Experimental result shows, has the antibody of high-caliber foot-and-mouth disease virus resistant in the recombined foot-and-mouth disease virus VP1 fusion protein immunization guinea pig serum.
In the neonatal rat of embodiment 9, recombined foot-and-mouth disease virus VP1 fusion protein immunization serum and the experiment
2 times of doubling dilution serum to be detected and hoof-and-mouth disease venom (100 TCID50/0.1ml) equal-volume are mixed, hatched 1 hour for 37 ℃.Through the above-mentioned mixed liquor of neonatal rat item back subcutaneous injection 0.2ml, observed each group morbidity of record, death condition continuously 3 days.
Carry out in Inner Mongol bio-pharmaceuticals factory with experiment in the neonatal rat, adopt " O " type hoof-and-mouth disease poison strain.The concentration of virus is 1 milliliter of 1000 LD50.The doubling dilution guinea pig serum.Different dilution factor guinea pig serum of 100 μ l and 100 μ l viral dilution liquid are mixed, hatched 1 hour for 37 ℃.At neonatal rat back injection said mixture.Observed 48 hours.The neonatal rat number of record survival.
Result's (as accompanying drawing 3,4, shown in 5): the serum of recombined foot-and-mouth disease virus VP1 fusion protein immunization Cavia porcellus still can be protected neonatal rat fully being diluted at 1: 2048 o'clock.
Conclusion: recombined foot-and-mouth disease virus VP1 fusion rotein stimulates animal to produce neutralizing antibody, and this neutralizing antibody can prevent the zoogenetic infection foot and mouth disease virus.
Sequence table
＜110〉Beijing DiWeiHuaYu Biological Technology Co., Ltd
＜120〉recombined foot-and-mouth disease virus VP1 amalgamation protein vaccine
<160>9
<210>1
<211>186
<212>DNA
<213>artificial
<220>
＜223〉the VP1 BT1 fusogenic peptide encoding gene at both sides band restricted enzyme point of contact
<400>1
gaattcagat?ctggcggtgt?atctaacgta?cgtggtgacc?tgcaagtact?ggctcaaaaa 60
gcagaacgtg?ctctgccggg?tggcgaagaa?aactacggtg?gtgaaactca?ggttcagcgt 120
cgtcagcaca?ctgacatctc?cttcatcctg?gaccgtttcg?ttaaagttac?tccgggatcc 180
aagctt 186
<210>2
<211>54
<212>DNA
<213>artificial
<220>
＜223〉aminoacid sequence of foot and mouth disease virus VP1BT1 fusogenic peptide
<400>2
Gly?Gly?Val?Ser?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu?Ala?Gln
1 5 10 15
Lys?Ala?Glu?Arg?Ala?Leu?Pro?Gly?Gly?Glu?Glu?Asn?Tyr?Gly?Gly
20 25 30
Glu?Thr?Gln?Val?Gln?Arg?Arg?Gln?His?Thr?Asp?Ile?Ser?Phe?Ile
35 40 45
Leu?Asp?Arg?Phe?Val?Lys?Val?Thr?Pro
50 54
<210>3
<211>167
<212>DNA
<213>artificial
<220>
＜223〉the VP1 BT2 fusogenic peptide encoding gene at both sides band restricted enzyme point of contact
<400>3
ccatggaatt?cagatctggt?ggtgtttcta?acgttcgtgg?tgacctgcaa?gtactggctc 60
aaaaagctaa?acgtgctctg?ccgggtggtc?cgtctgacgc?tcgtcacaaa?cagaaaatcg 120
ttgctccggc?taaacagctg?ctgaacttcg?acctgggatc?caagctt 167
<210>4
<211>46
<212>
<213>artificial
<220>
＜223〉aminoacid sequence of foot and mouth disease virus VP1BT2 fusogenic peptide
<400>4
Gly?Gly?Val?Ser?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu?Ala?Gln
1 5 10 15
Lys?Ala?Lys?Arg?Ala?Leu?Pro?Gly?Gly?Pro?Ser?Asp?Ala?Arg?His
20 25 30
Lys?Gln?Lys?Ile?Val?Ala?Pro?Ala?Lys?Gln?Leu?Leu?Asn?Phe?Asp
35 40 45
Leu
46
<210>5
<211>330
<212>DNA
<213>artificial
<220>
＜223〉BT1 fusogenic peptide encoding gene-BT2 fusogenic peptide encoding gene fusion gene sequence
<400>5
gaattcagat?ctggcggtgt?atctaacgta?cgtggtgacc?tgcaagtact?ggctcaaaaa 60
gcagaacgtg?ctctgccggg?tggcgaagaa?aactacggtg?gtgaaactca?ggttcagcgt 120
cgtcagcaca?ctgacatctc?cttcatcctg?gaccgtttcg?ttaaagttac?tccgggatct 180
ggtggtgttt?ctaacgttcg?tggtgacctg?caagtactgg?ctcaaaaagc?taaacgtgct 240
ctgccgggtg?gtccgtctga?cgctcgtcac?aaacagaaaa?tcgttgctcc?ggctaaacag 300
ctgctgaact?tcgacctggg?atccaagctt 330
<210>6
<211>642
<212>DNA
<213>artificial
<220>
＜223〉2 aggressiveness gene order＜400 of VP1 BT1 fusogenic peptide encoding gene-BT2 fusogenic peptide encoding gene fusion gene〉6
gaattcagat?ctggcggtgt?atctaacgta?cgtggtgacc?tgcaagtact?ggctcaaaaa 60
gcagaacgtg?ctctgccggg?tggcgaagaa?aactacggtg?gtgaaactca?ggttcagcgt 120
cgtcagcaca?ctgacatctc?cttcatcctg?gaccgtttcg?ttaaagttac?tccgggatct 180
ggtggtgttt?ctaacgttcg?tggtgacctg?caagtactgg?ctcaaaaagc?taaacgtgct 210
ctgccgggtg?gtccgtctga?cgctcgtcac?aaacagaaaa?tcgttgctcc?ggctaaacag 300
ctgctgaact?tcgacctggg?atctggcggt?gtatctaacg?tacgtggtga?cctgcaagta 360
ctggctcaaa?aagcagaacg?tgctctgccg?ggtggcgaag?aaaactacgg?tggtgaaact 420
caggttcagc?gtcgtcagca?cactgacatc?tccttcatcc?tggaccgttt?cgttaaagtt 480
actccgggat?ctggtggtgt?ttctaacgtt?cgtggtgacc?tgcaagtact?ggctcaaaaa 540
gctaaacgtg?ctctgccggg?tggtccgtct?gacgctcgtc?acaaacagaa?aatcgttgct 600
ccggctaaac?agctgctgaa?cttcgacctg?ggatccaagc?tt 642
<210>7
<211>954
<212>DNA
<213>artificial
<220>
＜223〉gene order of 3 aggressiveness of VP1 BT1 fusogenic peptide encoding gene-VP1 BT2 fusogenic peptide encoding gene fusion gene
<400>7
gaattcagat?ctggcggtgt?atctaacgta?cgtggtgacc?tgcaagtact?ggctcaaaaa 60
gcagaacgtg?ctctgccggg?tggcgaagaa?aactacggtg?gtgaaactca?ggttcagcgt 120
cgtcagcaca?ctgacatctc?cttcatcctg?gaccgtttcg?ttaaagttac?tccgggatct 180
ggtggtgttt?ctaacgttcg?tggtgacctg?caagtactgg?ctcaaaaagc?taaacgtgct 240
ctgccgggtg?gtccgtctga?cgctcgtcac?aaacagaaaa?tcgttgctcc?ggctaaacag 300
ctgctgaact?tcgacctggg?atctggcggt?gtatctaacg?tacgtggtga?cctgcaagta 360
ctggctcaaa?aagcagaacg?tgctctgccg?ggtggcgaag?aaaactacgg?tggtgaaact 420
caggttcagc?gtcgtcagca?cactgacatc?tccttcatcc?tggaccgttt?cgttaaagtt 480
actccgggat?ctggtggtgt?ttctaacgtt?cgtggtgacc?tgcaagtact?ggctcaaaaa 540
gctaaacgtg?ctctgccggg?tggtccgtct?gacgctcgtc?acaaacagaa?aatcgttgct 600
ccggctaaac?agctgctgaa?cttcgacctg?ggatctggcg?gtgtatctaa?cgtacgtggt 660
gacctgcaag?tactggctca?aaaagcagaa?cgtgctctgc?cgggtggcga?agaaaactac 720
ggtggtgaaa?ctcaggttca?gcgtcgtcag?cacactgaca?tctccttcat?cctggaccgt 780
ttcgttaaag?ttactccggg?atctggtggt?gtttctaacg?ttcgtggtga?cctgcaagta 840
ctggctcaaa?aagctaaacg?tgctctgccg?ggtggtccgt?ctgacgctcg?tcacaaacag 900
aaaatcgttg?ctccggctaa?acagctgctg?aacttcgacc?tgggatccaa?gctt 954
<210>8
<211>1092
<212>DNA
<213>artificial
<220>
＜223〉recombined foot-and-mouth disease virus VP1 fusion rotein coding gene sequence
<400>8
atgggcagca?gccatcatca?tcatcatcac?agcagcggcc?tggtgccgcg?cggcagccat 60
atggctagca?tgactggtgg?acagcaaatg?ggtcgcggat?ccgaattcag?atctggcggt 120
gtatctaacg?tacgtggtga?cctgcaagta?ctggctcaaa?aagcagaacg?tgctctgccg 180
ggtggcgaag?aaaactacgg?tggtgaaact?caggttcagc?gtcgtcagca?cactgacatc 240
tccttcatcc?tggaccgttt?cgttaaagtt?actccgggat?ctggtggtgt?ttctaacgtt 300
cgtggtgacc?tgcaagtact?ggctcaaaaa?gctaaacgtg?ctctgccggg?tggtccgtct 360
gacgctcgtc?acaaacagaa?aatcgttgct?ccggctaaac?agctgctgaa?cttcgacctg 420
ggatctggcg?gtgtatctaa?cgtacgtggt?gacctgcaag?tactggctca?aaaagcagaa 480
cgtgctctgc?cgggtggcga?agaaaactac?ggtggtgaaa?ctcaggttca?gcgtcgtcag 540
cacactgaca?tctccttcat?cctggaccgt?ttcgttaaag?ttactccggg?atctggtggt 600
gtttctaacg?ttcgtggtga?cctgcaagta?ctggctcaaa?aagctaaacg?tgctctgccg 600
ggtggtccgt?ctgacgctcg?tcacaaacag?aaaatcgttg?ctccggctaa?acagctgctg 720
aacttcgacc?tgggatctgg?cggtgtatct?aacgtacgtg?gtgacctgca?agtactggct 780
caaaaagcag?aacgtgctct?gccgggtggc?gaagaaaact?acggtggtga?aactcaggtt 840
cagcgtcgtc?agcacactga?catctccttc?atcctggacc?gtttcgttaa?agttactccg 900
ggatctggtg?gtgtttctaa?cgttcgtggt?gacctgcaag?tactggctca?aaaagctaaa 960
cgtgctctgc?cgggtggtcc?gtctgacgct?cgtcacaaac?agaaaatcgt?tgctccggct 1020
aaacagctgc?tgaacttcga?cctgggatcc?aagcttgcgg?ccgcactcga?gcaccaccac 1080
caccaccact?ga 1092
<210>9
<211>363
<212>
<213>artificial
<220>
＜223〉aminoacid sequence of recombined foot-and-mouth disease virus VP1 fusion rotein
<400>9
Met?Gly?Ser?Ser?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val
1 5 10 15
Pro?Arg?Gly?Ser?His?Met?Ala?Ser?Met?Thr?Gly?Gly?Gln?Gln?Met
20 25 30
Gly?Arg?Gly?Ser?Glu?Phe?Arg?Ser?Gly?Gly?Val?Ser?Asn?Val?Arg
35 40 45
Gly?Asp?Leu?Gln?Val?Leu?Ala?Gln?Lys?Ala?Glu?Arg?Ala?Leu?Pro
50 55 60
Gly?Gly?Glu?Glu?Asn?Tyr?Gly?Gly?Glu?Thr?Gln?Val?Gln?Arg?Arg
65 70 75
Gln?His?Thr?Asp?Ile?Ser?Phe?Ile?Leu?Asp?Arg?Phe?Val?Lys?Val
80 85 90
Thr?Pro?Gly?Ser?Gly?Gly?Val?Ser?Asn?Val?Arg?Gly?Asp?Leu?Gln
95 100 105
Val?Leu?Ala?Gln?Lys?Ala?Lys?Arg?Ala?Leu?Pro?Gly?Gly?Pro?Ser
110 115 120
Asp?Ala?Arg?His?Lys?Gln?Lys?Ile?Val?Ala?Pro?Ala?Lys?Gln?Leu
125 130 135
Leu?Asn?Phe?Asp?Leu?Gly?Ser?Gly?Gly?Val?Ser?Asn?Val?Arg?Gly
140 145 150
Asp?Leu?Gln?Val?Leu?Ala?Gln?Lys?Ala?Glu?Arg?Ala?Leu?Pro?Gly
155 160 165
Gly?Glu?Glu?Asn?Tyr?Gly?Gly?Glu?Thr?Gln?Val?Gln?Arg?Arg?Gln
170 175 180
His?Thr?Asp?Ile?Ser?Phe?Ile?Leu?Asp?Arg?Phe?Val?Lys?Val?Thr
185 190 195
Pro?Gly?Ser?Gly?Gly?Val?Ser?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val
200 205 210
Leu?Ala?Gln?Lys?Ala?Lys?Arg?Ala?Leu?Pro?Gly?Gly?Pro?Ser?Asp
215 220 225
Ala?Arg?His?Lys?Gln?Lys?Ile?Val?Ala?Pro?Ala?Lys?Gln?Leu?Leu
230 235 240
Asn?Phe?Asp?Leu?Gly?Ser?Gly?Gly?Val?Ser?Asn?Val?Arg?Gly?Asp
245 250 255
Leu?Gln?Val?Leu?Ala?Gln?Lys?Ala?Glu?Arg?Ala?Leu?Pro?Gly?Gly
260 265 270
Glu?Glu?Asn?Tyr?Gly?Gly?Glu?Thr?Gln?Val?Gln?Arg?Arg?Gln?His
275 280 285
Thr?Asp?Ile?Ser?Phe?Ile?Leu?Asp?Arg?Phe?Val?Lys?Val?Thr?Pro
290 295 300
Gly?Ser?Gly?Gly?Val?Ser?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu
305 310 315
Ala?Gln?Lys?Ala?Lys?Arg?Ala?Leu?Pro?Gly?Gly?Pro?Ser?Asp?Ala
320 325 330
Arg?His?Lys?Gln?Lys?Ile?Val?Ala?Pro?Ala?Lys?Gln?Leu?Leu?Asn
335 340 345
Phe?Asp?Leu?Gly?Ser?Lys?Leu?Ala?Ala?Ala?Leu?Glu?His?His?His
350 355 360
His?His?His
363
Claims (4)
1, a kind of recombined foot-and-mouth disease virus VP1 fusion rotein, it is that the fusion rotein encoding gene of foot and mouth disease virus VP1 peptide section and 6 polyhistidyl encoding genes are merged, at the recombination fusion protein that escherichia coli produce, VP1 peptide section fusion rotein encoding gene wherein is 3 aggressiveness genes of VP1BT1 fusogenic peptide encoding gene-VP1BT2 fusogenic peptide encoding gene fusion gene with the nucleotide sequence shown in the SEQ ID NO5.
2, according to the described recombined foot-and-mouth disease virus VP1 of claim 1 fusion rotein, it is coded or have an aminoacid sequence shown in the SEQ ID NO.9 by the nucleotide sequence shown in the SEQ ID NO.8.
3, according to the described recombined foot-and-mouth disease virus VP1 of claim 1 fusion rotein, there are 6 polyhistidyl sequences foot and mouth disease virus VP1 peptide section fusion rotein both sides wherein.
4, according to the purposes of the described recombined foot-and-mouth disease virus VP1 of claim 1 fusion rotein in the vaccine that preparation prevention animal foot and mouth disease virus infects.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101001751A CN1290579C (en) | 2003-10-15 | 2003-10-15 | Recombinant foot-and-mouth disease virus VP1 confluent protein vaccine |
| PCT/CN2004/001168 WO2005034993A1 (en) | 2003-10-15 | 2004-10-14 | Vp1 fusion protein vaccin of recombinant foot-and-mouth disease virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101001751A CN1290579C (en) | 2003-10-15 | 2003-10-15 | Recombinant foot-and-mouth disease virus VP1 confluent protein vaccine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1607004A CN1607004A (en) | 2005-04-20 |
| CN1290579C true CN1290579C (en) | 2006-12-20 |
Family
ID=34427783
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2003101001751A Expired - Fee Related CN1290579C (en) | 2003-10-15 | 2003-10-15 | Recombinant foot-and-mouth disease virus VP1 confluent protein vaccine |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1290579C (en) |
| WO (1) | WO2005034993A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212137A (en) * | 2010-04-07 | 2011-10-12 | 高地 | Genetically engineered recombinant polypeptide for cloven-hoofed animals |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106987595B (en) * | 2009-11-02 | 2021-07-06 | 宾夕法尼亚大学托管会 | Foot and Mouth Disease Virus (FMDV) consensus proteins, their coding sequences and vaccines prepared therefrom |
| GB201111183D0 (en) * | 2011-06-30 | 2011-08-17 | Animal Health Inst | Peptide |
| CN102372766B (en) * | 2011-07-13 | 2013-12-25 | 青岛红桥明勤生物科技有限公司 | O-type foot-and-mouth disease multi-epitope vaccine |
| CN104474542A (en) * | 2014-11-18 | 2015-04-01 | 天津瑞普生物技术股份有限公司 | Preparation method of bi-combined inactivated vaccine |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6107021A (en) * | 1998-06-20 | 2000-08-22 | United Biomedical, Inc. | Synthetic peptide vaccines for foot-and-mouth disease |
| FR2810888B1 (en) * | 2000-06-29 | 2004-07-30 | Merial Sas | VACCINE AGAINST FOOT AND MOUTH DISEASE |
-
2003
- 2003-10-15 CN CNB2003101001751A patent/CN1290579C/en not_active Expired - Fee Related
-
2004
- 2004-10-14 WO PCT/CN2004/001168 patent/WO2005034993A1/en active Application Filing
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212137A (en) * | 2010-04-07 | 2011-10-12 | 高地 | Genetically engineered recombinant polypeptide for cloven-hoofed animals |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2005034993A1 (en) | 2005-04-21 |
| CN1607004A (en) | 2005-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1179973C (en) | HBV core antigen particles with multiple immunogenic components attached via peptide ligands | |
| CN1073118C (en) | Hepatitis C diagnostics and vaccines | |
| CN1282656C (en) | HIV peptides from conserved regions in GAGP 17 and 924 and their application in e.g. vaccines | |
| CN1246038C (en) | Compositions and methods for systemic delivery of oral vaccines and therapeutic agents | |
| CN1354674A (en) | Synthetic peptide vaccines for foot-and-mouth disease | |
| CN1884303A (en) | SARS-CoV virus structural protein infusion protein and its high yield expression and purification and uses | |
| CN1913920A (en) | Influenza immunogen and vaccine | |
| CN1556857A (en) | Novel peptides of the respiratory syncytial virus (RSV) G protein and their use in a vaccine | |
| CN101080239A (en) | Stabilized HBc chimer particles as therapeutic vaccine for chronic hepatitis | |
| CN1609617A (en) | Composition and method for diagnosing and preventing serious acute respiratory syndrome(SARS) | |
| CN1198933C (en) | Epitopes of protein pre-M/M of dengue virus, synthetic peptides | |
| CN101031647A (en) | Process for producing erysipelothrix rhusiopathiae surface protective antigen mutant in escherichia coli | |
| CN101033468A (en) | Helicobacter pylori urease B subunit B cell antigen epitope polypeptide, identification method and application | |
| CN1458936A (en) | HIV peptides from Tat, Rev and Nef conserved regions and their application as E.G. vaccine components | |
| CN1255540C (en) | Vaccine-induced hepatitis B viral strain and uses thereof | |
| CN87105411A (en) | The T cell in S district and B cell antigen decision base before the hepatitis B virus surface antigen | |
| CN1290579C (en) | Recombinant foot-and-mouth disease virus VP1 confluent protein vaccine | |
| CN1238011A (en) | Immunogenic peptides of foot-and-mouth disease viruses | |
| CN101039955A (en) | Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with sars corona virus spike protein | |
| CN1122162A (en) | Diagnostic kit and method for the simultaneous diagnosis of hepatitis B and C | |
| CN1531548A (en) | Chimeric chains that code for proteins that induce effects directed against viruses | |
| CN1273593C (en) | Gene cloning of polyepitope antigen of hepatitis C virus and its coding sequence | |
| CN1763203A (en) | Gene recombinant human cytomegalovirus fusion protein pp150/MDBP, preparation process and application thereof | |
| CN1603346A (en) | Protein, products containing same, and their use in diagnosis, treatment and /or prevention of SARS related disease | |
| CN1218749C (en) | Recombination protein vaccine for preventing animal aphtha virus infection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: JINYU BAOLING BIOPHARMACEUTICAL CO., LTD. |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20100906 Address after: 100088, A-337, Hua Hui Road, 6, Beijing, Haidian District, May Co-patentee after: Jinyu Baoling Bio-pharmaceutical Co., Ltd. Patentee after: Diweihuayu Bio-Technology Co., Ltd., Beijing Address before: 100088, A-337, Hua Hui Road, 6, Beijing, Haidian District, May Patentee before: Diweihuayu Bio-Technology Co., Ltd., Beijing |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20061220 Termination date: 20191015 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |