CN101579522A - Novel peptide-based vaccine used for domestic animal and preparation method thereof - Google Patents

Abstract

The invention discloses a novel peptide-based vaccine for resisting foot-and-mouth disease virus (FMDV) infection and a preparation method thereof. After being immunized with the peptide-based vaccine, susceptible animals can resist the FMDV infection. The peptide-based vaccine combines the dominant B cell epitope of a capsid protein VP1 of FMDV and a powerful foreign T cell epitope of FMDV, has an amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:2, and can be prepared by a chemical synthetic method and a genetic engineering method. The product has stable quality, good immunization effect and excellent safety, and can be used for protecting susceptible animals such as cows, pigs and the like from being infected by the FMDV.

Description

A kind of novel peptide-based vaccine used for domestic animal and preparation method thereof

Technical field

The present invention relates to a kind of foot and mouth disease chemosynthesis peptide vaccine for animals and preparation method thereof.

Background technology

Foot and mouth disease (foot and mouth disease is called for short FMD) is a kind of height contact artiodactylous, heat generation, acute infectious disease, in the countries in the world widespread distribution.It is propagated rapidly, infects horse, cattle, pig, domestic animals such as sheep cause young stock death, and adults production capacity sharply descends, the production and supply of serious harm Developing of Animal Industry and meat and livestock products thereof is produced for the animal husbandry of popular countries and regions and is caused enormous economic loss.At present, foot and mouth disease has been eliminated or controlled well in many countries, yet in recent years, it brings enormous economic loss to aquaculture once again.More than 600 pig farm brought disaster in China's Taiwan foot and mouth disease great outburst in 1997, causes enormous economic loss in Russia outburst in 2000.Caused again giving a heavy blow to continue bovine spongiform encephalopathy and swine fever two big epidemic situations infringements after for Britain aquaculture in the outburst of Britain and popular February calendar year 2001, its influence even expand to whole Europe.In China, A type FMDV, O type FMDV, AsiaI type FMDV have generation, it is popular that AsiaI type FMDV only is confined to the Yunnan Province of China border area in history, but successively broken out AsiaI type foot and mouth disease at 2004-2006, had a strong impact on local Developing of Animal Industry on Xinjiang of China, Gansu, Jiangsu, Beijing, Henan and other places.

Foot and mouth disease virus (FMDV) belongs to picorna virus, and the virus of this genus has characteristics such as pleiomorphism, changeableness.There is A, O, C, Sat1, Sat2, a Sat3 and AsiaI7 serotype in the whole world, and hypotype has more than 70, does not have cross immunity between each serotype, and virus easily morphs, and this has just caused very big difficulty for the control of FMD.At present, China carries out mandatory immunity to foot and mouth disease, and vaccine immunity is the main means of preventing and treating foot and mouth disease.And the foot-and-mouth disease vaccine of the existing use of China mainly is a viral inactivation vaccine; but there are many hidden danger in deactivation; as causing easily that owing to careless the escape and the deactivation of virus not exclusively cause breaking out once again of FMD, can not provide effective protection etc. between veriform strain in the production of vaccine process.A lot of in the world at present countries have stopped using inactivated vaccine, also forbid from using the national import livestock products of inactivated vaccine.In order to overcome self deficiency of inactivated vaccine, the various new generation vaccines of FMD continue to bring out as raising recombinant vaccine, protein carrier vaccine, gene-deleted vaccine, live vector vaccine, nucleic acid vaccine, synthetic peptide vaccine etc., but all there are problems in it at safety, immune effect, process aspect, has influenced the use of these new generation vaccines.

Studies show that: the hoof-and-mouth disease virus capsid protein is made up of each 60 of four kinds of structural protein VP1, VP2, VP3 and VP4, and VP1-VP3 is positioned at the outside of capsid protein, form capsomere, and VP4 is positioned at the inside of virion.VP1 is main protective antigen; containing FMDV major antigen binding site on it can induce body to produce neutralizing antibody; studies show that, the G-H ring between VP1 134-158 aminoacid, it is the main immunizing antigen site of inducing neutralizing antibody that C holds the 200-213 amino acid residue.In the present invention, selected the advantage B cell epitope of O type, A type and the last 130-168 of AsiaI type FMDV structural protein VP1 position.Simultaneously since foot and mouth disease mainly based on humoral immunization, effectively vaccine should be able to be induced the immunoreation of taking as the leading factor with Th2.DiMarchi etc. reported once that after being in series with the synthetic peptide immune cattle of FMDV VP1 140-160 and 200-213 amino acid residue, animal capable was resisted the infection of virus.But studies confirm that subsequently when this type of synthetic peptide vaccine is used on a large scale, but only can provide limited protection in the susceptible animal group.Trace it to its cause, the shortage of potent t cell epitope may be a factor that influences this vaccine effect.The tandem polypeptide that carries B cell antigen epi-position and T cell antigen epitope simultaneously has been proved to be cattle has been shown good immunoreactivity.Studies confirm that, on the FMDV capsid protein, have a plurality of can be by the t cell epitope of cattle and pig lymphocyte identification, but the identification of these t cell epitopes has been subjected to the restriction of host MHC polymorphism significantly, and then has also limited their application in vaccine.Discover that simultaneously endogenic t cell epitope is not enough to stimulate the susceptible animal immune system that foot and mouth disease virus is produced effectively protection.So it is to study the key of efficient peptide Seedling with the auxiliary immunne response that strengthens susceptible animal that potent external source t cell epitope is provided.

Summary of the invention

Therefore, the purpose of this invention is to provide a kind of peptide vaccine that can resist the foot and mouth disease virus infection.

Another object of the present invention provides a kind of method of producing above-mentioned peptide vaccine.

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

The invention provides a kind of peptide vaccine of foot-and-mouth disease virus resistant, it is characterized in that peptide antigen has the aminoacid sequence shown in SEQ ID NO:1, SEQ ID NO:2.

On the basis that foot and mouth disease virus antigen epitope is fully understood, obtain advantage B cell antigen epi-position and the optimum composition method thereof of foot and mouth disease virus by the experimental animal screening, and then by a large amount of laboratory tests at the potent external source t cell epitope that has found a section can strengthen advantage B cell epitope on the foot and mouth disease VP1 on the PPR virus, by certain joint aminoacid potent external source t cell epitope is connected with advantage B cell epitope then, made up the polypeptide vaccine that contains complementary potent external source t cell epitope and FMDV major antigen epitope gene, and adhered to specification fully by computer simulation.

Above-mentioned peptide Seedling, it can adopt the method for chemosynthesis to prepare antigenic synthetic peptide.

The present invention also provides the preparation method of this peptide vaccine, mainly may further comprise the steps:

(1) chemosynthesis of antigenic peptides;

(2) cracking of antigenic peptides, precipitation;

(4) high pressure liquid chromatography separation and purification polypeptide antigen;

(5) configuration of polypeptide vaccine.

The chemosynthesis of antigenic peptides is to be raw material with amino resins and FMOC aminoacid in the above-mentioned preparation method, progressively connects aminoacid, and per step is sealed unreacted active ammonia cardinal extremity with acetyl imidazole after connecing reactive polypeptide, mainly comprises the steps:

(a) the FMOC group removes: removed the FMOC group of protecting on the amino with the DMF solution that contains the 15%-35% hexahydropyridine at 20-26 ℃ of conditioned response 20-40 minute, nitrogen dries up, the DMF washing resin;

(b) connect reactive polypeptide: the aminoacid that adds HOBT, HBTU, DIEA and FMOC protection was at 20-28 ℃ of conditioned response 0.5-2.5 hour, and nitrogen dries up, the DMF washing resin;

(c) capping: the acetyl imidazole that uses 1.5%-3.5% was at 20-26 ℃ of conditioned response 15-30 minute.

In the above-mentioned preparation method in the antigenic peptides cracking precipitation operation employed lytic reagent be: trifluoroacetic acid (TFA), phenol (Phenol) and water (H2O), their ratio is: 90/8/2, the time of lytic response is 1-3 hour.

High pressure liquid chromatography is mainly used in the antigenic separation and purification of peptide in the above-mentioned preparation method, and chromatographic condition is as follows:

(a) C ₁₈Reversed phase chromatographic column, specification: 19mm * 250mm * 5um, aperture 300 dusts;

(b) for containing the water of 0.5%TFA, B contains the acetonitrile of 0.5%TFA to mobile phase: A mutually mutually.

The method of vaccine preparation comprises as follows in the above-mentioned preparation method:

(a) with water for injection with the peptide antigen diluent to 30-200ug/ml;

(b) with standby behind the oily adjuvant autoclaving.

(c) under 20-28 ℃ of condition, antigen water and oily adjuvant are carried out emulsifying according to 5: 5 ratio, promptly obtain polypeptide vaccine after the packing.

The present invention further provides above-mentioned peptide and composition thereof and prevented and/or treated purposes in the medicine of foot and mouth disease in preparation.

The present invention is at detailed understanding foot and mouth disease virus molecular structure and biological function thereof; analyze the branch subconstiuent that virus suppresses host immune response; and activate on the molecular basis of host immune cell generation protective immune response; at the popular present situation of China's foot and mouth disease; go up the dominant antigen epitope gene based on O type, A type and AsiaI type FMDV primary structure albumen VP1, designed new and effective peptide Seedling in conjunction with potent external source Th cell epitope.Anti-FMDV peptide vaccine of the present invention can successfully manage the antigenic variation of foot and mouth disease virus, not have biological safety, is easy to extensive synthesizing, and has a good application prospect.

The present invention is described further below in conjunction with specific embodiment.

Example I. peptide antigen solid state chemistry is synthetic

The antigenic preparation of peptide of the present invention can use full-automatic polypeptide synthetic instrument to use classical solid state chemistry synthetic method, employing be FMOC protection aminoacid, what solid phase carrier adopted is amino resins.

Production process is divided into cracking, antigen purification and the degerming of the antigenic solid phase synthesis of peptide, peptide usually and preserves.Concrete steps are as follows:

1.1 the antigenic solid phase synthesis of peptide

1.1.1 the preparation of synthesis material

The sequence of antigenic synthetic peptide as: shown in SEQ ID NO:1, the SEQ ID NO:2.

Prepare suitable protection aminoacid according to above-mentioned antigenic sequence and synthetic scale, join in the corresponding amino demijohn.Weighing amino resins is put into reaction chamber equally on request, and up and down lid is tightened, label, record title, the tare weight of reaction chamber and the weight of alleged resin of synthetic peptide.With the reaction chamber synthesizer of packing into.The preparation synthetic agent comprises that DMF, AIM (hexanoyl imidazoles), PIP (piperidines), methanol etc. are placed in the corresponding reagent bottle.

1.1.2 synthesizer status detection

Check whether normally operation of synthetic instrument, after the start, the operation self-check program, whether the every index of instrument self checking is normal.Check in addition whether nitrogen is sufficient, and 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.Send test program to synthesizer, measure or observe,, then regulate valve pressure up and down, until reaching requirement if flow is improper.

Reagent	The bottle number	Test program	The testing standard scope
				Piperidine	1	I	1.0-1.2ml
AIM	5	E	1.0-1.2ml
				MeOH	9	D	3.5-4.0ml
DIC	8	S	0.45-0.55g
				DMF	10	A	2.6-2.8ml

1.1.3 the antigenic synthetic beginning of peptide

Will synthetic aminoacid sequence in the program of synthesizer and synthetic method send on the synthesizer.The sequence of the synthetic peptide of editor is preserved.Check whether synthetic method is correct, whether sequence sets synthesis cycle by being deposited name; Determine the back preservation, then working procedure.

1.1.4 peptide is antigenic synthetic

As above-mentioned peptide sequence, be to begin end in the time of synthetic to N from the C end, the program according to setting constantly is repeated below synthesis step successively:

(1) removes the FMOC blocking group: use the DMF solution that contains 25% hexahydropyridine to remove the FMOC group of protecting on the amino in 30 minutes at 25 ℃ of conditioned responses;

(2) washing resin: nitrogen dries up resin, uses DMF washing resin 5 times;

(3) connect reactive polypeptide: add aminoacid that HOBT, HBTU, DIEA and FMOC protect 25 ℃ of conditioned responses 1.5 hours, nitrogen dries up resin, then the DMF washing resin;

(4) washing resin: nitrogen dries up resin, uses the DMF washing resin 5 times;

(5) capping: use 2.5% acetyl imidazole 25 ℃ of conditioned responses 30 minutes, the unreacted active amino is sealed;

(6) the synthetic post processing of polypeptide antigen: take off reactor from Peptide synthesizer behind the end of synthesis, reuse methanol wash peptide resin 5 times, the back dries up in ventilating kitchen, then polypeptide resin is transferred in the brown bottle, puts into-20 ℃ of refrigerators, and it is standby to seal film phonograph seal.

1.2, the antigenic cracking of peptide and evaluation

1.2.1, the antigenic cracking of peptide

Proportionally (TFA/Phenol/H2O=90/8/2) prepares lysate, in refrigerator, take out synthetic good polypeptide resin then, put into round-bottomed flask, in ventilating kitchen, in flask, add lysate and the magnetic stick for preparing, stably be placed on then on the magnetic stirring apparatus, continue to stir 2 hours under the room temperature to reacting completely.After reaction finishes, use the Rotary Evaporators of band cold-trap to continue the TFA that evaporation was removed in the thick product in 80 minutes.Use dimethylformamide (DMF) repeatedly to clean the crude product of polypeptide antigen then, at last the resin that mixes is filtered out with the sand core funnel, both obtained polypeptide antigen.

1.2.2, the evaluation of synthetic antigen

Peptide antigen is synthetic, and the back that finishes flies to try time mass spectrum method (MALDL-TOF) and reversed-phase high pressure liquid chromatography (RP-HPLC) carries out qualitative and quantitative analysis with substance assistant laser desorpted.

1.3, the antigenic separation and purification of peptide

High pressure lipuid chromatography (HPLC) is adopted in the antigenic separation and purification of peptide, and chromatographic condition is: permaphase ODS C ₁₈(post specification 19mm * 250mm * 5 μ, 300A, Hypersil); Mobile phase A (0.5%TFA/H ₂O), Mobile phase B (0.5%TFA/ acetonitrile); Condition of gradient elution is to change to 50%B from 10%B in the 30min; Detect wavelength 216nm; Flow velocity is 1.0mL/min; Sample size: 20ul.

1.4, the preparation of peptide vaccine

1.4.1, peptide antigen water preparation

Peptide antigen lyophilization that will be synthetic good, various antigen takes by weighing antigen according to 1: 1: 1 mass ratio and mixes during preparation multivalence peptide vaccine, with sterilized water for injection antigenic synthetic peptide is diluted to 180 μ g/ml; During preparation unit price peptide vaccine this type peptide antigen is diluted to 60 μ g/ml with sterilized water for injection.The filter that with peptide antigenic solution via hole diameter is 0.2 μ m then filters degerming.

1.4.2, the preparation of vaccine oil phase

Oily adjuvant through 121 ℃, was sterilized in 30 minutes, standby.

1.4.3, the emulsifying of peptide vaccine

It in oil phase and antigen water 5: 5 ratio, earlier oil phase is added in the emulsion tank, after starting motor and stirring 5 minutes with the 200r/m slow rotation, slowly add water antigen simultaneously, adding the back stirred 15 minutes, with 5000r/m high-speed stirred 10 minutes, left standstill 10 minutes again, make vaccine be emulsified into water in oil single-phase vaccine.

The safety testing of example II peptide vaccine

According to the foregoing description synthetic shown in SEQ ID NO:1, SEQ ID NO:2 the polypeptide antigen of aminoacid sequence, according to the polypeptide vaccine of the foregoing description preparation foot-and-mouth disease virus resistant, every batch of vaccine antigen content is 60ug/ml then.10 of the Cavia porcelluss of every crowd of vaccine usefulness body weight 350～450g, every subcutaneous injection 2ml; With 10 of the mices of body weight 18～22g, every subcutaneous injection 0.5ml.Observed 7 continuously, judge dead or tangible local untoward reaction or the general reaction that cause because of vaccinate whether occur.Observed result sees Table 2.

Table 2: peptide vaccine is to the safety testing result of Cavia porcellus and mice

From this result of the test as can be seen, observed 7 continuously behind peptide vaccine immune guinea pig of the present invention and the mice, dead or tangible local untoward reaction or the general reaction that cause because of vaccinate all do not occur.In entire test, the phenomena of mortality all do not appear in Cavia porcellus and mice.This illustrates that peptide vaccine of the present invention is safe for Cavia porcellus and mice.

The immune effect research of EXAMPLE III peptide vaccine

Healthy guinea pig about body weight 450 grams is divided into five groups at random, and every group of six Cavia porcelluss carry out multiple spot intramuscular injection experimental vaccine at back leg, and each organizes the injections of antigens kind and dosage sees Table 1.The immunity second time was carried out in immunity for the first time in back 28 days, the situation that anti-foot and mouth disease specific antibody of blood sampling mensuration then and neutralizing antibody produce.

Table 1: each organizes Cavia porcellus immunizing antigen type and dosage

1, indirect elisa method detects the foot and mouth disease virus specific antibody

Be envelope antigen (1: 10 dilution) with O type foot and mouth disease virus on 96 hole ELISA Plate, tested guinea pig serum is one anti-, and the anti-Cavia porcellus IgG of the rabbit of peroxidase labelling is two anti-, measures the OD value that each learns the final proof product at wavelength 492nm place, and the while returns to zero with blank well.

Take a blood sample 3 at random for every group,, get the meansigma methods (seeing Table 2) of every group of indirect ELISA method measurement result with 1: 32 dilution guinea pig serum.Table 2 shows that except that the 4th group, second week of the specific antibody level of each immune group Cavia porcellus after the immunity first time obviously improves, i.e. the specific antibody level of resisting O-type foot and mouth disease virus synthetic peptide vaccine group obviously improving in second week behind first time immune guinea pig.Simultaneously, its specific antibody level is higher than the inactivated vaccine group.

Table 2: indirect ELISA detection guinea pig serum FMDV specific antibody result (X ± SE)

2, the detection of guinea pig serum neutralizing antibody

Select immunity preceding (0 day) respectively for use, the immunity back is the 28th day for the first time, and the guinea pig serum of immune for the second time back the 3rd week (the 49th day) is used for microneutralization test, surveys 2 Cavia porcelluss for every group.After the guinea pig serum deactivation with the normal saline dilution, make a series of doubling dilutions with diluent on the trace Tissue Culture Plate of 96 holes, each dilution factor is done 4 holes, and every hole adds 50 μ l virus liquid (100TCID ₅₀), every hole adds 100 μ l cell suspension in 37 ℃ of incubators and behind the 1h, places 37 ℃ of cultivations of 5%CO2 incubator, begins to observe from 48h, declares eventually to 144h.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 dilution factor is the NAT of this part serum.

The result is as shown in table 3, except that the 4th group, neutralizing antibody all appears in each test group after immunity for the first time, and second group, the 3rd group NAT is higher than first group, and promptly the NAT that produces after the synthetic peptide vaccine immunity of resisting O-type foot and mouth disease virus is higher than the NAT that inactivated vaccine produces.

Table 3: the neutralizing antibody level of Cavia porcellus

By The above results as can be seen, peptide vaccine is after immunity on the Cavia porcellus, and the specific antibody level of generation and neutralizing antibody level all are higher than the foot and mouth disease inactivated vaccine, is higher than the antibody horizontal that the adjuvant group produces far away.This has embodied the potential actual application value of peptide vaccine.

3, peptide vaccine lymphocyte proliferation assay

Test is prepared:

96 orifice plates, incubator, Bio-Rad microplate reader, Biohazard Safety Equipment

3-(4,5-dimethylthiazole-2)-2, the preparation of 5-diphenyl tetrazole bromine salt (MTT): MTT 50mg is dissolved in the mixed solution of the hyclone (FCS) of 10ml RPMI1640 and 0.1ml, promptly obtains the MTT. of 5mg/ml

Test antigen:

According to antigen sequence shown in the following table 4 according to the foregoing description antigenic synthetic peptide.

Test method and result:

Aseptic separating guinea pig spleen lymphocyte, adjusting cell concentration is 106/mL, and cell suspension 0.1ml is added 96 porocyte culture plates, adds 30ug peptide antigen more respectively, each antigen is provided with 3 repetitions.After cultivating 48h, 37 ℃ of stimulations add MTT, continue to cultivate 4h, add again and be preheating to 37 ℃ dimethyl sulfoxide (DMSO), persistent oscillation 5min, survey the OD570/OD630 absorbance value with the Bio-Rad microplate reader, (Stimulation Index, SI): SI=treats gaging hole OD value/blank hole OD value, result of calculation such as following table 1 to calculate stimulation index by following formula.

Table 4: lymphocyte proliferation assay result: stimulation index SI

Last table explanation: all there is significant difference (P＜0.05) in immune group (first group to the 3rd group) with matched group (the 4th group).Each immune group is compared, and second group, the 3rd group stimulation index will be higher than first group, illustrates that the relative inactivated vaccine of this synthetic peptide vaccine has stronger stimulation to mouse lymphocyte.

Sequence table

＜110〉Tao Yu, Shao Guohu

＜120〉a kind of novel peptide-based vaccine used for domestic animal and preparation method thereof

＜130〉a kind of novel peptide-based vaccine used for domestic animal and preparation method thereof

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<170>PatentIn version 3.3

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<212>PRT

＜213〉artificial sequence

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Gly Leu Ser Ile Leu Phe Leu Ser Glu Ile Lys Gly Val Leu Val His

1 5 10 15

Lys Ile Glu Lys Lys Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser

20 25 30

Pro Val Ala Asn Val Arg Gly Asp Leu Gln Val Leu Thr Pro Lys Ala

35 40 45

Ala Arg Thr Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile

50 55 60

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<211>61

<212>PRT

＜213〉artificial sequence

<400>2

Gly Leu Ser Ile Leu Phe Leu Ser Glu Ile Lys Gly Val Leu Val His

1 5 10 15

Lys Ile Glu Lys Lys Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser

20 25 30

Pro Val Thr Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala

35 40 45

Ala Arg Thr Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile

50 55 60

Claims (7)

1, a kind of peptide vaccine of foot-and-mouth disease virus resistant is characterized in that peptide antigen has the aminoacid sequence shown in SEQ IDNO:1, SEQ ID NO:2.

2, vaccine described in the claim 1 is characterized in that, described vaccine also comprises corresponding adjuvant.

3, described polypeptide of claim and composition thereof prevents and/or treats purposes in the medicine of foot and mouth disease in preparation.

4, a kind of method according to the peptide antigen preparation vaccine described in the claim 1 mainly may further comprise the steps:

(1) chemosynthesis of antigenic peptides;

(2) cracking of antigenic peptides, precipitation;

(3) high pressure liquid chromatography separation and purification polypeptide antigen;

(4) vaccine configuration.

5, vaccine production method according to claim 4, the chemosynthesis of antigenic peptides is characterized in that, be to be raw material with amino resins and 9 fluorenylmethyloxycarbonyls (Fmoc) aminoacid, progressively connect aminoacid, per step is sealed unreacted active ammonia cardinal extremity with acetyl imidazole after connecing reactive polypeptide, mainly comprises the steps:

(a) the FMOC group removes: removed the FMOC group of protecting on the amino with dimethylformamide (DMF) solution that contains the 15%-35% hexahydropyridine at 20-26 ℃ of conditioned response 20-40 minute, nitrogen dries up, dimethylformamide (DMF) washing resin;

(b) connect reactive polypeptide: add 1-hydroxyl phenylpropyl alcohol triazole (HOBT), BTA-N, N, N ', the aminoacid of N '-tetramethylurea hexafluorophosphoric acid ester (HBTU), diisopropylethylamine (DIEA) and FMOC protection was at 20-28 ℃ of conditioned response 0.5-2.5 hour, nitrogen dries up, dimethylformamide (DMF) washing resin;

(c) capping: the acetyl imidazole that uses 1.5%-3.5% was at 20-26 ℃ of conditioned response 15-30 minute.

6, according to the preparation method of the described vaccine of claim 4, wherein said polypeptide antigen cracking agents useful for same is: trifluoroacetic acid (TFA)/phenol (Phenol)/H2O=90/8/2, the cracking time is 1-3 hour.

According to the preparation method of the described vaccine of claim 4, be characterised in that 7, the method for vaccine preparation comprises as follows:

(a) with water for injection with the peptide antigen diluent to 30-200ug/ml;

(b) with standby behind the oily adjuvant autoclaving.

(c) under 20-28 ℃ of condition, antigen water and oily adjuvant are carried out emulsifying according to 5: 5 ratio, promptly obtain peptide vaccine after the packing.