CN103214560A - Polypeptide used for preparing bovine foot and mouth disease type O peptide vaccine, and preparation method and application thereof - Google Patents
Polypeptide used for preparing bovine foot and mouth disease type O peptide vaccine, and preparation method and application thereof Download PDFInfo
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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
Technical field
The invention belongs to medical technical field, particularly, the present invention relates to a kind of polypeptide that is used to prepare ox foot and mouth disease O type peptide vaccine, and contain the vaccine of 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 is taken place.The infectivity height of foot and mouth disease is propagated rapidly, will cause cub death behind the livestocks such as infected pigs, ox, sheep, and adult animals throughput sharply descends, so the production and supply of serious harm Developing of Animal Industry and meat and livestock product thereof.At present, foot and mouth disease makes the market circulation of animal and animal product and international trade be subjected to blocking greatly and restriction, produces for the livestock industry of popular countries and regions and causes enormous economic loss.
Foot and mouth disease is infected by foot and mouth disease virus (FMDV) and causes.Foot and mouth disease virus belongs to picornavirus, has characteristics 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).In these principal modes each is divided some hypotypes again, at present the existing kind more than 70 of the hypotype of finding.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 that 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, have now found that O type foot and mouth disease has 3 main antigen sites to be positioned on the VP1, and wherein 133-160 and 200-213 position have constituted VP1 and go up most important and the protective antigen site of easy variation.
Antigenicity difference between foot and mouth disease virus is various, immunity alternately each other.And the degree of antigenic difference is also very big in a kind of serotype, may not have protectiveness at the another kind of hypotype in the same serotype to such an extent as to can resist a kind of aftosa vaccine of hypotype effectively.In addition, the antigenicity of foot and mouth disease strain is also constantly changing, and As time goes on, the effectiveness of original vaccine weakens even disappears, and has brought very big difficulty therefore for the preventing and controlling of foot and mouth disease.
Current in China cows the popular foot and mouth disease mainly be 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 mainly is a viral inactivation vaccine, there is problems such as biological safety is poor, side reaction big, unstable product quality.By contrast, a lot of in the world at present countries have stopped using inactivated vaccine, also forbid from using the national import livestock product of inactivated vaccine.This shows that aspect foot and mouth disease prevented and treated, China had lagged behind the 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 has influenced the use of these new generation vaccines all existing problems aspect immune effect, the biological safety.In addition, often effect is relatively poor for the current epidemic isolates that has morphed for these vaccines, can not watch for animals effectively.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 that is used to prepare ox foot and mouth disease O type synthetic peptide vaccine, and the vaccine that contains this polypeptide.
Another object of the present invention provides the preparation method of a kind of aforementioned polypeptides and vaccine.
Another purpose of the present invention provides 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 that is used to prepare ox foot and mouth disease O type peptide vaccine, described polypeptide comprises the aminoacid sequence 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 at foot and mouth disease virus.
SEQ?ID?NO.?1：
YNGSCKYGDASANNVRGDLQVLALKTEKCLPTSFNYGAIK
" polypeptide " mentioned among the present invention and " synthetic peptide " for identical concept, all are meant the peptide material that obtains by the solid phase organic synthesis.
Preferably, described polypeptide also comprises the endogenous or exogenous t cell epitope aminoacid sequence of the foot and mouth disease with enhancing immunity effect, and wherein, described aminoacid sequence is selected from one or more among SEQ ID NO. 2, SEQ ID NO. 3 and the SEQ ID NO. 4.In the present invention, by the t cell epitope of computer forecast foot and mouth disease O virus, utilize the new t cell epitope peptide of software design then, again by in-vitro evaluation animal cell immunity level, screening obtains the polypeptide of above-mentioned aminoacid sequence thus, and it can assist the 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 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, amino acid sequence of polypeptide of the present invention is shown in SEQ ID NO.5, SEQ ID NO. 6 or SEQ ID NO. 7;
Preferably, the sulfydryl of two halfcystines in the described amino acid sequence of polypeptide can be joined together to form disulfide linkage through oxidation;
Further preferably, can react between the head and the tail amino-acid residue of described amino acid sequence of polypeptide form covalently bound.Sequence wherein provided herein is to hold the C end from N, and the residue reaction of N end and C end just forms and is connected.Particularly, reaction forms covalently bound between the carboxyl of the head and the tail amino-acid residue of described amino acid sequence of polypeptide and amino or carboxyl and the hydroxyl.
Aforementioned polypeptides provided by the invention can be directly obtains to the customization of commercial peptide Synesis Company, perhaps 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 may further comprise the steps:
(1) with aminoresin being starting raw material, is monomer with the amino acid by the 9-fluorenylmethyloxycarbonyl protection, connects amino acid with synthetic described polypeptide according to described aminoacid sequence condensation successively, seals unreacted aminoterminal with acetyl imidazole after per step condensation reaction;
(2) the synthetic back that finishes adds lytic reagent, thereby the cracking from the aminoresin of described polypeptide is got off;
(3) use the described polypeptide of ether sedimentation; With
(4) described polypeptide is carried out ultrafiltration purification, carry out aseptically process then.
In above-mentioned preparation method, described step (1) specifically may further comprise the steps:
(1-a) deprotection reaction: it is N-Methyl pyrrolidone (NMP) solution of the hexahydropyridine of 15-30% that aminoresin is placed volume percent, reaction is 25-40 minute under 20-28 ℃ of condition, thereby removes the 9-fluorenylmethyloxycarbonyl blocking group on the aminoresin;
(1-b) washing: nitrogen dries up, and washs aminoresin with N-Methyl pyrrolidone then;
(1-c) condensation reaction: add 1-hydroxyl azimidobenzene (HOBT), dicyclohexylcarbodiimide (DCC) and the amino acid of protecting by 9-fluorenylmethyloxycarbonyl, under 20-28 ℃ of condition, reacted 0.5-2.5 hour then;
(1-d) washing: nitrogen dries up, and washs aminoresin with N-Methyl pyrrolidone then; With
(1-e) capping: adding percent weight in volume is N-Methyl pyrrolidone (NMP) solution of the acetyl imidazole of 1.5-4%, and reaction is 20-40 minute under 20-28 ℃ of condition.
In above-mentioned preparation method, the component of lytic reagent is that volume ratio is the trifluoroacetic acid of 85:8:6:1 in the described step (2): tri isopropyl silane: phenol: H
2O; And the cracking time of described step (2) is 1-4 hour.
In above-mentioned preparation method, described step (3) specifically comprises:
(3-a) use the described polypeptide of ether sedimentation, wash with dimethyl formamide then;
(3-b) account under dimethyl sulfoxide (DMSO) (DMSO) situation of reaction system cumulative volume 10%, make to precipitate in the amino acid sequence of polypeptide that obtains to form disulfide linkage between two halfcystines in adding; With
(3-c) make that reaction forms covalently bound between the head and the tail amino-acid residue of described amino acid sequence of polypeptide; Preferably, account for the carboxyl of the head and the tail amino-acid residue that makes described amino acid sequence of polypeptide under the DIC (DIC) of reaction system cumulative volume 1% and 0.5% 1-hydroxyl-7-azo benzotriazole (HOAT) situation with amino or at adding 0.1M H in adding
2SO
4It is covalently bound that reaction is formed.
In above-mentioned preparation method, described step (4) specifically may further comprise the steps:
(4-a) use the tangential flow filtration film to wrap in the described polypeptide of ultrafiltration under the 20-28 ℃ of condition, thereby remove small molecular weight impurity; With
(4-b) use 0.2 micron 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 among white oil, 50V, the 50VII one or more.Further preferably, the polypeptide that is comprised in the 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 may further comprise the steps:
(1) is the concentration of 50 μ g/ml with water for injection with described polypeptide dilution, thereby obtains the polypeptide antigen water;
(2) adjuvant was sterilized 30 minutes under 121 ℃ of conditions; With
(3) under 20-28 ℃ of condition, volume ratio according to described polypeptide antigen water and described adjuvant 1:1, earlier adjuvant is added in the emulsion tank, stirred 1.5-3 minute down at 90-150 rev/min, slowly add the polypeptide antigen water then, stirred 20-30 minute, stirred 15-30 minute down at 8000-10000 rev/min again, left standstill packing 5 minutes.
Also on the one hand, the invention provides aforementioned polypeptides or peptide vaccine are used for preventing the medicine of ox foot and mouth disease O type in preparation purposes.
Particularly, the inventor by to domestic foot and mouth disease recently epidemic isolates sequencing and in conjunction with aftosa vaccine strain sequence, the variation situation in research foot and mouth disease major antigen site, add up the frequency of its variation at the amino acid sites of main variation, simultaneously carry out the analyses and prediction in foot-and-mouth disease antigen site in conjunction with area of computer aided, possible antigen site peptide section is carried out chemosynthesis, promptly at the variation frequency of easy variant sites according to statistics, use different amino acid in these sites, obtain containing multiple candidate's polypeptide antigen of the possible variant sites of current institute.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 height, the polypeptide antigen of the attack of avoiding the foot and mouth disease epidemic isolates of can be good at watching for animals.In addition, the inventor optimizes the foot-and-mouth disease virus antigen site according to the screening experiment result, and has effectively made up t cell epitope and B cell epitope, has strengthened the immune effect of polypeptide antigen.
Peptide vaccine is renderd a service experiment, the safety experiment result shows, ox foot and mouth disease O type synthetic peptide vaccine provided by the invention has good immune efficacy, and can not cause problems such as heating that traditional vaccine exists, redness, therefore vaccine of the present invention can successfully manage the antigenic variation of present foot and mouth disease virus, not have biological safety, be easy to extensive synthesizing, have a good application prospect.
Embodiment
Followingly the present invention is described with reference to specific embodiment.It will be appreciated by those skilled in the art that these embodiment only are used to illustrate the present invention, the scope that it does not limit the present invention in any way.
Experimental technique among the following embodiment if no special instructions, is ordinary method.Used medicinal raw material, reagent material etc. if no special instructions, are commercially available purchase product among the following embodiment.
The solid phase synthesis of 1 N of foot and mouth disease O of embodiment type antigenic synthetic peptide
Antigenic synthetic peptide of the present invention can use 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 available from U.S. Sigma company.Production process generally includes the solid phase synthesis of polypeptide antigen, cracking, antigen purification and the degerming of polypeptide preserved.
1.1 the solid phase synthesis of antigenic synthetic peptide
1.1.1 synthesis material is prepared
The sequence of synthetic polypeptide antigen is respectively shown in SEQ ID NO.5, SEQ ID NO. 6 or SEQ ID NO. 7.
According to the synthetic scale of antigenic sequence and 1mmol, prepare the amino acid (available from Shanghai gill biochemistry) that suitable Fmoc modifies, add in the corresponding amino acid bottle.Weighing Rink Amide mbha resin is put into reaction chamber equally on request, and up and down lid is tightened, label, record title, lot number, 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 N-Methyl pyrrolidone (NMP), acetyl imidazole (AIM), piperidines (PIP), methyl alcohol etc. are placed in the corresponding reagent bottle.
1.1.2 synthesizer state-detection
Check whether normally operation of Peptide synthesizer.After the start, operation Run Self Test program, whether the every index of instrument self checking is normal.Check N in addition
2Whether sufficient, whether system's gauge pressure is normal.The performance of reply instrument is had gained some understanding before synthetic, so will measure the flow velocity of every kind of synthetic agent.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, then regulate lower valve pressure, until reaching requirement.
1.1.3 the synthetic beginning of antigenic synthetic peptide
The method Std Fmoc 1.0 Sol DIC90 that will synthesize needs in the program of synthesizer send on the synthesizer.File-New-Sequence-edits the sequence of synthetic peptide, preserves.File-New-Run checks Chemistry; Whether Sequence is by being deposited name; Set Cycles; Preserve.Send on the synthesizer at last.
Main Menu-Cycle Monitor-begin brings into operation.
1.1.4 antigenic synthetic peptide is synthetic
As above-mentioned peptide sequence, be to begin end in the time of synthetic to N from the C end, according to given order, constantly be repeated below synthesis step successively:
(1) deprotection reaction: it is the nmp solution of the hexahydropyridine of 15%-30% that above-mentioned aminoresin is placed volume percent, the Fmoc blocking group that reaction removed on the aminoresin in 25-40 minute under 20-28 ℃ of condition;
(2) washing: nitrogen dries up, and NMP washs aminoresin;
(3) condensation reaction: the amino acid that adds HOBT, DCC and Fmoc protection reacted 0.5-2.5 hour 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%, and reaction is 20-40 minute under 20-28 ℃ of condition.
1.1.5 antigenic synthetic peptide end of synthesis
Synthesizer will stop automatically behind the antigen end of synthesis.Take off reactor from Peptide synthesizer then, use 100% methanol wash polypeptide resin 3 times again, dry up in stink cupboard then, polypeptide resin is transferred in the brown bottle, put into-20 ℃ of refrigerators, it is standby to seal film phonograph seal.
1.2 the cracking of antigenic synthetic peptide 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, in refrigerator, take out the synthetic polypeptide resin then, put into round-bottomed flask, in stink cupboard, in flask, add lysate and the magnetic stick for preparing, stably be placed on then on the magnetic stirring apparatus, continue to stir 1 hour under the room temperature until 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 30 to 120 minutes.Then use ether to collect, precipitate polypeptide, use dimethyl formamide (DMF) repeatedly to clean the crude product of polypeptide antigen then, at last the resin that mixes is filtered out with sand core funnel, promptly obtain polypeptide antigen.
1.2.2 the evaluation of antigenic synthetic peptide
Qualitative and quantitative analysis is carried out with substance assistant laser desorpted time-of-flight mass spectrometry (TOFMS) (MALDL-TOF) and reversed-phase high pressure liquid chromatography (RP-HPLC) in the synthetic back that finishes of polypeptide antigen.
1.3 the conformation of antigenic synthetic peptide forms
With 15%DMSO polypeptide antigen is mixed with the polypeptide solution that concentration is 2mg/ml, adjust pH value=8.5 of initial gross separation polypeptide solution then with 0.1N NaOH or 0.1N HCl, being to place 48 hours on the shaking table of 110rpm at rotating speed under 25 ℃ the environment, make the formation disulfide linkage.
And then carry out the 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 " and " OH " is reacted and form ring texture is referring to Chem.Soc 1970.92:3771-3777 such as Mmenhofer.Obtain thus can simulated virus particle native conformation the polypeptide cyclized structure.
1.4 the purifying degerming of antigenic synthetic peptide
Antigenic synthetic peptide uses circulating tangential flow filtration film to wrap in to carry out ultrafiltration (Tangential Flow Device circulating tangential flow filtration film bag and the peristaltic pump supporting with it) under the 20-28 ℃ of condition, 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 then can pass through filter membrane.And then be 0.2 μ m pot strainer degerming by the aperture, the solution branch that obtains is at last installed in the aseptic plastic bottle, labelled.Indicate title, numbering, product batch number, concentration, date manufactured, storage life and the preservation condition of polypeptide on the label, it is standby after the packing, to be stored in-20 ℃ or-40 ℃.
Needs for the ease of transportation and prolonged preservation carry out lyophilize to obtain the polypeptide of solid state with polypeptide antigen.Take out freezing good polypeptide antigen in advance, on the Labconco freeze drier, carry out drying, obtain the polypeptide antigen of solid state.Simultaneously labelled.Indicate title, numbering, product batch number, concentration, date manufactured, storage life and the preservation condition of polypeptide on the label.
The preparation of embodiment 2 synthetic peptide vaccines
2.1 the preparation of antigen water
Take by weighing respectively according to the sequence of the embodiment 1 preparation antigenic synthetic peptide shown in SEQ ID NO.5, SEQ ID NO. 6 or SEQ ID NO. 7 respectively, then with sterilized water for injection with antigenic synthetic peptide concentration dilution to 50 μ g/ml.With gained antigenic solution via hole diameter is the strainer filtration of 0.2 μ m, degerming.
2.2 oil phase adjuvant preparation
Oil phase adjuvant 50V is through 121 ℃ of sterilizations 30 minutes, standby.
2.3 the emulsification of synthetic peptide vaccine
Distilled water 2000ml with sterilization cleans the IKA emulsifying device 3 times, under 20-28 ℃ of condition, be the volume ratio of 1:1 then by oil phase adjuvant and antigen water, earlier oil phase is added in the emulsion tank, after starting motor and stirring 2 minutes with 90～150r/m slow rotation, add simultaneously slowly water antigen, add the back and stirred 30 minutes, again with 10000r/m high-speed stirring 20 minutes, left standstill 5 minutes, and made vaccine be emulsified into water in oil single-phase vaccine.
3 Ns of foot and mouth disease O of embodiment type synthetic peptide vaccine potency test
1. materials and methods
1.1 synthetic peptide vaccine
According to the polypeptide antigen of embodiment 1 preparation sequence shown in SEQ ID NO.5, SEQ ID NO. 6 or SEQ ID NO. 7, the corresponding lot number of preparing respectively according to embodiment 2 is then: the O type aftosa synthetic peptide vaccine of ZM01, ZM02 and ZM03.
1.2 experimental animal
Select 6 monthly ages, 37 of the healthy oxes (available from cattle farm, area, Lanzhou) of foot-and-mouth disease antibody feminine gender (suckling mouse NAT≤1: 4).
1.3 seed culture of viruses OS/99
Measure and adjust malicious valency with 3-4 age in days suckling mouse, place-25 ℃ of freezing preservations standby.
1.4 test method
With 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 provides lot number 1112001 by biological pharmaceutical factory, Zhongmu Stocks Trading Co. Lanzhou) as positive control simultaneously, 5 oxen of immunity.2 oxen of negative control.Adopt the injection of posterior auricular muscle meat during immunity, injected dose is the 2ml/ ox.After the 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, contains 10000ID
50).Attack poison after 10 days, the observed and recorded test-results.
1.5 the result judges
Bubble or ulcer appear in contrast Niu Junying at least 3 hoof.Immune cattle only bubble or ulcer occur and other position is judged to protection when not having pathology at lingual surface, is judged to when typical foot and mouth disease bubble or ulcer appear in arbitrary position except that lingual surface and does not protect.
2. test-results and discussion
2.1 test-results
After the 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, contains 10000ID
50).Attack poison after 10 days, the result sees table 1 for details.
Table 1 ox foot and mouth disease O type synthetic peptide vaccine potency test result
2.2 discussion of results
From this test-results as can be seen, ox foot and mouth disease O type synthetic peptide vaccine of the present invention protection ratio behind this animal of immunity ox is up to 100% protection between 80% to 100%.The ox foot and mouth disease O type synthetic peptide vaccine that proves these antigen preparations thus has good immune efficacy and clinical application potentiality.
The safety testing of 4 Ns of foot and mouth disease O of embodiment type synthetic peptide vaccine
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 with 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.Observed 7 continuously, dead or tangible local untoward reaction or the systemic reaction that cause because of vaccinate all must not occur.
1.3.2 with 18 of the healthy susceptible oxen (foot and mouth disease cell NAT is not higher than 1:8) at least 6 monthly ages, divide 20 some vaccinate 2ml in every cow tongue intracutaneous, every some 0.1ml observed 4 day by day at least.Afterwards, every ox intramuscular injection vaccine 9ml continues to observe day by day 6.The toxic reaction that the foot and mouth disease symptom all must not occur or significantly cause because of vaccinate.
2. test-results
2.1 vaccine is to the security of cavy and mouse
12 of cavys, every subcutaneous injection vaccine 2ml; 30 of mouse, every subcutaneous injection 0.5ml.Observed 7 continuously, dead or tangible local untoward reaction or systemic reaction, concrete outcome such as the following table 2 that cause because of vaccinate all do not occur.
The vaccine safety test-results of table 2 cavy and mouse
2.2. vaccine is to the security of healthy susceptible ox
With synthetic peptide vaccine take out equilibrate to room temperature after, divide 20 some vaccinate 2ml in every cow tongue intracutaneous, every some 0.1ml observed 4 day by day at least.Afterwards, every ox intramuscular injection vaccine 9ml continues to observe day by day 6.Concrete outcome sees 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 to have side reaction problems such as heating, redness the traditional vaccine, so have good promotion prospect and marketable value.
More than specific description of embodiments of the present invention does not limit the present invention, those skilled in the art can make various changes or distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the scope of claims of the present invention.
Claims (12)
1. a polypeptide that is used to prepare ox foot and mouth disease O type peptide vaccine is characterized in that described polypeptide comprises the aminoacid sequence shown in SEQ ID NO. 1.
2. polypeptide according to claim 1, it is characterized in that, described polypeptide also comprises the endogenous or exogenous t cell epitope aminoacid sequence of the foot and mouth disease with enhancing immunity effect, wherein, described aminoacid sequence is selected from one or more among SEQ ID NO. 2, SEQ ID NO. 3 and the SEQ ID NO. 4;
Preferably, described polypeptide comprises the aminoacid sequence shown in SEQ ID NO.5, SEQ ID NO. 6 or SEQ ID NO. 7.
3. polypeptide according to claim 2 is characterized in that, described amino acid sequence of polypeptide is shown in SEQ ID NO.5, SEQ ID NO. 6 or SEQ ID NO. 7;
Preferably, the sulfydryl of two halfcystines in the described amino acid sequence of polypeptide is joined together to form disulfide linkage through oxidation;
Further preferably, reaction forms covalently bound between the head and the tail amino-acid residue of described amino acid sequence of polypeptide;
More preferably, reaction forms covalently bound between the carboxyl of the head and the tail amino-acid residue of described amino acid sequence of polypeptide and amino or carboxyl and the hydroxyl.
4. according to the preparation method of each described polypeptide in the claim 1 to 3, it is characterized in that described preparation method may further comprise the steps:
(1) with aminoresin being starting raw material, is monomer with the amino acid by the 9-fluorenylmethyloxycarbonyl protection, connects amino acid with synthetic described polypeptide according to described aminoacid sequence condensation successively, seals unreacted aminoterminal with acetyl imidazole after per step condensation reaction;
(2) the synthetic back that finishes adds lytic reagent, thereby the cracking from the aminoresin of described polypeptide is got off;
(3) use the described polypeptide of ether sedimentation; With
(4) described polypeptide is carried out ultrafiltration purification, carry out aseptically process then.
5. preparation method according to claim 4 is characterized in that, described step (1) may further comprise the steps:
(1-a) deprotection reaction: it is the N-Methyl pyrrolidone solution of the hexahydropyridine of 15-30% that aminoresin is placed volume percent, and reaction is 25-40 minute under 20-28 ℃ of condition, thereby removes the 9-fluorenylmethyloxycarbonyl blocking group on the aminoresin;
(1-b) washing: nitrogen dries up, and washs aminoresin with N-Methyl pyrrolidone then;
(1-c) condensation reaction: the amino acid that adds 1-hydroxyl azimidobenzene, dicyclohexylcarbodiimide and protected by 9-fluorenylmethyloxycarbonyl, reacted 0.5-2.5 hour under 20-28 ℃ of condition then;
(1-d) washing: nitrogen dries up, and washs aminoresin with N-Methyl pyrrolidone then; With
(1-e) capping: adding percent weight in volume is the N-Methyl pyrrolidone solution of the acetyl imidazole of 1.5-4%, and reaction is 20-40 minute under 20-28 ℃ of condition.
6. preparation method according to claim 4 is characterized in that, the component of lytic reagent is that volume ratio is the trifluoroacetic acid of 85:8:6:1 in the described step (2): tri isopropyl silane: phenol: H
2O;
Preferably, the cracking time of described step (2) is 1-4 hour.
7. preparation method according to claim 4 is characterized in that, described preparation method's step also comprises in (3):
(3-a) use the described polypeptide of ether sedimentation, wash with dimethyl formamide then;
(3-b) account under the dimethyl sulfoxide (DMSO) situation of reaction system cumulative volume 10%, make to precipitate in the amino acid sequence of polypeptide that obtains to form disulfide linkage between two halfcystines in adding; With
(3-c) make that reaction forms covalently bound between the head and the tail amino-acid residue of described amino acid sequence of polypeptide; Preferably, account for the carboxyl of the head and the tail amino-acid residue that makes described amino acid sequence of polypeptide under the DIC of reaction system cumulative volume 1% and 0.5% the 1-hydroxyl-7-azo benzotriazole situation with amino or at adding 0.1M H in adding
2SO
4It is covalently bound that reaction is formed.
8. preparation method according to claim 4 is characterized in that, described step (4) may further comprise the steps:
(4-a) use the tangential flow filtration film to wrap in the described polypeptide of ultrafiltration under the 20-28 ℃ of condition, thereby remove small molecular weight impurity; With
(4-b) use 0.2 micron online filter degerming to preserve.
9. an ox foot and mouth disease O type peptide vaccine is characterized in that, described peptide vaccine comprises one or more according to each described polypeptide in the claim 1 to 3.
10. peptide vaccine according to claim 9 is characterized in that described peptide vaccine also comprises adjuvant;
Preferably, described adjuvant is to be selected among white oil, 50V, the 50VII one or more;
Further preferably, the polypeptide that is comprised in the described peptide vaccine and the volume ratio of adjuvant are 1:1.
11. the preparation method according to claim 9 or 10 described peptide vaccines is characterized in that, described preparation method may further comprise the steps:
(1) is the concentration of 50 μ g/ml with water for injection with described polypeptide dilution, thereby obtains the polypeptide antigen water;
(2) adjuvant was sterilized 30 minutes under 121 ℃ of conditions; With
(3) under 20-28 ℃ of condition, volume ratio according to described polypeptide antigen water and described adjuvant 1:1, earlier adjuvant is added in the emulsion tank, stirred 1.5-3 minute down at 90-150 rev/min, slowly add the polypeptide antigen water then, stirred then 20-30 minute, stirred 15-30 minute down at 8000-10000 rev/min again, left standstill packing 5 minutes.
12. according to each described polypeptide in the claim 1 to 3 or be used for preventing the purposes of the medicine of ox foot and mouth disease O type in preparation according to claim 9 or 10 described peptide vaccines.
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| CN101565457A (en) * | 2009-06-04 | 2009-10-28 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN101659695A (en) * | 2008-08-27 | 2010-03-03 | 中牧实业股份有限公司 | O-type aftosa synthetic peptide vaccine |
| US20110206718A1 (en) * | 2008-10-24 | 2011-08-25 | Ray Vaccine Limited | Anti-fmd vaccine composition and preparation and use thereof |
| CN102580076A (en) * | 2011-12-23 | 2012-07-18 | 广州自远生物科技有限公司 | Synthetic peptide vaccine for O-type foot and mouth disease of swine and preparation method thereof |
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|---|---|---|---|---|
| CN101659695A (en) * | 2008-08-27 | 2010-03-03 | 中牧实业股份有限公司 | O-type aftosa synthetic peptide vaccine |
| US20110206718A1 (en) * | 2008-10-24 | 2011-08-25 | Ray Vaccine Limited | Anti-fmd vaccine composition and preparation and use thereof |
| CN101565457A (en) * | 2009-06-04 | 2009-10-28 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN102580076A (en) * | 2011-12-23 | 2012-07-18 | 广州自远生物科技有限公司 | Synthetic peptide vaccine for O-type foot and mouth disease of swine and preparation method thereof |
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| 杨亮等: "模拟牛O型口蹄疫病毒天然表位的重组蛋白疫苗构建、表达及效果检测", 《南开大学学报（自然科学版）》, vol. 45, no. 3, 20 June 2012 (2012-06-20), pages 44 - 49 * |
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