CN102406929B - Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine - Google Patents
Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine Download PDFInfo
- Publication number
- CN102406929B CN102406929B CN 201110379549 CN201110379549A CN102406929B CN 102406929 B CN102406929 B CN 102406929B CN 201110379549 CN201110379549 CN 201110379549 CN 201110379549 A CN201110379549 A CN 201110379549A CN 102406929 B CN102406929 B CN 102406929B
- Authority
- CN
- China
- Prior art keywords
- vaccine
- foot
- mouth disease
- type
- asia
- 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.)
- Active
Links
- 241000710198 Foot-and-mouth disease virus Species 0.000 title claims abstract description 100
- 229960005486 vaccine Drugs 0.000 title claims abstract description 93
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 title claims abstract description 70
- 239000002671 adjuvant Substances 0.000 title claims abstract description 61
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 57
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 235000018102 proteins Nutrition 0.000 claims description 48
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 230000002265 prevention Effects 0.000 claims description 7
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- 102000039446 nucleic acids Human genes 0.000 claims description 6
- 150000007523 nucleic acids Chemical class 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 40
- 241001465754 Metazoa Species 0.000 abstract description 34
- 229920001184 polypeptide Polymers 0.000 abstract description 27
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 27
- 241000700605 Viruses Species 0.000 abstract description 19
- 238000000034 method Methods 0.000 abstract description 13
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 abstract description 12
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 7
- 230000003053 immunization Effects 0.000 abstract description 6
- 230000001681 protective effect Effects 0.000 abstract description 5
- 108010047761 Interferon-alpha Proteins 0.000 abstract description 4
- 102000006992 Interferon-alpha Human genes 0.000 abstract description 4
- 230000000890 antigenic effect Effects 0.000 abstract description 4
- 101710132601 Capsid protein Proteins 0.000 abstract 1
- 101710197658 Capsid protein VP1 Proteins 0.000 abstract 1
- 101710164702 Major outer membrane protein Proteins 0.000 abstract 1
- 101710081079 Minor spike protein H Proteins 0.000 abstract 1
- 101710136297 Protein VP2 Proteins 0.000 abstract 1
- 101710118046 RNA-directed RNA polymerase Proteins 0.000 abstract 1
- 101710108545 Viral protein 1 Proteins 0.000 abstract 1
- 230000001524 infective effect Effects 0.000 abstract 1
- 241000283690 Bos taurus Species 0.000 description 34
- 210000004027 cell Anatomy 0.000 description 30
- 239000000427 antigen Substances 0.000 description 24
- 108091007433 antigens Proteins 0.000 description 24
- 102000036639 antigens Human genes 0.000 description 24
- 231100000614 poison Toxicity 0.000 description 21
- 238000012360 testing method Methods 0.000 description 19
- 210000003719 b-lymphocyte Anatomy 0.000 description 18
- 239000003440 toxic substance Substances 0.000 description 17
- 229940031416 bivalent vaccine Drugs 0.000 description 15
- 108010050904 Interferons Proteins 0.000 description 14
- 102000014150 Interferons Human genes 0.000 description 14
- 229940079322 interferon Drugs 0.000 description 14
- 239000012634 fragment Substances 0.000 description 12
- 238000000855 fermentation Methods 0.000 description 11
- 230000004151 fermentation Effects 0.000 description 11
- 241000282898 Sus scrofa Species 0.000 description 9
- 238000013461 design Methods 0.000 description 9
- 230000036541 health Effects 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 208000030059 frontometaphyseal dysplasia 2 Diseases 0.000 description 8
- 230000036039 immunity Effects 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 239000012528 membrane Substances 0.000 description 8
- 241000700199 Cavia porcellus Species 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 230000036760 body temperature Effects 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- 238000009781 safety test method Methods 0.000 description 7
- 231100000041 toxicology testing Toxicity 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 101710116435 Outer membrane protein Proteins 0.000 description 6
- 230000036528 appetite Effects 0.000 description 6
- 235000019789 appetite Nutrition 0.000 description 6
- 244000309466 calf Species 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 230000001804 emulsifying effect Effects 0.000 description 6
- 230000003862 health status Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 230000003472 neutralizing effect Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 206010037660 Pyrexia Diseases 0.000 description 5
- 206010058874 Viraemia Diseases 0.000 description 5
- 230000000840 anti-viral effect Effects 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 230000002209 hydrophobic effect Effects 0.000 description 5
- 230000002757 inflammatory effect Effects 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 230000003902 lesion Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 230000002787 reinforcement Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 101900061472 Foot-and-mouth disease virus Capsid protein VP1 Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000002163 immunogen Effects 0.000 description 4
- 229940031551 inactivated vaccine Drugs 0.000 description 4
- 210000003292 kidney cell Anatomy 0.000 description 4
- 238000006386 neutralization reaction Methods 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000002574 poison Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000004153 renaturation Methods 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 238000002255 vaccination Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 230000007850 degeneration Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 230000008676 import Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 208000030194 mouth disease Diseases 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000010254 subcutaneous injection Methods 0.000 description 3
- 239000007929 subcutaneous injection Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 230000029812 viral genome replication Effects 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 208000031295 Animal disease Diseases 0.000 description 2
- 101800001311 Capsid protein VP2 Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 235000002937 Clintonia borealis Nutrition 0.000 description 2
- 240000002814 Clintonia borealis Species 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
- 238000002965 ELISA Methods 0.000 description 2
- 208000025309 Hair disease Diseases 0.000 description 2
- 108010033040 Histones Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 241000712079 Measles morbillivirus Species 0.000 description 2
- 208000009525 Myocarditis Diseases 0.000 description 2
- 235000007602 Opuntia linguiformis Nutrition 0.000 description 2
- 241000711975 Vesicular stomatitis virus Species 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000011149 active material Substances 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 235000013877 carbamide Nutrition 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 150000002460 imidazoles Chemical class 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000000568 immunological adjuvant Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000002560 nonimmunologic effect Effects 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000005498 polishing Methods 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011265 semifinished product Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 1
- 235000017491 Bambusa tulda Nutrition 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 206010062746 Carditis Diseases 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 241000606153 Chlamydia trachomatis Species 0.000 description 1
- 241000710777 Classical swine fever virus Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108010047524 Deoxyribonuclease HindIII Proteins 0.000 description 1
- 208000014770 Foot disease Diseases 0.000 description 1
- 101900061530 Foot-and-mouth disease virus Capsid protein VP2 Proteins 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 101000904173 Homo sapiens Progonadoliberin-1 Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 101150103227 IFN gene Proteins 0.000 description 1
- 206010021703 Indifference Diseases 0.000 description 1
- 206010024264 Lethargy Diseases 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 201000005505 Measles Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101710203389 Outer membrane porin F Proteins 0.000 description 1
- 101710160102 Outer membrane protein B Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 244000082204 Phyllostachys viridis Species 0.000 description 1
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 1
- 102100024028 Progonadoliberin-1 Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241001493546 Suina Species 0.000 description 1
- 101000996723 Sus scrofa Gonadotropin-releasing hormone receptor Proteins 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 241000710914 Totivirus Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 230000027645 antigenic variation Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000000680 avirulence Effects 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 108091006116 chimeric peptides Proteins 0.000 description 1
- 229940038705 chlamydia trachomatis Drugs 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- -1 connect etc. Proteins 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000006052 feed supplement Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009432 framing Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- XLXSAKCOAKORKW-UHFFFAOYSA-N gonadorelin Chemical compound C1CCC(C(=O)NCC(N)=O)N1C(=O)C(CCCN=C(N)N)NC(=O)C(CC(C)C)NC(=O)CNC(=O)C(NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC1=CC=C(O)C=C1 XLXSAKCOAKORKW-UHFFFAOYSA-N 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 210000000003 hoof Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000006303 immediate early viral mRNA transcription Effects 0.000 description 1
- 229960001438 immunostimulant agent Drugs 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 208000021646 inflammation of heart layer Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000007689 inspection 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
- 244000144972 livestock Species 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000007246 mechanism Effects 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
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 229940124551 recombinant vaccine Drugs 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- GOLXNESZZPUPJE-UHFFFAOYSA-N spiromesifen Chemical compound CC1=CC(C)=CC(C)=C1C(C(O1)=O)=C(OC(=O)CC(C)(C)C)C11CCCC1 GOLXNESZZPUPJE-UHFFFAOYSA-N 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940126577 synthetic vaccine Drugs 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000012646 vaccine adjuvant Substances 0.000 description 1
- 229940124931 vaccine adjuvant Drugs 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to preparation and application of a molecular adjuvant enhanced Asia 1 and A foot and mouth disease divalent vaccine. The vaccine comprises a molecular adjuvant interferon-alpha (IFN-alpha) polypeptide, a two-section T cell auxiliary antigenic epitope polypeptide, a six-section antigenic epitope polypeptide related to Asia 1 and A foot and mouth disease major outer membrane protein VP1 and VP2, and a killer T cell epitope polypeptide. The invention also relates to a preparation method and a using method for the vaccine. An animal experiment proves that the virus attack protective efficacy of the molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine is obviously higher than that of a non-molecular adjuvant enhanced vaccine. By the recommended immunizing dose of the molecular adjuvant enhanced vaccine, a tested animal can resist attack of 10,000 median infective doses (ID50) of virulent Asia 1 and A foot and mouth disease virus, and the efficacy experiment also proves that the molecular adjuvant enhanced divalent vaccine for each animal at least contains 4 median protective doses (PD50).
Description
Technical field
The invention belongs to biotechnology genetic engineering field, relate generally to a kind of prevention Asia 1 type of and molecule adjuvant coexpression and the preparation and application of A type fmd protein engineered vaccine.Particularly, utilize gene recombination technology, with different Asia 1 type and A type foot and mouth disease major outer membrane albumen VP1 and many B cell antigen epi-positions of VP2, killer T cell epitope (CTL epi-position), a plurality of T cells assist epitope (Th epi-position) to connect with molecule adjuvant polypeptide (IFN α), and be cloned into carrier, transform the host bacterium, by fermentation, purification, emulsifying process preparation obtain immune effect enhancement mode foot and mouth disease bivalence protein engineering vaccine and the application of this vaccine in the great Animal diseases foot and mouth disease of prevention of coexpression.
Background technology
Foot and mouth disease is one of global most important economic animal disease.This disease hyperinfection is by contact or air bamboo telegraph.Because the characteristic of foot and mouth disease hyperinfection, in order to forbid the intake fever aphthous and to participate in national trade, no foot and mouth disease country limits quarantine and import animal that epidemic situation country keeps strict.
In the popular country of foot and mouth disease, to responsive domestic animal inoculation efficient, safety, cost effectively vaccine be the basic demand of control foot and mouth disease.Foot and mouth disease can be controlled by animal health measure and vaccination, but owing to there is multiple cause of disease serotype, comprises multiple host and the extreme infectivity of wildlife, and the control foot and mouth disease becomes very difficult.Although modern intensive livestock culturing system does not allow foot and mouth disease to take place, foot and mouth disease is not fatal usually, and in the not preferentially control of many developing countries, this has just stayed the source of infection for the propagation of foot and mouth disease.Preferably diagnostic method especially preferably vaccine can improve the control ability of not having foot and mouth disease country and part epidemic situation country in the world significantly.Particularly, the vaccine of high thermal stability and long duration of immunity will help to control disease, reduce the dependence (David J.Paton et al, 2009) to advanced person's infrastructure for animals.The mankind consciously and attempt to control foot and mouth disease and can trace back to before the several centuries, Loeffler and Frosch studies show that foot and mouth disease virus is first mammalian virus disease pathogen (Blancou, 2002).
The totivirus inactivated vaccine is traditional foot and mouth disease control plan vaccine that is used for, and is obtaining great success aspect the control eqpidemic disease.Yet; the problem relevant with present vaccine comprises producing the requirement of the high preventer of vaccine virus; numerous serotypes and blood serum subtype that antigenic variation causes take place; and vaccine can not produce immune protective efficiency fast; if inactivation of virus is not thorough or attenuation is insufficient; will cause that foot and mouth disease breaks out increase etc., impel research worker to remove to develop more effective vaccine, for the viral infection commitment provides better protection.
Synthetic foot-and-mouth disease vaccine will be eliminated the deficiency of the each side of virus treated in the traditional vaccine preparation process, and the immunity inoculation synthetic vaccine is considered to be optimal selection.Still there are many problem and shortage in designed and many recombinant vaccines that studying at present.Still contain a large amount of in the antigenic component of many vaccine design and specific immunity is induced irrelevant composition, as immunopathogenesis compositions such as immunosuppressant and autoimmune, and these compositions cause the key factor of part or systemic adverse reactions just.
Have scholar's monoclonal antibody technique to study a plurality of antigen sites of VP1, further antigen site is from VP2 and VP3.Therefore, all merit attention (Reddy G R, 1999 of all hoof-and-mouth disease virus capsid protein coded sequence; Stram Y, 1994).VP1 albumen becomes the first-selected albumen of considerable research and structural analysis, has been found that VP1 can cause neutralizing antibody reaction in the pig body, and can protect pig and cattle to avoid the infection of FMDV.Kleid D.G. (1981) etc. has reported that biosynthesis fused polypeptide TRP LE and many immunoprotection cattle of VP1 and pig can resist the FMDV counteracting toxic substances.Corresponding FMDV VP1 polypeptide 141-160 and 200-213 regional chemistry have synthesized polypeptide and have shown that these peptides can make Cavia porcellus and rabbit produce high-level neutralizing antibody (WO8303547,1983).
Having the scholar that existing foot and mouth disease virus separated strain has been carried out the sequence contrast finds: the nucleocapsid protein coded sequence of Asia 1 type separated strain is all observed codon and is changed, but the variation of coding VP1 albumen is frequent, seldom variation (Acharya R et al, 1989 of coding VP4 albumen; Lea S et al, 1994).VP1 constitutes the major part of virus surface, and a lot of aminoacid replacement portions can not limit by structural damage, and VP4 is positioned at virion inside, owing to be subjected to structural limitations, aminoacid sequence is guarded between type.But Asia 1 type VP4 albumen lacks the 6th or the 7th residue once in a while, this point and A, O and SAT type foot and mouth disease inconsistent (Reddy G R, 1999; Stram Y, 1994).
About the VP1 epi-position, related to B-C, E-F and G-H are bad and C-is terminal.G-H goes bad and C-terminal is main antigen site (Brown F, 1995).The space conformation (Parry N, 1990) that B-C bad influence G-H is bad.The inside typical differences of VP2 mainly concentrates on B-C badly and β A2 and α Z place (Mateu MG, 1995) in E-F district, and they exist with helix A and G-sheet form, are A, O, in the SAT type foot and mouth disease virus and site.The inside typical differences of VP3 relate generally to the bad and G-H evil idea of E-F and β D district, be A, O, the antigen site of SAT type, the sequence of VP1 and VP3 one of carbon tip is heterogeneous further to be increased, and this adds mouth for the protease posttranslational protein is very significant (Ryan M D, 1997).The trypsin-resistant of antigen site shows that it is positioned at the outside of VP1.Its active relevant with the bad antigen site sequence of VP2B-C (Marquart O et al, 2000).The terminal discovery of the nitrogen of VP2 is unordered in virus crystals, shows it at virus surface, and this is the prerequisite of trypsin sensitivity.N-terminal is to have conserved sequence between the type of lysine in position 2 and 3.In SDS-PAGE result, the VP2 albumen that trypsin treatment is crossed is different from untreated VP2 (Strohmaier K, 1982).Zhang Z W et al (2010) uses bioinformatics and molecular biology associated methods, 6 VP1-VP4 epitopes of screening and affirmation Asia 1 type foot and mouth disease virus VP1 albumen, and VP1:
1TTTTGESADPVT
12, VP1:
17NYGGETQTARRLH
29, VP1:
194TTQDRRKQEIIAPEKQTL
211, VP2:
40EDAVSGPNTSG
50VP3:
26YGKVSNPPRTSFPG
39, VP4:
30YQNSMDTQLGDN
41
Still need at present a kind of bivalence based on synthetic peptide or the above FMDV vaccine of bivalence, be better than classical vaccine aspect effectiveness and the range.The purpose of this patent just provides a kind of synthetic peptide bivalence FMD vaccine and satisfies this demand.The method that is used for realizing this goal is as follows: (1) determines the effective procedure of high affine epi-position; (2) by chemical synthesis process, the composite coding foot-and-mouth disease vaccine is as the B cell that enlarges and whole polypeptide-nucleic acid sequences of t cell epitope; (3) be combined to strengthen the immunogenicity of B cell by B cell target epi-position and potent t helper cell epi-position, regulate the auxiliary epi-position of T simultaneously and adapt to the populational variation immunoreation.(4) make desirable space conformation feature stable by introducing the circulation constraint.
Mix the Th epi-position and can excite T cell assosting effect effectively, can the B cell epitope a little less than immunogenicity own be combined and form peptide based immunogens effectively.The mixing Th/B cell epitope chimeric peptide of sticking heart design can bring out that Th replys and express the antibody response that thereupon produces in the diversified group member of diversified MHC monoploid heredity at majority.This mixing Th can derive by effective virus or bacterial origin immunogen specific sequence, comprises Measles virus F albumen, hepatitis B virus surface antigen, chlamydia trachomatis outer membrane protein etc.Many known Th have shown and have strengthened weak immunogen (U.S.pat.No.5759551) effectively.Potent Th epi-position magnitude range is 15-50 amino acid residue (U.S.pat.No.5759551), has common architectural feature usually, may comprise special significant sequence.For example, a common characteristic is exactly the both sexes spirals, for having the α spiral of hydrophobic amino acid residue, controls an aspect of spiral, has the residue of electric charge and polarity, other aspects (Cease etal, 1987) of control spiral.Epi-position generally includes other main amino acid templates, for example adds Gly or charged residue after two or three hydrophobic amino acid residues, and this template definition is the Rothbard sequence.These epi-positions are also followed 1,4,5,8 rules, and namely hydrophobic amino acid residues is behind the residue charged residue of positive charge 4,5 and 8.Because these all structures all are by common hydrophobic, electrically charged and polar amino acid is formed, so can there be (Partidos et al, 1991) in the single Th epi-position simultaneously in each structural portion.If just great majority, rather than all t cell epitope mixing, so above the description has a kind of being fit at least.These features may be included the design in desirable artificial T h site in, comprise SSAL Th epi-position, and it can provide degeneracy in the site relevant with the different MHC molecules of identification and the site that remains unchanged.Meister et al has listed mutable site and preferably can be used for the aminoacid of MHC binding motif (Meister et al, 1995).For example, the Th degeneracy epi-position that WO95/11998 describes is imitated Measles virus F albumen exactly and is mixed epi-position (Partidos et al, 1991) as SSAL Th1.LHRH target peptide series system is used in the design of SSAL Th1 epi-position, and as the measles epi-position, SSAL Th1 meets Rothbard sequence and 1,4,5,8 rules.
Peptide based immunogens is more flexible than albumen usually, trends towards the structure that keeps optional preferred.Therefore, by introducing loop limit, use it for the stabilized peptide immunogen.A correct evil ideaization peptide antigen can imitate and keep the space conception of target epi-position, thereby excites activated antibody (Moore et al, 1992) at real molecular locus place.This point is passed through to add the cysteine residue at amino terminal and one of carbon tip, and with two cysteine residue oxidation evil ideaizations, realizes (WO083/03547) at FMDV VP1 advantage immunity evil ideaization district representative peptide.
I type interferon, α and interferon-or α/interferon-be known to have antiviral activity, is the first road barrier (vilcek and sen, 1996) that host cell is resisted virus infection.Virus infection cell induction expression-secretion α/interferon-, interferon is combined with the specific receptor of flanking cell again, activates the event that stimulated gene (ISGs) activates by a series of IFN α/β that cause, and starts antiviral state.The product of these genes influences the different phase of virus replication circulation, and different virus is to different ISG product sensitivities.The ISGs example has feature widely, comprises the protein kinase (PKR) that double-stranded RNA relies on, a kind of synzyme/RNA enzyme L and Mx.
FMD copies IFN α and β extremely sensitive, comprises in the supernatant suppressing pig and the Bov IFN (chinsangaran et al, 1999) that foot and mouth disease virus is copied.In order more directly to study the influence of the foot and mouth disease virus replication of IFN α and β, use the consensus sequence of specificity separately at IFN α and β as primer, amplify IFN α and the β of different animals from porcine kidney cell (PK) and embryo's bovine kidney cells (EBK).Each IFN clone checks order, and with escherichia coli expression (chinsangaram etal, 2001).Detect the biologic activity of interferon behind the serial dilution, pig is copied the FMDV from the allied species cell with β with cattle IFN α has similar inhibitory action, for example porcine kidney cell line (IBRS2) and bovine kidney cells (EBK).The cattle of expressing or pig IFN α and β are also to vesicular stomatitis virus (VSV), and the encephalomyo-carditis disease has similar antiviral activity with swine fever virus.And, find that pig and cattle IFN α and β also copy inhibited (chinsangaram et al, 2001) to the FMDV in other zooblasts.
Because FMDV IFN α and β are handled in cell culture is responsive, I type interferon can be used for anti-FMDV reagent in the body.The effect of I type interferon is rapid, considers the foot-and-mouth disease virus antigen multiformity, uses interferon to provide protection for all FMDV serotypes and hypotype.
Summary of the invention
The present invention is according to major outer membrane albumen VP1 and the VP2 aminoacid sequence of at present main Asia 1 type and the domestic epidemic strain of A type foot and mouth disease and Southeast Asia epidemic strain, utilize associated biomolecule informatics software to the hydrophilic of Asia 1 type and A type foot and mouth disease virus major outer membrane albumen, antigenicity, plasticity, the secondary structure of surface accessibility and Garnier-Robson is analyzed, again according to the conservative design oligonucleotides fragment of epi-position B cell and CTL epi-position position and aminoacid sequence, introduce effective versatility Th cell epitope (PADRE) and molecule adjuvant interferon I type simultaneously, with designed Asia 1 type and A type foot and mouth disease virus B cell epitope, the CTL epi-position, Th cell epitope and interferon I type polypeptide series connection back coexpression in escherichia coli, by fermentation, purification, technologies such as emulsifying obtain to have desirable immunogenic reinforced Asia 1 type and A type foot and mouth disease bivalence protein engineering vaccine.The vaccine that utilizes the present invention to prepare can effectively prevent the infection of Asia 1 type and A type foot and mouth disease.
One of purpose of the present invention has been to provide a kind of new coexpression molecule adjuvant reinforced protein engineering vaccine polypeptide and the vaccine combination thereof that can be used for prevention popular Asia 1 type and A type foot and mouth disease virus strain infection.
Two of purpose of the present invention has been to provide structure and the preparation method of described coexpression protein engineering vaccine.
Three of purpose of the present invention has been to provide the engineering strain that can express described foot and mouth disease polyepitope vaccines.
Four of purpose of the present invention has been to provide the preparation method of described foot and mouth disease bivalence protein engineering vaccine.
Five of purpose of the present invention has been to provide the purposes of described protein engineering vaccine in prevention Asia 1 type and A type foot and mouth disease.
In first aspect, the invention provides a kind of coexpression molecule adjuvant reinforced protein engineering bivalent vaccine polypeptide and compositions thereof for prevention Asia 1 type and the infection of A type hoof-and-mouth disease strain.It contains 6 main Asia 1 types and A type foot and mouth disease epidemic isolates AF/72, LC/96, AKT/03, Asia-1-JSL, KZ/03, major outer membrane albumen VP1, VP2B cell epitopes such as HeNzk/06 strain, 1 killer T cell epi-position, 2 T cell skeptophylaxis epitopes, and I type interferon molecule adjuvant.Described protein engineering vaccine comprises a plurality of B cells, killer T cell, Thelper epitope and molecule adjuvant be connected in series together formed albumen or polypeptide or pharmaceutically acceptable salt and coexpression epitope and the needed carrier of molecule adjuvant polypeptide protein.Carrier also can comprise the sequence of independent each epitope of coding, perhaps independent coding molecule adjuvant IFN α or IFN β, perhaps IFN α/β binding sequence.Series connection can be undertaken by genetic engineering method, in the protein engineering vaccine except containing major outer membrane albumen VP1, VP2B cell antigen epitope, outside killer T cell epitope and Thelper cell antigen epitope and the I type interferon molecule, also comprise non-immunologic active material.Described non-immunologic active material is the coupling part of each polypeptide, does not have the immunogenicity of epitope, does not also have any adjuvanticity, mainly contains purification tag, joint peptide, chemical modification part, N end signal peptide and C end polyadenylic acid etc.Described pharmaceutically acceptable salt refers to avirulence, stimulation and allergy, is applicable to the salt of human or animal tissues.Inert matter and pharmaceutically acceptable salt are well known to those skilled in the art.
Preferably be selected from Asia 1 type and A type foot and mouth disease virus major outer membrane albumen VP1 and VP2B cell, killer T cell and Thelper cell epitope respectively at the major outer membrane proteantigen epitope sequences described in the polyepitope vaccines polypeptide of first aspect present invention, molecule adjuvant is α and interferon-or the α/interferon-polypeptide with antiviral activity.Appropriate IFN gene is I type IFN α/β, from any animal to the foot and mouth disease sensitivity, comprises any artiodactyl such as pig, cattle and sheep.
Preferred in addition, the described polyepitope vaccines amino acid sequence of polypeptide of first aspect present invention is as follows:
(1) A type foot and mouth disease virus VP1B cell antigen epitope 135aa～173aa (ABe1):
KYSAASGRTRGDLGQLAARVAAQLPASFNFGAVRATTIHELL
(2) A type foot and mouth disease virus VP1B cell antigen epitope 199aa～213aa (ABe2):
DRHKQKIIAPAKQLL；
(3) A type foot and mouth disease virus VP1 B cell antigen epi-position 40aa～62aa (ABe3):
VKIGNTSPTHVIDLMQTHQHGLV
(4) Asia 1 type foot and mouth disease virus VP1 B cell antigen epi-position 133aa～158aa (A1Be1):
KTTYGEESSRRGDLAALARRVNNRL
(5) Asia 1 type foot and mouth disease virus VP1 B cell antigen epi-position 197aa～211aa (A1Be2):
DRRKQEIIAPEKQTL
(6) Asia 1 type foot and mouth disease virus VP2 B cell antigen epi-position 65aa～81aa (A1Be3):
HLFDWTPNLAFGHCHYL；
(7) killer T cell epi-position (Tce) aminoacid sequence is: KYKEAKEWL;
(8) the auxiliary epitope of T cell is general T helper epi-position: Thel:ISITEIKGVIV HRIETILF and The2:KFVAAWTL KAAA.
(9) molecule adjuvant is cattle alpha interferon, and sequence is (maIFN α):
RQLRRVSPSSCLQDRNDFAFPQEALGGSQLQKAQAISVLHEVTQHTFQLFS
TEGSAAAWDESLLDKLRTALDQQLIDLQACLRQEEGLPGAPLLKEDSSLAV
RKYFHRLTLYLQEKRHSPCAWEVVRAQVMRAFSSSTNLQESFRRKD
Preferred in addition, comprise 1-3 Asia 1 type foot and mouth disease outer membrane protein advantage B cell epitope at the described polypeptide fragment of first aspect, 1-3 A type foot and mouth disease outer membrane protein advantage B cell epitope, and killer T cell epi-position.Further be a killer T cell epi-position Tce7,1 Asia 1 type foot and mouth disease outer membrane protein V P2B cell epitope, 2 Asia 1 type foot and mouth disease outer membrane protein V P1B cell epitopes and 3 A type foot and mouth disease outer membrane protein V P1B cell epitopes.Contain 2 Thelper cell antigen epitopes and molecule adjuvant cattle IFN α in addition.
Above-mentioned different outer membrane protein B cell and the permutation and combination method of killer T cell epitope and the auxiliary epitope of T cell have the hundreds of thousands kind in theory, consider based on vaccine challenge mechanism and the biochemical characteristic of vaccine own, be preferably as follows order:
Preferably, polypeptide fragment combination order is: maIFN α-The1-The2-ABe1-ABe2-ABe3-A1Be1-A1Be2-A1Be3-Tce, The1-The2-Be1-Be2-Be3-Be-Tc4-Be5-Be6-Tce7-maIFN α, maIFN α-Be1-Be2-Be3-Be-Tc4-Be5-Be6-Tce7-The1-The2, Be1-Be2-Be3-Be-Tc4-Be5-Be6-Tce7-The1-The2-maIFN α, more preferably, polypeptide fragment combination order is: maIFN α-The1-The2-ABe1-ABe2-ABe3-A1 Be1-A1Be2-A1Be3-Tce.
In second aspect, the invention provides a kind of nucleic acid molecule, its coding first aspect present invention described molecule adjuvant reinforced Asia 1 type and A type foot and mouth disease bivalence protein engineering vaccine polypeptide.Nucleotide can be rna form among the present invention, dna form, by the synthetic former epi-position tandem sequence of multi-resistance of synthetic mode and molecule adjuvant sequence, be operatively connected rear clone through genetic engineering then and go into carrier, be transformed into escherichia coli, obtain reinforced bivalence protein engineering vaccine polypeptide behind screening, fermentation, the purification.Can carry out conventional molecular biology operation to this nucleic acid in the present invention, as: PCR, digestion with restriction enzyme, connect etc., nucleic acid design 5 ' end and 3 ' end all add restriction enzyme site.Nucleotide sequence among preferred the present invention is as follows:
GGA TCC CGA CAA CTG AGG AGG GTC TCC CCT TCC TCC TGC CTG CAG GACAGA AAT GAC TTT GCA TTC CCC CAG GAG GCG TTG GGT GGC AGC CAG TTG CAAAAG GCT CAA GCC ATC TCT GTA CTC CAC GAG GTG ACC CAA CAC ACC TTC CAGCTT TTC AGC ACA GAG GGC TCG GCC GCT GCG TGG GAT GAG AGC CTC CTG GACAAG CTC CGC ACT GCA CTG GAT CAG CAG CTC ATT GAC CTG CAA GCC TGT CTGAGG CAG GAG GAG GGG CTG CCA GGG GCT CCC CTG CTC AAG GAG GAC TCC AGCCTG GCT GTG AGG AAA TAC TTC CAC AGA CTC ACT CTC TAT CTG CAA GAG AAGAGA CAC AGC CCT TGT GCC TGG GAG GTT GTC AGA GCA CAA GTC ATG AGA GCCTTC TCT TCC TCA ACA AAC TTG CAG GAG AGT TTC AGG AGA AAG GAC GGT GAT ATCCCA GGT TGC AAG ATC AGC ATC ACC GAG ATC AAA GGT GTG ATT GTT CAT CGC ATTGAA ACC ATT CTG TTT GGT GGT GCA AAG TTC GTT GCA GCA TGG ACC CTG AAAGCA GCA GCA GGT GGT CCA TCT TGT AAG TAC TCT GCT GCA TCT GGT CGT ACT CGTGGT GAT CTT GGT CAG CTG GCA GCA CGT GTT GCA GCA CAG CTG CCA GCA TCTTTC AAC TTT GGT GCA GTT CGT GCA ACT ACC ATT CAT GAA CTG TTG GGT TCTGGT GAT CGT CAC AAA CAG AAG ATC ATT GCA CCA GCG AAA CAG CTG TTG GGT TCTGGT GTG AAG ATT GGT AAC ACC TCT CCA ACC CAT GTG ATT GAT CTG ATG CAA ACCCAT CAG CAT GGT CTG GTT TGT GCA GCA TAC AAG ACT ACC TAT GGT GAA GAA AGCTCT CGT CGT GGT GAT CTT GCA GCA CTT GCA CGT CGT GTG AAC AAC CGT CTG CCA GGT TCT GGT GAT CGT CGC AAA CAG GAA ATC ATT GCA CCA GAG AAA CAG ACCTTG GGT TCT GGT CAT CTG TTT GAT TGG ACT CCG AAC CTT GCA TTT GGT CAT TGCCAC TAT CTG TGT GCA GCA TAC AAG TAC AAA GAA GCG AAA GAA TGG CTG AAGCTT
In the third aspect, the invention provides a kind of carrier, it is connected except containing the described coding foot and mouth disease of second aspect present invention bivalence albumen Seedling nucleic acid molecule, also containing with this nucleotide sequence is exercisable, at the required expression control element of procaryotic cell expression (transcribe and translate).The most basic expression control element comprises promoter, transcription terminator, enhancer, selected marker etc., and these controlling elements are known in the art.In a preferred embodiment, described expression vector is coli expression carrier.
In fourth aspect, the invention provides a kind of host cell, it contains the described carrier of third aspect present invention.Host cell is through transforming or transfection contains the gene order of encoding proteins of the present invention, then have good heredity and expression stability after testing after, can be used for fermentation expression and produce required Asia 1 type and A type foot and mouth disease bivalence protein engineering vaccine polypeptide.
Aspect the 5th, the invention provides the preparation method of the reinforced Asia of a kind of molecule adjuvant 1 type and A type foot and mouth disease bivalence protein engineering vaccine, it may further comprise the steps: engineering bacterium fermentation expressing protein engineered vaccine polypeptide, through thick purification and polishing purification technology and follow-up emulsifying process, obtain needed polypeptide.The method that wherein relates to includes, but are not limited to the washing of bacterial cell disruption, inclusion body, centrifugal, degeneration, affinity chromatograph, hydrophobic chromatography, anion-exchange chromatography, reversed phase chromatography, renaturation, emulsifying etc.The preparation method that relates among the present invention is well known to those skilled in the art.
Aspect the 6th, the invention provides a kind of bivalence protein engineering vaccine for prevention Asia 1 type and the infection of A type foot and mouth disease virus, it comprises the described polypeptide of first aspect present invention and pharmaceutically acceptable carrier.Described foot-and-mouth disease vaccine can prevent infecting of Asia 1 type and A type foot and mouth disease epidemic isolates.Pharmaceutically acceptable carrier of the present invention is immunostimulant or immunological adjuvant, and preferred immunological adjuvant is the import white-oil adjuvant.
Aspect the 7th, the invention provides the application of the 6th aspect described coexpression molecule adjuvant reinforced Asia 1 type and A type fmd protein engineered vaccine.Vaccine is the effective dose intramuscular injection necessarily, Intradermal or subcutaneous injection or intranasal vaccination injection animal, and the IFN that can express q.s provides antiviral activity, watches for animals to avoid the attack of foot and mouth disease virulent strain.Consider size and body weight and the IFN type of immune animal, immunizing dose can change according to immune animal.Determine that by testing effective dose is 1 part 4ml (seeing embodiment six).In addition; in embodiments of the invention; by vaccine being carried out target animals counteracting toxic substances contrast test, laboratory safety test etc.; show that bivalence protein engineering vaccine of the present invention is safe (seeing embodiment four); watch for animals and avoid Asia 1 type and the infection of A type foot and mouth disease virus, result of the test shows that also I type interferon has immune strengthening effect (embodiment five, six) to bivalence protein engineering vaccine.
In addition, it is pointed out that on the basis of the application's contextual disclosure that the aspect that other have a substantive distinguishing features of the present invention is apparent to the ordinary skill people of this area.In addition, the present invention has also used open source literature, and their full text content is all included this paper in and carried out reference.
Description of drawings
Following accompanying drawing is used for explanation specific embodiments of the present invention, and is not used in the scope of the invention that restriction is defined by claims.
Fig. 1 is Asia 1 type and A type fmd protein engineering bivalent vaccine peptide coding expression carrier pPRSETB-FMD (the As1/A)-IFN α sketch map that contains molecule adjuvant IFN α.
Fig. 2 is not for containing Asia 1 type and A type fmd protein engineering bivalent vaccine peptide coding expression carrier pPRSETB-FMD (As1/A) sketch map of molecule adjuvant IFN α.
Fig. 3 identifies electrophoretogram for digestion with restriction enzyme.1:DL2000 DNA Marker; 2: Unreinforced foot and mouth disease bivalent vaccine plasmid double digestion; 3: the reinforced foot and mouth disease bivalent vaccine of molecule adjuvant IFN α plasmid double digestion.
Fig. 4 is vaccine-induced expression SDS-PAGE electrophoretogram.1: Unreinforced bivalence Seedling abduction delivering (arrow is destination protein); 2: the contrast bacterium of abduction delivering not; 3: molecular weight of albumen Marker; 4: the reinforced bivalent vaccine abduction delivering of molecule adjuvant IFN α (the arrow indication is destination protein).
Fig. 5 is fermented sample western blot hybridization figure.1: dye molecular weight of albumen Marker in advance.2: Unreinforced bivalence Seedling; 3: blank; 4: the reinforced bivalence Seedling of molecule adjuvant IFN α.
Fig. 6 is purifying protein Western Blot trace result.1: dye Marker in advance; 2: Unreinforced bivalence Seedling protein purification result; 3: the reinforced bivalence Seedling of molecule adjuvant IFN α protein purification result; 4: blank.
Fig. 7 is blank group inflammatory lesion score value figure
Fig. 8 is the reinforced vaccine inflammatory lesion of molecule adjuvant IFN α score value figure
Fig. 9 is the reinforced vaccine inflammatory lesion of non-molecule adjuvant score value figure
Figure 10 is blank and the reinforced vaccine virus mass formed by blood stasis of molecule adjuvant IFN α testing result: 1:DL2000 DNAMarker; The 2:AF/72 positive control; The 3:LC/96 positive control; 4: negative control; 5:AF-CK1-1D; 6:AF-CK1-2D; 7:AF-CK1-3D; 8:AF-CK1-4D; 9:AF-CK2-1D; 10:AF-CK2-2D; 11:AF-CK2-3D; 12:AF-CK2-4D; 13:LC-CK1-1D; 14:LC-CK1-2D; 15:LC-CK1-3D; 16:LC-CK1-4D; 17:LC-CK2-1D; 18:LC-CK2-2D; 19:LC-CK2-3D; 20:LC-CK2-4D; 21:LC-IFN α 2-1D; 22:LC-IFN α 2-2D; 23:LC-IFN α 2-3D; 24:LC-IFN α 2-4D;
Figure 11 is the reinforced vaccine virus mass formed by blood stasis of non-molecule adjuvant testing result.1:DL2000 DNA Marker; The 2:AF/72 positive control; The 3:LC/96 positive control; 4: negative control: 5:AF-FMD2-1D; 6:AF-FMD2-1D; 7:AF-FMD2-1D; 8:AF-FMD2-1D; : 9:AF-FMD3-1D; 10:AF-FMD3-1D; 11:AF-FMD3-1D; 12:AF-FMD3-1D; 13:LC-FMD1-1D; 14:LC-FMD1-2D; 15:LC-FMD1-3D; 16:LC-FMD1-4D; 17:LC-FMD2-1D; 18:LC-FMD2-2D; 19:LC-FMD2-3D; 20:LC-FMD2-4D; 21:LC-FMD4-1D; 22:LC-FMD4-2D; 23:LC-FMD4-3D; 24:LC-FMD4-4D;
Figure 12 is that the reinforced vaccine antibody titre of molecule adjuvant IFN α changes
Figure 13 is that the reinforced vaccine antibody titre of non-molecule adjuvant changes
The specific embodiment
It only is exemplary description that concrete test method described in the embodiment is described, and is used for elaborating the present invention, but does not constitute limitation of the scope of the invention, knows according to many those skilled in the art of being changed to of the present invention.
Embodiment a part adjuvant is strengthened the mentality of designing of Asia 1 type and A type foot and mouth disease bivalence protein engineering vaccine peptide coding albumen
Comprehensive domestic and Southeast Asia a plurality of Asia 1 type and A type foot and mouth disease virus epidemic strain AF/72, LC/96, AKT/03, Asia-1-JSL, KZ/03, the genome sequence of strains such as HeNzk/06, antigenic structure, epidemiological study progress have been carried out optimal design to recombined foot-and-mouth disease bivalence protein engineering vaccine.The present invention utilizes bioinformatics software to its major outer membrane albumen VP1, the hydrophilic that VP2 carries out, antigenicity, plasticity, the secondary structure of surface accessibility and Garnier-Robson is analyzed, predict on the basis of possible B cell antigen epi-position and killer T cell epi-position, similarity according to epi-position position and aminoacid sequence, analyze the total and specific antigen epi-position of two kinds of a plurality of strains of serotype, and with reference to the sequence information among the GenBank, epitope to prediction is compared, further analyze the conservative of epitope in the different virus strain, thereby determine each 3 sections of the B cell dominant antigen epitope polypeptides relevant with A type foot and mouth disease virus with Asia 1 type, 1 section of killer T cell dominant antigen epi-position; 2 sections of the auxiliary epitope dominant antigen epi-positions of T cell are with the framing structure of all epi-position series connection back formation bivalent vaccines, simultaneously at the terminal molecule adjuvant that adds of skeleton nitrogen.The population structure of this vaccine is:
Molecular Adjuvant(IFNα)-T Helper Epitope 1-T Helper Epitope 2-B Cell Epitope 1(A)-B Cell Epitope 2(A)-B Cell Epitope 3(A)-B Cell Epitope1(Asia1)-B Cell Epitope 2(Asia1)-B Cell Epitope 3(Asia1)-Tc Cell Epitope
The structure of embodiment two coli expression carriers and expression strain
It is synthetic that the peptide coding nucleotide that designs among the embodiment one is served extra large handsome biotech company, the nucleotide fragments two ends have been designed BamH I (5 ' end) and HindIII (3 ' end) restriction enzyme site respectively, the multi-epitope nucleosides in series acid fragment that will not add the molecule adjuvant simultaneously also designs also EcoRI (5 ' end) and HindIII (3 ' end) restriction enzyme site respectively for two sections, it is synthetic to serve extra large handsome biotech company simultaneously, in contrast.Be cloned into respectively on the pMD18T carrier after synthetic these 2 fragments, sequencing confirms to insert genetic fragment consistent with implementation sequence (seeing sequence table).With recombiant plasmid difference called after pMD18T-The/A-Asia1B/Tc and pMD18T-IFN α-The/A-Asia1B/Tc.With corresponding restricted enzyme two kinds of plasmids being carried out enzyme action handles, coli expression carrier is selected the pRSETB plasmid of Invitrogen company for use, also use identical restricted enzyme to handle, enzyme action condition: 10 μ l reaction systems, add 2 μ l plasmids in the system, restricted enzyme is 5 active units (New England biolabs), adds 10 * buffer, 1 μ l, the deionized water polishing, 37 ℃ of enzyme action 1.5 hours.Enzyme action finishes the back and adds 1 μ l 200mM EDTA cessation reaction.In 1% agarose gel electrophoresis, electrophoresis 30 minutes.Under the uviol lamp The/A-Asia1B/Tc fragment and 1089bp IFN α-The/A-Asia1B/Tc fragment of 2.9kb pRSETB plasmid, 625bp are downcut, reclaim the test kit description according to Qiagen company gel and carry out the glue recovery.According to carrier: the ratio of fragment 1: 2-3 with the multi-epitope nucleotide fragments separately and the expression vector mixing, reaction system 15 μ l, connected by the T4DNA ligase, 16 ℃ of connections are spent the night, obtain recombiant plasmid called after pRSETB-FMD (As1/A) and pRSETB-FMD (As1/A)-IFN α respectively, (see Fig. 1, Fig. 2), transformed competence colibacillus e. coli bl21 (DE3) pLysS.
Transform: pRSETB-FMD (As1/A) and pRSETB-FMD (As1/A)-IFN α are put thawing on ice, add 2 μ l link reactant liquor, mixing again, ice-water bath 30 minutes, 42 ℃ 30 seconds, put back to ice bath then rapidly 1.5 minutes, add 1ml LB culture fluid, 37 ℃, leave standstill and cultivated 1 hour, the 4000g low-temperature centrifugation is abandoned supernatant 10 seconds, with the resuspended thalline of 200 μ lLB culture medium; Bacterium liquid is evenly coated on the LB agar culture plate that contains 100 μ g/mL ampicillin, be inverted in 37 ℃ of calorstats and cultivated 12-16 hour, form until the clone.
Identify: the monoclonal on the picking flat board is to the LB culture medium, 37 ℃, the 180rpm concussion was cultivated 12 hours, extract plasmid, use restriction endonuclease BamHI and HindIII and EcoRI and HindIII to carry out double digestion respectively, can cut out the clone of corresponding foot-and-mouth disease vaccine gene size fragment, molecule adjuvant IFN α is reinforced to be 1089bp, Unreinforced is 625bp, can tentatively be defined as the positive colony (see figure 3); Positive colony carries out determined dna sequence and further verifies its correctness (seeing sequence table).
Abduction delivering.Be about to the positive colony incubated overnight, cultivate 3 hour after by switching in 1: 100 morning next day, adds 0.5mMIPTG, continues to cultivate 3 hours the preparation sample; Conventional SDS-PAGE testing goal protein expression situation---in 44.6KD and 27.8KD molecular weight place (see figure 4), see specific band and be correct clone; Get correct clone, amplification culture after SDS-PAGE confirms to express correctly, uses conventional Western-blot further to confirm its expression accuracy (see figure 5); Behind above-mentioned structure and evaluation program, the positive colony of selecting can be carried out the foundation of original species word bank as engineering bacteria, strain called after pRSETB-FMD (As1/A)/BL21 (DE3, Plys) and pRSETB-FMD (As1/A)-IFN α/BL21 (DE3, Plys).
Fermentation, purification and the emulsifying of embodiment three engineering bacterias
The production strain is got in fermentation, is inoculated in the 2ml LB fluid medium (to contain 100 μ g/ml ampicillin), and 37 ℃, 12 hours activated spawn of 180rpm shaken cultivation.Shake bottle with 1: 100 inoculum concentration access again, 37 ℃ of shaken cultivation can be inoculated into fermentation tank in 10% ratio to OD600=3.Fermentation is semisynthetic medium with culture medium, with the distilled water preparation, does not wherein contain any antibiotic.Proofread and correct dissolved oxygen and pH value electrode, open tank body and stir, revolution is 300rpm, the online sterilization of tank body, and when treating that a jar interior culture-liquid temp is down to 37.0 ℃, demarcation pH and dissolved oxygen (OD) zero point.Fermentation temperature is 37.0 ± 0.1 ℃, dissolved oxygen control is about 20%, pH control is 7.0, thalline OD600=1.0～1.2 o'clock flow feeding 500ml are cultivated in the inoculation back, 1 hour adding IPTG (final concentration is 0.5mM) abduction delivering after the feed supplement, continuous induction after fermentation in 6 hours finishes, and SDS-PAGE calibrating expression is done in sampling.
Purification is with the thalline of collecting, and is ultrasonic with carrying out behind occlusion body washing liquid I (1%Triton X-100, the 20mMTris-cl PH8.0) suspendible, 2000W ultrasonic degradation 1 hour.4 ℃, the centrifugal collection occlusion body of 12000rpm, and with occlusion body washing liquid II (1%DOC, 4M carbamide, 20mMTris-cl PH8.0) suspendible twice ultrasonic washing occlusion body, the secondary low-temperature centrifugation is collected occlusion body.The occlusion body precipitation is used 8M carbamide, 0.3% β-ME, and 20mM Tris-cl (pH=8.00) mixing, stirring at room 4 hours, 8000rpm low-temperature centrifugation 30min discards precipitation.Denatured protein dilution in 1: 100, renaturation solution adds the 0.3M arginine with Tris (PH8.0) buffer system, and 4 ℃ were stirred renaturation 24 hours.The renaturation solution 20mM phosphate buffer of pH=8.0,0.5M sodium chloride, the 20mM imidazoles, affinity column on the balance is with the 20mM phosphate buffer of pH=8.0,0.5M sodium chloride, 0.5M imidazoles eluting; Reuse 1.5M (NH
4)
2The 10mM Na of SO4,100mM EDTA, pH=8.5
2HPO
4Hydrophobic chromatography post on the balance, reequilibrate is with the 10mM Na of pH=8.5
2HPO
4Eluting namely gets recombined foot-and-mouth disease vaccine semi-finished product stock solution.Do whether SDS-PAGE and Western blot marking calibrating purified product is target protein (Fig. 6).
Emulsifying is diluted to 200 μ g/ml with the semi-finished product of purification with the PBS that sterilizes.Get import white mineral oil adjuvant DUOPRIME (pharmaceutical grade) through 121 ℃, sterilized 15 minutes, standby.In oil phase: the ratio preparation of water=50: 50, earlier oil phase is added in the emulsion tank, start blender and slowly stir with the speed of 80-100r/min, slowly add water, add back restir 2min, with 5500r/min high speed circulating emulsion 9min, make the single-phase vaccine of Water-In-Oil then.
Embodiment quadruple histone engineering bivalent vaccine safety testing
Material
Vaccine: reinforced Asia 1 type of recombinating and A type fmd protein engineering bivalent vaccine, lot number is 20100512,20100515,20100518.
8 of experimental animal (1) body weight 350-450g Cavia porcelluss, 17 of 18-22g white mice are available from Qingdao City medicine inspecting institute.(2) detect more than 6 monthly ages that serum is the foot-and-mouth disease antibody feminine gender 8 of healthy cattle through the neonatal rat neutralization test.
Method
Vaccine is to the safety of white mice
The subcutaneous injection white mice, every injection 0.5ml, 15 white mice are injected in 5 of the every batch of vaccine injections altogether, set 2 negative controls simultaneously, observe the health status of observation white mice continuously 10 days.
Vaccine is to the safety of Cavia porcellus
Cavia porcellus use in subcutaneous injection test, injects 1ml for every, and 2 of the every batch of vaccine injections are injected 6 altogether.Simultaneously respectively set 2 negative controls, observed continuously 10 days, the health status of observation Cavia porcellus.
Vaccine is to the safety of calf
Detect 8 of the cattle that serum is the foot-and-mouth disease antibody feminine gender through the neonatal rat neutralization test.Wherein 14 are used for the recombiant protein engineering bivalent vaccine that the injection center provides, 2 cattle of every batch of vaccine injection, every incidence intramuscular injection 4ml vaccine.Set up 2 of negative controls simultaneously, every injection white-oil adjuvant 2ml, clinical observation 10 days.
The result
Vaccine is to the safety testing of white mice
Result such as table 1, second day body temperature of 1 animal subject of 20100515 immune group slightly raises, and recovers normal on the 3rd day, appetite and health status are no abnormal, and is consistent with matched group, do not have dead the generation, as seen recombined foot-and-mouth disease protein engineering bivalent vaccine is safe to white mice, sees Table 1.
Table 1 vaccine is to the safety testing result of white mice
| Group | Size of animal | Body temperature | Appetite | Health status | Dead quantity |
| 20150512 | 5 | 1 fervescence, other are normal. | Normally | Be in a good state of |
0 |
| 20100515 | 5 | Normally | Normally | Be in a good state of |
0 |
| 02100518 | 5 | Normally | Normally | Be in a good state of |
0 |
| |
2 | Normally | Normally | Be in a good state of |
0 |
Vaccine is to the safety testing of Cavia porcellus
Result such as table 2, immune animal body temperature, appetite and health status and matched group indifference do not have dead.
Vaccine is to the safety testing of calf
Result such as table 3, in 10 days whole test observation periods, all immunity totally 66 the monthly age calf, body temperature and appetite are all normal, any clinical abnormal phenomena do not occur, after the off-test, 6 calves are strong living all.2 cattle of contrast adjuvant group are also without any untoward reaction.This explanation, recombined foot-and-mouth disease protein engineering bivalent vaccine is safe to calf.
Table 2 vaccine is to the safety testing result of Cavia porcellus
| Group | Size of animal | Body temperature | Appetite | Health status | Dead quantity |
| 20150512 | 2 | Normally | Normally | Be in a good state of |
0 |
| 20100515 | 2 | Normally | Normally | Be in a good state of |
0 |
| 02100518 | 2 | Normally | Normally | Be in a good state of |
0 |
| |
2 | Normally | Normally | Be in a good state of |
0 |
Table 3 vaccine is to the safety testing result of calf
| Group | Size of animal | Body temperature | Appetite | Unusual condition | Dead quantity |
| 20150512 | 2 | Normally | Normally | Do not have | 0 |
| 20100515 | 2 | Normally | Normally | Do not have | 0 |
| 02100518 | 2 | Normally | Normally | Do not have | 0 |
| |
2 | Normally | Normally | Do not have | 0 |
The reinforced bivalence FMD vaccine of embodiment five molecule adjuvants and the contrast of Unreinforced bivalent vaccine immune efficacy
Discovery I type interferon such as Marvin J have the obvious suppression effect to the foot and mouth disease virus replication; and utilize I type interferon and foot and mouth disease inactivated vaccine combined immunization target animals pig; show under the situation of participation of I type interferon; compared with the control; IFN/FMD vaccine group animal counteracting toxic substances A24 strain; obtain full guard (100%), immune animal neutralizing antibody level height, and clinical pathological changes and inflammatory reaction (U.S.pat.No.0171314A1) do not take place.The report of the evaluation of effect of the foot and mouth disease bivalence of Shang Weiyou protein engineering Seedling molecule adjuvant, the present invention estimates the immunologic enhancement of foot and mouth disease bivalence protein engineering vaccine as molecule adjuvant with regard to I type interferon.
Material
The reinforced fmd protein engineered vaccine of vaccine adjuvant, the reinforced bivalence protein engineering of non-adjuvant Seedling.
Experimental animal is selected kind, the source is consistent, detects 6 monthly ages, 24 of the healthy cattle that serum is the foot-and-mouth disease antibody feminine gender through the neonatal rat neutralization test.
Method
20 cattle are equally divided into two organize greatly, every big group is further divided into two groups, adjuvant reinforcement group and non-adjuvant reinforcement group, and 5 every group, and matched group, 2 every group.After the vaccination 21 days, together with 4 not immune contrast cattle, every cow tongue face divided 2 intradermal injection 10000ID
50The strong malicious AF/72 of homology foot and mouth disease and LC/96.Every treated animal isolated rearing, the clinical side reaction of observing animal during the whole test is as fever, lethargy etc.Continue behind the counteracting toxic substances to observe 14 days, comprise body temperature, inflammatory lesion, serology viremia and FMDV antibody titer production.PCR method is adopted in the viremia monitoring.AF/72 primer P1:gtgcgtcacgatgacaactt, P2:caggagttgctttgcaggtg (PCR product 402bp); LC/96 primer P1:accacggttgagaactacgg, P2:cggtcttgtgtggtgtcaag (PCR product 557bp).PCR overall reaction system is 25 μ l, and response parameter is: 95 ℃ of pre-degeneration 5min, and 94 ℃ of degeneration 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 40s, totally 35 circulations, last 72 ℃ are extended 10min.1% agarose gel on the PCR product.The antibody titer detection uses foot and mouth disease Asia I type antibody liquid to block the ELISA detection kit mutually and foot and mouth disease A type antibody liquid is blocked the ELISA detection kit mutually.
The result:
4 animals of blank group were all developed into high-caliber viremia in first day behind counteracting toxic substances, cotton swab shows as low virus titer, has continued 2-3 days always, and occurred inflammatory reaction behind counteracting toxic substances in second day.All animals showed as serious clinical onset symptom in 3 days behind counteracting toxic substances.4 days animal serums transform behind counteracting toxic substances.Can detect viremia (Figure 10) in continuous 4 days, behind the counteracting toxic substances the 3rd day, all animals of blank group showed as serious clinical onset symptom.AF/72 has an animal to have a fever for three days on end, only has a fever one day for other one.LC/96 blank group, two cattle is all had a fever, and the persistent period is respectively 2 days and 3 days, and a head of cattle 6 days dead (see figure 7)s behind counteracting toxic substances are arranged.Cut open inspection and tissue slice analysis and find the myocarditis that it exists typical FMDV to cause, the cause of death is myocardial necrosis.
5 cattle of AF/72 virus group IFN alpha molecule adjuvant reinforcement group all be protected (5/5 protection); 4 cattle of LC/96 virus group IFN alpha molecule adjuvant reinforcement group be protected (4/5 protection); and the independent immune group of FMD albumen; 5 cattle performance clinical onset symptoms; AF/72 virus group 2 hair diseases (3/5 protection), LC/96 organizes 3 hair diseases (2/5 protection) (seeing Fig. 8, Fig. 9).All morbidity immune animals are not all developed into viremia (seeing Figure 10, Figure 11).In immunity back 7 days, detect the reaction of FMDV specificity neutralizing antibody, whole neutralizing antibodies detection positives in 7 days are organized in IFN α reinforcement, non-immune strengthening group part animal's antibody tests positive, back 14 days antibody horizontals of immunity obviously raise, and continued to increase to immunity back 21 days, and adjuvant booster immunization group neutralizing antibody level is apparently higher than non-booster immunization group always.This and counteracting toxic substances result and inflammatory lesion reaction result coincide mutually (seeing Figure 12, Figure 13).
The dosage of embodiment sixfold histone engineering bivalent vaccine is determined and potency test
Material
Vaccine reorganization adjuvant reinforced Asia 1 type and A type fmd protein engineering bivalent vaccine are provided by the biological research and development centre of Bao Maide, and lot number is 20100512,20100515,20100518.
Experimental animal is selected kind, the source is consistent, detects more than 6 monthly ages that serum is the foot-and-mouth disease antibody feminine gender 94 of healthy cattle through the neonatal rat neutralization test.
Method
94 cattle are equally divided into two to be organized greatly, AF/72 counteracting toxic substances group and LC/96 counteracting toxic substances group, every big group is further divided into 4 groups, different lot number groups and matched group (2), each lot number group is by above healthy susceptible cattle of 6 monthly ages of 15 of 1 using dosages, 1/3 dosage, 1/9 dose inoculation, and 5 cattle of every dosage musculi colli inoculation inoculate back 28 days, together with 2 not immune contrast cattle, every cow tongue face divides 2 intradermal injection 10000ID
50Foot and mouth disease by force the poison, observed simultaneously 10 days, the contrast every of cattle must the foot and mouth disease typical cytopathic appear 3 above hoofs, immune cattle any foot and mouth disease pathological changes must not occur and be judged to protection except lingual surface.Quantity according to each dosage morbidity cattle is calculated PD
50, determine effective immunizing dose simultaneously.
The result
The results are shown in Table 4, behind 28 days counteracting toxic substances of vaccination, the matched group laboratory animal is all morbidities successively since second day.1/9 group began morbidity at the 3rd day, every group all has 3～4 cattle morbidities.1/3 dosage, every group all has 1～2 cattle morbidity, does not occur dead.As seen, recombined foot-and-mouth disease bivalence protein engineering vaccine 1/3 dosage of producing can make immune animal avoid the attack of strong poison when using.According to the using dosage of recommending, every part vaccine contains 4 PD at least
50Dosage.
Table 4 counteracting toxic substances protection number and PD
50Value
The Ministry of Agriculture has formulated the unified quality standard of foot and mouth disease inactivated vaccine according to the OIE proposed standard.Cattle Asia 1 type and A type foot and mouth disease inactivated vaccine can be resisted 10000ID
50The attack of the strong poison an of/homotype can be thought qualified products.From this result of the test as can be seen, the reinforced foot and mouth disease bivalence of recombinant molecule adjuvant protein engineering vaccine can be resisted 10000ID
50The attack of the strong poison an of/AF/72 and LC/96; except the former multiple dose counteracting toxic substances of 20100518AF/72 counteracting toxic substances group protective rate is 80%; all the other 5 groups of immune cattles all reach 100% (25/25 protection) to the protective rate of strong virus attack, and 100% (4/4) morbidity of adjuvant matched group.
Claims (6)
1. one kind is used for the reinforced bivalence fmd protein of the molecule adjuvant engineered vaccine albumen that prevention Asia 1 type and A type foot and mouth disease epidemic isolates infect, and the aminoacid sequence of described albumen is SEQ ID No.2.
2. nucleic acid molecules, nucleotide sequence is SEQ ID No.1, its coding claim 1 reinforced bivalence protein engineering of described molecule adjuvant vaccine protein aminoacid sequence.
3. carrier, it contains the described nucleic acid molecules of claim 2.
4. host cell, it contains the described carrier of claim 3.
5. the reinforced bivalence protein engineering of a molecule adjuvant vaccine that is used for A type and Asia 1 type foot and mouth disease immunoprotection comprises the described vaccine protein of claim 1 and pharmaceutically acceptable carrier.
6. the application of the described albumen of claim 1 in the reinforced bivalence fmd protein of preparation molecule adjuvant engineered vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110379549 CN102406929B (en) | 2011-11-25 | 2011-11-25 | Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110379549 CN102406929B (en) | 2011-11-25 | 2011-11-25 | Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102406929A CN102406929A (en) | 2012-04-11 |
| CN102406929B true CN102406929B (en) | 2013-09-04 |
Family
ID=45909464
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110379549 Active CN102406929B (en) | 2011-11-25 | 2011-11-25 | Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102406929B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104892734B (en) * | 2015-04-30 | 2019-09-13 | 中国科学院过程工程研究所 | The method of hydrophobic interaction chromatography purification aftosa inactivation of viruses antigen |
| US10172933B2 (en) * | 2016-10-31 | 2019-01-08 | The United States Of America, As Represented By The Secretary Of Agriculture | Mosaic vaccines for serotype a foot-and-mouth disease virus |
| CN108117583B (en) * | 2018-01-03 | 2020-06-09 | 中牧实业股份有限公司 | Immunopotentiator and application thereof |
| CN110606875A (en) * | 2019-09-20 | 2019-12-24 | 中国农业科学院兰州兽医研究所 | Intramolecular adjuvant for preparing foot-and-mouth disease vaccine, application thereof and foot-and-mouth disease vaccine |
-
2011
- 2011-11-25 CN CN 201110379549 patent/CN102406929B/en active Active
Non-Patent Citations (5)
| Title |
|---|
| 冉波等.O型口蹄疫病毒VP1表位重组蛋白疫苗的构建，表达和转化.《免疫学杂志》.2006,第22卷(第3期),全文. * |
| 国外口蹄疫流行现状分析及防治策略;高飞等;《北京农学院学报》;20061231;第21卷(第1期);全文 * |
| 王新国等.用转基因植物生产基因工程疫苗.《中国生物工程杂志》.1998,第18卷(第1期),全文. |
| 用转基因植物生产基因工程疫苗;王新国等;《中国生物工程杂志》;19981231;第18卷(第1期);全文 * |
| 高飞等.国外口蹄疫流行现状分析及防治策略.《北京农学院学报》.2006,第21卷(第1期),全文. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102406929A (en) | 2012-04-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111560074B (en) | Novel coronavirus S protein single-region subunit nano vaccine based on helicobacter pylori ferritin | |
| CN103172749B (en) | Preparation of African swine fever protein engineering vaccine | |
| CN113512096B (en) | Weever rhabdovirus recombinant G2 protein and application thereof | |
| CN109182380B (en) | Preparation method and application of baculovirus-expressed classical swine fever E2 subunit vaccine | |
| CN113337478B (en) | Cat parvovirus strain and application thereof | |
| CN113943375B (en) | Recombinant fusion protein derived from HR region of novel coronavirus S2 protein and application thereof | |
| CN113293145B (en) | New measles virus live vector corona vaccine | |
| CN102406929B (en) | Co-expressed molecular adjuvant enhanced divalent foot and mouth disease protein engineering vaccine | |
| CN104628865B (en) | A kind of pseudo- mad dog epitope polypeptide recombinant vaccine | |
| CN115960262A (en) | Canine parvovirus-like particle for displaying CDV epitope as well as construction method and application thereof | |
| CN106279431B (en) | A kind of pig circular ring virus subunit inactivated vaccine | |
| CN103539839A (en) | Neutralizing epitope peptide of enterovirus 71-type VP2 antigen and application thereof | |
| CN102058881B (en) | Gene recombinant vaccine for preventing enterovirus 71 infection and preparation method thereof | |
| CN109021115A (en) | A kind of pig circular ring virus trivalent subunit vaccine | |
| CN104804099B (en) | A kind of reinforced polyepitope vaccines of recombination H9N2 subtype avian influenza | |
| CN110437318A (en) | The antigen and its preparation method and application of immune horse production anti-rabies virus antibody | |
| CN114807059B (en) | PCV3 virus strain and PCV3 genetic engineering subunit vaccine | |
| CN103992408A (en) | Preparation of blue ear disease protein engineering vaccine | |
| CN105267988A (en) | Novel echinococosis granulosis vaccine with CPG DNA (deoxyribonucleic acid) immune adjuvants | |
| CN105267989A (en) | Novel water-soluble echinococosis granulosis vaccine with immune adjuvants | |
| CN109053896A (en) | A kind of pig circular ring virus bivalent genetic engineering vaccine | |
| CN102372766B (en) | O-type foot-and-mouth disease multi-epitope vaccine | |
| CN102218139A (en) | Medicament for treating and/or preventing viral infection | |
| CN106397602A (en) | A molecular adjuvant enhanced type protein engineered vaccine for chicken Marek's disease | |
| CN109517044B (en) | Porcine epidemic diarrhea virus genetic engineering antigen and antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: QINGDAO HONGQIAO MINGQIN BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: QINGDAO BIOMADE TECHNOLOGY CO., LTD. Effective date: 20130725 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20130725 Address after: 266061 Shandong City, Laoshan Province, Zhuzhou Road, No. 153, high-level personnel entrepreneurship center, building, room 1, unit B702 Applicant after: QINGDAO HONGQIAO MINGQIN BIOLOGICAL TECHNOLOGY Co.,Ltd. Address before: 266061 Shandong City, Laoshan Province, Zhuzhou Road, No. 153, high-level personnel entrepreneurship center, building, room 1, unit B702 Applicant before: QINGDAO BAOMAIDE BIOLOGICAL MEDICAL TECHNOLOGY Co.,Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 266061 Shandong City, Laoshan Province, Zhuzhou Road, No. 153, high-level personnel entrepreneurship center, building, room 1, unit B702 Patentee after: QINGDAO MINGQIN BIOTECHNOLOGY CO.,LTD. Address before: 266061 Shandong City, Laoshan Province, Zhuzhou Road, No. 153, high-level personnel entrepreneurship center, building, room 1, unit B702 Patentee before: QINGDAO HONGQIAO MINGQIN BIOLOGICAL TECHNOLOGY Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230926 Address after: Room 402, 4th Floor, Chuanyan Building, No. A7 Jiaomen Road, Fengtai District, Beijing, 100068 Patentee after: Beijing Resources and Source Biotechnology Co.,Ltd. Address before: Room B702, East Unit, Building 1, High Level Talent Entrepreneurship Center, No. 153 Zhuzhou Road, Laoshan District, Qingdao City, Shandong Province, 266061 Patentee before: QINGDAO MINGQIN BIOTECHNOLOGY CO.,LTD. |