CN101659695A - O-type aftosa synthetic peptide vaccine - Google Patents
O-type aftosa synthetic peptide vaccine Download PDFInfo
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- CN101659695A CN101659695A CN200810118967A CN200810118967A CN101659695A CN 101659695 A CN101659695 A CN 101659695A CN 200810118967 A CN200810118967 A CN 200810118967A CN 200810118967 A CN200810118967 A CN 200810118967A CN 101659695 A CN101659695 A CN 101659695A
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Abstract
The invention provides O-type aftosa synthetic peptide vaccine, and in particular relates to polypeptide or polypeptide polymer thereof used in the vaccine as well as the vaccine containing the polypeptide or the polypeptide polymer thereof and a preparation method thereof. The polypeptide has amino acid sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3. The O-type aftosa synthetic peptide vaccine carries out chemical synthesis of potential antigen site peptide segments by carrying out sequencing of domestic aftosa epidemic strains to study the variation of the main antigen sites ofaftosa and combining with computer assistant to carry out antigen site analytical prediction. Candidate polypeptide antigens are screened out by carrying out large numbers of animal experiments and aftosa virus antigen sites are optimized according to the screening result; and T cell epitope and B cell epitope are effectively combined to improve the immune effects of the polypeptide antigens. TheO-type aftosa synthetic peptide vaccine can effectively cope with the antigen variation of aftosa virus and has ideal biosafety and easy large-scale synthesis, thereby having a good application prospect.
Description
Technical field
The present invention relates to a kind of polypeptide or its polypeptide polymer that is used for aftosa synthetic peptide vaccine, and contain the vaccine of this polypeptide or its polypeptide polymer and their preparation method, be specifically related to a kind of polypeptide or its polypeptide polymer of the O of being used for type aftosa synthetic peptide vaccine, and contain the vaccine of this polypeptide or its polypeptide polymer and their preparation method.
Background technology
Foot and mouth disease (foot and mouth disease is called for short FMD) is a kind of acute, highly contact, infectious fever, worldwide extensively distribution artiodactylous that betide.Because its infectivity height is propagated rapidly, livestocks such as infected pigs, ox, sheep cause cub death, and adult animals throughput sharply descends, the production and supply of serious harm Developing of Animal Industry and meat and livestock product thereof.Can make the market circulation and the international trade of animal and animal product be subjected to blocking greatly and restriction simultaneously, produce for the livestock industry of popular countries and regions and cause enormous economic loss.Foot and mouth disease is infected by foot and mouth disease virus (FMDV) and causes that the Hostis picornavirus has characteristics such as polytypism, volatility.At present, there are the foot and mouth disease virus of 7 kinds of serotypes: A, O, C, Sat1 (South Africa I type), Sat2 (South Africa II type), Sat3 (South Africa III type) and AsiaI (Asia I type) in the known whole world.And every kind of principal mode divides some hypotypes, at present the existing kind more than 70 of the hypotype of finding.Serotype A, O, C and Asia I type are the most common, and wherein the mutation of serotype A virus is maximum, have 30 kinds of hypotypes of surpassing.Result of study shows: the capsid protein of foot and mouth disease virus is to be made up of four kinds of structural protein VP1, VP2, VP3 and VP4 (Logan D etc., 1993), every 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, have now found that O type foot and mouth disease has 3 main antigen sites to be positioned on the VP1, and wherein 133-160 and 200-213 position have constituted VP1 and go up most important and the protective antigen site of easy variation.
Current is O type and Asia I type at China's main popular foot and mouth disease strain.Antigenicity difference between foot and mouth disease virus is various, immunity alternately each other.And; the degree of antigenic variation is also very big in a kind of serotype; may not have protectiveness at the another kind of hypotype in the same serotype to such an extent as to can resist a kind of aftosa vaccine of hypotype effectively, brought very big difficulty therefore for 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 of preventing and treating foot and mouth disease.And the aftosa vaccine of the existing use of China mainly is a viral inactivation vaccine, there is problems such as biological safety is poor, side reaction big, unstable product quality.A lot of in the world at present countries have stopped using inactivated vaccine, also forbid from using the national import livestock product of inactivated vaccine.Aspect the research of foot and mouth disease new generation vaccine, successively there are genetic engineering subunit vaccine, foot and mouth disease virus carrier bacterin, foot-and-mouth disease gene engineering to modify the research report of vaccine, but it has influenced the use of these new generation vaccines all existing problems aspect immune effect, the biological safety.
Summary of the invention
Therefore, the object of the present invention is to provide a kind of polypeptide or its polypeptide polymer of the O of being used for type aftosa synthetic peptide vaccine, and the vaccine that contains this polypeptide or its polypeptide polymer, especially for polypeptide or its polypeptide polymer of pig O type aftosa synthetic peptide vaccine, and the vaccine that contains this polypeptide or its polypeptide polymer.
Another object of the present invention provides the preparation method of a kind of aforementioned polypeptides and above-mentioned vaccine.
For achieving the above object, the present invention has adopted following technical scheme:
A kind of polypeptide that is used for O type aftosa synthetic peptide vaccine, wherein said polypeptide have the aminoacid sequence shown in SEQ IDNO.1, SEQ ID NO.2 or the SEQ ID NO.3.
One or more Xie Ansuan can be substituted by norvaline in the above-mentioned aminoacid sequence; And/or one or more leucine can be substituted by nor-leucine.Preferably, whole Xie Ansuans are substituted by norvaline in the above-mentioned aminoacid sequence; And/or all leucine is substituted by nor-leucine.
The sulfydryl of two halfcystines in the above-mentioned aminoacid sequence can be joined together to form disulfide linkage through oxidation.
It is covalently bound to react formation between the head and the tail amino-acid residue of above-mentioned aminoacid sequence.Specifically, reaction forms covalently bound between the carboxyl of the head and the tail amino-acid residue of above-mentioned aminoacid sequence and amino or carboxyl and the hydroxyl.
A kind of aftosa synthetic peptide vaccine is comprising one or more aforementioned polypeptides or its polypeptide polymer.For example: polypeptide or its polypeptide polymer with the aminoacid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or the SEQ ID NO.3.Above-mentioned vaccine can also comprise adjuvant.
The present invention also provides the preparation method of aforementioned polypeptides, comprising following steps:
(1) with aminoresin be starting raw material, the amino acid of protecting with 9-fluorenylmethyloxycarbonyl is monomer, connects amino acid according to described aminoacid sequence condensation successively, seals unreacted aminoterminal with acetyl imidazole after per step condensation reaction;
(2) the synthetic back that finishes adds lytic reagent cracking polypeptide;
(3) use ether to collect, precipitate polypeptide;
(4) carry out aseptically process behind the ultrafiltration purification polypeptide.
In above-mentioned preparation method, the component volume ratio of described lytic reagent is trifluoroacetic acid (TFA): tri isopropyl silane (TIS): phenol: H
2O=85: 8: 6: 1; The described cracking time is 1-4 hour.
In above-mentioned preparation method, described step (1) specifically may further comprise the steps:
(a) deprotection reaction: it is N-Methyl pyrrolidone (NMP) solution of the hexahydropyridine of 15-30% that aminoresin is placed volume percent, the 9-fluorenylmethyloxycarbonyl blocking group that reaction removed on the aminoresin in 25-40 minute under 20-28 ℃ of condition;
(b) washing: nitrogen dries up, N-Methyl pyrrolidone washing aminoresin;
(c) condensation reaction: the amino acid that adds 1-hydroxyl azimidobenzene (HOBT), dicyclohexylcarbodiimide (DCC) and 9-fluorenylmethyloxycarbonyl protection reacted 0.5-2.5 hour under 20-28 ℃ of condition;
(d) washing: nitrogen dries up, N-Methyl pyrrolidone washing aminoresin;
(e) capping: adding percent weight in volume is N-Methyl pyrrolidone (NMP) solution of the acetyl imidazole of 1.5-4%, and reaction is 20-40 minute under 20-28 ℃ of condition.
In above-mentioned preparation method, described step (4) specifically may further comprise the steps:
(a) use the tangential flow filtration film to wrap in the polypeptide that ultrafiltration makes under the 20-28 ℃ of condition, remove small molecular weight impurity;
(b) use 0.2 micron online filter degerming to preserve.
The present invention also provides the preparation method of above-mentioned vaccine, comprising following steps:
(1) with water for injection aforementioned polypeptides or its polypeptide polymer are diluted to 50 μ g/ml and make the antigen water;
(2) adjuvant is standby through sterilization in 121 ℃, 30 minutes;
(3) under 20-28 ℃ of condition, according to antigen water and 1: 1 volume ratio of adjuvant, earlier adjuvant is added in the emulsion tank, 90-150 rev/min was stirred at a slow speed 1.5-3 minute, slowly add water, add the back and stirred 20-30 minute, high speed 8000-10000 rev/min was stirred 15-30 minute again, left standstill 5 minutes, after the packing promptly.
The present invention further provides aforementioned polypeptides or its polypeptide polymer, vaccine and treated and/or prevented purposes in the medicine of O type foot and mouth disease in preparation.Wherein, described O type foot and mouth disease is preferably pig O type foot and mouth disease.
The present invention is by the sequencing to domestic foot and mouth disease epidemic isolates, the variation situation in research foot and mouth disease major antigen site, add up the frequency of its variation at the amino acid sites of main variation, simultaneously carry out the analyses and prediction in foot-and-mouth disease antigen site in conjunction with area of computer aided, possible antigen site peptide section is carried out chemosynthesis, promptly at the variation frequency of easy variant sites according to statistics, use different amino acid in these sites, obtain containing multiple candidate's polypeptide antigen of the possible variant sites of current institute.And then by a large amount of animal experiments these candidate's polypeptide antigens are screened, obtain causing the immune response of animal, and immune response level height, the polypeptide antigen of the attack of avoiding the foot and mouth disease epidemic isolates of can be good at watching for animals.The present invention optimizes the foot-and-mouth disease virus antigen site according to the screening experiment result, and has effectively made up t cell epitope and B cell epitope, has strengthened the immune effect of polypeptide antigen.This foot and mouth disease O type synthetic peptide vaccine can successfully manage the antigenic variation of present foot and mouth disease virus, not have biological safety, is easy to extensive synthesizing, and has a good application prospect.
Embodiment
The antigenic solid phase synthesis of embodiment 1 aftosa synthetic peptide
Polypeptide antigen of the present invention can use ABI 433A full-automatic polypeptide synthetic instrument, utilizes the preparation of Merrifield solid-phase synthesis, the amino acid that has wherein adopted 9-fluorenylmethyloxycarbonyl (Fmoc) to modify, and solid phase carrier is a Rink Amide mbha resin.Production process generally includes the solid phase synthesis of polypeptide antigen, cracking, antigen purification and the degerming of polypeptide preserved.
1.1 the solid phase synthesis of polypeptide antigen
1.1.1 the preparation of synthesis material
The sequence of synthetic polypeptide antigen is respectively shown in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3.
According to antigenic sequence and synthetic scale is that 1mmol prepares the amino acid that suitable Fmoc modifies, and adds in the corresponding amino acid bottle.Weighing Rink Amide mbha resin is put into reaction chamber equally on request, and up and down lid is tightened, label, record title, lot number, the tare weight of reaction chamber and the weight of alleged resin of synthetic peptide.With the reaction chamber synthesizer of packing into.The preparation synthetic agent comprises that N-Methyl pyrrolidone (NMP), acetyl imidazole (AIM), piperidines (PIP), methyl alcohol etc. are placed in the corresponding reagent bottle.
1.1.2 synthesizer status detection
Check the whether normally operation of 433A Peptide synthesizer.After the start, operation Run Self Test program, whether the every index of instrument self checking is normal.Check N in addition
2Whether sufficient, whether system's gauge pressure is normal.The performance of reply instrument is had gained some understanding before synthetic, so will measure the flow velocity of every kind of synthetic agent.433A synthesizer: send Flow Rate1-18 to synthesizer, select Main Menu-Module Test-to look for Module A, ModuleD, ModuleI, ModuleI, Module A-to measure or observe by more by Start-by Prer or next, if flow is improper, then regulate lower valve pressure, until reaching requirement.
1.1.3 the synthetic beginning of polypeptide antigen
The method Std Fmoc 1.0Sol DIC90 that will synthesize needs in the program of 433A synthesizer sends on the synthesizer.File-New-Sequence-edits the sequence of synthetic peptide, preserves.File-New-Run checks Chemistry; Whether Sequence is by being deposited name; Set Cycles; Preserve.Send on the synthesizer at last.
Main Menu-Cycle Monitor-begin brings into operation.
1.1.4 polypeptide antigen is synthetic
As above-mentioned peptide sequence, be to begin end in the time of synthetic to N from the C end, according to given order, constantly be repeated below synthesis step successively:
(1) deprotection reaction: it is the nmp solution of the hexahydropyridine of 15%-30% that above-mentioned aminoresin is placed volume percent, the Fmoc blocking group that reaction removed on the aminoresin in 25-40 minute under 20-28 ℃ of condition;
(2) washing: nitrogen dries up, and NMP washs aminoresin;
(3) condensation reaction: the amino acid that adds HOBT, DCC and Fmoc protection reacted 0.5-2.5 hour under 20-28 ℃ of condition;
(4) washing: nitrogen dries up, and NMP washs aminoresin;
(5) capping: adding percent weight in volume is the nmp solution of the acetyl imidazole of 1.5%-4%, and reaction is 20-40 minute under 20-28 ℃ of condition.
1.1.5 polypeptide antigen end of synthesis
Synthesizer will stop automatically behind the antigen end of synthesis.Take off reactor from Peptide synthesizer then, use 100% methanol wash polypeptide resin 3 times again, dry up in stink cupboard then, polypeptide resin is transferred in the brown bottle, put into-20 ℃ of refrigerators, it is standby to seal film phonograph seal.
1.2 the cracking of polypeptide antigen and evaluation
1.2.1 the cracking of polypeptide antigen
According to volume ratio (TFA/TIS/ phenol/H
2O=85/8/6/1) preparation lysate, in refrigerator, take out the synthetic polypeptide resin then, put into round-bottomed flask, in stink cupboard, in flask, add lysate and the magnetic stick for preparing, stably be placed on then on the magnetic stirring apparatus, continue to stir 1 hour under the room temperature until reacting completely.After reaction finishes, use the Rotary Evaporators of band cold-trap to continue the TFA that evaporation was removed in the thick product in 30 to 120 minutes.Then use ether to collect, precipitate polypeptide, use dimethyl formamide (DMF) repeatedly to clean the crude product of polypeptide antigen then, at last the resin that mixes is filtered out with sand core funnel, promptly obtain polypeptide antigen.
1.2.2 the evaluation 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) in the synthetic back that finishes of polypeptide antigen.
1.3 the cyclisation of polypeptide antigen
With 10%DMSO polypeptide antigen is mixed with the polypeptide solution that concentration is 2mg/ml, adjust pH value=8.5 of initial gross separation polypeptide solution then with 0.1N NaOH or 0.1N HCl, be to place 48 hours on the shaking table of 110rpm at rotating speed under 25 ℃ the environment, make the formation disulfide linkage, can obtain can simulated virus particle native conformation the polypeptide cyclized structure.
1.4 the purifying degerming of polypeptide antigen
Polypeptide antigen uses circulating tangential flow filtration film to wrap in to carry out under the 20-28 ℃ of condition ultrafiltration (Tangential Flow Device circulating tangential flow filtration film bag and the peristaltic pump supporting with it produced with PALL company), polypeptide antigen is that macromole can not be by the filter membrane of certain pore size, and the small molecular weight impurity that building-up process and later stage cyclization formed or introduced early stage then can pass through filter membrane.And then be 0.2 μ m pot strainer degerming by the aperture, the solution branch that obtains is at last installed in the aseptic plastic bottle, labelled.Indicate title, numbering, product batch number, concentration, date manufactured, storage life and the preservation condition of polypeptide on the label, it is standby after the packing, to be stored in-20 ℃ or-40 ℃.
Needs for the ease of transportation and prolonged preservation carry out lyophilize to obtain the polypeptide of solid state with polypeptide antigen.Take out freezing good polypeptide antigen in advance, on the Labconco freeze drier, carry out drying, obtain the polypeptide antigen of solid state.Simultaneously labelled.Indicate title, numbering, product batch number, concentration, date manufactured, storage life and the preservation condition of polypeptide on the label.
The preparation of embodiment 2. synthetic peptide vaccines
2.1 the preparation of antigen water
Take by weighing at first, respectively according to the foregoing description 1 synthetic three peptide species antigens; Then, with sterilized water for injection with antigenic synthetic peptide concentration dilution to 50 μ g/ml; With the antigenic solution via hole diameter is the strainer filtration of 0.2 μ m, degerming.
2.2 oil phase adjuvant preparation
The oil phase adjuvant is through sterilization in 121 ℃, 30 minutes, standby.
2.3 the emulsification of synthetic peptide vaccine
Distilled water 2000ml with sterilization cleans the IKA emulsifying device 3 times, under 20-28 ℃ of condition be 1: 1 volume ratio then by oil phase adjuvant and antigen water, earlier oil phase is added in the emulsion tank, after starting motor and stirring 2 minutes with 90～150r/m slow rotation, add simultaneously slowly water antigen, add the back and stirred 30 minutes, again with 10000r/m high-speed stirring 20 minutes, left standstill 5 minutes, and made vaccine be emulsified into water in oil single-phase vaccine.
The potency test of embodiment 3 Schweineseuche O-shaped synthetic peptide vaccines
1. materials and methods
1.1 synthetic peptide vaccine
Have SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 polypeptide of sequence antigen according to the foregoing description is synthetic, the corresponding with it lot number of preparation is respectively then: the Schweineseuche O-shaped synthetic peptide vaccine of ZM433A01, ZM433A02, ZM433A03.
In addition, with all Xie Ansuan is alternative with norvaline in the aminoacid sequence among the SEQ ID NO:1; All leucines substitute with nor-leucine, and it is synthetic that the method that provides according to the foregoing description is carried out antigen, and preparation vaccine lot number is: ZM433A04.
Use the dimer antigen of synthetic this sequence of conventional synthetic peptide technology according to SEQ ID NO:1 sequence, the preparation vaccine obtains lot number and is: the synthetic peptide vaccine of ZM433A05.
1.2 experimental animal
It is identical to select kind, about 4 monthly ages, body weight 40Kg, and 77 of the healthy feeder pigs that the foot and mouth disease neutralizing antibody is negative.
1.3 seed culture of viruses OR/80MF
8
With OR/80MF
6After suckling mouse passed for 2 generations, collect virus.Suckling mouse is surveyed ℃ freezing preservation of poison qualified being placed on-20, and is standby.
1.4 test method
At 15 of the healthy susceptible feeder pigs (measuring no foot and mouth disease neutralizing antibody) about every group of vaccine employing body weight 40kg, be divided into 3 groups, 5 every group through the suckling mouse neutralization test.Each batch synthetic peptide vaccine to be checked is divided into 1 part, 1/3 part, 3 dosage groups of 1/9 part, each dosage group respectively at the basal part of the ear after 5 pigs of intramuscular injection.Inoculate after 28 days, together with 2 of the identical contrast pigs of condition, 1000 ID of intramuscular injection behind every pig basal part of the ear
50The strong malicious OR/80MF of Schweineseuche O C-type virus C
8, observed continuously 10.The bubble pathology appears in all at least one hoof of contrast pig.Immune swine any foot and mouth disease symptom occurs and promptly is judged to and does not protect.According to the protection number of immune swine, calculate the PD of tested vaccine by the Reed-Muench method
50
1.5 the result judges
Foot and mouth disease typical case bubble appears in the above or snout of pig one hoof, then is judged to morbidity.Any position of pig does not have the foot and mouth disease typical water and bubbles out the existing protection that then is judged to.
2. test-results and discussion
2.1 test-results
After the immunity 28 days, with 1000 ID
50The OR/80MF of/head part
8Behind the strong virus attack 10 days, the result sees table 1 for details.
Table 1 pig O type aftosa synthetic peptide vaccine potency test
2.2 discussion of results
From this test-results as can be seen, 3 PD have all been reached behind the Schweineseuche O-shaped synthetic peptide vaccine immune animal of the present invention
50More than, satisfied the aftosa vaccine requirement of Ministry of Agriculture's regulation.Simultaneously, discover and carried out that amino acid is replaced and antigen polymeric synthetic peptide vaccine has better immune effect.The Schweineseuche O-shaped synthetic peptide vaccine that these antigen preparations are described has good immune efficacy and good clinical using value.
Sequence table
＜110〉Zhongmu Industry Co.,Ltd
＜120〉O type aftosa synthetic peptide vaccine
<130>DIC08110081
<160>3
<170>PatentIn?version?3.3
<210>1
<211>61
<212>PRT
＜213〉artificial sequence
<400>1
Ile?Ser?Ile?Ser?Glu?Ile?Gly?Lys?Val?Ile?Val?Lys?His?Ile?Glu?Gly
1 5 10 15
Ile?Phe?Leu?Leu?Val?Tyr?Asn?Gly?Asn?Cys?Lys?Tyr?Gly?Glu?Asn?Ala
20 25 30
Val?Thr?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu?Ala?Gln?Lys?Ala?Ala
35 40 45
Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys
50 55 60
<210>2
<211>61
<212>PRT
＜213〉artificial sequence
<400>2
Ile?Ser?Ile?Thr?Glu?Ile?Arg?Thr?Val?Ile?Val?Arg?Thr?Ile?Glu?Thr
1 5 10 15
Ile?Phe?Leu?Leu?Val?Tyr?Asn?Gly?Asn?Cys?Lys?Tyr?Gly?Glu?Asn?Ala
20 25 30
Val?Thr?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu?Ala?Gln?Lys?Ala?Ala
35 40 45
Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys
50 55 60
<210>3
<211>61
<212>PRT
＜213〉artificial sequence
<400>3
Ile?Ser?Ile?Thr?Glu?Ile?Gly?Lys?Val?Ile?Val?Lys?Thr?Ile?Glu?Gly
1 5 10 15
Ile?Phe?Leu?Leu?Val?Tyr?Asn?Gly?Asn?Cys?Lys?Tyr?Gly?Glu?Asn?Ala
20 25 30
Val?Thr?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu?Ala?Gln?Lys?Ala?Ala
35 40 45
Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys
50 55 60
Claims (16)
1. polypeptide that is used for O type aftosa synthetic peptide vaccine, wherein said polypeptide has the aminoacid sequence shown in SEQID NO.1, SEQ ID NO.2 or the SEQ ID NO.3.
2. polypeptide according to claim 1 is characterized in that one or more Xie Ansuan is substituted by norvaline in the described aminoacid sequence; And/or one or more leucine is substituted by nor-leucine.
3. polypeptide according to claim 2 is characterized in that, whole Xie Ansuans are substituted by norvaline in the described aminoacid sequence; And/or all leucine is substituted by nor-leucine.
4. according to each described polypeptide in the claim 1 to 3, it is characterized in that the sulfydryl of two halfcystines in the described aminoacid sequence is joined together to form disulfide linkage through oxidation.
5. according to each described polypeptide in the claim 1 to 4, it is characterized in that reaction forms covalently bound between the head and the tail amino-acid residue of described aminoacid sequence.
6. polypeptide according to claim 5 is characterized in that, reaction forms covalently bound between the carboxyl of the head and the tail amino-acid residue of described aminoacid sequence and amino or carboxyl and the hydroxyl.
7. an aftosa synthetic peptide vaccine is selected from each described polypeptide or its polypeptide polymer in the claim 1 to 6 comprising one or more.
8. vaccine according to claim 7 is characterized in that described vaccine comprises adjuvant.
9. preparation method according to each described polypeptide in the claim 1 to 6, comprising following steps:
(1) with aminoresin be starting raw material, the amino acid of protecting with 9-fluorenylmethyloxycarbonyl is monomer, connects amino acid according to described aminoacid sequence condensation successively, seals unreacted aminoterminal with acetyl imidazole after per step condensation reaction;
(2) the synthetic back that finishes adds lytic reagent cracking polypeptide;
(3) use ether to collect, precipitate polypeptide;
(4) carry out aseptically process behind the ultrafiltration purification polypeptide.
10. preparation method according to claim 9 is characterized in that, the component volume ratio of described lytic reagent is a trifluoroacetic acid: tri isopropyl silane: phenol: H
2O=85: 8: 6: 1.
11., it is characterized in that the described cracking time is 1-4 hour according to claim 9 or 10 described preparation methods.
12., it is characterized in that described step (1) may further comprise the steps according to each described preparation method in the claim 9 to 11:
(a) deprotection reaction: it is N-Methyl pyrrolidone (NMP) solution of the hexahydropyridine of 15-30% that aminoresin is placed volume percent, the 9-fluorenylmethyloxycarbonyl blocking group that reaction removed on the aminoresin in 25-40 minute under 20-28 ℃ of condition;
(b) washing: nitrogen dries up, N-Methyl pyrrolidone washing aminoresin;
(c) condensation reaction: the amino acid that adds 1-hydroxyl azimidobenzene (HOBT), dicyclohexylcarbodiimide (DCC) and 9-fluorenylmethyloxycarbonyl protection reacted 0.5-2.5 hour under 20-28 ℃ of condition;
(d) washing: nitrogen dries up, N-Methyl pyrrolidone washing aminoresin;
(e) capping: adding percent weight in volume is N-Methyl pyrrolidone (NMP) solution of the acetyl imidazole of 1.5-4%, and reaction is 20-40 minute under 20-28 ℃ of condition.
13., it is characterized in that described step (4) may further comprise the steps according to each described preparation method in the claim 9 to 12:
(a) use the tangential flow filtration film to wrap in ultrafiltration polypeptide antigen under the 20-28 ℃ of condition, remove small molecular weight impurity;
(b) use 0.2 micron online filter degerming to preserve.
14. the preparation method according to claim 7 or 8 described vaccines, comprising following steps:
(1) with water for injection aforementioned polypeptides or its polypeptide polymer are diluted to 50 μ g/ml and make the antigen water;
(2) adjuvant is standby through sterilization in 121 ℃, 30 minutes;
(3) under 20-28 ℃ of condition, according to antigen water and 1: 1 volume ratio of adjuvant, earlier adjuvant is added in the emulsion tank, 90-150 rev/min was stirred at a slow speed 1.5-3 minute, slowly add water, add the back and stirred 20-30 minute, high speed 8000-10000 rev/min was stirred 15-30 minute again, left standstill 5 minutes, after the packing promptly.
15. treat and/or prevent purposes in the medicine of O type foot and mouth disease in preparation according to each described polypeptide or its polypeptide polymer, claim 7 or 8 described vaccines in the claim 1 to 6.
16. purposes according to claim 15 is characterized in that, described O type foot and mouth disease is a pig O type foot and mouth disease.
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| CN200810118967A CN101659695B (en) | 2008-08-27 | 2008-08-27 | O-type aftosa synthetic peptide vaccine |
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| CN101659695B CN101659695B (en) | 2012-08-29 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838658A (en) * | 2010-04-30 | 2010-09-22 | 中国农业科学院哈尔滨兽医研究所 | O type foot-and-mouth disease virus variant as well as coding gene and application thereof |
| CN103214560A (en) * | 2013-03-25 | 2013-07-24 | 中国牧工商(集团)总公司 | Polypeptide used for preparing bovine foot and mouth disease type O peptide vaccine, and preparation method and application thereof |
| CN103408641A (en) * | 2013-08-13 | 2013-11-27 | 中牧实业股份有限公司 | Foot and mouth disease virus structural protein antibody enzyme-linked immunosorbent assay kit |
| CN103864905A (en) * | 2013-12-30 | 2014-06-18 | 大连大学 | CTL (Cytotoxic T Lymphocyte) epitope peptide of foot-and-mouth disease virus type O and screening method of CTL epitope peptide |
| CN105820217A (en) * | 2014-03-07 | 2016-08-03 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN110885362A (en) * | 2019-11-22 | 2020-03-17 | 中牧实业股份有限公司 | O-type synthetic peptide vaccine for foot-and-mouth disease and preparation method and application thereof |
| CN110894214A (en) * | 2019-11-22 | 2020-03-20 | 中牧实业股份有限公司 | Foot-and-mouth disease O-type epitope polypeptide and preparation method and application thereof |
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| US6107021A (en) * | 1998-06-20 | 2000-08-22 | United Biomedical, Inc. | Synthetic peptide vaccines for foot-and-mouth disease |
| CN1287858C (en) * | 2002-09-16 | 2006-12-06 | 复旦大学 | Foot-and-mouth disease gene engineering polypeptide vaccine and its preparing method |
| CN100381170C (en) * | 2003-09-03 | 2008-04-16 | 上海华谊生物技术有限公司 | Foot and mouth disease bivalent polypeptide vaccine and its preparation method and use |
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| CN103408641B (en) * | 2013-08-13 | 2015-06-03 | 中牧实业股份有限公司 | Foot and mouth disease virus structural protein antibody enzyme-linked immunosorbent assay kit |
| CN103864905A (en) * | 2013-12-30 | 2014-06-18 | 大连大学 | CTL (Cytotoxic T Lymphocyte) epitope peptide of foot-and-mouth disease virus type O and screening method of CTL epitope peptide |
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| CN105820217A (en) * | 2014-03-07 | 2016-08-03 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN105820217B (en) * | 2014-03-07 | 2019-06-07 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN110885362A (en) * | 2019-11-22 | 2020-03-17 | 中牧实业股份有限公司 | O-type synthetic peptide vaccine for foot-and-mouth disease and preparation method and application thereof |
| CN110894214A (en) * | 2019-11-22 | 2020-03-20 | 中牧实业股份有限公司 | Foot-and-mouth disease O-type epitope polypeptide and preparation method and application thereof |
| CN111803626A (en) * | 2020-07-08 | 2020-10-23 | 申联生物医药（上海）股份有限公司 | Bivalent synthetic peptide vaccine for O-type and A-type pig foot-and-mouth disease and preparation method and application thereof |
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