CN104961808B

CN104961808B - It is used to prepare the polypeptide and its preparation method and application of the O-shaped peptide vaccine of ox aftosa

Info

Publication number: CN104961808B
Application number: CN201510115360.0A
Authority: CN
Inventors: 巴利民; 齐鹏; 肖进; 栗利芳; 宋芳; 董春娜; 赵洪涛; 郑应华; 郑明举; 张爱民; 马爱荣
Original assignee: CHINA ANIMAL HUSBANDRY COMMERICAL (GROUP) GEN CORP; China Animal Husbandry Industry Co Ltd
Current assignee: CHINA ANIMAL HUSBANDRY COMMERICAL (GROUP) GEN CORP; China Animal Husbandry Industry Co Ltd
Priority date: 2013-03-25
Filing date: 2013-03-25
Publication date: 2018-09-04
Anticipated expiration: 2033-03-25
Also published as: CN104961807A; CN103183728A; CN103183728B; CN104961808A; CN104961807B

Abstract

The present invention provides a kind of polypeptide being used to prepare the O-shaped peptide vaccine of ox aftosa, and the polypeptide includes the amino acid sequence as shown in SEQ ID NO.1 and contains the peptide vaccine of the polypeptide.The O-shaped synthetic peptide vaccine of ox aftosa of offer has good immune efficacy, and the problems such as existing fever of traditional vaccine, redness will not be caused, therefore the vaccine of the present invention can successfully manage the antigenic variation of current foot and mouth disease virus, biological safety is not present, it is easy to synthesize on a large scale, has a good application prospect.The present invention also provides the preparation methods and its pharmaceutical applications of the polypeptide and peptide vaccine.

Description

It is used to prepare the polypeptide and its preparation method and application of the O-shaped peptide vaccine of ox aftosa

Technical field

The invention belongs to pharmaceutical technology fields, specifically, the present invention relates to one kind being used to prepare the O-shaped peptide epidemic disease of ox aftosa The polypeptide of seedling, and the vaccine containing these polypeptides and their preparation method.

Background technology

Aftosa (foot and mouth disease, abbreviation FMD) be a kind of artiodactyl occur it is acute, highly connect Property, infectious fever are touched, it is worldwide widely distributed.The infectiousness of aftosa is high, propagates rapid, infected pigs, ox, sheep Cub will be caused dead after equal livestocks, adults production capacity drastically declines, therefore seriously endangers development and the meat of animal husbandry The production and supply of food and its livestock products.Currently, aftosa make animal and animal's products market circulation and international trade by Greatly block and limitation, huge economic loss is caused to popular countries and regions Animal husbandry production.

Aftosa is caused by being infected by foot and mouth disease virus (FMDV).Foot and mouth disease virus belongs to picornavirus, Have the characteristics that polymorphism, mutability.At present it is known that there is the foot and mouth disease virus of 7 kinds of serotype in the whole world：A, (the south O, C, Sat1 Non- I types), Sat2 (South Africa II types), Sat3 (South Africa type III) and Asia I (Asia I type).Each in these principal modes is divided into Several hypotypes, presently found hypotype have more than 70 kinds.Serotypes A, the foot and mouth disease virus of O, C and Asia I types are most commonly seen, The mutation of wherein serotypes A virus is most, has more than 30 kinds hypotypes.Result of study shows the capsid protein of foot and mouth disease virus It is to be made of four kinds of Structural protein VP1s, VP2, VP3 and VP4 (Logan D etc., 1993), each each 60.VP1-VP3 groups At capsomere, it is located at the outside of capsid protein, and VP4 is located at the inside of virion.VP1 is main protectiveness Antigen, it has now been found that O-shaped aftosa has 3 main antigen sites to be located on VP1, wherein 133-160 and 200-213 compositions Protective antigens site that is most important and being easiest to variation on VP1.

Antigenicity between foot and mouth disease virus is various is different, is unable to Immunogenicity each other.Moreover, in same serum The degree of antigenic difference is also very big in type so that can be reasonably resistant to a kind of aftosa vaccine of hypotype may be directed to it is same Another hypotype in serotype does not have protectiveness.In addition, aftosa strain antigenicity also constantly changing, with when Between passage, the efficacy wanes of original vaccine even disappear, therefore bring prodigious difficulty to the preventing and controlling of aftosa.

Currently aftosa popular in China cows is mainly O-shaped, ASIA I types and A type aftosas.China's counterpart hoof Epidemic disease implementation is mandatory immune, and vaccine immunity is the main means for preventing aftosa.But the existing aftosa vaccine used in China Mainly viral inactivation vaccine there is the problems such as biological safety is poor, side reaction is big, unstable product quality.Compared to it Under, many countries have stopped using inactivated vaccine in the world at present, also forbid from the national import livestock products for using inactivated vaccine Product.It can be seen that in terms of aftosa prevention, China has lagged behind world development situation.

In terms of the research of aftosa new generation vaccine, successively there are genetic engineering subunit vaccine, hoof-and-mouth disease poisonous carrier epidemic disease Seedling, foot-and-mouth disease gene engineering modification vaccine research report, but its all there is all in terms of immune effect, biological safety More problems affect the use of these new generation vaccines.In addition, these vaccines are past for currently having occurred and that the popular strain of variation It is poor toward effect, animal cannot be effectively protected.Therefore, this field still has for the novel epidemic disease of safe and effective aftosa The demand of seedling.

Invention content

Therefore, the purpose of the present invention is to provide a kind of polypeptides being used to prepare the O-shaped synthetic peptide vaccine of ox aftosa, and Vaccine containing the polypeptide.

It is a further object to provide a kind of aforementioned polypeptides and the preparation methods of vaccine.

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

To achieve the above object, present invention employs following technical schemes：

On the one hand, the present invention provides a kind of polypeptide being used to prepare the O-shaped peptide vaccine of ox aftosa, and the polypeptide includes such as Amino acid sequence shown in SEQ ID NO.1.This sequence is the core B cell epitope sequences of aftosa O viruses, is responsible for generating needle To the neutralizing antibody of foot and mouth disease virus.

SEQ ID NO.1：

YNGNCKYGENAVTNVRGDLQVLAQKAARCLPTSFNYGAIK

" polypeptide " and " synthetic peptide " that is referred in the present invention is identical concept, each means and is obtained by Solid-phase organic synthesis Peptide material.

Preferably, the polypeptide also includes to have the aftosa of enhancing immunization endogenous or foreign T-cell epitopes ammonia Base acid sequence, wherein one in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 of the amino acid sequence Or it is multiple.In the present invention, by the t cell epitope of computer forecast aftosa O viruses, the T for then utilizing Software for Design new is thin Born of the same parents' epitope peptide, then it is horizontal by in-vitro evaluation animal cell immunity, thus screening obtains the polypeptide of above-mentioned amino acid sequence, energy Enough Help B Cells epitopes generate neutralizing antibody.

SEQ ID NO.2：

AGLAGVMVTESVAFRKKV

SEQ ID NO.3：

VAQHNLSTEAIPLVVHESLMIVAQSIAGELKV

SEQ ID NO.4：

SPGQVVYNRPHNSAYKV

It is further preferred that the polypeptide includes as shown in SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 Amino acid sequence.

SEQ ID NO.5：

AGLAGVMVTESVAFRKKVYNGNCKYGENAVTNVRGDLQVLAQKAARCLPTSFNYGAIK

SEQ ID NO.6：

VAQHNLSTEAIPLVVHESLMIVAQSIAGELKVYNGNCKYGENAVTNVRGDLQVLAQKAARCLPTSFNYG AIK

SEQ ID NO.7：

SPGQVVYNRPHNSAYKVYNGNCKYGENAVTNVRGDLQVLAQKAARCLPTSFNYGAIK

Specific implementation mode according to the present invention, amino acid sequence such as SEQ ID NO.5, the SEQ ID of polypeptide of the invention Shown in NO.6 or SEQ ID NO.7；

Preferably, the sulfydryl of two cysteines in the amino acid sequence of the polypeptide oxidized can link together Form disulfide bond；

It is further preferred that can react to be formed covalently between the head and the tail amino acid residue of the amino acid sequence of the polypeptide Connection.Sequence wherein provided herein is from N-terminal to C-terminal, that is, N-terminal and the residue of C-terminal react to form connection.It is specific and Speech, the carboxyl of the head and the tail amino acid residue of the amino acid sequence of the polypeptide with shape is reacted between amino or carboxyl and hydroxyl At covalent linkage.

Aforementioned polypeptides provided by the invention can be customized directly to business peptide synthesis company and be obtained, or using commercially available Automatic synthesizer is synthesized according to the operating instruction of manufacturer.

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

(1) using amino resins as starting material, using the amino acid protected by 9-fluorenylmethyloxycarbonyl as monomer, according to the ammonia Base acid sequence is condensed successively connects amino acid to synthesize the polypeptide, often walk closed with acetyl imidazole after condensation reaction it is unreacted Aminoterminal；

(2) lytic reagent is added after synthesizing, to which the polypeptide to be cleaved from amino resins；

(3) polypeptide is precipitated using ether；With

(4) ultrafiltration purification is carried out to the polypeptide, then carries out aseptic process.

In the above preparation method, the step (1) specifically includes following steps：

(1-a) deprotection reaction：Amino resins is placed in the N- methyl pyrroles for the hexahydropyridine that percent by volume is 15-30% It in pyrrolidone (NMP) solution, is reacted 25-40 minutes under the conditions of 20-28 DEG C, to remove the 9- fluorenes methoxy carbonyls on amino resins Base blocking group；

(1-b) is washed：Nitrogen dries up, and then washs amino resins with N-Methyl pyrrolidone；

(1-c) condensation reaction：1- hydroxyls azimidobenzene (HOBT), dicyclohexylcarbodiimide (DCC) is added and by 9- fluorenes The amino acid of methoxycarbonyl group protection, then reacts 0.5-2.5 hours under the conditions of 20-28 DEG C；

(1-d) is washed：Nitrogen dries up, and then washs amino resins with N-Methyl pyrrolidone；With

(1-e) capping：The N-Methyl pyrrolidone for the acetyl imidazole that percent weight in volume is 1.5-4% is added (NMP) solution reacts 20-40 minutes under the conditions of 20-28 DEG C.

In the above preparation method, it is 85 that the group of lytic reagent, which is divided into volume ratio, in the step (2)：8：6：1 trifluoro Acetic acid：Tri isopropyl silane：Phenol：H₂O；Also, the pyrolysis time of the step (2) is 1-4 hours.

In the above preparation method, the step (3) specifically includes：

(3-a) precipitates the polypeptide using ether, is then washed with dimethylformamide；

(3-b) accounts for the dimethyl sulfoxide (DMSO) (DMSO) of reaction system total volume 10% in the case that being added, and precipitation is made to obtain In the amino acid sequence of polypeptide disulfide bond is formed between two cysteines；With

(3-c) makes between the head and the tail amino acid residue of the amino acid sequence of the polypeptide reaction be formed to be covalently attached；It is preferred that Ground, in the 1- hydroxyl -7- azo benzos that the diisopropylcarbodiimide (DIC) and 0.5% that account for reaction system total volume 1% is added Make the carboxyl of the head and the tail amino acid residue of the amino acid sequence of the polypeptide in the case of triazole (HOAT) with amino or is adding Enter 0.1M H₂SO₄In the case of make to react formation between carboxyl and hydroxyl and be covalently attached.

In the above preparation method, the step (4) specifically includes following steps：

(4-a) uses tangential flow filtration film packet polypeptide described in ultrafiltration under the conditions of 20-28 DEG C, to remove small molecular weight impurity； With

(4-b) is preserved using 0.2 micron of online filter degerming.

Another aspect, the present invention provides a kind of peptide vaccine, the peptide vaccine includes one or more aforementioned polypeptides.And And the peptide vaccine preferably also includes adjuvant；Preferably, the adjuvant is one kind or more in white oil, 50V, 50VII Kind.It is further preferred that the volume ratio of polypeptide and adjuvant included in the peptide vaccine is 1:1.

In another aspect, the present invention provides the preparation method of the peptide vaccine, the preparation method comprises the following steps：

(1) polypeptide is diluted to the concentration of 50 μ g/ml with water for injection, to obtain polypeptide antigen water phase；

(2) adjuvant is sterilized 30 minutes under the conditions of 121 DEG C；With

(3) under the conditions of 20-28 DEG C, according to the polypeptide antigen water phase and the adjuvant 1：1 volume ratio, first by adjuvant It is added in emulsion tank, is stirred 1.5-3 minutes under 90-150 revs/min, be then slowly added into polypeptide antigen water phase, stir 20- 30 minutes, then stirred 15-30 minutes under 8000-10000 revs/min, 5 minutes are stood, packing.

Further aspect, the present invention provides aforementioned polypeptides or peptide vaccine to prepare the drug O-shaped for preventing ox aftosa In purposes.

Specifically, the present inventor by the sequencing of popular strain and combining aftosa epidemic disease recently to domestic aftosa Seedling strain sequence studies the variation situation of aftosa major antigenic sites, its change is counted for the amino acid sites mainly to make a variation Different frequency, the analysis that foot-and-mouth disease antigen site is carried out in combination with area of computer aided is predicted, to possible antigen site peptide fragment Chemical synthesis is carried out, that is, is directed to variation frequency of the easy variant sites according to statistics, different amino acid is used in these sites, obtains To a variety of candidate polypeptide antigens for covering current be possible to variant sites.In turn, these are waited by a large amount of animal experiment It selects polypeptide antigen to be screened, obtains the immune response that can cause animal, and immune response is horizontal high, can be good at protecting Protect polypeptide antigen of the animal from the attack of aftosa prevalence strain.In addition, the present inventor is according to screening experiment result counterpart hoof Epidemic disease virus antigenic site is optimized, and efficient combination t cell epitope and B cell epitope, enhances exempting from for polypeptide antigen Epidemic disease effect.

Peptide vaccine potency test, safety experiment the result shows that, the O-shaped synthetic peptide vaccine tool of ox aftosa provided by the invention The problems such as having good immune efficacy, and the existing fever of traditional vaccine, redness will not be caused, therefore the vaccine of the present invention can To successfully manage the antigenic variation of current foot and mouth disease virus, biological safety is not present, it is easy to synthesize on a large scale, has good Application prospect.

Specific implementation mode

The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only For illustrating the present invention, do not limit the scope of the invention in any way.

Experimental method in following embodiments is unless otherwise specified conventional method.Medicine as used in the following examples Material raw material, reagent material etc. are commercially available products unless otherwise specified.

The synthesis in solid state of 1 N of O-shaped antigenic synthetic peptide of aftosa of embodiment

The antigenic synthetic peptide of the present invention can use 433 type full-automatic polypeptide synthetic instrument of ABI companies, utilize Merrifield Prepared by solid-phase synthesis, wherein using the amino acid modified by 9-fluorenylmethyloxycarbonyl (Fmoc), solid phase carrier is purchased from the U.S. The Rink Amide MBHA resins of Sigma companies.Production process generally include the synthesis in solid state of polypeptide antigen, polypeptide cracking, Antigen purification is preserved with degerming.

The synthesis in solid state of 1.1 antigenic synthetic peptides

1.1.1 synthesis material prepares

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 synthesis scale of 1mmol, the amino acid for preparing suitable Fmoc modifications (is purchased from Shanghai Gill is biochemical), it is added in corresponding amino acid bottle.RinkAmide MBHA resins are equally weighed as required, are put into reaction chamber In, upper and lower lid is tightened, is labelled, the title of synthesized peptide, the weight of lot number, the tare weight of reaction chamber and alleged resin are recorded Amount.Reaction chamber is packed into synthesizer.Prepare synthetic agent, including N-Methyl pyrrolidone (NMP), acetyl imidazole (AIM), piperidines (PIP), methanol etc. is placed into corresponding reagent bottle.

1.1.2 synthesizer state-detection

Check Peptide synthesizer whether normal operation.After booting, Run Self Test programs are run, instrument self checking is every Whether index is normal.In addition N is checked₂Whether sufficient, whether system gauge pressure is normal.The performance of instrument has been coped with before synthesis Solution, so to be measured to the flow velocity of each synthetic agent.Flow Rate1-18 are sent to synthesizer, select Main Menu-Module Test-look for ModuleA, ModuleD, ModuleI, ModuleI, ModuleA-to press by Prer or next Start-is measured or is observed by more, if flow is improper, adjusts lower valve pressure, until reaching requirement.

1.1.3 the synthesis of antigenic synthetic peptide starts

The 1.0 Sol DIC90 of method Std Fmoc that synthesis needs are sent on synthesizer in the program of synthesizer. The sequence of File-New-Sequence- Edit and Compose peptides preserves.File-New-Run checks Chemistry；Sequence is It is no to be deposited name；Set Cycles；It preserves.It is finally sent on synthesizer.

Main Menu-Cycle Monitor-begin, bring into operation.

1.1.4 the synthesis of antigenic synthetic peptide

Such as above-mentioned polypeptide sequence, synthesis when is to N-terminal since C-terminal, according to given sequence, successively constantly Repeat following synthesis step：

(1) deprotection reaction：Above-mentioned amino resins is placed in the NMP for the hexahydropyridine that percent by volume is 15%-30% In solution, the Fmoc blocking groups on 25-40 minutes removing amino resins are reacted under the conditions of 20-28 DEG C；

(2) it washs：Nitrogen dries up, and NMP washs amino resins；

(3) condensation reaction：HOBT, DCC is added and reacts 0.5-2.5 under the conditions of 20-28 DEG C with fmoc-protected amino acid Hour；

(4) it washs：Nitrogen dries up, and NMP washs amino resins；

(5) capping：The nmp solution for the acetyl imidazole that percent weight in volume is 1.5%-4% is added, in 20-28 It is reacted 20-40 minutes under the conditions of DEG C.

1.1.5 antigenic synthetic peptide synthesis terminates

Synthesizer will be automatically stopped after antigen synthesizes.Then reactor is removed from Peptide synthesizer, then with 100% Methanol washs polypeptide resin 3 times, is then dried up in draught cupboard, polypeptide resin is transferred in brown bottle, be put into -20 DEG C of refrigerators Interior, sealed membrane sealing is spare.

The cracking and identification of 1.2 antigenic synthetic peptides

1.2.1 the cracking of antigenic synthetic peptide

According to volume ratio (trifluoroacetic acid (TFA)/tri isopropyl silane (TIS)/phenol/H₂O=85/8/6/1 it) prepares Then lysate takes out the polypeptide resin of synthesis out of refrigerator, is put into round-bottomed flask, be added and match into flask in draught cupboard The lysate and magnetic stick made, is then stably placed on magnetic stirring apparatus, is persistently stirred 1 hour at room temperature until anti- It should be complete.After reaction, 30 to 120 minutes TFA removed in crude product are persistently evaporated using the Rotary Evaporators with cold-trap. Then it uses ether to collect, precipitated polypeptide, the crude product of polypeptide antigen is then cleaned multiple times with dimethylformamide (DMF), finally The resin mixed is filtered out with sand core funnel to get to polypeptide antigen.

1.2.2 the identification of antigenic synthetic peptide

Polypeptide antigen is high with substance assistant laser desorpted time-of-flight mass spectrometry (TOFMS) (MALDL-TOF) and reverse phase after synthesizing Pressure liquid chromatography (RP-HPLC) carries out qualitative and quantitative analysis.

The conformation of 1.3 antigenic synthetic peptides is formed

Polypeptide antigen is configured to the polypeptide solution of a concentration of 2mg/ml with 15%DMSO, then use 0.1N NaOH or PH value=8.5 of 0.1N HCl adjustment initial gross separation polypeptide solutions, in the environment of 25 DEG C on the shaking table that rotating speed is 110rpm It places 48 hours, makes to form disulfide bond.

And then head and the tail cyclisation is carried out, "-COOH " and the "-NH of head and the tail amino acid₂" cyclization method is referring to Mengfen etc. Peptide Protein Reserch 1996.48:229-239；"-the COOH " of head and the tail amino acid is set to be reacted with "-OH " And the method for cyclic structure is formed referring to the Chem.Soc such as Mmenhofer 1970.92：3771-3777.Thus obtaining being capable of mould The polypeptide cyclisation structure of pseudovirus particle native conformation.

The purifying degerming of 1.4 antigenic synthetic peptides

Antigenic synthetic peptide carries out ultrafiltration (Tangential using circulating tangential flow filtration film packet under the conditions of 20-28 DEG C The circulating tangential flow filtration film packets of Flow Device and the peristaltic pump mating with it), polypeptide antigen is that macromolecular cannot be by one The filter membrane of set aperture, and the small molecular weight impurity that building-up process early period and later stage cyclization are formed or introduced can then pass through filter Film.Then again by aperture be 0.2 μm of pot strainer degerming, the solution finally obtained is dispensed into aseptic plastic bottle, paste Upper label.Title, number, product batch number, concentration, date of manufacture, pot-life and the preservation condition of polypeptide are indicated on label, point After dress, be stored in -20 DEG C or -40 DEG C it is spare.

For the ease of transporting the needs with long-term preservation, polypeptide antigen is freeze-dried to obtain the more of solid state Peptide.The polypeptide antigen frozen in advance is taken out, is dried on Labconco freeze driers, obtains the polypeptide of solid state Antigen.It is labelled simultaneously.Indicated on label the title of polypeptide, number, product batch number, concentration, the date of manufacture, the pot-life and Preservation condition.

The preparation of 2 synthetic peptide vaccine of embodiment

The preparation of 2.1 antigen water phases

The sequence prepared according to embodiment 1 is weighed respectively respectively such as SEQ ID NO.5, SEQ ID NO.6 or SEQ ID Antigenic synthetic peptide shown in NO.7, then with sterilized water for injection by antigenic synthetic peptide concentration dilution to 50 μ g/ml.Gained is resisted The filter that original solution via hole diameter is 0.2 μm filters, degerming.

It is prepared by 2.2 oil phase adjuvants

Oil phase adjuvant 50V is sterilized 30 minutes through 121 DEG C, it is spare.

The emulsification of 2.3 synthetic peptide vaccines

IKA emulsifying devices are cleaned with the distilled water 2000ml of sterilizing 3 times, and oil phase adjuvant is then pressed under the conditions of 20-28 DEG C It is 1 with antigen water phase：First oil phase is added in emulsion tank for 1 volume ratio, starts motor and is stirred with 90~150r/m slow rotations After 2 minutes, while it being slowly added to water phase antigen, stirred 30 minutes after adding, then with 10000r/m high-speed stirreds 20 minutes, stood 5 minutes, vaccine is made to be emulsified into the single-phase vaccine of Water-In-Oil.

The O-shaped synthetic peptide vaccine potency test of 3 Ns of aftosas of embodiment

1. materials and methods

1.1 synthetic peptide vaccine

It is anti-that sequence polypeptide as shown in SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 is prepared according to embodiment 1 Original, the correspondence lot number then prepared respectively according to embodiment 2 are：The O-shaped aftosa synthetic peptide vaccine of ZM01, ZM02 and ZM03.

1.2 experimental animal

Select negative (suckling mouse neutralizing antibody titers≤1: 6 monthly age health oxes 37 4) (are purchased from Lanzhou of foot-and-mouth disease antibody Regional cattle farm).

1.3 seed culture of viruses OS/99

Malicious valence is measured and adjusted with 3-4 age in days suckling mouses, and it is spare to be placed in -25 DEG C of freezen protectives.

1.4 test method

Every group of vaccine in 3 groups of experiment peptide vaccines is immunized 5 oxen respectively, while with routine inactivated vaccine (ox aftosa O Type bivalent inactivated vaccine is provided, lot number 1112001 by Zhongmu Stocks Trading Co. Lanzhou biology pharmaceutical factory) it is used as positive control, 5 oxen are immunized. 2 oxen of negative control.It is injected using posterior auricular muscle meat when immune, injection dosage is 2ml/ oxen.After 21 days immune, together with condition phase Same control ox 2,2 O-shaped Virus OS/99 of intracutaneous injection ox aftosa, every point are divided in every cow tongue upper surface both sides (total 0.2ml contains 10000ID to 0.1ml₅₀).Poison is attacked after 10 days, observes and records test result.

1.5 result judgement

There is bubble or ulcer in control Niu Junying at least 3 hoof.Only there is bubble or ulcer and other portions in lingual surface in immune cattle It is judged to protect when position is without lesion, any position occurs being judged to not protect when typical aftosa bubble or ulcer in addition to lingual surface.

2. test result and discussion

2.1 test result

After 21 days immune, together with the identical control ox of condition 2,2 intracutaneous injection oxen are divided in every cow tongue upper surface both sides The O-shaped Virus OS/99 of aftosa, (total 0.2ml contains 10000ID to every 0.1ml₅₀).Poison is attacked after 10 days, the results detailed in Table 1.

The O-shaped synthetic peptide vaccine efficacy test results of 1 N of aftosa of table

2.2 discussion of results

The O-shaped synthetic peptide vaccine of ox aftosa that can be seen that the present invention from this test result is protected after immune this animal ox Shield rate is between 80% to 100%, up to 100% protection.Thus the O-shaped conjunction of ox aftosa that these antigens are prepared is proved There are good immune efficacy and clinical application potentiality at peptide vaccine.

The safety testing of 4 Ns of O-shaped synthetic peptide vaccines of aftosa of embodiment

1. materials and methods

1.1 synthetic peptide vaccine

With embodiment 3.

1.2 experimental animal

From the cavy of numerous 350~450g；The mouse of 18~22g；The susceptible ox of health at least 6 monthly ages.

1.3 test method

1.3.1 the cavy 12 of 350~450g of weight is used, every is subcutaneously injected vaccine 2ml；It is small with 18~22g of weight Mouse 30, every is subcutaneously injected vaccine 0.5ml.It is observed continuously 7, it is there is not allowed that dead or apparent caused by vaccinating Local adverse reaction or general reaction.

1.3.2 with the susceptible ox of health at least 6 monthly ages, (aftosa cell neutralize antibody titers are not higher than 1:8) 18, in Intradermal point of 20 points of every cow tongue vaccinate 2ml, every 0.1ml, observe day by day at least 4 days.Later, every ox intramuscular injection Vaccine 9ml continues to observe 6 day by day.There is not allowed that aftosa symptom or the apparent toxic reaction caused by vaccinating.

2. test result

The safety of 2.1 vaccine in guinea pigs and mouse

Cavy 12, every is subcutaneously injected vaccine 2ml；Mouse 30, every hypodermic injection 0.5ml.It is observed continuously 7, Without there is dead because vaccinating caused by or apparent part adverse reaction or general reaction, concrete outcome such as the following table 2.

The vaccine safety test result of 2 cavy of table and mouse

2.2. safety of the vaccine to the susceptible ox of health

After synthetic peptide vaccine taking-up is equilibrated to room temperature, 2ml, every point are vaccinated in intradermal point of 20 points of every cow tongue 0.1ml is observed at least 4 days day by day.Later, every ox intramuscular injection vaccine 9ml continues to observe 6 day by day.Concrete outcome is shown in Table 2。

The vaccine safety test result of the 3 susceptible ox of health of table

The above results illustrate that these O-shaped synthetic peptide vaccines of ox aftosa are safe to cavy, mouse and ox, unlike tradition There are the side reactions such as fever, redness in vaccine, so having good promotion prospect and market value like that.

Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this Invention is variously modified or deforms, and without departing from the spirit of the present invention, should all belong to the model of appended claims of the present invention It encloses.

Claims

1. a kind of polypeptide being used to prepare the O-shaped peptide vaccine of ox aftosa, which is characterized in that the amino acid sequence of the polypeptide is such as Shown in SEQ ID NO.7, the oxidized shape that links together of sulfydryl of two cysteines in the amino acid sequence of the polypeptide At disulfide bond, also, the carboxyl of the head and the tail amino acid residue of the amino acid sequence of the polypeptide and amino or carboxyl and hydroxyl Between reaction formed be covalently attached.

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

(1) using amino resins as starting material, using the amino acid protected by 9-fluorenylmethyloxycarbonyl as monomer, according to the amino acid Sequence is condensed successively connects amino acid to synthesize the polypeptide, and unreacted amino is closed with acetyl imidazole after often step condensation reaction End；

(3) polypeptide is precipitated using ether；

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

(1-a) deprotection reaction：Amino resins is placed in the N- crassitudes for the hexahydropyridine that percent by volume is 15-30% It in ketone solution, is reacted 25-40 minutes under the conditions of 20-28 DEG C, to remove the 9-fluorenylmethyloxycarbonyl protecting group on amino resins Group；

(1-c) condensation reaction：1- hydroxyls azimidobenzene, dicyclohexylcarbodiimide and the ammonia protected by 9-fluorenylmethyloxycarbonyl is added Base acid, then reacts 0.5-2.5 hours under the conditions of 20-28 DEG C；

(1-d) is washed：Nitrogen dries up, and then washs amino resins with N-Methyl pyrrolidone；

(1-e) capping：The N-Methyl pyrrolidone solution for the acetyl imidazole that percent weight in volume is 1.5-4% is added, It is reacted 20-40 minutes under the conditions of 20-28 DEG C.

4. preparation method according to claim 2, which is characterized in that the group of lytic reagent is divided into body in the step (2) Product is than being 85：8：6：1 trifluoroacetic acid：Tri isopropyl silane：Phenol：H₂O。

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

6. preparation method according to claim 2, which is characterized in that further include in (3) the step of the preparation method：

(3-b) accounts for the dimethyl sulfoxide (DMSO) of reaction system total volume 10% in the case that being added, and makes the amino for the polypeptide that precipitation obtains In acid sequence disulfide bond is formed between two cysteines；

(3-c) makes between the head and the tail amino acid residue of the amino acid sequence of the polypeptide reaction be formed to be covalently attached.

7. preparation method according to claim 6, which is characterized in that account for the two different of reaction system total volume 1% being added Propyl carbodiimide and 0.5% 1- hydroxyl -7- azo benzotriazole in the case of make the polypeptide amino acid sequence head The carboxyl of tail amino acid residue is with amino or 0.1MH is being added₂SO₄In the case of to make to react formation between carboxyl and hydroxyl covalent Connection.

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

(4-a) uses tangential flow filtration film packet polypeptide described in ultrafiltration under the conditions of 20-28 DEG C, to remove small molecular weight impurity；

(4-b) is preserved using 0.2 micron of online filter degerming.

9. a kind of O-shaped peptide vaccine of ox aftosa, which is characterized in that the peptide vaccine includes polypeptide described in claim 1.

10. peptide vaccine according to claim 9, which is characterized in that the peptide vaccine also includes adjuvant.

11. peptide vaccine according to claim 10, which is characterized in that the adjuvant is in white oil, 50V, 50VII It is one or more.

12. the peptide vaccine according to claim 10 or 11, which is characterized in that polypeptide and assistant included in the peptide vaccine The volume ratio of agent is 1:1.

13. the preparation method of the peptide vaccine described in any one of claim 9 to 12, which is characterized in that the preparation method packet Include following steps：

(2) adjuvant is sterilized 30 minutes under the conditions of 121 DEG C；

(3) under the conditions of 20-28 DEG C, according to the polypeptide antigen water phase and the adjuvant 1：1 volume ratio, first adjuvant is added In emulsion tank, is stirred 1.5-3 minutes under 90-150 revs/min, be then slowly added into polypeptide antigen water phase, then stir 20- 30 minutes, then stirred 15-30 minutes under 8000-10000 revs/min, 5 minutes are stood, packing.

14. polypeptide described in claim 1 or claim 9 to 12 any one of them peptide vaccine are being prepared for preventing ox Application in the O-shaped drug of aftosa.