CN101565457B - Synthetic peptide vaccine and preparation method thereof - Google Patents
Synthetic peptide vaccine and preparation method thereof Download PDFInfo
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- CN101565457B CN101565457B CN200910143571.XA CN200910143571A CN101565457B CN 101565457 B CN101565457 B CN 101565457B CN 200910143571 A CN200910143571 A CN 200910143571A CN 101565457 B CN101565457 B CN 101565457B
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Abstract
The invention provides aftosa synthetic peptide vaccine, more specifically to polypeptide or polypeptide polymer used for O type aftosa synthetic peptide vaccine, vaccine containing the polypeptide orpolypeptide polymer and a preparation method thereof. The polypeptide has amino acid sequence represented by SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3. By sequencing of recent aftosa strain in China an d combination of aftosa vaccine strain OS/99 and OZK/93 sequence, the invention researches variation case of main antigen site, carries out antigen site analysis and forecast assisted by computer, andcarries out chemical synthesis to possible antigen site peptide segment. The invention screens polypeptide antigen through a large amount of animal tests, optimizes aftosa virus antigen site accordin g to screening result, effectively combines T cell epitope and B cell epitope, and enhances immunization effect of the polypeptide antigen. The O type aftosa synthetic peptide vaccine can effectively reply antigenic variation of aftosa virus, has good biological safety, is easy for large scale production, and has better application prospect.
Description
Technical field
The present invention relates to a kind of polypeptide for aftosa synthetic peptide vaccine or its polypeptide polymer, and the vaccine that contains this polypeptide or its polypeptide polymer and their preparation method, be specifically related to a kind of polypeptide for O type aftosa synthetic peptide vaccine or its polypeptide polymer, and the vaccine that contains 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 is high, to propagate rapidly, the livestocks such as infected pigs, ox, sheep cause cub dead, and adult animals throughput sharply declines, the production and supply of the development of serious harm livestock industry and meat and livestock product thereof.Can make market circulation and the international trade of animal and animal's products be subject to blocking greatly and restriction simultaneously, produce to the livestock industry of popular countries and regions and cause huge financial loss.Foot and mouth disease is infected and is caused that Hostis picornavirus has the features such as polytypism, volatility by foot and mouth disease virus (FMDV).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, the existing kind more than 70 of hypotype of finding at present.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 comprised 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, has now found that O type foot and mouth disease has 3 main antigen sites to be positioned at VP1 upper, and wherein 133-160 and 200-213 position have formed the upper protective antigen site most important and that the most easily make a variation of VP1.
The current foot and mouth disease at China's Major Epidemic is O type and Asia I type foot and mouth disease.Antigenicity between foot and mouth disease virus is various is different, each other can not Immunogenicity.And the degree of antigenic difference is also very large in same serotype, to such an extent as to the aftosa vaccine that can effectively resist 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 effect of original vaccine weakens even disappearance, has brought 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 of preventing and treating foot and mouth disease.And the aftosa vaccine of the existing use of China is mainly viral inactivation vaccine, there is the problems such as biological safety is poor, side reaction large, unstable product quality.A lot of countries have stopped using inactivated vaccine in the world at present, 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,, all existing problems aspect immune effect, biological safety, has affected the use of these new generation vaccines.In addition, often effect is poor for the current epidemic isolates having 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 or its polypeptide polymer, and the vaccine that contains this polypeptide or its polypeptide polymer, especially for polypeptide or its polypeptide polymer of O type aftosa synthetic peptide vaccine, and the vaccine that contains this polypeptide or its polypeptide polymer.
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, the present invention has adopted following technical scheme:
For a polypeptide for O type aftosa synthetic peptide vaccine, wherein said polypeptide has the aminoacid sequence shown in SEQ IDNO.1, SEQ ID NO.2 or SEQ ID NO.3.
In above-mentioned aminoacid sequence, one or more α-amino-isovaleric acid can be substituted by norvaline; And/or one or more leucine can be substituted by nor-leucine.Preferably, in above-mentioned aminoacid sequence, all α-amino-isovaleric acid is substituted by norvaline; And/or all leucine is substituted by nor-leucine.
The sulfydryl of two halfcystines in above-mentioned aminoacid sequence can be joined together to form disulfide linkage through oxidation.
Between the head and the tail amino-acid residue of above-mentioned aminoacid sequence, can react formation covalently bound.Specifically, between the carboxyl of the head and the tail amino-acid residue of above-mentioned aminoacid sequence and amino or carboxyl and hydroxyl, react formation covalently bound.
An aftosa synthetic peptide vaccine, 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 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) take aminoresin as starting raw material, the amino acid of the 9-fluorenylmethyloxycarbonyl of take protection is monomer, according to described aminoacid sequence successively condensation, connects amino acid, after every step condensation reaction, with acetyl imidazole, seals unreacted aminoterminal;
(2) after synthetic, add lytic reagent cracking polypeptide;
(3) use ether to collect, precipitate polypeptide;
(4) after ultrafiltration purification polypeptide, carry out aseptically process.
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; 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 to the N-Methyl pyrrolidone that volume percent is the hexahydropyridine of 15-30% (NMP) solution, reacts the 9-fluorenylmethyloxycarbonyl blocking group removing for 25-40 minute on aminoresin under 20-28 ℃ of condition;
(b) washing: nitrogen dries up, N-Methyl pyrrolidone washing aminoresin;
(c) condensation reaction: add I-hydroxybenzotriazole (HOBT), dicyclohexylcarbodiimide (DCC) to react 0.5-2.5 hour with the amino acid of 9-fluorenylmethyloxycarbonyl protection 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%, reacts 20-40 minute under 20-28 ℃ of condition.
In above-mentioned preparation method, described step (4) specifically comprises the following steps:
(a) use tangential flow filtration film to wrap in the polypeptide that under 20-28 ℃ of condition, ultrafiltration makes, remove small molecular weight impurity;
(b) use 0.2 micron of 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 antigen water;
(2) adjuvant is standby through sterilizing in 121 ℃, 30 minutes;
(3) under 20-28 ℃ of condition, according to the volume ratio of antigen water 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 water, add rear stirring 20-30 minute, then high speed 8000-10000 rev/min is stirred 15-30 minute, standing 5 minutes, after packing and get final product.
The present invention further provides aforementioned polypeptides or its polypeptide polymer, vaccine and treated and/or prevented the 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 domestic foot and mouth disease sequencing combined mouth fever aphthous vaccine strain OS/99 and the OZK/93 sequence of epidemic isolates recently, the variation situation in research foot and mouth disease major antigen site, for the amino acid sites of main variation, add up the frequency of its variation, in conjunction with area of computer aided, carry out the analyses and prediction in foot-and-mouth disease antigen site simultaneously, possible antigen site peptide section is carried out to chemosynthesis, for easy variant sites according to statistics variation frequency, in these sites, use different amino acid, obtain containing current likely multiple candidate's polypeptide antigen of variant sites.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 is high, the polypeptide antigen of the attack of avoiding foot and mouth disease epidemic isolates of can be good at watching for animals.The present invention is optimized foot-and-mouth disease virus antigen site according to screening experiment result, and has effectively combined 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 current foot and mouth disease virus, not have biological safety, is easy to extensive synthesizing, and has a good application prospect.
Embodiment
The solid phase synthesis of embodiment 1 aftosa synthetic peptide antigen
Polypeptide antigen of the present invention can be used 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 Rink Amide mbha resin.Production process generally includes the solid phase synthesis of polypeptide antigen, the cracking of polypeptide, antigen purification and degerming are preserved.
The solid phase synthesis of 1.1 polypeptide antigens
1.1.1 synthesis material is prepared
The sequence of synthetic polypeptide antigen is respectively as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3.
Sequence and synthetic scale according to antigen are that 1mmol prepares the amino acid that suitable Fmoc modifies, and add in corresponding amino acid bottle.Weigh equally on request Rink Amide mbha resin, put into reaction chamber, upper and lower lid is tightened, label, record title, lot number, the tare weight of reaction chamber and the weight of alleged resin of synthesized peptide.Pack reaction chamber into synthesizer.Preparation synthetic agent, comprises that N-Methyl pyrrolidone (NMP), acetyl imidazole (AIM), piperidines (PIP), methyl alcohol etc. are placed in corresponding reagent bottle.
1.1.2 synthesizer state-detection
Check whether normally operation of Peptide synthesizer.After start, operation Run Self Test program, whether instrument self checking indices is normal.Check in addition N
2whether sufficient, whether system gauge pressure is normal.Before synthetic, the performance of reply instrument is had gained some understanding, so will measure the flow velocity of every kind of synthetic agent.Send Flow Ratc1-18 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, regulate lower valve pressure, until reach requirement.
1.1.3 the synthetic beginning of polypeptide antigen
In the program of synthesizer, the synthetic method Std Fmoc 1.0Sol DIC90 needing 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; Set Cycles; Preserve.Finally send on synthesizer.
Main Menu-Cycle Monitor-begin, brings into operation.
1.1.4 polypeptide antigen is synthetic
Peptide sequence described above, is to start the end to N from C end in the time of synthetic, according to given order, is constantly repeated below successively synthesis step:
(1) deprotection reaction: above-mentioned aminoresin is placed in to the nmp solution that volume percent is the hexahydropyridine of 15%-30%, reacts the Fmoc blocking group removing for 25-40 minute on aminoresin under 20-28 ℃ of condition;
(2) washing: nitrogen dries up, and NMP washs aminoresin;
(3) condensation reaction: add HOBT, DCC to react 0.5-2.5 hour with the amino acid of Fmoc protection 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%, reacts 20-40 minute under 20-28 ℃ of condition.
1.1.5 polypeptide antigen end of synthesis
After antigen end of synthesis, synthesizer will stop automatically.Then from Peptide synthesizer, take off reactor, then use 100% methanol wash polypeptide resin 3 times, then in stink cupboard, dry up, polypeptide resin is transferred in brown bottle, put into-20 ℃ of refrigerators, sealed membrane sealing is standby.
The cracking of 1.2 polypeptide antigens 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, then in refrigerator, take out synthetic polypeptide resin, put into round-bottomed flask, in stink cupboard, in flask, add lysate and the magnetic stick preparing, then be stably placed on magnetic stirring apparatus, under room temperature, continue to stir 1 hour until react completely.After reaction finishes, use with the lasting evaporation of Rotary Evaporators of cold-trap and within 30 to 120 minutes, remove the TFA in thick product.Then use ether to collect, precipitate polypeptide, then use dimethyl formamide (DMF) repeatedly to clean the crude product of polypeptide antigen, finally the resin mixing is filtered out with sand core funnel, obtain polypeptide antigen.
1.2.2 the evaluation of synthetic antigen
After polypeptide antigen is synthetic, with substance assistant laser desorpted time-of-flight mass spectrometry (TOFMS) (MALDL-TOF) and reversed-phase high pressure liquid chromatography (RP-HPLC), carry out qualitative and quantitative analysis.
The conformation of 1.3 polypeptide antigens forms
With 15%DMSO, polypeptide antigen is mixed with to the polypeptide solution that concentration is 2mg/ml, then with 0.1N NaOH or 0.1N HCl, adjust pH value=8.5 of initial gross separation polypeptide solution, on the shaking table that is 110rpm at rotating speed under the environment of 25 ℃, place 48 hours, make to form disulfide linkage.
And then carrying out head and the tail cyclisation, " COOH " of head and the tail is shown in the Peptide Protein Reserch 1996.48:229-239 such as Mengfen with " NH2 " cyclization method; " COOH " of head and the tail reacts with " OH " and forms ring texture and see the Chem.Soc 1970.92:3771-3777 such as Mmenhofer.Can obtain can simulated virus particle native conformation polypeptide cyclized structure.
The purifying degerming of 1.4 polypeptide antigens
Polypeptide antigen is used circulating tangential flow filtration film to wrap under 20-28 ℃ of condition, to carry out ultrafiltration (Tangential Flow Device circulating tangential flow filtration film bag and the peristaltic pump supporting with it), 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 can pass through filter membrane.And then be 0.2 μ m pot strainer degerming by aperture, the solution finally obtaining is divided and installed in aseptic plastic bottle, labelled.On label, indicate title, numbering, product batch number, concentration, date manufactured, storage life and the preservation condition of polypeptide, after packing, be stored in-20 ℃ or-40 ℃ standby.
For the ease of transportation and the long-term needs of preserving, polypeptide antigen is carried out to lyophilize to obtain the polypeptide of solid state.The polypeptide antigen having frozen is in advance taken out, on Labconco freeze drier, be dried, obtain the polypeptide antigen of solid state.Simultaneously labelled.On label, indicate title, numbering, product batch number, concentration, date manufactured, storage life and the preservation condition of polypeptide.
The preparation of embodiment 2. synthetic peptide vaccines
The preparation of 2.1 antigen waters
First, take respectively according to three synthetic peptide species antigens of above-described embodiment 1; Then, with sterilized water for injection by antigenic synthetic peptide concentration dilution to 50 μ g/ml; The strainer that is 0.2 μ m by antigenic solution via hole diameter filters, degerming.
2.2 oil phase adjuvant preparations
Oil phase adjuvant is through sterilizing in 121 ℃, 30 minutes, standby.
The emulsification of 2.3 synthetic peptide vaccines
With the distilled water 2000ml of sterilizing, clean IKA emulsifying device 3 times, then the volume ratio that is 1: 1 by oil phase adjuvant and antigen water under 20-28 ℃ of condition, first oil phase is added in emulsion tank, starting motor stirs after 2 minutes with 90～150r/m slow rotation, simultaneously slowly add water antigen, add rear stirring 30 minutes, then with 10000r/m high-speed stirring 20 minutes, standing 5 minutes, make vaccine be emulsified into water in oil single-phase vaccine.
The potency test of embodiment 3 synthetic peptide vaccines
1. materials and methods
1.1 synthetic peptide vaccine
According to the synthetic polypeptide antigen with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 sequence of above-described embodiment, then respectively preparation with it corresponding lot number be: the foot and mouth disease O type synthetic peptide vaccine of ZM433A01, ZM433A02, ZM433A03.
In addition, by aminoacid sequence in SEQ ID NO:1, all α-amino-isovaleric acid is alternative with norvaline; All leucine substitutes with nor-leucine, and it is synthetic that the method providing according to above-described embodiment is carried out antigen, and preparation vaccine lot number is: ZM433A04.
According to SEQ ID NO:1 sequence, use the dimer antigen of synthetic this sequence of conventional synthetic peptide technology, preparation vaccine, obtains lot number and is: the synthetic peptide vaccine of ZM433A05.
1.2 experimental animal
Select kind identical, 4 monthly ages, body weight 40Kg left and right, 27 of the health frame piglings that foot and mouth disease neutralizing antibody is negative.
1.3 seed culture of viruses OR/80MF
8
By OR/80MF
6after suckling mouse passed for 2 generations, collect virus.Suckling mouse is surveyed ℃ freezing preservation of poison qualified being placed on-20, standby.
1.4 test method
For 5 of the healthy susceptible feeder pigs (measuring without foot and mouth disease neutralizing antibody through suckling mouse neutralization test) about every group of vaccine employing body weight 40kg.By each batch of synthetic peptide vaccine to be checked respectively at the basal part of the ear after intramuscular injection 2ml.Inoculate after 28 days, together with 2 of the identical contrast pigs of condition, 1000 ID of intramuscular injection 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 contrast pig all at least one hoof.Immune swine occurs that any foot and mouth disease symptom is judged to and does not protect.
1.5 results are judged
There is foot and mouth disease typical case bubble in the above or snout of pig one hoof, is judged to morbidity.Any position of pig bubbles out the existing protection that is judged to without foot and mouth disease typical water.
2. test-results and discussion
2.1 test-results
After immunity 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
From this test-results, can find out, after this animal of foot and mouth disease O type synthetic peptide vaccine immunity pig of the present invention, be up to 100% protection.Meanwhile, research finds that the synthetic peptide vaccine that has carried out amino acid substitution and antigen polymerization has better immune effect.The foot and mouth disease O type synthetic peptide vaccine that these antigen preparations are described has good immune efficacy and clinical value.
The safety testing of embodiment 4 synthetic peptide vaccines
1, test method
10 of the cavys of 1.1 use body weight 350～450g, every subcutaneous injection vaccine 2ml; With 25 of the mouse of body weight 18～22g, every subcutaneous injection vaccine 0.5ml., all must not there is dead or obvious local untoward reaction or the systemic reaction that because of vaccinate, cause in Continuous Observation 7 days.
10 of the piglets of 1.2 use 30～40 ages in days (measuring without foot and mouth disease neutralizing antibody through suckling mouse neutralization test), after each two Herba Houttuyniae, intramuscular injection vaccine 2ml (every side 1ml), observes 14 day by day.All must not there is foot and mouth disease symptom or the toxic reaction significantly causing because of vaccinate.
2, test-results
The security of 2.1 vaccines to cavy and mouse
10 of cavys, every subcutaneous injection vaccine 2ml; 25 of mouse, every subcutaneous injection 0.5ml., all there is not dead or obvious local untoward reaction or the systemic reaction that cause because of vaccinate in Continuous Observation 7 days, concrete outcome is as following table 1.
The result of table 1 vaccine to cavy and mouse safety testing
The security of 2.2 vaccines to piglet
Synthetic peptide vaccine is taken out and equilibrated to after room temperature, to susceptible piglet (measuring without foot and mouth disease neutralizing antibody through suckling mouse neutralization test), 2 part vaccines of intramuscular injection after each two Herba Houttuyniae, a side 1ml, observes 14 day by day.All there is not foot and mouth disease symptom or the toxic reaction significantly causing because of vaccinate.Concrete outcome is in Table 2.
Table 2 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 has the side reaction problems such as heating, redness, so have good promotion prospect and marketable value.
Sequence table
<110> Zhongmu Industry Co.,Ltd
<120> synthetic peptide vaccine and preparation method thereof
<130> synthetic peptide vaccine and preparation method thereof
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Claims (13)
1. for a polypeptide for O type aftosa synthetic peptide vaccine, the aminoacid sequence of wherein said polypeptide is as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.
2. for a polypeptide for O type aftosa synthetic peptide vaccine, wherein said polypeptide is by whole α-amino-isovaleric acids in the aminoacid sequence as shown in SEQ ID NO.1 are substituted and obtained with nor-leucine with alternative, the whole leucines of norvaline.
3. an aftosa synthetic peptide vaccine, it comprises that one or more are selected from polypeptide claimed in claim 1.
4. an aftosa synthetic peptide vaccine, it comprises polypeptide according to claim 2; Or the dimer that comprises the polypeptide of aminoacid sequence as shown in SEQ ID NO.1.
5. according to the vaccine described in claim 3 or 4, it is characterized in that, described vaccine comprises adjuvant.
6. a preparation method for polypeptide according to claim 1 and 2, comprising following steps:
(1) take aminoresin as starting raw material, the amino acid of the 9-fluorenylmethyloxycarbonyl of take protection is monomer, according to described aminoacid sequence successively condensation, connects amino acid, after every step condensation reaction, with acetyl imidazole, seals unreacted aminoterminal;
(2) after synthetic, add lytic reagent cracking polypeptide;
(3) use ether to collect, precipitate polypeptide;
(4) after ultrafiltration purification polypeptide, carry out aseptically process.
7. preparation method according to claim 6, is characterized in that, the component volume ratio of described lytic reagent is trifluoroacetic acid: tri isopropyl silane: phenol: H
2o=85:8:6:1.
8. according to the preparation method described in claim 6 or 7, it is characterized in that, described pyrolysis time is 1-4 hour.
9. according to the preparation method described in claim 6 or 7, it is characterized in that, described step (1) comprises the following steps:
(a) deprotection reaction: aminoresin is placed in to the N-Methyl pyrrolidone that volume percent is the hexahydropyridine of 15-30% (NMP) solution, reacts the 9-fluorenylmethyloxycarbonyl blocking group removing for 25-40 minute on aminoresin under 20-28 ℃ of condition;
(b) washing: nitrogen dries up, N-Methyl pyrrolidone washing aminoresin;
(c) condensation reaction: add I-hydroxybenzotriazole (HOBT), dicyclohexylcarbodiimide (DCC) to react 0.5-2.5 hour with the amino acid of 9-fluorenylmethyloxycarbonyl protection 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%, reacts 20-40 minute under 20-28 ℃ of condition.
10. according to the preparation method described in claim 6 or 7, it is characterized in that, described step (4) comprises the following steps:
(a) use tangential flow filtration film to wrap in ultrafiltration polypeptide antigen under 20-28 ℃ of condition, remove small molecular weight impurity;
(b) use 0.2 micron of online filter degerming to preserve.
The preparation method of 11. 1 kinds of vaccines according to claim 3, comprising following steps:
(1) with water for injection, one or more being selected to polypeptide claimed in claim 1 is diluted to 50 μ g/ml and makes antigen water;
(2) adjuvant is standby through sterilizing in 121 ℃, 30 minutes;
(3) under 20-28 ℃ of condition, according to the volume ratio of antigen water 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 water, add rear stirring 20-30 minute, then high speed 8000-10000 rev/min is stirred 15-30 minute, standing 5 minutes, after packing and get final product.
12. 1 kinds according to the preparation method of the vaccine described in claim 4 or 5, comprising following steps:
(1) with the dimer of water for injection polypeptide as shown in SEQ ID NO.1 by polypeptide according to claim 2 or aminoacid sequence, be diluted to 50 μ g/ml and make antigen water;
(2) adjuvant is standby through sterilizing in 121 ℃, 30 minutes;
(3) under 20-28 ℃ of condition, according to the volume ratio of antigen water 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 water, add rear stirring 20-30 minute, then high speed 8000-10000 rev/min is stirred 15-30 minute, standing 5 minutes, after packing and get final product.
The purposes of vaccine in the dimer of 13. polypeptide according to claim 1 and 2 or the aminoacid sequence polypeptide as shown in SEQ ID NO.1, claim 3 to 5 as described in any one in the medicine of preparation prevention O type foot and mouth disease, wherein said O type foot and mouth disease is pig O type foot and mouth disease.
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| CN103214560B (en) * | 2013-03-25 | 2014-10-01 | 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 |
| 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 |
| CN104961809B (en) * | 2015-07-10 | 2018-06-26 | 中牧实业股份有限公司 | The O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of ox aftosa |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1354674A (en) * | 1998-06-20 | 2002-06-19 | 美国联合生物医学公司 | Synthetic peptide vaccines 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 |
-
2009
- 2009-06-04 CN CN200910143571.XA patent/CN101565457B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1354674A (en) * | 1998-06-20 | 2002-06-19 | 美国联合生物医学公司 | Synthetic peptide vaccines 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 |
Non-Patent Citations (2)
| Title |
|---|
| 口蹄疫研究进展;朗洪武等;《中国兽药杂志》;20051231;第39卷(第10期);第16-21页 * |
| 朗洪武等.口蹄疫研究进展.《中国兽药杂志》.2005,第39卷(第10期),第16-21页. |
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| Publication number | Publication date |
|---|---|
| CN101565457A (en) | 2009-10-28 |
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