CN103214560B - Polypeptide used for preparing bovine foot and mouth disease type O peptide vaccine, and preparation method and application thereof - Google Patents

Abstract

The invention provides a polypeptide used for preparing bovine foot and mouth disease type O peptide vaccine. The polypeptide comprises an amino acid sequence represented by SEQ ID NO.1. The invention also provides a peptide vaccine comprising the polypeptide. The provided bovine foot and mouth disease type O synthetic peptide vaccine has good immunization effect, and does not cause problems such as fever and red swelling of traditional vaccines. Therefore, the vaccine provided by the invention is effective against current antigenic variations of foot and mouth disease, and has no biological safety problem. The vaccine is suitable for large-scale synthesis, and has good application prospect. The invention also provides preparation methods of the polypeptide and the peptide vaccine, and a pharmaceutical application thereof.

Description

For the preparation of polypeptide of ox foot and mouth disease O type peptide vaccine and its production and use

Technical field

The invention belongs to medical technical field, particularly, the present invention relates to a kind of polypeptide for the preparation of ox foot and mouth disease O type peptide vaccine, and the vaccine that contains these polypeptide and their preparation method.

Background technology

Foot and mouth disease (foot and mouth disease is called for short FMD) is acute, highly contact, infectious fever, the worldwide extensively distribution that a kind of artiodactyl occurs.The infectivity of foot and mouth disease is high, propagates rapidly, will cause cub dead after the livestocks such as infected pigs, ox, sheep, and adult animals throughput sharply declines, so the production and supply of the development of serious harm livestock industry and meat and livestock product thereof.At present, foot and mouth disease makes the market circulation of animal and animal's products and international trade be subject to blocking greatly and restriction, produces to the livestock industry of popular countries and regions and causes huge financial loss.

Foot and mouth disease is infected and is caused by foot and mouth disease virus (FMDV).Foot and mouth disease virus belongs to picornavirus, has the features 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 in the known whole world), Sat2(South Africa II type), Sat3(South Africa III type) and Asia I(Asia I type).Each in these principal modes is divided again some hypotypes, the existing kind more than 70 of hypotype of finding at present.The foot and mouth disease virus of serotype A, O, C and Asia I type is the most common, and wherein the mutation of serotype A virus is maximum, has 30 kinds of hypotypes of surpassing.Result of study shows, the capsid protein of foot and mouth disease virus is by four kinds of structural protein VP1, VP2, VP3 and VP4(Logan D etc., 1993) form, 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.

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.

Current foot and mouth disease popular in China cows is mainly O type, Asia I type and A type foot and mouth disease.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.But 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.By contrast, 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.As can be seen here, aspect foot and mouth disease prevents and treats, China has lagged behind world development situation.

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.Therefore still there is the demand for safe and effective foot and mouth disease new generation vaccine in this area.

Summary of the invention

Therefore, the object of the present invention is to provide a kind of polypeptide for the preparation of ox foot and mouth disease O type synthetic peptide vaccine, and the vaccine that contains this polypeptide.

Another object of the present invention is to provide the preparation method of a kind of aforementioned polypeptides and vaccine.

Another object of the present invention is to provide the purposes of aforementioned polypeptides and vaccine.

For achieving the above object, the present invention has adopted following technical scheme:

On the one hand, the invention provides a kind of polypeptide for the preparation of ox foot and mouth disease O type peptide vaccine, described polypeptide comprises the aminoacid sequence as shown in SEQ ID NO. 1.This sequence is the core B cell epitope sequence of foot and mouth disease O virus, is responsible for producing the neutralizing antibody for foot and mouth disease virus.

SEQ?ID?NO.?1：

YNGSCKYGDASANNVRGDLQVLALKTEKCLPTSFNYGAIK

" polypeptide " mentioned in the present invention and " synthetic peptide " are identical concept, all refer to the peptide material obtaining by solid phase organic synthesis.

Preferably, described polypeptide also comprises to have and strengthens the endogenous or exogenous t cell epitope aminoacid sequence of the foot and mouth disease of immunization, and wherein, described aminoacid sequence is selected from one or more in SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.In the present invention, by the t cell epitope of computer forecast foot and mouth disease O virus, then utilize the t cell epitope peptide that software design is new, then by in-vitro evaluation animal cell immunity level, screening obtains the polypeptide of above-mentioned aminoacid sequence thus, and it can assist B cell epitope to produce neutralizing antibody.

SEQ?ID?NO.?2：

AGLAGVMVTESVAFRKKV

SEQ?ID?NO.?3：

HLEFNNFTVLRVPSFWKVSASKV

SEQ?ID?NO.?4：

SHLEFNNFTVSVPKVFWLRSAKV

Further preferably, described polypeptide comprises the aminoacid sequence as shown in SEQ ID NO.5, SEQ ID NO. 6 or SEQ ID NO. 7.

SEQ?ID?NO.?5：

AGLAGVMVTESVAFRKKVYNGSCKYGDASANNVRGDLQVLALKTEKCLPTSFNYGAIK

SEQ?ID?NO.?6：

HLEFNNFTVLRVPSFWKVSASKVYNGSCKYGDASANNVRGDLQVLALKTEKCLPTSFNYGAIK

SEQ?ID?NO.?7：

SHLEFNNFTVSVPKVFWLRSAKVYNGSCKYGDASANNVRGDLQVLALKTEKCLPTSFNYGAIK

According to the specific embodiment of the present invention, the aminoacid sequence of polypeptide of the present invention is as shown in SEQ ID NO.5, SEQ ID NO. 6 or SEQ ID NO. 7;

Preferably, the sulfydryl of two halfcystines in the aminoacid sequence of described polypeptide can be joined together to form disulfide linkage through oxidation;

Further preferably, between the head and the tail amino-acid residue of the aminoacid sequence of described polypeptide, can react form covalently bound.Wherein sequence provided herein is to hold C end from N, and the namely residue reaction of N end and C end forms and is connected.Particularly, between the carboxyl of the head and the tail amino-acid residue of the aminoacid sequence of described polypeptide and amino or carboxyl and hydroxyl, react formation covalently bound.

Aforementioned polypeptides provided by the invention can directly obtain to the customization of business peptide Synesis Company, or adopts commercially available automatic DNA synthesizer DNA, synthetic according to the working specification of manufacturers.

On the other hand, the present invention also provides the preparation method of aforementioned polypeptides, and described preparation method comprises the following steps:

(1) take aminoresin as starting raw material, the amino acid of being protected by 9-fluorenylmethyloxycarbonyl of take is monomer, according to described aminoacid sequence successively condensation, connects amino acid to synthesize described polypeptide, after every step condensation reaction, with acetyl imidazole, seals unreacted aminoterminal;

(2) after synthetic, add lytic reagent, thereby the cracking from aminoresin of described polypeptide is got off;

(3) polypeptide described in use ether sedimentation; With

(4) described polypeptide is carried out to ultrafiltration purification, then carry out aseptically process.

In above-mentioned preparation method, described step (1) specifically comprises the following steps:

(1-a) deprotection reaction: aminoresin is placed in to the N-Methyl pyrrolidone that volume percent is the hexahydropyridine of 15-30% (NMP) solution, under 20-28 ℃ of condition, react 25-40 minute, thereby remove the 9-fluorenylmethyloxycarbonyl blocking group on aminoresin;

(1-b) washing: nitrogen dries up, and then washs aminoresin with N-Methyl pyrrolidone;

(1-c) condensation reaction: the amino acid that adds 1-hydroxyl azimidobenzene (HOBT), dicyclohexylcarbodiimide (DCC) and protected by 9-fluorenylmethyloxycarbonyl then reacts 0.5-2.5 hour under 20-28 ℃ of condition;

(1-d) washing: nitrogen dries up, and then washs aminoresin with N-Methyl pyrrolidone; With

(1-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, in described step (2), the component of lytic reagent is that volume ratio is the trifluoroacetic acid of 85:8:6:1: tri isopropyl silane: phenol: H ₂o; And the pyrolysis time of described step (2) is 1-4 hour.

In above-mentioned preparation method, described step (3) specifically comprises:

(3-a) polypeptide described in use ether sedimentation, then washs with dimethyl formamide;

(3-b) adding in dimethyl sulfoxide (DMSO) (DMSO) situation that accounts for reaction system cumulative volume 10%, making between two halfcystines, to form disulfide linkage in the aminoacid sequence of the polypeptide that obtains of precipitation; With

(3-c) make reaction between the head and the tail amino-acid residue of aminoacid sequence of described polypeptide form covalently bound; Preferably, at the carboxyl that adds the head and the tail amino-acid residue that accounts for the aminoacid sequence that makes described polypeptide in the DIC (DIC) of reaction system cumulative volume 1% and 1-hydroxyl-7-azo benzotriazole (HOAT) situation of 0.5% with amino or adding 0.1M H ₂sO ₄in situation, make to react between carboxyl and hydroxyl form covalently bound.

In above-mentioned preparation method, described step (4) specifically comprises the following steps:

(4-a) use tangential flow filtration film to wrap under 20-28 ℃ of condition polypeptide described in ultrafiltration, thereby remove small molecular weight impurity; With

(4-b) use 0.2 micron of online filter degerming to preserve.

Another aspect, the invention provides a kind of peptide vaccine, and described peptide vaccine comprises one or more aforementioned polypeptides.And described peptide vaccine preferably also comprises adjuvant; Preferably, described adjuvant is to be selected from one or more in white oil, 50V, 50VII.Further preferably, the polypeptide comprising in described peptide vaccine and the volume ratio of adjuvant are 1:1.

Again on the one hand, the invention provides the preparation method of this peptide vaccine, described preparation method comprises the following steps:

(1) with water for injection, by described polypeptide dilution, be the concentration of 50 μ g/ml, thereby obtain polypeptide antigen water;

(2) by adjuvant sterilizing 30 minutes under 121 ℃ of conditions; With

(3) under 20-28 ℃ of condition, according to the volume ratio of described polypeptide antigen water and described adjuvant 1:1, first adjuvant is added in emulsion tank, under 90-150 rev/min, stir 1.5-3 minute, then slowly add polypeptide antigen water, stir 20-30 minute, then stir 15-30 minute under 8000-10000 rev/min, standing 5 minutes, packing.

Also on the one hand, the invention provides aforementioned polypeptides or the peptide vaccine purposes in the medicine for the preparation of prevention ox foot and mouth disease O type.

Particularly, the inventor is by domestic foot and mouth disease sequencing the combined mouth fever aphthous vaccine strain 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.In addition, the inventor 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.

Peptide vaccine potency test, safety experiment result show, ox foot and mouth disease O type synthetic peptide vaccine provided by the invention has good immune efficacy, and can not cause the problems such as heating that traditional vaccine exists, redness, therefore vaccine of the present invention can successfully manage the antigenic variation of current foot and mouth disease virus, not have biological safety, be easy to extensive synthesizing, have a good application prospect.

Embodiment

Referring to specific embodiment, the present invention is described.It will be appreciated by those skilled in the art that these embodiment are only for the present invention is described, the scope that it does not limit the present invention in any way.

Experimental technique in following embodiment, if no special instructions, is ordinary method.In following embodiment, medicinal raw material used, reagent material etc., if no special instructions, be commercially available purchase product.

the solid phase synthesis of 1 N of foot and mouth disease O type antigenic synthetic peptide of embodiment

Antigenic synthetic peptide of the present invention can be used ABI company 433 type full-automatic polypeptide synthetic instruments, utilize the preparation of Merrifield solid-phase synthesis, wherein adopted the amino acid of being modified by 9-fluorenylmethyloxycarbonyl (Fmoc), solid phase carrier is the Rink Amide mbha resin purchased from U.S. Sigma company.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 antigenic synthetic peptides

1.1.1 synthesis material is prepared

The sequence of synthetic polypeptide antigen is respectively as shown in SEQ ID NO.5, SEQ ID NO. 6 or SEQ ID NO. 7.

According to the sequence of antigen and the synthetic scale of 1mmol, prepare the amino acid (biochemical purchased from Shanghai gill) that suitable Fmoc modifies, 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 Rate1-18 to synthesizer, select Main Menu-Module Test-look for ModuleA, ModuleD, ModuleI, ModuleI, ModuleA-by Start-measure or observe by more by Prer or next, if flow is improper, regulate lower valve pressure, until reach requirement.

1.1.3 the synthetic beginning of antigenic synthetic peptide

In the program of synthesizer, the synthetic method Std Fmoc 1.0 Sol DIC90 that need are 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 antigenic synthetic peptide 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 antigenic synthetic peptide 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 antigenic synthetic peptides and evaluation

1.2.1 the cracking of antigenic synthetic peptide

According to volume ratio (trifluoroacetic acid (TFA)/tri isopropyl silane (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 antigenic synthetic peptide

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 antigenic synthetic peptides 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 carry out head and the tail cyclisation, head and the tail amino acid whose " COOH " and " NH ₂" cyclization method is referring to Peptide Protein Reserch 1996.48:229-239 such as Mengfen; The method that head and the tail amino acid whose " COOH " is reacted with " OH " and form ring texture is referring to Chem.Soc 1970.92:3771-3777 such as Mmenhofer.Obtain thus can simulated virus particle native conformation polypeptide cyclized structure.

The purifying degerming of 1.4 antigenic synthetic peptides

Antigenic synthetic peptide 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

Take respectively according to the sequence of the embodiment 1 preparation antigenic synthetic peptide as shown in SEQ ID NO.5, SEQ ID NO. 6 or SEQ ID NO. 7 respectively, 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 gained antigenic solution via hole diameter filters, degerming.

2.2 oil phase adjuvant preparations

Oil phase adjuvant 50V is through 121 ℃ of sterilizings 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.

3 Ns of foot and mouth disease O type synthetic peptide vaccine potency tests of embodiment

1. materials and methods

1.1 synthetic peptide vaccine

According to the polypeptide antigen of embodiment 1 preparation sequence as shown in SEQ ID NO.5, SEQ ID NO. 6 or SEQ ID NO. 7, the corresponding lot number of then preparing respectively according to embodiment 2 is: the O type aftosa synthetic peptide vaccine of ZM01, ZM02 and ZM03.

1.2 experimental animal

Select foot-and-mouth disease antibody feminine gender (suckling mouse NAT≤1: healthy 37 of oxes (purchased from cattle farm, Lanzhou) of 6 monthly ages 4).

1.3 seed culture of viruses OS/99

With 3-4 age in days suckling mouse, measure and adjust malicious valency, be placed in-25 ℃ of freezing saving backup.

1.4 test method

By respectively immune 5 oxen of every group of vaccine in 3 groups of test peptides vaccines, use conventional inactivated vaccine (ox foot and mouth disease O type bivalent inactivated vaccine is provided lot number 1112001 by biological pharmaceutical factory, Zhongmu Stocks Trading Co. Lanzhou) as positive control, 5 oxen of immunity simultaneously.2 oxen of negative control.During immunity, adopt the injection of posterior auricular muscle meat, injected dose is 2ml/ ox.After immunity 21 days, together with 2 of the identical contrast oxen of condition, 2 strong malicious OS/99 of intradermal injection ox foot and mouth disease O C-type virus C are divided in every cow tongue upper surface both sides, and every some 0.1ml(is 0.2ml altogether, containing 10000ID ₅₀).Attack poison after 10 days, observed and recorded test-results.

1.5 results are judged

There is bubble or ulcer in contrast Niu Junying at least 3 hoof.Only there is bubble or ulcer and other position is judged to protection during without pathology at lingual surface in immune cattle, when typical foot and mouth disease bubble or ulcer appear in arbitrary position except lingual surface, is judged to and does not protect.

2. test-results and discussion

2.1 test-results

After immunity 21 days, together with 2 of the identical contrast oxen of condition, 2 strong malicious OS/99 of intradermal injection ox foot and mouth disease O C-type virus C are divided in every cow tongue upper surface both sides, and every some 0.1ml(is 0.2ml altogether, containing 10000ID ₅₀).Attack poison after 10 days, the results detailed in Table 1.

Table 1 ox foot and mouth disease O type synthetic peptide vaccine potency test result

2.2 discussion of results

From this test-results, can find out, ox foot and mouth disease O type synthetic peptide vaccine of the present invention protection ratio after this animal of immunity ox, between 80% to 100%, is up to 100% protection.The ox foot and mouth disease O type synthetic peptide vaccine that proves thus these antigen preparations has good immune efficacy and clinical application potentiality.

the safety testing of 4 Ns of foot and mouth disease O type synthetic peptide vaccines of embodiment

1. materials and methods

1.1 synthetic peptide vaccine

With embodiment 3.

1.2 experimental animal

Cavy from numerous 350 ~ 450g; The mouse of 18 ~ 22g; The healthy susceptible ox at least 6 monthly ages.

1.3 test method

1.3.1 use 12 of the cavys of body weight 350 ~ 450g, every subcutaneous injection vaccine 2ml; With 30 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.

1.3.2 use 18 of the healthy susceptible oxen (foot and mouth disease cell NAT is not higher than 1:8) at least 6 monthly ages, in every cow tongue intracutaneous, divide 20 some vaccinate 2ml, every some 0.1ml, observes at least 4 day by day.Afterwards, every ox intramuscular injection vaccine 9ml, continues to observe day by day 6.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

12 of cavys, every subcutaneous injection vaccine 2ml; 30 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 2.

The vaccine safety test-results of table 2 cavy and mouse

2.2. the security of vaccine to healthy susceptible ox

Synthetic peptide vaccine is taken out and equilibrated to after room temperature, in every cow tongue intracutaneous, divide 20 some vaccinate 2ml, every some 0.1ml, observes at least 4 day by day.Afterwards, every ox intramuscular injection vaccine 9ml, continues to observe day by day 6.Concrete outcome is in Table 2.

The vaccine safety test-results of the healthy susceptible ox of table 3

The above results illustrates that these ox foot and mouth disease O type synthetic peptide vaccine is safe to cavy, mouse and ox, does not resemble traditional vaccine and has the side reaction problems such as heating, redness, so have good promotion prospect and marketable value.

Specific description of embodiments of the present invention above does not limit the present invention, and those skilled in the art can make according to the present invention various changes or distortion, only otherwise depart from spirit of the present invention, all should belong to the scope of claims of the present invention.

Claims (14)

1. for the preparation of a polypeptide for ox foot and mouth disease O type peptide vaccine, it is characterized in that, the aminoacid sequence of described polypeptide is as shown in SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7; And, the sulfydryl of two halfcystines in the aminoacid sequence of described polypeptide is joined together to form disulfide linkage through oxidation, and it is covalently bound between the carboxyl of the head and the tail amino-acid residue of the aminoacid sequence of described polypeptide and amino or carboxyl and hydroxyl, to react formation.

2. the preparation method of polypeptide according to claim 1, is characterized in that, described preparation method comprises the following steps:

(1) take aminoresin as starting raw material, the amino acid of being protected by 9-fluorenylmethyloxycarbonyl of take is monomer, according to described aminoacid sequence successively condensation, connects amino acid to synthesize described polypeptide, after every step condensation reaction, with acetyl imidazole, seals unreacted aminoterminal;

(2) after synthetic, add lytic reagent, thereby the cracking from aminoresin of described polypeptide is got off;

(3) polypeptide described in use ether sedimentation; With

(4) described polypeptide is carried out to ultrafiltration purification, then carry out aseptically process.

3. preparation method according to claim 2, is characterized in that, described step (1) comprises the following steps:

(1-a) deprotection reaction: aminoresin is placed in to the N-Methyl pyrrolidone solution that volume percent is the hexahydropyridine of 15-30%, reacts 25-40 minute under 20-28 ℃ of condition, thereby remove the 9-fluorenylmethyloxycarbonyl blocking group on aminoresin;

(1-b) washing: nitrogen dries up, and then washs aminoresin with N-Methyl pyrrolidone;

(1-c) condensation reaction: the amino acid that adds 1-hydroxyl azimidobenzene, dicyclohexylcarbodiimide and protected by 9-fluorenylmethyloxycarbonyl then reacts 0.5-2.5 hour under 20-28 ℃ of condition;

(1-d) washing: nitrogen dries up, and then washs aminoresin with N-Methyl pyrrolidone; With

(1-e) capping: adding percent weight in volume is the N-Methyl pyrrolidone solution of the acetyl imidazole of 1.5-4%, reacts 20-40 minute under 20-28 ℃ of condition.

4. preparation method according to claim 2, is characterized in that, in described step (2), the component of lytic reagent is that volume ratio is the trifluoroacetic acid of 85:8:6:1: tri isopropyl silane: phenol: H ₂o.

5. preparation method according to claim 2, is characterized in that, the pyrolysis time of described step (2) is 1-4 hour.

6. preparation method according to claim 2, is characterized in that, described preparation method's step also comprises in (3):

(3-a) polypeptide described in use ether sedimentation, then washs with dimethyl formamide;

(3-b) adding in the dimethyl sulfoxide (DMSO) situation that accounts for reaction system cumulative volume 10%, making between two halfcystines, to form disulfide linkage in the aminoacid sequence of the polypeptide that obtains of precipitation; With

(3-c) make reaction between the head and the tail amino-acid residue of aminoacid sequence of described polypeptide form covalently bound.

7. preparation method according to claim 6, it is characterized in that, at the carboxyl that adds the head and the tail amino-acid residue that accounts for the aminoacid sequence that makes described polypeptide in the DIC of reaction system cumulative volume 1% and 1-hydroxyl-7-azo benzotriazole situation of 0.5% with amino or adding 0.1M H ₂sO ₄in situation, make to react between carboxyl and hydroxyl form covalently bound.

8. preparation method according to claim 2, is characterized in that, described step (4) comprises the following steps:

(4-a) use tangential flow filtration film to wrap under 20-28 ℃ of condition polypeptide described in ultrafiltration, thereby remove small molecular weight impurity; With

(4-b) use 0.2 micron of online filter degerming to preserve.

9. an ox foot and mouth disease O type peptide vaccine, is characterized in that, described peptide vaccine comprises polypeptide according to claim 1.

10. peptide vaccine according to claim 9, is characterized in that, described peptide vaccine also comprises adjuvant.

11. peptide vaccines according to claim 10, is characterized in that, described adjuvant is to be selected from one or more in white oil, 50V, 50VII.

12. according to the peptide vaccine described in claim 10 or 11, it is characterized in that, the polypeptide comprising in described peptide vaccine and the volume ratio of adjuvant are 1:1.

The preparation method of the peptide vaccine in 13. claims 9 to 12 described in any one, is characterized in that, described preparation method comprises the following steps:

(1) with water for injection, by described polypeptide dilution, be the concentration of 50 μ g/ml, thereby obtain polypeptide antigen water;

(2) by adjuvant sterilizing 30 minutes under 121 ℃ of conditions; With

(3) under 20-28 ℃ of condition, according to the volume ratio of described polypeptide antigen water and described adjuvant 1:1, first adjuvant is added in emulsion tank, under 90-150 rev/min, stir 1.5-3 minute, then slowly add polypeptide antigen water, then stir 20-30 minute, then stir 15-30 minute under 8000-10000 rev/min, standing 5 minutes, packing.

The purposes of peptide vaccine in 14. polypeptide claimed in claim 1 or claim 9 to 12 described in any one in the medicine for the preparation of prevention ox foot and mouth disease O type.