CN103848902B - A kind of synthetic peptide vaccine and preparation method thereof - Google Patents
A kind of synthetic peptide vaccine and preparation method thereof Download PDFInfo
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- CN103848902B CN103848902B CN201410081709.9A CN201410081709A CN103848902B CN 103848902 B CN103848902 B CN 103848902B CN 201410081709 A CN201410081709 A CN 201410081709A CN 103848902 B CN103848902 B CN 103848902B
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- 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
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- 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
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- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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- 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
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- 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
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- 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
Abstract
The invention provides a kind of aftosa synthetic peptide vaccine.It is particularly used for polypeptide or its polypeptide polymer of O type aftosa synthetic peptide vaccine, and vaccine containing this polypeptide or its polypeptide polymer and their preparation method, described polypeptide has the aminoacid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.These candidate polypeptide antigens are screened by the present invention by a large amount of animal experiments, according to the selection result, foot and mouth disease virus antigen site are optimized, and efficient combination t cell epitope and B cell epi-position, enhance the immune effect of polypeptide antigen.This O type aftosa synthetic peptide vaccine can successfully manage the antigenic variation of foot and mouth disease virus, and biological safety is good, it is easy to synthesize on a large scale, has a good application prospect.
Description
Technical field
The present invention relates to a kind of polypeptide for aftosa synthetic peptide vaccine or its polypeptide polymer, and contain vaccine and their preparation method of this polypeptide or its polypeptide polymer, it is specifically related to a kind of polypeptide for O type aftosa synthetic peptide vaccine or its polypeptide polymer, and contains vaccine and their preparation method of this polypeptide or its polypeptide polymer.
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 widely distributed.Owing to its infectiousness is high, propagating rapidly, the domestic animal such as infected pigs, cattle, sheep causes young stock dead, and adults production capacity sharply declines, the development of serious harm animal husbandry and carnivorous and livestock products production and supply.Market circulation and the international trade that can make animal and animal's products are subject to block greatly and restriction simultaneously, cause huge economic 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 picorna virus has the feature such as pleiomorphism, changeableness.At present it is known that there is the foot and mouth disease virus of 7 kinds of serotypes in the 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 every kind of principal mode divides some hypotypes, the hypotype having now been found that has kind more than 70.Serotypes A, O, C and AsiaI type are most commonly seen, 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, every kind each 60.VP1-VP3 forms capsomere, is positioned at the outside of capsid protein, and VP4 is positioned at the inside of virion.VP1 is main protective antigen, it has now been 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 protective antigen site that is most important on VP1 and that be easiest to 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 big, to such an extent as to the foot-and-mouth disease vaccine that can be reasonably resistant to a kind of hypotype is likely to do not have protectiveness for the another kind of hypotype in same serotype.Additionally, the antigenicity of foot and mouth disease strain is also constantly changing, As time goes on, the efficacy wanes of original vaccine even disappears, and brings very big difficulty therefore to the preventing and controlling of foot and mouth disease.
At present, foot and mouth disease is carried out mandatory immunity by China, and vaccine immunity is the Main Means preventing and treating foot and mouth disease.And the foot-and-mouth disease vaccine of the existing use of China is mainly viral inactivation vaccine, there is the problem such as biological safety difference, big, the unstable product quality of side reaction.A lot of countries have stopped using inactivated vaccine in the world at present, also forbid from the national import livestock products 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.Additionally, these vaccines are poor for the epidemic isolates often effect currently having occurred and that variation, it is impossible to effectively protect animal.
Summary of the invention
It is an object of the invention to provide a kind of polypeptide for aftosa synthetic peptide vaccine or its polypeptide polymer, and the vaccine containing this polypeptide or its polypeptide polymer, especially for polypeptide or its polypeptide polymer of O type aftosa synthetic peptide vaccine, and the vaccine containing this polypeptide or its polypeptide polymer.
It is a further object to provide a kind of aforementioned polypeptides and the preparation method of above-mentioned vaccine.
For achieving the above object, present invention employs techniques below scheme:
A kind of polypeptide for O type aftosa synthetic peptide vaccine, wherein said polypeptide has the aminoacid sequence shown in SEQIDNO.1, SEQIDNO.2 or SEQIDNO.3.
In above-mentioned aminoacid sequence, one or two above valines can be substituted by norvaline；And/or one or two above leucines can be substituted by nor-leucine.Preferably, in above-mentioned aminoacid sequence, whole valines are substituted by norvaline；And/or all leucine is substituted by nor-leucine.
The sulfydryl of any two cysteine in above-mentioned aminoacid sequence oxidized can be joined together to form disulfide bond.
Formation can be reacted covalently bound between the head and the tail amino acid residue of above-mentioned aminoacid sequence.Specifically, between carboxyl and amino or carboxyl and the hydroxyl of the head and the tail amino acid residue of above-mentioned aminoacid sequence, reaction is formed covalently bound.
A kind of aftosa synthetic peptide vaccine, including one or more aforementioned polypeptides or its polypeptide polymer.Such as: there is polypeptide or its polypeptide polymer of the aminoacid sequence shown in SEQIDNO.1, SEQIDNO.2 or SEQIDNO.3 or their any two kinds or two or more combinations.Above-mentioned vaccine can also comprise adjuvant.
The preparation method that present invention also offers aforementioned polypeptides, including following steps:
(1) with amino resins for initiation material, with the aminoacid of 9-fluorenylmethyloxycarbonyl protection for monomer, connect aminoacid according to described aminoacid sequence successively condensation, often close unreacted aminoterminal with acetyl imidazole after step condensation reaction；
(2) lytic reagent cracking polypeptide is added after synthesis；
(3) ether collection, precipitated polypeptide are used；
(4) aseptic process is carried out after ultrafiltration purification polypeptide.
In above-mentioned preparation method, the volume components of described lytic reagent is than for trifluoroacetic acid (TFA): tri isopropyl silane (TIS): phenol: H2O=85:8:6:1；Described pyrolysis time is 1-4 hour.
In above-mentioned preparation method, described step (1) specifically includes following steps:
A () deprotection reaction: be placed in by amino resins in N-Methyl pyrrolidone (NMP) solution of the hexahydropyridine that percent by volume is 15-30%, reacts the 9-fluorenylmethyloxycarbonyl blocking group on 25-40 minute elimination amino resins under 20-28 DEG C of condition；
B () washs: nitrogen dries up, N-Methyl pyrrolidone washing amino resins；
(c) condensation reaction: the aminoacid adding 1-hydroxyl azimidobenzene (HOBT), dicyclohexylcarbodiimide (DCC) and 9-fluorenylmethyloxycarbonyl protection reacts 0.5-2.5 hour under 20-28 DEG C of condition；
D () washs: nitrogen dries up, N-Methyl pyrrolidone washing amino resins；
(e) capping: add N-Methyl pyrrolidone (NMP) solution of the acetyl imidazole that percent weight in volume is 1.5-4%, react 20-40 minute under 20-28 DEG C of condition.
In above-mentioned preparation method, described step (4) specifically includes following steps:
A () uses tangential flow filtration film to wrap in the polypeptide that under 20-28 DEG C of condition, ultrafiltration prepares, remove small molecular weight impurity；
B () uses 0.2 micron of degerming preservation of online filter.
The preparation method that present invention also offers above-mentioned vaccine, including following steps:
(1) with water for injection, aforementioned polypeptides or its polypeptide polymer are diluted to 50 μ g/ml and prepare antigen aqueous phase；
(2) adjuvant is standby through sterilizing in 121 DEG C, 30 minutes；
(3) under 20-28 DEG C of condition, volume ratio according to antigen aqueous phase Yu adjuvant 1:1, first adjuvant is added in emulsion tank, 90-150 rev/min of low rate mixing 1.5-3 minute, it is slowly added to aqueous phase, stirs 20-30 minute after adding, then high speed 8000-10000 rev/min is stirred 15-30 minute, stand 5 minutes, after subpackage and get final product.
Invention further provides aforementioned polypeptides or its polypeptide polymer, the vaccine purposes in the medicine of preparation treatment and/or prevention O type foot and mouth disease.Wherein, described O type foot and mouth disease is preferably pig O type foot and mouth disease.
The present invention passes through the sequencing to domestic foot and mouth disease epidemic isolates recently combined mouth fever aphthous vaccine strain MYA/98 and OZK/93 sequence, the variation situation of research foot and mouth disease major antigenic sites, the frequency of its variation is added up for the main amino acid sites made a variation, the analyses and prediction in foot-and-mouth disease antigen site are carried out in combination with area of computer aided, possible antigen site peptide fragment is carried out chemosynthesis, namely for easy variant sites according to the variation frequency added up, different aminoacid is used in these sites, obtain containing the multiple candidate polypeptide antigen of current likely variant sites.And then by substantial amounts of animal experiment, these candidate polypeptide antigens are screened, obtain causing the immunoreation of animal, and immunoreation level is high, it is possible to well protection animal is from the polypeptide antigen of the attack of foot and mouth disease epidemic isolates.Foot and mouth disease virus antigen site is optimized by the present invention according to screening experiment result, and efficient combination t cell epitope and B cell epi-position, enhances the immune effect of polypeptide antigen.This foot and mouth disease O type synthetic peptide vaccine can successfully manage the antigenic variation of current foot and mouth disease virus, be absent from biological safety, it is easy to synthesizes on a large scale, has a good application prospect.
Detailed description of the invention
Method in following embodiment, if no special instructions, is conventional method.
Percentage composition in following embodiment, if no special instructions, is weight/mass percentage composition.
Embodiment 1, embodiment 1 aftosa synthetic peptide antigen solid phase synthesis
The present invention passes through the sequencing to domestic foot and mouth disease epidemic isolates recently combined mouth fever aphthous vaccine strain MYA/98 and OZK/93 sequence, the variation situation of research foot and mouth disease major antigenic sites, the frequency of its variation is added up for the main amino acid sites made a variation, the analyses and prediction in foot-and-mouth disease antigen site are carried out in combination with area of computer aided, possible antigen site peptide fragment is carried out chemosynthesis, namely for easy variant sites according to the variation frequency added up, different aminoacid is used in these sites, obtain containing the multiple candidate polypeptide antigen of current likely variant sites.And then by substantial amounts of animal experiment, these candidate polypeptide antigens are screened, obtain causing the immunoreation of animal, and immunoreation level is high, it is possible to well protection animal is from the polypeptide antigen of the attack of foot and mouth disease epidemic isolates.
The 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 aminoacid 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.
1, the solid phase synthesis of polypeptide antigen
1) synthesis material prepares
The sequence of synthetic polypeptide antigen is respectively as shown in SEQIDNO:1, SEQIDNO:2, SEQIDNO:3.
Sequence and synthesis scale according to antigen are that 1mmol prepares the suitable Fmoc aminoacid modified, and add in corresponding aminoacid bottle.Weigh RinkAmideMBHA resin equally on request, put in reaction chamber, upper and lower lid is tightened, labels, the weight of the synthesized title of peptide of record, lot number, the tare weight of reaction chamber and alleged resin.Reaction chamber is loaded synthesizer.Preparation synthetic agent, is placed in corresponding reagent bottle including N-Methyl pyrrolidone (NMP), acetyl imidazole (AIM), piperidines (PIP), methanol etc..
2) synthesizer state-detection
Check that whether Peptide synthesizer is properly functioning.After start, running RunSelfTest program, whether instrument self checking indices is normal.Additionally checking that whether N2 is sufficient, whether system gauge pressure is normal.Before synthesis, the performance of reply instrument is had gained some understanding, so the flow velocity of every kind of synthetic agent to be measured.Send FlowRate1-18 to synthesizer, MainMenu ModuleTest is selected to look for ModuleA, ModuleD, ModuleI, ModuleI, ModuleA or to observe by more measurement by Start by Prer or next, if flow is improper, then regulate lower valve pressure, until it reaches requirement.
3) synthesis of polypeptide antigen starts
The method StdFmoc1.0SolDIC90 that synthesis needs is sent on synthesizer by the program of synthesizer.The sequence of File-New-Sequence-Edit and Compose peptide, preserves.File-New-Run, checks Chemistry；Whether Sequence is by being deposited name；Set Cycles；Preserve.It is finally sent on synthesizer.
MainMenu CycleMonitor begin, brings into operation.
4) synthesis of polypeptide antigen
Peptide sequence described above, is start to N end from C end when synthesis, according to given order, is repeated continuously following synthesis step successively:
(1) deprotection reaction: be placed in by above-mentioned amino resins in the nmp solution of the hexahydropyridine that percent by volume is 15%-30%, reacts the Fmoc blocking group on 25-40 minute elimination amino resins under 20-28 DEG C of condition；
(2) washing: nitrogen dries up, and NMP washs amino resins；
(3) condensation reaction: the aminoacid adding HOBT, DCC and Fmoc protection reacts 0.5-2.5 hour under 20-28 DEG C of condition；
(4) washing: nitrogen dries up, and NMP washs amino resins；
(5) capping: add the nmp solution of the acetyl imidazole that percent weight in volume is 1.5%-4%, react 20-40 minute under 20-28 DEG C of condition.
5) polypeptide antigen end of synthesis
After antigen end of synthesis, synthesizer will be automatically stopped.Then taking off reactor from Peptide synthesizer, then wash polypeptide resin 3 times with 100% methanol, then dry up in fume hood, be transferred in brown bottle by polypeptide resin, put into-20 DEG C of refrigerators, sealed membrane seals standby.
2, the cracking of polypeptide antigen and qualification
1) cracking of polypeptide antigen
According to volume ratio (TFA/TIS/ phenol/H2O=85/8/6/1) preparation lysate, then in refrigerator, take out the polypeptide resin of synthesis, put into round-bottomed flask, in fume hood, in flask, add the lysate prepared and magnetic stick, then being stably placed on magnetic stirring apparatus, room temperature with constant stirs 1 hour until reacting completely.After reaction terminates, the Rotary Evaporators with cold-trap is used to continue to evaporate the TFA removed in thick product 30 to 120 minutes.It is then used by ether collection, precipitated polypeptide, then repeatedly cleans the crude product of polypeptide antigen with dimethylformamide (DMF), finally the resin sand core funnel mixed is filtered out, namely obtain polypeptide antigen.
2) 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.
3) conformation of polypeptide antigen is formed
With 15%DMSO, polypeptide antigen is configured to 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, place 48 hours on the shaking table that rotating speed is 110rpm under the environment of 25 DEG C, make formation disulfide bond.
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 reacts and forms circulus and see the Chem.Soc1970.92:3771-3777 such as Mmenhofer.Can obtain can the polypeptide cyclized structure of simulated virus particle native conformation.
4) purification of polypeptide antigen is degerming
The polypeptide antigen circulating tangential flow filtration film of use wraps in and carries out ultrafiltration (TangentialFlowDevice circulating tangential flow filtration film bag and the peristaltic pump supporting with it) under 20-28 DEG C of condition, polypeptide antigen is that macromole can not by the filter membrane of certain pore size, and the small molecular weight impurity that early stage building-up process and later stage cyclization are formed or introduce then can pass through filter membrane.Then it is that 0.2 μm of pot strainer is degerming again through aperture, the solution finally obtained is dispensed in aseptic plastic bottle, labelled.Label indicates the title of polypeptide, numbering, product batch number, concentration, date of manufacture, pot-life and preservation condition, after subpackage, be stored in-20 DEG C or-40 DEG C standby.
For the ease of transport and the long-term needs preserved, polypeptide antigen is carried out lyophilization to obtain the polypeptide of solid state.The polypeptide antigen frozen in advance is taken out, Labconco freezer dryer is dried, obtains the polypeptide antigen of solid state.Simultaneously labelled.Label indicates the title of polypeptide, numbering, product batch number, concentration, date of manufacture, pot-life and preservation condition.
Embodiment 2, synthetic peptide vaccine preparation
1, the preparation of antigen aqueous phase
First, the three peptide species antigens according to above-described embodiment 1 synthesis are weighed respectively；Then, with sterilized water for injection by antigenic synthetic peptide concentration dilution to 50 μ g/ml；The filter that antigenic solution via hole diameter is 0.2 μm is filtered, degerming.
2, prepared by oil phase adjuvant
By oil phase adjuvant ISA50V through sterilizing in 121 DEG C, 30 minutes, standby.
3, the emulsifying of synthetic peptide vaccine
IKA emulsifying device is cleaned (purchased from IKA company with the distilled water 2000ml of sterilizing, article No. 200603) 3 times, then it is 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 motor, after 90～150r/m slow rotation stirring 2 minutes, to be slowly added aqueous phase antigen, stir 30 minutes after adding, again with 10000r/m high-speed stirred 20 minutes, stand 5 minutes, make vaccine be emulsified into water in oil single-phase vaccine.
Embodiment 3, synthetic peptide vaccine potency test
One, materials and methods
1, synthetic peptide vaccine
There is the polypeptide antigen of SEQIDNO:1, SEQIDNO:2, SEQIDNO:3 sequence according to above-described embodiment 1 synthesis, the oxidized disulfide bond that is joined together to form of sulfydryl of these two cysteine of polypeptide, and between head and the tail aminoacid carboxyl and hydroxyl, reaction is formed covalently bound.These are then according to the method for embodiment 2 prepares corresponding lot number respectively is: the foot and mouth disease O type synthetic peptide vaccine of ZM433A01, ZM433A02, ZM433A03.
Additionally, valine norvalines whole in aminoacid sequence in SEQIDNO:1 are substituted；Whole leucine nor-leucines substitute, the method provided according to above-described embodiment carries out antigen synthesis, in this antigen, the sulfydryl of same two cysteine is oxidized is joined together to form disulfide bond, and reaction is formed covalently bound between head and the tail aminoacid carboxyl and hydroxyl, by preparing vaccine lot number according to the method for embodiment 2 it is: ZM433A04.
The peptide technology that is conventionally synthesized described in embodiment 1 is used to synthesize the dimer antigen of this sequence according to SEQIDNO:1 sequence, wherein, in composition dimer, the sulfydryl of two cysteine of every polypeptide is oxidized is joined together to form disulfide bond, and reaction is formed covalently bound between head and the tail aminoacid carboxyl and hydroxyl, preparing vaccine according to the method for embodiment 2, obtaining lot number is: the synthetic peptide vaccine of ZM433A05.
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 27 (two-way cross, silver pig farm, Lanzhou).
3, seed culture of viruses OR/80MF8
By OR/80MF8(offer of biology pharmaceutical factory, Lanzhou) surveys malicious qualified-20 DEG C of freezen protective that are placed on through neonatal rat, standby.
4, test method
For the healthy susceptible feeder pig (measuring without foot and mouth disease neutralizing antibody through neonatal rat neutralization test) 5 often organizing vaccine employing about body weight 40kg.By each batch of synthetic peptide vaccine intramuscular injection 2ml after the basal part of the ear to be checked.After inoculating 28, together with the comparison pig 2 that condition is identical, 1000 ID of intramuscular injection after every pig basal part of the ear50Foot and mouth disease O type Virus OR/80MF8, Continuous Observation 10 days.There is blister pathological changes in comparison all at least one hoof of pig.Immune swine occurs that namely any foot and mouth disease symptom is judged to and does not protect.
5, result judges
More than pig one hoof or snout foot and mouth disease typical case's blister occurs, then be judged to morbidity.Any position of pig bubbles out without foot and mouth disease typical water and is now then judged to protection.
Two, result of the test and discussion
1, result of the test
After immunity 28 days, with 1000 ID50The 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, discussion of results
From this result of the test it can be seen that the present invention foot and mouth disease O type synthetic peptide vaccine immunity this animal pig after be up to 100% protection.Meanwhile, research finds that having carried out the synthetic peptide vaccine that aminoacid is replaced and antigen is polymerized has better immune effect.Illustrate that the foot and mouth disease O type synthetic peptide vaccine that these antigen is prepared has good immune efficacy and clinical value.
Embodiment 4, synthetic peptide vaccine safety testing
One, test method
1, with the Cavia porcellus 10 of body weight 350-450g, every subcutaneous injection vaccine 2ml；With the mice 25 of body weight 18-22g, every subcutaneous injection vaccine 0.5ml.Continuous Observation 7 days, all must not occur because vaccinating the death caused or significantly local untoward reaction or general reaction.
2, with the piglet (measuring without foot and mouth disease neutralizing antibody through neonatal rat neutralization test) 10 of 30-40 age in days, the every side 1ml of intramuscular injection vaccine 2ml(after each two Herba Houttuyniae), observe 14 day by day.Foot and mouth disease symptom all must not occur or significantly because vaccinating the toxic reaction caused.
Two, result of the test
1, the safety of vaccine in guinea pigs and mice
Cavia porcellus 10, every subcutaneous injection vaccine 2ml；Mice 25, every subcutaneous injection 0.5ml.Continuous Observation 7 days, does not all occur that concrete outcome is table 1 below such as because vaccinating the death caused or significantly local untoward reaction or general reaction.
The result of table 1 vaccine in guinea pigs and mice safety testing
2, the vaccine safety to piglet
Synthetic peptide vaccine is taken out after equilibrating to room temperature, to susceptible piglet (measuring without foot and mouth disease neutralizing antibody through neonatal rat neutralization test), 2 part vaccines of intramuscular injection after each two Herba Houttuyniae, side 1ml, observe 14 day by day.Foot and mouth disease symptom does not all occur or significantly because vaccinating the toxic reaction caused.Concrete outcome is in Table 2.
The 2-in-1 one-tenth peptide vaccine of table is to piglet safety testing result
The above results illustrates that Cavia porcellus, mice and piglet are safe by these synthetic peptide vaccines, there is the side reaction problems such as heating, redness like that not as traditional vaccine, so having good promotion prospect and market value.
Claims (17)
1., for a polypeptide for O type aftosa synthetic peptide vaccine, its aminoacid sequence is such as shown in SEQIDNO.1.
2. polypeptide according to claim 1, it is characterised in that: in the aminoacid sequence of described polypeptide, one or more valine is substituted by norvaline；And/or one or more leucine is substituted by nor-leucine.
3. polypeptide according to claim 2, it is characterised in that: in the aminoacid sequence of described polypeptide, whole valines are substituted by norvaline；And/or all leucine is substituted by nor-leucine.
4. polypeptide according to any one of claim 1 to 3, it is characterised in that: the sulfydryl of any two cysteine in the aminoacid sequence of described polypeptide is oxidized is joined together to form disulfide bond.
5. polypeptide according to any one of claim 1 to 3, it is characterised in that: between the head and the tail amino acid residue of the aminoacid sequence of described polypeptide, reaction is formed covalently bound.
6. polypeptide according to claim 5, it is characterised in that: between carboxyl and amino or carboxyl and the hydroxyl of the head and the tail amino acid residue of the aminoacid sequence of described polypeptide, reaction is formed covalently bound.
7. an aftosa synthetic peptide vaccine, including the polypeptide shown in SEQIDNO.1 or its polypeptide polymer.
8. aftosa synthetic peptide vaccine according to claim 7, wherein also includes the polypeptide shown in the polypeptide of SEQIDNO.2 or its polypeptide polymer, SEQIDNO.3 or the polypeptide polymer connected into by least one polypeptide in SEQIDNO.1, SEQIDNO.2 and SEQIDNO.3.
9. aftosa synthetic peptide vaccine according to claim 7, it is characterised in that: in the aminoacid sequence of described polypeptide, one or more valine is substituted by norvaline；And/or one or more leucine is substituted by nor-leucine.
10. aftosa synthetic peptide vaccine according to claim 9, it is characterised in that: in the aminoacid sequence of described polypeptide, whole valines are substituted by norvaline；And/or all leucine is substituted by nor-leucine.
11. the aftosa synthetic peptide vaccine according to any one of claim 7 to 10, it is characterised in that: the sulfydryl of any two cysteine in the aminoacid sequence of described polypeptide is oxidized is joined together to form disulfide bond.
12. the aftosa synthetic peptide vaccine according to any one of claim 7 to 10, it is characterised in that: between the head and the tail amino acid residue of the aminoacid sequence of described polypeptide, reaction is formed covalently bound.
13. aftosa synthetic peptide vaccine according to claim 12, it is characterised in that: between carboxyl and amino or carboxyl and the hydroxyl of the head and the tail amino acid residue of the aminoacid sequence of described polypeptide, reaction is formed covalently bound.
14. aftosa synthetic peptide vaccine according to claim 7, it is characterised in that described vaccine comprises adjuvant.
15. a preparation method for the polypeptide according to any one of claim 1 to 6, including following steps:
(1) with amino resins for initiation material, with the aminoacid of 9-fluorenylmethyloxycarbonyl protection for monomer, connect aminoacid according to described aminoacid sequence successively condensation, often close unreacted aminoterminal with acetyl imidazole after step condensation reaction；
(2) lytic reagent cracking polypeptide is added after synthesis；
(3) ether collection, precipitated polypeptide are used；
(4) aseptic process is carried out after ultrafiltration purification polypeptide.
16. polypeptide according to any one of claim 1 to 6 or vaccine described in any one application in the medicine of preparation prevention O type foot and mouth disease in its polypeptide polymer, claim 7-14.
17. application according to claim 16, it is characterised in that: described O type foot and mouth disease is pig O type foot and mouth disease.
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| CN107827958A (en) * | 2017-11-11 | 2018-03-23 | 中牧实业股份有限公司 | Canine parvovirus synthetic peptide vaccine and its preparation method and application |
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| CN106021974B (en) * | 2016-05-19 | 2019-05-03 | 中牧智合（北京）生物技术有限公司 | The composition of the method and the peptide library thus prepared that optimized to t cell epitope |
| CN106986925B (en) * | 2017-05-08 | 2020-08-25 | 中牧实业股份有限公司 | O-type and A-type bivalent synthetic peptide vaccine for bovine foot and mouth disease and preparation method and application thereof |
| CN107827986B (en) * | 2017-05-09 | 2019-09-24 | 青岛明勤生物科技有限公司 | Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine |
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| US6107021A (en) * | 1998-06-20 | 2000-08-22 | United Biomedical, Inc. | Synthetic peptide vaccines for foot-and-mouth disease |
| 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 |
| 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 |
| CN102580076A (en) * | 2011-12-23 | 2012-07-18 | 广州自远生物科技有限公司 | Synthetic peptide vaccine for O-type foot and mouth disease of swine and preparation method thereof |
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| CN107827958A (en) * | 2017-11-11 | 2018-03-23 | 中牧实业股份有限公司 | Canine parvovirus synthetic peptide vaccine and its preparation method and application |
| CN107827958B (en) * | 2017-11-11 | 2020-08-25 | 中牧实业股份有限公司 | Canine parvovirus synthetic peptide vaccine and preparation method and application thereof |
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| CN105906693A (en) | 2016-08-31 |
| CN103848902A (en) | 2014-06-11 |
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