CN101643500B - Asia I synthetic peptide vaccine of foot and mouth disease - Google Patents
Asia I synthetic peptide vaccine of foot and mouth disease Download PDFInfo
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- CN101643500B CN101643500B CN2009100843228A CN200910084322A CN101643500B CN 101643500 B CN101643500 B CN 101643500B CN 2009100843228 A CN2009100843228 A CN 2009100843228A CN 200910084322 A CN200910084322 A CN 200910084322A CN 101643500 B CN101643500 B CN 101643500B
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Abstract
The invention provides an Asia I synthetic peptide vaccine of a foot and mouth disease, in particular to a polypeptide or a polypeptide polymer thereof for the Asia I synthetic peptide vaccine of the foot and mouth disease, the vaccine containing the polypeptide or the polypeptide polymer thereof, and a preparation method of both, wherein, the amino acid sequence of the polypeptide is shown as SEQ ID NO:1. In the invention, variation situation of main antigen sites of the foot and mouth disease is researched by determining a sequence of an epidemic strain of the foot and mouth disease, and meanwhile analysis prediction of the antigen sites of the foot and mouth disease is performed followed by chemical synthesis on possible peptide segments of the antigen sites combined with a computer auxiliary method; and candidate polypeptide antigens are screened through a large quantity of animal experiments to finally obtain the polypeptide antigens with high immune reaction level which can protect well an animal from being attacked by the epidemic strain of the foot and mouth disease. The vaccine prepared from the polypeptide antigens has the advantages of no biological safety problem, easy large-scale synthesis, good application prospect and good ability of effectively dealing with antigen variation of foot and mouth disease virus.
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 that is used for Asia I-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, be called for short FMD) is a kind of acute, height contact artiodactylous, infectious fever of betiding, and it is very wide worldwide to distribute, and belongs to the global epidemic infectious diseases of international mutual propagation.Foot and mouth disease is caused that by foot and mouth disease virus this virus belongs to the Picornaviridae Hostis, and is spherical in shape, and diameter is 30nm, no cyst membrane.Foot and mouth disease virus is simple in structure, is made up of singlestranded RNA and protein, and its content in ill domestic animal blister intracutaneous and lymph liquid is maximum, and virulence is strong.Foot and mouth disease virus has 7 kinds of serotypes (O, A, C, SAT1, SAT2, SAT3, Asia I), and every kind of serotype is divided into a plurality of hypotypes again, does not have cross-immunity between the different subtype.
In recent years, it is popular that foot-and-mouth disease virus Asia I (FMDV) is region on South East Asia and the India peninsula, and be the middle mind-set Middle East and the diffusion of surrounding area, South East Asia with these two areas.For the prevention of this disease, most of developing countries adopt viral inactivation vaccine to carry out immunity at present.In the world; Most manufacturing enterprises take the training method of cell suspension or cell rolling bottle when producing aftosa vaccine; And pass through divinyl imines (BEI) with inactivation of virus, adopt aluminium hydroxide gel to prepare vaccine as emulsification adjuvant as sorbent material, MO.Though the foot and mouth disease inactivated vaccine has stronger immune efficacy; But since poor, the product of its biological safety stable not good enough, be easy to generate many unfavorable factors such as allergic side reactions; And World Organization for Animal Health (OIE) special stipulation does not have the country forbidding inactivated vaccine of foot and mouth disease, so the research of development of new aftosa vaccine has just been put on schedule.
Along with the fast development of modern biotechnology, people are more and more deep to the immunne response Study on Mechanism of foot and mouth disease virus itself and virus and animal, for development recombinant vaccine, the horizontal vaccine of chemosynthesis peptide vaccine equimolecular provide technical guarantee.Discover: FMDV is made up of a RNA nucleic acid chains and capsid protein, and the about 8.5kb of geneome RNA total length can be directly as messenger RNA(mRNA).The FMDV geneome RNA is made up of 5 ' UTR, 3 ' UTR and OFR.5 ' UTR is about 1300nt, contains (IRES) such as VPg, secondary structure, P10 gene, Poly (c) section and internal ribosome entry sites.3 ' UTR forms long 172nt by 92 nt residues between Poly (A) tail and big ORF 3 ' end and Poly (A) section 5 ' end.Big ORF is made up of L gene (P20a), P1 structural protein gene, P2 and P3 nonstructural protein gene and initiator codon and terminator codon; And be arranged in order with the order of L, P1, P2 and P3; Be about 6.5Kb; Wherein genomic 3D section is the RNA polymerase of coding FMDV, is indispensable composition in the virus replication, is called as VIA; It is commonly used to detect animal with Nonstructural Protein 3A, 3B, 3C etc. and whether contacts, infects the FMDV (Long Yongjin etc., 2003) that makes a living.P1 structural protein gene coding 4 kinds of main structural protein VP1, VP2, VP3 and VP4 (Logan D, Abu-Ghazaleh R, Blakemore W etc., 1993), their each 60 coat protein of forming FMDV; VP1～VP3 forms capsomere, and 1 protein subunit constitutes a face of 20 bodies of virus particle, and VP4 is positioned at the inside of virion.Capsid protein VP1～VP4 gene is all participated in the formation of antigen site.
Bachrach equals from foot and mouth disease virus, to isolate capsid protein VP1 in 1975, is equipped with Freund's incomplete adjuvant and processes a kind of protein subunit vaccine, has certain immune effect, for the development recombinant vaccine provides foundation.Since then, many researchers also focus on goal in research in the proteic research of VP1.After the eighties in 20th century; External many scholars combine DNA recombinant technology and the molecular biological achievement in research of FMDV; Produce part-structure albumen or the entire infrastructure albumen of foot and mouth disease virus with dust Xi Shi intestinal bacteria, yeast, baculovirus, poxvirus, mammalian cell even plant expression system, the development recombinant vaccine.China has also carried out deep research to the foot and mouth disease recombinant vaccine, and has obtained interim achievement.2000, Yang Zhijun etc. prepared with intestinal bacteria and have expressed the VP1 recombinant vaccine that contains T cell and B cell epitope resisting O-type FMDV.Test shows that this vaccine can induce cavy and pig to produce neutralizing antibody, has certain immune effect.But, therefore be difficult to improve immune efficacy because the amount of gene expression product is very limited, and can't obtain antigenic three-dimensional arrangement.2003; Zheng Zhaoxin etc. contact the gene of t cell epitope on VP1 and the VP4 and B cell epitope and have developed the recombinant vaccine to Asia 1 type foot and mouth disease; Can not reach 100% protection under the strong virus attack but test-results is illustrated in, simultaneously as vaccine, the recombinant vaccine post-processed is too complicated; Cost is high, and it is low to tire.So far, also there is not a kind of gene engineering polypeptide vaccine of commercially producing in the world.
Chemically synthesized polypeptide vaccine (synthetic peptide vaccine) is exactly to adopt the method for chemosynthesis epitope aminoacid sequence to prepare the polypeptide vaccine that protective effect is similar to the natural antigen determinant.This vaccine does not contain nucleic acid, is ideal security vaccine, is one of the main direction of the vaccine of development prevention at present and sense of control metachromia disease yet.The crucial part of development aftosa synthetic peptide vaccine is on the capsid protein of virus particle, to find topmost antigenic determinant, and the vaccine of the synthetic peptide of ideal generally should comprise antigenic B cell site and helper cell site.At present; Foot-and-mouth disease virus Asia I is one of main popular foot and mouth disease virus of China just, and the antigenicity of its foot and mouth disease strain is also constantly changing, As time goes on; The effectiveness of original vaccine weakens even disappears, and has brought very big difficulty therefore for the preventing and controlling of foot and mouth disease.And the degree of antigenic difference is also very big in a kind of serotype, possibly not have protectiveness to 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.Therefore, develop a kind of resistance different, render a service, the synthetic peptide vaccine of safe, stable processing technique, foot-and-mouth disease virus Asia I that cost is low become a urgent and important task.
Summary of the invention
Therefore; The shortcoming that the objective of the invention is to overcome above-mentioned prior art is with not enough; Provide a kind of and can successfully manage antigenic variation, and have the effectiveness that is superior to existing vaccine, security is good, stable processing technique, the foot-and-mouth disease virus Asia I vaccine based on synthetic peptide that cost is low.
Another object of the present invention is to provide a kind of method for preparing above-mentioned foot-and-mouth disease virus Asia I vaccine.
The technical scheme that realizes above-mentioned purpose of the present invention is following:
A kind of polypeptide that is used for Asia I-type aftosa synthetic peptide vaccine, this amino acid sequence of polypeptide are the aminoacid sequence shown in the SEQ ID NO:1.One or more Xie Ansuan in the above-mentioned aminoacid sequence can be substituted by norvaline; And/or one or more leucine can be substituted by nor-leucine.Preferably, the whole Xie Ansuans in the above-mentioned aminoacid sequence are substituted by norvaline; 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.
The present invention also provides a kind of Asia I-type aftosa synthetic peptide vaccine, and this vaccine comprises aforementioned polypeptides or its polypeptide polymer.
This vaccine can also comprise adjuvant, and for example: white oil is preferably white oil 50V.
The present invention also provides the preparation method of aforementioned polypeptides, comprising following steps:
(1) protective reaction: in volume percent is reaction 25-40 minute under 20-28 ℃ of condition in the N-Methyl pyrrolidone solution of hexahydropyridine of 15%-30%; Remove 9-fluorenylmethyloxycarbonyl (Fmoc) blocking group on aminoresin or the peptide resin; Nitrogen dries up, the N-Methyl pyrrolidone washing;
(2) amino acid whose activation: each amino acid that has the 9-fluorenylmethyloxycarbonyl blocking group and the 1-hydroxyl azimidobenzene reaction synthesizing amino acid-1-hydroxyl azimidobenzene ester that will synthesize usefulness;
(3) building-up reactions: use Peptide synthesizer will above-mentioned each seed amino acid and resin and DIC add to automatically in the reactor drum, under 20-28 ℃ of condition, reacted 0.5-2.5 hour, nitrogen dries up, the N-Methyl pyrrolidone washing resin;
(4) acetylization reaction: the operating weight volume percent is that the N-Methyl pyrrolidone solution and the middle resin that obtains of step (3) of the acetyl imidazole of 1.5%-4% reacted 20-40 minute under 20-28 ℃ of condition, and nitrogen dries up, the methanol wash resin;
(5) building-up process is held the end to N by C, according to the continuous repeating step of aminoacid sequence (1)～(4), after reaction is accomplished, cleans with N-Methyl pyrrolidone, obtains the exsiccant polypeptide resin;
(6) separating of polypeptide and resin: in the exsiccant polypeptide resin, add lytic reagent, at the uniform velocity stir and be placed on 0 ℃ of reaction 10 minutes down in 1-4 hour, return to room temperature; Fling to trifluoroacetic acid; T-butyl methyl ether and diethyl ether are added in the polypeptide solution, and agitator treating is crossed and is filtered polypeptide solution;
(7) cyclization: polypeptide solution was at room temperature placed 42-47 hour, per hour stirred once;
(8) ultrafiltration purification polypeptide and aseptically process: use film to wrap in ultrafiltration polypeptide under the 20-28 ℃ of condition, use 0.2 micron online filter degerming to preserve.
In above-mentioned preparation method, the component volume ratio of said lytic reagent is specially trifluoroacetic acid: tri isopropyl silane: dithioglycol: phenol: water=85: 8: 3: 3: 1.
In addition, the present invention also provides a kind of 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;
(2) under 20-28 ℃ of condition, according to polypeptide or its polypeptide polymer solution: the volume ratio of adjuvant=1: 1, earlier adjuvant is added in the emulsion tank, 90-150 rev/min was stirred at a slow speed 1.5-3 minute;
(3) slowly add polypeptide or its polypeptide polymer solution, stirred 20-30 minute;
(4) left standstill 5 minutes after high-speed stirring 15-30 minute, promptly get after the packing.
In above-mentioned preparation method, said high-speed stirring is 8000-10000 rev/min.
The present invention also provides aforementioned polypeptides or its polypeptide polymer, vaccine to treat and/or prevent the purposes in the medicine of Asia I-type aftosa in preparation.
The present invention studies the variation situation in foot and mouth disease major antigen site through the sequencing to the foot and mouth disease epidemic isolates.Amino acid sites to main variation; Add up the frequency of its variation; Combine computer-aid method to carry out the analyses and prediction in foot-and-mouth disease antigen site simultaneously, possible antigen site peptide section is carried out chemosynthesis, promptly according to the variation frequency of adding up; Be prone to use different amino acid on the variant sites, obtaining containing multiple candidate's polypeptide antigen of the possible variant sites of current institute.And then these candidate's polypeptide antigens are screened through a large amount of animal experiments, obtain causing the immunoreation of animal, and the immunoreation level is high, the polypeptide antigen of the attack of avoiding the foot and mouth disease epidemic isolates of can watching for animals well.The present invention has carried out optimum combination to the foot-and-mouth disease virus antigen site on the basis of this The selection result, and has effectively made up t cell epitope and B cell epitope, has prepared the synthetic peptide vaccine of foot-and-mouth disease virus Asia I.This vaccine can successfully manage the antigenic variation of foot and mouth disease virus, does not also have the biological safety problem, is easy to extensive synthesizing, and has a good application prospect.
Embodiment
The concrete embodiment of following reference explains the present invention.It will be appreciated by those skilled in the art that these embodiment only are used to explain the present invention, the scope that it does not limit the present invention in any way.
Embodiment 1: the solid phase synthesis of synthetic peptide vaccine polypeptide antigen
Polypeptide antigen of the present invention can pass through ABI 433A full-automatic polypeptide synthetic instrument, utilizes the preparation of Merrifield solid-phase synthesis, the amino acid that has wherein adopted 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, the preparation of synthesis material
Synthetic peptide vaccine polypeptide antigen sequence is shown in SEQ ID NO:1.Comprise following polypeptide in addition:
Aminoacid sequence is the peptide of the aminoacid sequence after the leucine in the SEQ ID NO:1 sequence and Xie Ansuan are all replaced by nor-leucine and norvaline.
Aminoacid sequence is the dimer of the polypeptide of aminoacid sequence shown in the SEQ ID NO:1.
Synthetic scale according to aforementioned polypeptides antigen sequence and 1mmol is prepared the amino acid that suitable Fmoc modifies, and adds among the corresponding C artridge (adorning amino acid whose bottle).Weighing Rink Amide mbha resin is put into reaction chamber equally on request, and lid is tightened up and down, labels the title of record institute synthetic peptide, lot number, the weight of the tare weight of reaction chamber and the resin that takes by weighing.With the reaction chamber synthesizer of packing into.The preparation synthetic agent comprises N-Methyl pyrrolidone (NMP), acyl imidazoles (AIM), piperidines (PIP), methyl alcohol etc. and be placed in the corresponding reagent bottle.
2, synthesizer status detection
Normally whether inspection 433A Peptide synthesizer device operation, after the start, and operation Run Self Test program, whether instrument self checking each item index 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 Ratel-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.
3, the preparation of synthetic peptide vaccine polypeptide antigen
(1) protective reaction: in volume percent is reaction 30 minutes under 22 ℃ of conditions in N-Methyl pyrrolidone (NMP) solution of 20% hexahydropyridine, removes the Fmoc blocking group on aminoresin or the peptide resin, and nitrogen dries up, the NMP washing resin;
(2) amino acid whose activation: each amino acid that has Fmoc and 1-hydroxyl azimidobenzene (HOBT) reaction synthesizing amino acid-HOBT ester that will synthesize usefulness;
(3) building-up reactions: start the automatic synthesis program of Peptide synthesizer, use synthesizer automatically above-mentioned amino acid, resin and DIC to be added in the reactor drum, reaction is 2 hours under 22 ℃ of conditions, and nitrogen dries up, the NMP washing resin;
(4) acetylization reaction: use the acetyl imidazole of 2.5% weight and the middle resin that obtains of step (3) under 22 ℃ of conditions, to react 30 minutes, nitrogen dries up, the methanol wash resin;
(5) building-up process is held the end to N by C, constantly repeats (1)～(4) step according to aminoacid sequence, after reaction is accomplished, cleans with NMP, removes remaining amino acid, obtains the exsiccant polypeptide resin;
(6) separating of polypeptide and resin: exsiccant polypeptide-resin is put into Glass Containers, with the lytic reagent for preparing (trifluoroacetic acid: tri isopropyl silane: dithioglycol: phenol: water=85: 8: 3: 3: 1) add in the container, at the uniform velocity stirred 3 hours; Place 0 ℃ to react 10 minutes down Glass Containers; Taking-up returns to room temperature, flings to trifluoroacetic acid (TFA), and t-butyl methyl ether and diethyl ether are added in the polypeptide solution; Agitator treating filters to isolate polypeptide solution;
(7) cyclization: polypeptide solution was at room temperature placed 45 hours, per hour stirred once;
(8) carry out aseptically process behind the ultrafiltration purification polypeptide antigen: use film to wrap in ultrafiltration polypeptide antigen under 22 ℃ of conditions, use 0.2 micron online filter degerming to preserve.
Embodiment 2: the preparation of synthetic peptide vaccine
With water for injection the three peptide species antigenic solutions that embodiment 1 prepares are diluted to 50 μ g/ml respectively, as water; The 50V white-oil adjuvant through 121 ℃, was sterilized in 30 minutes, subsequent use as oil phase.Under 22 ℃ of conditions, the volume ratio according to the 50V of antigen water and sterilization is 1: 1 adds oil phase in the emulsion tank earlier; 100 rev/mins were stirred at a slow speed 2 minutes, slowly added water, added the back and stirred 30 minutes; High speed stirred 20 minutes for 9000 rev/mins again, left standstill then 5 minutes, promptly got the foot-and-mouth disease virus Asia I synthetic peptide vaccine after the packing; Be SEQ ID NO:1 polypeptide vaccine, SEQ ID NO:1 replaces polypeptide vaccine and SEQ ID NO:1 polymerization polypeptide vaccine.
Embodiment 3: synthetic peptide vaccine is to the immune effect research of cavy
300～500g healthy guinea pig is divided into five groups at random, and every group of six cavys carry out intramuscular injection at back leg, and each group injection reagent and dosage are seen table 1.Carry out the immunity second time around the immunity back for the first time, poison was attacked in the 3rd week in the immunity back for the second time.
Table 1: each test group immunizing antigen and immunizing dose
1, the detection of FMDV specific antibody
Measure the OD of serum antibody with indirect elisa method
492Value is envelope antigen (1: 10 dilution) with Asia 1 type FMDV Jiangsu poison on 96 hole enzyme plates, and seized GPS is one anti-, and the anti-cavy IgG of the rabbit of peroxidase labelling is two anti-, and the OD value in the every hole of mensuration returns to zero with blank well at wavelength 492nm place.
Take a blood sample 3 at random for every group,, get the MV (seeing table 2) that every group of indirect ELISA method measured the result with 1: 32 dilution GPS.Table 2 shows that except that the E group, second week of the specific antibody level of each immune group cavy after the immunity first time obviously improves, i.e. the specific antibody level of foot-and-mouth disease virus Asia I synthetic peptide vaccine group obviously improving in second week behind first time immune guinea pig.Simultaneously, its specific antibody level is higher than the deactivation vaccine group.
Table 2: indirect ELISA detection GPS FMDV specific antibody result (X ± SE)
2, the detection of serum neutralizing antibody
Select for use respectively (the 0th day) before the immunity, for the first time after the immunity around (the 28th day), the GPS of immunity back the 3rd week (the 49th day) is used for microneutralization test for the second time, surveys 2 cavys for every group.After the GPS deactivation with the saline water dilution, on the trace Tissue Culture Plate of 96 holes, make a series of doubling dilutions with diluent, each extent of dilution is done 4 holes, and every hole adds 50ul virus liquid (100TCID
50), add 100 μ l cell suspensions with every hole after 1 hour in 37 ℃ of incubators, place 5%CO
237 ℃ of cultivations in the incubator began to observe from 48 hours, declared eventually to 144 hours.The contrast of the positive and negative serum, virus recurrence test, serum toxicity test, normal cell contrast are set.According to the Spearman-Karber method, calculate the cell that can protect in 50% hole and do not produce cytopathic serum dilution, this extent of dilution is the NAT of this part serum.
The result is as shown in table 3; Except that the E group; Neutralizing antibody all appears in each test group after immunity for the first time; And the NAT of B group, C group, D group is higher than the A group, and the NAT that promptly produces after the immunity of foot-and-mouth disease virus Asia I synthetic peptide vaccine is higher than the NAT that deactivation vaccine produces.
Table 3: the neutralizing antibody level of immune guinea pig
3, cavy T lymphocyte proliferation assay
The cavy spleen cell of aseptic collection is diluted to 2 * 10 with the RPMI RPMI-1640
7Individual/ml, place 96 porocyte culture plates, every hole adds 50 μ l cell suspensions and 50 μ l PHA solution (concentration 50 μ g/ml), three repeating holes is set as parallel, and other establishes control wells.Culture plate places 37 ℃, 5%CO
2Cultivate after 45 hours under the saturated humidity condition; Every hole adds MTT solution (5mg/ml) 10 μ l; Continuation was cultivated 3 hours and with 10%SDS-0.01mol/l.HCl solution stopped reaction, was treated to measure every hole OD value at wavelength 570nm place behind the resolution of precipitate, and the result representes with the MV in three holes.
For the first time (the 28th day) around the immunity back the, immunity back the 3rd week (the 49th day) cavy spleen lymphocyte of gathering is used for proliferation test for the second time.The result shows that proliferative response all appears in the splenic T lymphocyte of each immune group except that the E group.
4, synthetic peptide vaccine cavy challenge test
Get 4 cavys for every group,, count infective dose (ID at every Guinea Pig Left hind paw subcutaneous puncture and intradermal vaccination 0.2ml 100 sesquialters together with 4 of negative control group (F group) cavys
50) Asia 1 type FMDV Jiangsu poison, incidence is observed in 1 week in the inoculation back.The severity of pathology is expressed as " nothing " respectively, and promptly two hind paws all do not have pathology (bubble); " slightly ", promptly pathology (bubble) only betides single hind paw of injection; " seriously ", promptly pathology (bubble) all appears in two hind paws.The severity that occurs with pathology is as the judgement of protection effect: protection is not for any pathology (bubble) occurring fully; Partial protection is single the pin that pathology (bubble) only betides injection.The cavy number of protection ratio (%)=fully protection/every group of cavy sum * 100.
Table 4: the cavy anti-virus ability detects
The result shows (table 4), and the antiviral attacking ability of immune group cavy is much larger than E group and F group, and wherein B group, C group, D organize cavy all not morbidities after attacking poison, and its protection ratio reaches 75%, 100% and 100% respectively.This explanation foot-and-mouth disease virus Asia I synthetic peptide vaccine has good protection effect for cavy, and the vaccine after polymerization simultaneously and the replacement has better immune effect.
Embodiment 4: the cavy of synthetic peptide vaccine and mouse safety testing
With 60 of the cavys of body weight 350～450g, 10 of the every batch of vaccinations, every subcutaneous injection 2ml; With 60 of the mouse of body weight 18～22g, 10 of the every batch of vaccinations, every subcutaneous injection 0.5ml of vaccine.Observed 7 continuously, judge dead or tangible local untoward reaction or the systemic reaction that cause because of vaccinate whether occur.Observations is seen table 5.
Table 5: vaccine is to the safety testing result of cavy and mouse
Can find out from this test-results, observe 7 continuously behind foot-and-mouth disease virus Asia I synthetic peptide vaccine immune guinea pig of the present invention and the mouse, dead or tangible local untoward reaction or the systemic reaction that cause because of vaccinate all not occur.In entire test, the phenomena of mortality all do not appear in cavy and mouse.This explains that foot-and-mouth disease virus Asia I synthetic peptide vaccine of the present invention is safe for cavy and mouse.
Sequence table
< 110>Zhongmu Industry Co.,Ltd
< 120>a kind of Asia I-type aftosa synthetic peptide vaccine
<130>DIC09110033
<160>1
<170>PatentIn version 3.3
<210>1
<211>47
<212>PRT
< 213>artificial sequence
<400>1
Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu Glu Asp Arg Ile Leu Thr
1 5 10 15
Thr Arg Asn Gly His Thr Thr Ser Thr Thr Gln Ser Ser Val Gly Val
20 25 30
Thr Lys Phe Asp Trp Thr Pro Asp Leu Ser Phe Gly His Cys His
35 40 45
Claims (12)
1. polypeptide that is used for Asia I-type aftosa synthetic peptide vaccine, this amino acid sequence of polypeptide is the aminoacid sequence shown in the SEQ ID NO:1, said aminoacid sequence is that reaction forms covalently bound aminoacid sequence between the head and the tail amino-acid residue.
2. polypeptide according to claim 1 is characterized in that, the whole Xie Ansuans in the said aminoacid sequence are substituted by norvaline; Substituted by nor-leucine with whole leucines.
3. Asia I-type aftosa synthetic peptide vaccine, this vaccine comprises one or more claims 1 or 2 described polypeptide.
4. vaccine according to claim 3 is characterized in that said vaccine comprises adjuvant.
5. vaccine according to claim 4 is characterized in that, said adjuvant is a white oil.
6. vaccine according to claim 5 is characterized in that, said adjuvant is white oil 50V.
7. the preparation method of a polypeptide according to claim 1, comprising following steps:
(1) protective reaction: in volume percent is reaction 25-40 minute under 20-28 ℃ of condition in the N-Methyl pyrrolidone solution of hexahydropyridine of 15%-30%; Remove the 9-fluorenylmethyloxycarbonyl blocking group on the aminoresin; Nitrogen dries up, the N-Methyl pyrrolidone washing;
(2) amino acid whose activation: each amino acid that has the 9-fluorenylmethyloxycarbonyl blocking group and the 1-hydroxyl azimidobenzene reaction synthesizing amino acid-1-hydroxyl azimidobenzene ester that will synthesize usefulness;
(3) building-up reactions: use Peptide synthesizer will above-mentioned each seed amino acid and resin and DIC add to automatically in the reactor drum, under 20-28 ℃ of condition, reacted 0.5-2.5 hour, nitrogen dries up, the N-Methyl pyrrolidone washing resin;
(4) acetylization reaction: the operating weight volume percent is that the N-Methyl pyrrolidone solution and the middle resin that obtains of step (3) of the acetyl imidazole of 1.5%-4% reacted 20-40 minute under 20-28 ℃ of condition, and nitrogen dries up, the methanol wash resin;
(5) building-up process is held the end to N by C, according to the continuous repeating step of aminoacid sequence (1)～(4), after reaction is accomplished, cleans with N-Methyl pyrrolidone, obtains the exsiccant polypeptide resin;
(6) separating of polypeptide and resin: in the exsiccant polypeptide resin, add lytic reagent, at the uniform velocity stir and be placed on 0 ℃ of reaction 10 minutes down in 1-4 hour, return to room temperature; Fling to trifluoroacetic acid; T-butyl methyl ether and diethyl ether are added polypeptide solution, and agitator treating is crossed and is filtered polypeptide solution;
(7) cyclization: polypeptide solution was at room temperature placed 42-47 hour, per hour stirred once;
(8) ultrafiltration purification polypeptide and aseptically process: use film to wrap in ultrafiltration polypeptide under the 20-28 ℃ of condition, use 0.2 micron online filter degerming to preserve.
8. preparation method according to claim 7 is characterized in that, the component volume ratio of said lytic reagent is a trifluoroacetic acid: tri isopropyl silane: dithioglycol: phenol: water=85: 8: 3: 3: 1.
9. preparation method according to each described vaccine in the claim 3 to 6, comprising following steps:
(1) with water for injection polypeptide is diluted to 50 μ g/ml;
(2) under 20-28 ℃ of condition, according to polypeptide solution: the volume ratio of adjuvant=1: 1, earlier adjuvant is added in the emulsion tank, 90-150 rev/min was stirred at a slow speed 1.5-3 minute;
(3) slowly add polypeptide solution, stirred 20-30 minute;
(4) left standstill 5 minutes after high-speed stirring 15-30 minute, promptly get after the packing.
10. the preparation method of vaccine according to claim 9 is characterized in that, said high-speed stirring is 8000-10000 rev/min.
11. claim 1 or 2 described polypeptide treat and/or prevent the purposes in the medicine of Asia I-type aftosa in preparation.
12. each described vaccine treats and/or prevents the purposes in the medicine of Asia I-type aftosa in preparation in the claim 3 to 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100843228A CN101643500B (en) | 2009-05-19 | 2009-05-19 | Asia I synthetic peptide vaccine of foot and mouth disease |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100843228A CN101643500B (en) | 2009-05-19 | 2009-05-19 | Asia I synthetic peptide vaccine of foot and mouth disease |
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| Publication Number | Publication Date |
|---|---|
| CN101643500A CN101643500A (en) | 2010-02-10 |
| CN101643500B true CN101643500B (en) | 2012-06-06 |
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| CN2009100843228A Active CN101643500B (en) | 2009-05-19 | 2009-05-19 | Asia I synthetic peptide vaccine of foot and mouth disease |
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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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999066954A1 (en) * | 1998-06-20 | 1999-12-29 | United Biomedical Inc. | Synthetic peptide vaccines for foot-and-mouth disease |
| CN1408349A (en) * | 2002-09-16 | 2003-04-09 | 复旦大学 | Foot-and-mouth disease gene engineering polypeptide vaccine and its preparing method |
| CN1589901A (en) * | 2003-09-03 | 2005-03-09 | 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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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999066954A1 (en) * | 1998-06-20 | 1999-12-29 | United Biomedical Inc. | Synthetic peptide vaccines for foot-and-mouth disease |
| CN1408349A (en) * | 2002-09-16 | 2003-04-09 | 复旦大学 | Foot-and-mouth disease gene engineering polypeptide vaccine and its preparing method |
| CN1589901A (en) * | 2003-09-03 | 2005-03-09 | 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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