CN105175516A - Peptide vaccine for animals and preparing thereof - Google Patents
Peptide vaccine for animals and preparing thereof Download PDFInfo
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- CN105175516A CN105175516A CN201510603614.3A CN201510603614A CN105175516A CN 105175516 A CN105175516 A CN 105175516A CN 201510603614 A CN201510603614 A CN 201510603614A CN 105175516 A CN105175516 A CN 105175516A
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- polypeptide
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- mouth disease
- antigen
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- 229940023041 peptide vaccine Drugs 0.000 title claims abstract description 27
- 241001465754 Metazoa Species 0.000 title abstract description 12
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 86
- 229920001184 polypeptide Polymers 0.000 claims abstract description 77
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 77
- 239000000427 antigen Substances 0.000 claims abstract description 50
- 102000036639 antigens Human genes 0.000 claims abstract description 50
- 108091007433 antigens Proteins 0.000 claims abstract description 50
- 241000710198 Foot-and-mouth disease virus Species 0.000 claims abstract description 43
- 229960005486 vaccine Drugs 0.000 claims abstract description 38
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 claims abstract description 33
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 19
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 17
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims description 20
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 16
- 229920003180 amino resin Polymers 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 235000001014 amino acid Nutrition 0.000 claims description 12
- 150000001413 amino acids Chemical class 0.000 claims description 12
- 239000002671 adjuvant Substances 0.000 claims description 11
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 239000008346 aqueous phase Substances 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 9
- -1 9-fluorenylmethyloxycarbonyl Chemical group 0.000 claims description 7
- VIHYIVKEECZGOU-UHFFFAOYSA-N N-acetylimidazole Chemical compound CC(=O)N1C=CN=C1 VIHYIVKEECZGOU-UHFFFAOYSA-N 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000006482 condensation reaction Methods 0.000 claims description 5
- 238000005336 cracking Methods 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 125000000539 amino acid group Chemical group 0.000 claims description 4
- 230000000903 blocking effect Effects 0.000 claims description 4
- 238000009295 crossflow filtration Methods 0.000 claims description 4
- 230000002101 lytic effect Effects 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 claims description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000010511 deprotection reaction Methods 0.000 claims description 3
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- 238000012856 packing Methods 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
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- 239000003814 drug Substances 0.000 claims description 2
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- 230000003647 oxidation Effects 0.000 claims description 2
- 238000007254 oxidation reaction Methods 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 238000000197 pyrolysis Methods 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 239000008215 water for injection Substances 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 3
- 230000001900 immune effect Effects 0.000 abstract description 3
- 210000003719 b-lymphocyte Anatomy 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
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- 108010033276 Peptide Fragments Proteins 0.000 abstract 1
- 210000001744 T-lymphocyte Anatomy 0.000 abstract 1
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 241000282898 Sus scrofa Species 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
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- 239000012071 phase Substances 0.000 description 5
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- 239000011347 resin Substances 0.000 description 5
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- 101710197658 Capsid protein VP1 Proteins 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 238000010255 intramuscular injection Methods 0.000 description 4
- 239000007927 intramuscular injection Substances 0.000 description 4
- 244000144972 livestock Species 0.000 description 4
- 230000003472 neutralizing effect Effects 0.000 description 4
- 238000010532 solid phase synthesis reaction Methods 0.000 description 4
- 238000010254 subcutaneous injection Methods 0.000 description 4
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- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
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- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 2
- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical group COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 description 2
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- 210000000003 hoof Anatomy 0.000 description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 229940031551 inactivated vaccine Drugs 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 150000003053 piperidines Chemical class 0.000 description 2
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- 238000007363 ring formation reaction Methods 0.000 description 2
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- UAJTZZNRJCKXJN-UHFFFAOYSA-M sodium;2-dodecoxy-2-oxoethanesulfonate Chemical compound [Na+].CCCCCCCCCCCCOC(=O)CS([O-])(=O)=O UAJTZZNRJCKXJN-UHFFFAOYSA-M 0.000 description 2
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- 241001494479 Pecora Species 0.000 description 1
- 241000709664 Picornaviridae Species 0.000 description 1
- 101710136297 Protein VP2 Proteins 0.000 description 1
- 101800003658 Protein VP4 Proteins 0.000 description 1
- 101150097559 Slc26a1 gene Proteins 0.000 description 1
- 101710106388 Structural protein VP1 Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
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- 238000010353 genetic engineering Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000009775 high-speed stirring Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 101710121537 mRNA (guanine-N(7))-methyltransferase Proteins 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
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- 239000002574 poison Substances 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 230000003612 virological effect Effects 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- 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
- 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
-
- 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
- C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Peptides Or Proteins (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
Abstract
The invention provides synthetic peptide vaccine for a foot-and-mouth disease, and particularly relates to polypeptide for the synthetic peptide vaccine for the foot-and-mouth disease, the vaccine with the polypeptide, a preparing method of the polypeptide and a preparing method of the vaccine. The polypeptide has the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. According to the polypeptide, the vaccine and the preparing methods, by determining the sequences of recent foot-and-mouth disease prevalent strains in China, the variation conditions of main antigen sites of the strains are researched in cooperation with the sequences of foot-and-mouth disease vaccine strains; meanwhile, analytical predication of the antigen sites is carried out in cooperation with computer assistance, and possible antigen site peptide fragments are chemically synthesized. Candidate polypeptide antigens are screened through a large quantity of animal experiments, the foot-and-mouth disease virus antigen sites are optimized according to the screening result, T-cell epitope and B-cell epitope are effectively combined, and the immune effect of the polypeptide antigens is improved. The synthetic peptide vaccine for the foot-and-mouth disease can effectively respond to foot-and-mouth disease virus antigen variations, is good in biological safety and beneficial for large-scale synthesis and has good application prospects.
Description
Technical field
The present invention relates to one group of polypeptide for aftosa synthetic peptide vaccine, and contain vaccine and their preparation method of these polypeptide, be specifically related to a kind of polypeptide for aftosa synthetic peptide vaccine, and contain vaccine and their preparation method of this polypeptide.
Background technology
Foot and mouth disease (footandmouthdisease is called for short FMD) is that one betides acute, high degree in contact, infectious fever artiodactylous, worldwide extensively distributes.Because its infectivity is high, propagate rapidly, the livestocks such as infected pigs, ox, sheep cause cub dead, and adult animals throughput sharply declines, the development of serious harm livestock industry and production and supply that is carnivorous and livestock product.The market circulation of animal and animal's products and international trade can be made simultaneously to be subject to blocking greatly and restriction, to cause huge financial loss to popular countries and regions Animal husbandry production.Foot and mouth disease is infected by foot and mouth disease virus (FMDV) and causes, and Hostis picornavirus, has the feature such as polytypism, volatility.At present, there is the foot and mouth disease virus of 7 kinds of serotypes in the known whole world: A, O, C, Sat1 (South Africa I type), Sat2 (South Africa II type), Sat3 (South Africa type III) and AsiaI (Asia I type).And often kind of principal mode divides some hypotypes, the existing kind more than 70 of the hypotype found at present.Serotypes A, O, C and AsiaI type are the most common, and wherein the mutation of serotypes A virus is maximum, has more than 30 kinds of hypotypes.Result of study shows: the capsid protein of foot and mouth disease virus is made up of four kinds of Structural protein VP1, VP2, VP3 and VP4 (LoganD etc., 1993), often kind each 60.VP1-VP3 forms capsomere, be positioned at the outside of capsid protein, and VP4 is positioned at the inside of virion.VP1 is main protective antigen, has now found that O type foot and mouth disease has 3 main antigen sites to be positioned on VP1, and wherein 133-160 and 200-213 position constitutes most important on VP1 and the protective antigen site the most easily made a variation.
The current foot and mouth disease at China's Major Epidemic is O type and AsiaI type foot and mouth disease.Antigenicity between foot and mouth disease virus is various is different, each other can not Immunogenicity.And in same serotype, the degree of antigenic difference is also very large, to such an extent as to the aftosa vaccine effectively can resisting a kind of hypotype may not have protectiveness for the another kind of hypotype in same serotype.In addition, the antigenicity of foot and mouth disease strain is also constantly changing, and As time goes on, the efficacy wanes of original vaccine even disappears, and brings very large difficulty therefore to the preventing and controlling of foot and mouth disease.
At present, China carries out mandatory immunity to foot and mouth disease, and vaccine immunity is the Main Means preventing and treating foot and mouth disease.And the aftosa vaccine mainly viral inactivation vaccine of the existing use of China, there is the problems such as biological safety difference, side reaction are large, unstable product quality.A lot of country stops using inactivated vaccine in the world at present, also forbids the national import livestock product from using inactivated vaccine.In the research of foot and mouth disease new generation vaccine, genetic engineering subunit vaccine, foot and mouth disease virus carrier bacterin, foot-and-mouth disease gene engineering is successively had to modify the research report of vaccine, but it all also exists problems in immune effect, biological safety, have impact on the use of these new generation vaccines.In addition, often effect is poor for the current epidemic isolates morphed for these vaccines, can not effectively watch for animals.
Summary of the invention
Therefore, the object of the present invention is to provide a kind of polypeptide for aftosa synthetic peptide vaccine, and the vaccine containing this polypeptide, especially for the polypeptide of aftosa synthetic peptide vaccine, and the vaccine containing this polypeptide.
Another object of the present invention is to provide the preparation method of a kind of aforementioned polypeptides and above-mentioned vaccine.
For achieving the above object, present invention employs following technical scheme:
For a polypeptide for aftosa synthetic peptide vaccine, wherein said polypeptide has the aminoacid sequence shown in SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4, SEQIDNO.5 and SEQIDNO.6.
The sulfydryl of two halfcystines in above-mentioned aminoacid sequence can be joined together to form disulfide linkage through oxidation.
Formation can be reacted covalently bound between the head and the tail amino-acid residue of above-mentioned aminoacid sequence.Specifically, carboxyl and the amino or react between carboxyl and hydroxyl of the head and the tail amino-acid residue of above-mentioned aminoacid sequence is formed covalently bound.
A kind of aftosa synthetic peptide vaccine, comprising one or more aforementioned polypeptides.Such as: the polypeptide with the aminoacid sequence shown in SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4, SEQIDNO.5 and SEQIDNO.6.Above-mentioned vaccine can also comprise adjuvant.
Present invention also offers the preparation method of aforementioned polypeptides, comprising following steps:
(1) take aminoresin as starting raw material, with the amino acid of 9-fluorenylmethyloxycarbonyl protection for monomer, connect amino acid according to described aminoacid sequence successively condensation, after often walking condensation reaction, close unreacted aminoterminal with acetyl imidazole;
(2) lytic reagent cracking polypeptide is added after synthesis;
(3) ether collection, precipitated polypeptide is used;
(4) aseptically process is carried out after ultrafiltration purification polypeptide.
In above-mentioned preparation method, the volume components of described lytic reagent is than being trifluoroacetic acid (TFA): tri isopropyl silane (TIS): phenol: H
2o=85: 8: 6: 1; Described pyrolysis time is 1-4 hour.
In above-mentioned preparation method, described step (1) specifically comprises the following steps:
(a) deprotection reaction: aminoresin is placed in N-Methyl pyrrolidone (NMP) solution that volume percent is the hexahydropyridine of 15-30%, reacts the 9-fluorenylmethyloxycarbonyl blocking group removed for 25-40 minute on aminoresin under 20-28 DEG C of condition;
B () washs: nitrogen dries up, N-Methyl pyrrolidone washing aminoresin;
(c) condensation reaction: add 1-hydroxyl azimidobenzene (HOBT), amino acid that dicyclohexylcarbodiimide (DCC) and 9-fluorenylmethyloxycarbonyl are protected reacts 0.5-2.5 hour under 20-28 DEG C of condition;
D () washs: nitrogen dries up, N-Methyl pyrrolidone washing aminoresin;
(e) capping: add N-Methyl pyrrolidone (NMP) solution that percent weight in volume is the acetyl imidazole of 1.5-4%, react 20-40 minute under 20-28 DEG C of condition.
In above-mentioned preparation method, described step (4) specifically comprises the following steps:
A () uses tangential flow filtration film to wrap in the obtained polypeptide of ultrafiltration under 20-28 DEG C of condition, removing small molecular weight impurity;
B () uses 0.2 micron of degerming preservation of online filter.
Present invention also offers the preparation method of above-mentioned vaccine, comprising following steps:
(1) with water for injection, aforementioned polypeptides is diluted to 50 μ g/ml and obtains antigen aqueous phase;
(2) adjuvant is for subsequent use through sterilizing in 121 DEG C, 30 minutes;
(3) under 20-28 DEG C of condition, according to the volume ratio of antigen aqueous phase and adjuvant 1: 1, first adjuvant is added in emulsion tank, 90-150 rev/min of low rate mixing 1.5-3 minute, slowly add aqueous phase, add rear stirring 20-30 minute, then high speed 8000-10000 rev/min is stirred 15-30 minute, leave standstill 5 minutes, after packing and get final product.
Invention further provides aforementioned polypeptides, the purposes treated and/or prevented in the medicine of foot and mouth disease prepared by vaccine.
The present invention is by the domestic foot and mouth disease sequencing of epidemic isolates combined mouth fever aphthous vaccine strain sequence recently, the variation situation of research foot and mouth disease major antigenic sites, amino acid sites for main variation adds up the frequency of its variation, carry out the analyses and prediction in foot-and-mouth disease antigen site in conjunction with area of computer aided simultaneously, chemosynthesis is carried out to possible antigen site peptide section, namely for the variation frequency of easy variant sites according to statistics, use different amino acid in these sites, obtain containing the multiple candidate polypeptide antigen of current likely variant sites.And then by a large amount of animal experiments, these candidate polypeptide antigens are screened, obtain the immune response that can cause animal, and immune response level is high, can be good at the polypeptide antigen watched for animals from the attack of foot and mouth disease epidemic isolates.The present invention is optimized foot and mouth disease virus antigen site according to screening experiment result, and efficient combination t cell epitope and B cell epi-position, enhance the immune effect of polypeptide antigen.This aftosa synthetic peptide vaccine can successfully manage current foot and mouth disease virus antigenic variation, there is not biological safety, be easy to extensive synthesis, have a good application prospect.
Embodiment
The solid phase synthesis of embodiment 1 aftosa synthetic peptide antigen
Polypeptide antigen of the present invention can use full-automatic polypeptide synthetic instrument, utilizes Merrifield solid-phase synthesis to prepare, and wherein have employed the amino acid that 9-fluorenylmethyloxycarbonyl (Fmoc) is modified, and solid phase carrier is RinkAmideMBHA resin.Production process generally includes the solid phase synthesis of polypeptide antigen, the cracking of polypeptide, antigen purification and degerming preservation.
The solid phase synthesis of 1.1 polypeptide antigens
1.1.1 synthesis material prepares
The sequence of synthetic polypeptide antigen is respectively as shown in SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4, SEQIDNO.5 and SEQIDNO.6.
The sequence of foundation antigen and synthesis scale are the amino acid that 1mmol prepares suitable Fmoc modification, add in corresponding amino acid bottle.Weigh RinkAmideMBHA resin equally on request, put into reaction chamber, upper and lower lid is tightened, label, record the title of synthesized peptide, lot number, the tare weight of reaction chamber and the weight of alleged resin.Reaction chamber is loaded synthesizer.Preparation synthetic agent, comprises N-Methyl pyrrolidone (NMP), acetyl imidazole (AIM), piperidines (PIP), methyl alcohol etc. and is placed in corresponding reagent bottle.
1.1.2 synthesizer state-detection
Check whether Peptide synthesizer normally runs.After start, run RunSelfTest program, whether instrument self checking indices is normal.Check N in addition
2whether sufficient, whether system gauge pressure is normal.Before synthesis, the performance of reply instrument is had gained some understanding, so will measure the flow velocity of often kind of synthetic agent.Send FloWRate1-18 to synthesizer, MainMenu-ModuleTest-is selected to look for ModuleA, ModuleD, ModuleI, ModuleI, ModuleA-to be undertaken measuring or observing by more by Start-by Prer or next, if flow is improper, then regulate lower valve pressure, until reach requirement.
1.1.3 the synthesis of polypeptide antigen starts
In the program of synthesizer, the method StdFmoc1.0SolDIC90 that synthesis needs is sent on synthesizer.The sequence of File-New-Sequence-Edit and Compose peptide, preserves.File-New-Run, checks Chemistry; Whether Sequence is by being deposited name; Setting Cycles; Preserve.Finally be sent on synthesizer.
MainMenu-CycleMonitor-begin, brings into operation.
1.1.4 the synthesis of polypeptide antigen
Peptide sequence described above, is hold to N from C end when synthesis, according to given order, is constantly repeated below synthesis step successively:
(1) deprotection reaction: above-mentioned aminoresin is placed in the nmp solution that volume percent is the hexahydropyridine of 15%-30%, reacts the Fmoc blocking group removed for 25-40 minute on aminoresin under 20-28 DEG C of condition;
(2) wash: nitrogen dries up, and NMP washs aminoresin;
(3) condensation reaction: the amino acid adding HOBT, DCC and Fmoc protection reacts 0.5-2.5 hour under 20-28 DEG C of condition;
(4) wash: nitrogen dries up, and NMP washs aminoresin;
(5) capping: add the nmp solution that percent weight in volume is the acetyl imidazole of 1.5%-4%, react 20-40 minute under 20-28 DEG C of condition.
1.1.5 polypeptide antigen end of synthesis
After antigen end of synthesis, synthesizer will stop automatically.Then take off reactor from Peptide synthesizer, then use 100% methanol wash polypeptide resin 3 times, then dry up in stink cupboard, be transferred to by polypeptide resin in brown bottle, put into-20 DEG C of refrigerators, sealed membrane sealing is for subsequent use.
The cracking of 1.2 polypeptide antigens and qualification
1.2.1 the cracking of polypeptide antigen
According to volume ratio (TFA/TIS/ phenol/H
2o=85/8/6/1) lysate is prepared, then in refrigerator, take out the polypeptide resin of synthesis, put into round-bottomed flask, in flask, the lysate prepared and magnetic stick is added in stink cupboard, then be stably placed on magnetic stirring apparatus, room temperature with constant stirs 1 hour until react completely.After reaction terminates, use the Rotary Evaporators of band cold-trap to continue evaporation and within 30 to 120 minutes, remove TFA in thick product.Then use ether collection, precipitated polypeptide, then use dimethyl formamide (DMF) repeatedly to clean the crude product of polypeptide antigen, finally the resin sand core funnel mixed is filtered out, namely obtain polypeptide antigen.
1.2.2 the qualification of synthetic antigen
Qualitative and quantitative analysis is carried out with substance assistant laser desorpted time-of-flight mass spectrometry (TOFMS) (MALDL-TOF) and reversed-phase high pressure liquid chromatography (RP-HPLC) after polypeptide antigen synthesis.
The conformation of 1.3 polypeptide antigens is formed
With 15%DMSO, polypeptide antigen is mixed with the polypeptide solution that concentration is 2mg/ml, then pH value=8.5 of initial gross separation polypeptide solution are adjusted with 0.1NNaOH or 0.1NHCl, under the environment of 25 DEG C at rotating speed be 110rpm shaking table on place 48 hours, make formation disulfide linkage.
And then carrying out head and the tail cyclisation, "-COOH " and "-NH2 " cyclization method of head and the tail is shown in the PeptideProteinReserch1996.48:229-239 such as Mengfen; "-the COOH " and "-OH " of head and the tail carries out reacting and forms ring texture and see the Chem.Soc1970.92:3771-3777 such as Mmenhofer.Can obtain can the polypeptide cyclized structure of simulated virus particle native conformation.
The purifying of 1.4 polypeptide antigens is degerming
Polypeptide antigen carries out ultrafiltration (TangentialFlowDevice circulating tangential flow filtration film bag and the peristaltic pump supporting with it) under using circulating tangential flow filtration film to wrap in 20-28 DEG C of condition, polypeptide antigen be macromole not by the filter membrane of certain pore size, early stage building-up process and the later stage cyclization formed or introduce small molecular weight impurity then can pass through filter membrane.And then be that 0.2 μm of pot strainer is degerming by aperture, the solution finally obtained is dispensed in aseptic plastic bottle, labelled.Label indicates the title of polypeptide, numbering, product batch number, concentration, date manufactured, storage life and preservation condition, after packing, be stored in-20 DEG C or-40 DEG C for subsequent use.
For the ease of transport and the long-term needs preserved, polypeptide antigen is carried out lyophilize to obtain the polypeptide of solid state.The polypeptide antigen frozen in advance is taken out, Labconco freeze drier carries out drying, obtains the polypeptide antigen of solid state.Simultaneously labelled.Label indicates the title of polypeptide, numbering, product batch number, concentration, date manufactured, storage life and preservation condition.
The preparation of embodiment 2. synthetic peptide vaccine
The preparation of 2.1 antigen aqueous phases
First, the three peptide species antigens synthesized according to above-described embodiment 1 are taken respectively; Then, with sterilized water for injection by antigenic synthetic peptide concentration dilution to 50 μ g/ml; By the metre filter that antigenic solution via hole diameter is 0.2 μm, degerming.
2.2 oil phase adjuvant preparations
By oil phase adjuvant through sterilizing in 121 DEG C, 30 minutes, for subsequent use.
The emulsification of 2.3 synthetic peptide vaccines
IKA emulsifying device is cleaned 3 times with the distilled water 2000ml of sterilizing, then be the volume ratio of 1: 1 by oil phase adjuvant and antigen aqueous phase under 20-28 DEG C of condition, first oil phase is added in emulsion tank, start after motor stirs 2 minutes with 90 ~ 150r/m slow rotation, simultaneously slowly add aqueous phase antigen, add rear stirring 30 minutes, then with 10000r/m high-speed stirring 20 minutes, leave standstill 5 minutes, make vaccine be emulsified into water in oil single-phase vaccine.
The potency test of embodiment 3 synthetic peptide vaccine
1. materials and methods
1.1 synthetic peptide vaccine
Have the polypeptide antigen of SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4, SEQIDNO.5 and SEQIDNO.6 sequence according to above-described embodiment synthesis, then preparing lot number corresponding with it is respectively: the aftosa synthetic peptide vaccine of ZM62601, ZM626A02, ZM626A03, ZM626A04, ZM626A05, ZM626A06.
1.2 experimental animal
Select kind identical, 4 monthly ages, about body weight 40Kg, the health frame pigling that foot and mouth disease neutralizing antibody is negative 32.
1.3 seed culture of viruses OR/80MF
8
By OR/80MF
6pass after 2 generations through suckling mouse, collect virus.Suckling mouse survey poison is qualified is placed on-20 DEG C of freezen protective, for subsequent use.
1.4 test method
The healthy susceptible feeder pig of about body weight 40kg (measuring without foot and mouth disease neutralizing antibody through suckling mouse neutralization test) 5 is adopted for often organizing vaccine.By each batch of synthetic peptide vaccine to be checked respectively at intramuscular injection 2ml after the basal part of the ear.Inoculate after 28 days, together with the contrast pig 2 that condition is identical, intramuscular injection 1000 ID after every pig basal part of the ear
50the strong malicious OR/80MF of foot and mouth disease O C-type virus C
8, Continuous Observation 10 days.There is bubble pathology in all at least one hoof of contrast pig.Immune swine occurs that namely any foot and mouth disease symptom is judged to and does not protect.
1.5 results judge
More than pig one hoof or snout occur foot and mouth disease typical case bubble, be then judged to morbidity.Any position of pig bubbles out without foot and mouth disease typical water and is now then judged to protection.
2. test-results and discussion
2.1 test-results
After immune 28 days, with 1000 ID
50the OR/80 strong virus attack of/head part is after 10 days, the results detailed in Table 1.
Table 1 aftosa synthetic peptide vaccine potency test
2.2 discussion of results
As can be seen from this test-results, after this animal of aftosa synthetic peptide vaccine immunity pig of the present invention, protection ratio is between 60% to 100%, is up to the protection of 100%.Illustrate that the aftosa synthetic peptide vaccine that these antigen is prepared has good immune efficacy and clinical application potentiality.
The safety testing of embodiment 4 synthetic peptide vaccine
1, test method
The cavy 12 of 1.1 use body weight 350 ~ 450g, every subcutaneous injection vaccine 2ml; With the mouse 30 of body weight 18 ~ 22g, every subcutaneous injection vaccine 0.5ml., all must not there is the death because vaccinate causes or significantly local untoward reaction or systemic reaction in Continuous Observation 7 days.
1.2 with the piglet of 30 ~ 40 ages in days (measuring without foot and mouth disease neutralizing antibody through suckling mouse neutralization test) 12, intramuscular injection vaccine 2ml (every side 1ml) after each two Herba Houttuyniae, day by day observation 14 days.All must not there is foot and mouth disease symptom or significantly because of toxic reaction that vaccinate causes.
2, test-results
The security of 2.1 vaccine in guinea pigs and mouse
Cavy 12, every subcutaneous injection vaccine 2ml; Mouse 30, every subcutaneous injection 0.5ml.Continuous Observation 7 days, all do not occur the death because vaccinate causes or significantly local untoward reaction or systemic reaction, concrete outcome is as following table 2.
The result of table 2 vaccine in guinea pigs and mouse safety testing
2.2 vaccines are to the security of piglet
Taken out by synthetic peptide vaccine after equilibrating to room temperature, to susceptible piglet (measuring without foot and mouth disease neutralizing antibody through suckling mouse neutralization test), intramuscular injection 2 part vaccines after each two Herba Houttuyniae, a routine 1ml, observes 14 day by day.All there is not foot and mouth disease symptom or significantly because of toxic reaction that vaccinate causes.
Concrete outcome is in table 3.
Table 3 synthetic peptide vaccine is to piglet safety testing result
The above results illustrates that these synthetic peptide vaccines are safe to cavy, mouse and piglet, does not resemble traditional vaccine and there is the side reaction problems such as heating, redness, so have good promotion prospect and marketable value.
Claims (13)
1., for a polypeptide for aftosa synthetic peptide vaccine, wherein said polypeptide has the aminoacid sequence shown in SEQIDNO.5 or SEQIDNO.6.
2. polypeptide according to claim 1, is characterized in that, the sulfydryl of two halfcystines in described aminoacid sequence is joined together to form disulfide linkage through oxidation.
3. polypeptide according to claim 1 and 2, is characterized in that, between the head and the tail amino-acid residue of described aminoacid sequence, reaction is formed covalently bound.
4. polypeptide according to claim 3, is characterized in that, the carboxyl of the head and the tail amino-acid residue of described aminoacid sequence and amino or react between carboxyl and hydroxyl is formed covalently bound.
5. an aftosa synthetic peptide vaccine, is selected from the polypeptide according to any one of Claims 1-4 comprising one or more.
6. vaccine according to claim 5, is characterized in that, described vaccine comprises adjuvant.
7. a preparation method for polypeptide according to any one of claim 1 to 4, comprising following steps:
(1) take aminoresin as starting raw material, with the amino acid of 9-fluorenylmethyloxycarbonyl protection for monomer, connect amino acid according to described aminoacid sequence successively condensation, after often walking condensation reaction, close unreacted aminoterminal with acetyl imidazole;
(2) lytic reagent cracking polypeptide is added after synthesis;
(3) ether collection, precipitated polypeptide is used;
(4) aseptically process is carried out after ultrafiltration purification polypeptide.
8. preparation method according to claim 7, is characterized in that, the volume components of described lytic reagent is than being trifluoroacetic acid: tri isopropyl silane: phenol: H
2o=85: 8: 6: 1.
9. the preparation method according to claim 7 or 8, is characterized in that, described pyrolysis time is 1-4 hour.
10. preparation method according to claim 7, is characterized in that, described step (1) comprises the following steps:
(a) deprotection reaction: aminoresin is placed in N-Methyl pyrrolidone (NMP) solution that volume percent is the hexahydropyridine of 15-30%, reacts the 9-fluorenylmethyloxycarbonyl blocking group removed for 25-40 minute on aminoresin under 20-28 DEG C of condition;
B () washs: nitrogen dries up, N-Methyl pyrrolidone washing aminoresin;
(c) condensation reaction: add I-hydroxybenzotriazole (HOBT), amino acid that dicyclohexylcarbodiimide (DCC) and 9-fluorenylmethyloxycarbonyl are protected reacts 0.5-2.5 hour under 20-28 DEG C of condition;
D () washs: nitrogen dries up, N-Methyl pyrrolidone washing aminoresin;
(e) capping: add N-Methyl pyrrolidone (NMP) solution that percent weight in volume is the acetyl imidazole of 1.5-4%, react 20-40 minute under 20-28 DEG C of condition.
11. preparation methods according to claim 7, is characterized in that, described step (4) comprises the following steps:
A () uses tangential flow filtration film to wrap in ultrafiltration polypeptide antigen under 20-28 DEG C of condition, removing small molecular weight impurity;
B () uses 0.2 micron of degerming preservation of online filter.
The preparation method of 12. 1 kinds of vaccines according to claim 5 or 6, comprising following steps:
(1) with water for injection, by one or more, the polypeptide be selected from according to any one of Claims 1-4 is diluted to 50 μ g/ml and obtains antigen aqueous phase;
(2) adjuvant is for subsequent use through sterilizing in 121 DEG C, 30 minutes;
(3) under 20-28 DEG C of condition, according to the volume ratio of antigen aqueous phase and adjuvant 1: 1, first adjuvant is added in emulsion tank, 90-150 rev/min of low rate mixing 1.5-3 minute, slowly add aqueous phase, add rear stirring 20-30 minute, then high speed 8000-10000 rev/min is stirred 15-30 minute, leave standstill 5 minutes, after packing and get final product.
13. polypeptide according to any one of claim 1 to 4, vaccine described in claim 5 or 6 purposes in the medicine of preparation prevention foot and mouth disease.
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| CN102775478B (en) * | 2012-08-10 | 2013-06-19 | 申联生物医药（上海）有限公司 | Foot and mouth disease virus antigen peptide and vaccine |
| CN102998460B (en) * | 2012-10-31 | 2014-12-10 | 广东海大畜牧兽医研究院有限公司 | Enzyme linked immunosorbent assay (ELISA) kit of foot-and-mouth disease virus antibody and preparation method thereof |
| CN103183728B (en) * | 2013-03-25 | 2015-06-10 | 中国牧工商(集团)总公司 | Polypeptide used for preparing O type peptide vaccine of cattle foot-and-mouth disease and preparation methods and applications thereof |
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| EP0090581A2 (en) * | 1982-03-26 | 1983-10-05 | Biogen N.V. | Small peptides with the specificity of foot and mouth disease viral antigens |
| US20020076405A1 (en) * | 1999-04-15 | 2002-06-20 | Xie Yong | Antigenized antibody vaccine for foot-and-mouth disease |
| CN1853721A (en) * | 2005-04-25 | 2006-11-01 | 申联生物医药（上海）有限公司 | Schweineseuche O-shaped synthetic peptide vaccine and preparation thereof |
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| CN101082051A (en) * | 2006-06-02 | 2007-12-05 | 广东出入境检验检疫局检验检疫技术中心 | Recombined foot-and-mouth disease virus polypeptide fragment and and uses thereof |
| CN101353373A (en) * | 1998-06-20 | 2009-01-28 | 美国联合生物医学公司 | Synthetic peptide vaccines for foot-and-mouth disease |
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| CN1321516A (en) * | 2000-04-28 | 2001-11-14 | 中牧实业股份有限公司兰州生物药厂 | Multistrain universal inactivated vaccine for bovine, ovine and porcine foot-and-mouth diseases |
| US8088388B2 (en) * | 2002-02-14 | 2012-01-03 | United Biomedical, Inc. | Stabilized synthetic immunogen delivery system |
| US7323546B2 (en) * | 2003-05-29 | 2008-01-29 | Academia Sinica | Apoptosis-inducing polypeptides |
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2009
- 2009-06-04 CN CN201410025274.6A patent/CN103936841B/en not_active Expired - Fee Related
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| EP0090581A2 (en) * | 1982-03-26 | 1983-10-05 | Biogen N.V. | Small peptides with the specificity of foot and mouth disease viral antigens |
| CN101353373A (en) * | 1998-06-20 | 2009-01-28 | 美国联合生物医学公司 | Synthetic peptide vaccines for foot-and-mouth disease |
| US20020076405A1 (en) * | 1999-04-15 | 2002-06-20 | Xie Yong | Antigenized antibody vaccine for foot-and-mouth disease |
| CN1853721A (en) * | 2005-04-25 | 2006-11-01 | 申联生物医药（上海）有限公司 | Schweineseuche O-shaped synthetic peptide vaccine and preparation thereof |
| CN101070348A (en) * | 2006-05-12 | 2007-11-14 | 北京宝麦德生物医药科技有限责任公司 | O-type foot-and-mouth disease virus multi-epitope mucous membrane immunization vaccine and use |
| CN101082051A (en) * | 2006-06-02 | 2007-12-05 | 广东出入境检验检疫局检验检疫技术中心 | Recombined foot-and-mouth disease virus polypeptide fragment and and uses thereof |
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| CN101565458A (en) | 2009-10-28 |
| CN103936841A (en) | 2014-07-23 |
| CN103936841B (en) | 2016-01-20 |
| CN101565458B (en) | 2014-01-08 |
| CN105175516B (en) | 2018-11-27 |
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