CN102363043A

CN102363043A - Swine C-type foot-and-mouth disease genetic engineering vaccine adjuvant, and preparation method thereof

Info

Publication number: CN102363043A
Application number: CN2011103033094A
Authority: CN
Inventors: 刘永生; 张�杰; 丁耀忠; 陈豪泰; 马丽娜; 周建华
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2011-10-09
Filing date: 2011-10-09
Publication date: 2012-02-29
Anticipated expiration: 2031-10-09
Also published as: CN102363043B

Abstract

The invention discloses a swine C-type foot-and-mouth disease genetic engineering vaccine adjuvant, and a preparation method thereof, in particular a C-type foot-and-mouth disease subunit vaccine adjuvant which consists of a mixture of VP1 and VP2 proteins of C-type foot-and-mouth diseases, and the preparation method of the vaccine adjuvant. The swine C-type foot-and-mouth disease genetic engineering vaccine adjuvant is recombinant swine alpha-interferon, particularly the recombinant swine alpha-interferon of which the eight front-end signal peptides of the interferon are rejected, and the nucleotide sequence of the vaccine adjuvant is SEQ ID No3.

Description

One boar is with C type foot-and-mouth disease gene engineering vaccine adjuvant and preparation method thereof

Technical field

The present invention relates to adjuvant and method for preparing that a kind of animal vaccine is used; Be that a boar is with C type foot-and-mouth disease gene engineering vaccine adjuvant and method for preparing exactly; The present invention more exactly is the adjuvant of the C type hoof-and-mouth disease subunit vaccine that constitutes of a kind of VP1 and VP3 protein mixture that is used for by C type foot and mouth disease, with and preparation method thereof.

Background technology

Foot and mouth disease (Foot-and-Mouth Disease; FMD) be a kind of infectious disease that can infect multiple artiodactylous a kind of acute, strong, the height contact that comprises main domestic animal such as cattle, sheep, pig; Its pathogen be foot and mouth disease virus (Foot-and-Mouth Disease Virus, FMDV).FMDV belongs to microRNA section (Picornaviridae), Hostis (Aphthovirus), comprises 7 serotypes (A, C, O, Asia I, SAT I, SAT II and SAT III) altogether, no cross protection power between each serotype.

The gene subunit engineered vaccine is owing to advantages such as safety is easy to well preserve, and is satisfactory for result have broad prospects.Yet gene subunit engineered vaccine immunogenicity relatively a little less than, for the immune system of excitating organism better, make it have enough resistances to foot and mouth disease virus, be employed in more and add adjuvant in the vaccine, for example oily adjuvant.

Summary of the invention

The present invention provides a kind of adjuvant of the C of being used for type foot-and-mouth disease gene engineering subunit vaccine.

Pig of the present invention is a recombinant IFN-alpha with C type foot-and-mouth disease gene engineering vaccine adjuvant.More exactly, pig of the present invention is the recombinant IFN-alpha that has weeded out 8 signal peptides of front end of interferon with C type foot-and-mouth disease gene engineering vaccine adjuvant, and its nucleotides sequence is classified SEQ ID No3 as.

Pig of the present invention with C type foot-and-mouth disease gene engineering vaccine adjuvant method for preparing is: use the fresh spleen of pig of behind foot and mouth disease virus counteracting toxic substances, butchering to extract the total RNA of spleen; Obtain pig α-IFN gene through reverse transcription method again; Resulting pig α-IFN gene is connected in constitutes plasmid on the prokaryotic expression carrier; Change prokaryotic expression system again over to and express, expression product obtains desired adjuvant after the renaturation purification process.

As preferred forms; Pig of the present invention is to use the fresh spleen of pig of behind foot and mouth disease virus counteracting toxic substances, butchering to extract the total RNA of spleen with C type foot-and-mouth disease gene engineering vaccine adjuvant method for preparing; Obtain pig α-IFN gene through reverse transcription method again; Resulting pig α-IFN gene is connected in constitutes plasmid on the prokaryotic expression carrier, change prokaryotic expression system again over to and express, expression product obtains desired adjuvant after the renaturation purification process.Here can adopt the urea liquid of variable concentrations during used renaturation is handled, perhaps guanidine hydrochloride or formic acid solution (as 70% formic acid solution).Used a pair of pig alpha-interferon genes uses the RT-PCR primer to be in aforementioned method for preparing:

The IFN-forward primer: 5 '-GACCTGGAAGCCTGTGTCAT-3 '

The IFN-reverse primer: 5 '-CTGTCTTGCAGGTTTGTGGA-3 ';

Used prokaryotic expression carrier is pET-30a; Used prokaryotic expression system is e. coli bl21 (DE3).

Pig of the present invention is used the guanidine hydrochloride solution that preferably contains DDT and EDTA in the C type foot-and-mouth disease gene engineering vaccine adjuvant method for preparing in product renaturation processing method as employing; Wherein the final concentration of guanidine hydrochloride is 1.5M; The final concentration of DDT is 1mM, and the final concentration of EDTA is 10mM.

α-IFN is a kind of broad-spectrum disease resistance toxic agent, though its direct killing or suppress virus not can make cell produce antiviral protein through the cell surface receptor effect, thereby suppress duplicating of virus; But also enhanced natural killer cell (NK cell), macrophage and the lymphocytic vigor of T simultaneously, thus play immunoregulation effect, and strengthen anti-virus ability.Interferon is one group of reactive protein (mainly being glycoprotein) with multiple function, is a kind of cytokine that is produced by mononuclear cell and lymphocyte.They have the broad-spectrum antiviral on allogenic cell, influence the cell growth, and differentiation, regulate multiple biological activity such as immunologic function.In recent years research shows that interferon helps the growth effect of CD8+ memory T cell, in viral disease, is widely used in must treating like hepatitis B, hepatitis C, vesicular stomatitis virus etc.IFN-α family is made up of at least 13 functional hypotypes, and they have same receptor system and similar biological activity.Discover that it has many important function aspect innate immunity and the adaptive immunity.Be shown in the report of Moraes (2003) the earliest as the research of foot-and-mouth disease vaccine adjuvant; Domestic research is shown in 2008; Be with live vector vaccine (the Veterinary Immunology and Immunopathology of interferon complete sequence (containing whole signal peptides) with foot and mouth disease virus VP1 marismortui structural protein sequence and adenovirus vector coexpression; 124,2008:274-283), but still there is risk at present in safety.

So adjuvant of the present invention is with in the hoof-and-mouth disease subunit vaccine, because there is not the of self-replication capacity in hoof-and-mouth disease subunit vaccine, can not be incorporated on the experimental animal genome, safety is very high.VP1 and the VP3 albumen that the present invention preferably adopts C type FMDV is based on following reason as the development object of subunit vaccine: at first; Its topmost structural protein of the VP1 of C type FMDV; The arginine-glycine-aspartic acid (Arg-Gly-Asp) that contains a high conservative on it is the G-H ring (G-H Loop) of motif (RGD); Be the main component of the adsorption site of cell, important again in and the site, generally be positioned at the 140-160 amino acids residue of VP1.Also have a main seriality antigen site at the C of VP1 end in addition, the inoculation animal can produce neutralizing antibody.Secondly, comprised a plurality of antigen sites of B-B knob on the VP3 of C type FMDV, and the same with other picornaviruss, and VP3 is that structure is the most conservative in the structural protein.Therefore, VP3 also can be used as the basic point of recombinant vaccine preparation.

It is pointed out that IFN-α preparation is used widely in more than 40 countries, is used to treat the cancer of type more than 14 kinds; Comprise some haematogenous malignant tumor; Like hairy cell leukemia, chronic myeloid leukemia, some B cells and t cell lymphoma and some solid tumour; Like melanoma, renal carcinoma, safety is good.The present invention be first with the prokaryotic expression product of IFN-as the subunit vaccine adjuvant, in particular as the adjuvant of the combined vaccine of the formation of foot and mouth disease VP1 and VP3 expression product.

Be adjuvant owing to having adopted the recombinant IFN-alpha of 8 signal peptides of front end that weeded out interferon in the present invention, the fore-end signal peptide that interferon is rejected in this utilization can improve proteic expression, more helps the preparation of adjuvant.

Adopted the guanidine hydrochloride solution that contains DDT and EDTA when to the prokaryotic expression product time, carrying out renaturation among the present invention.Reducing agent DTT can open all disulfide bond in the cysteine fusion rotein as much as possible, but EDTA chelating Cu ²⁺, Fe ³⁺Deng the sulfydryl generation oxidation reaction of metal ion and reducing condition, thereby make the renaturation yield of fusion rotein reach the highest.Simultaneously DDT and EDTA can farthest reduce in the fused protein solution various ion-types, nonionic surfactant etc. must interferential effect; Renaturation manipulation is simple, does not need specific apparatus, and agents useful for same is stable, can stablize usually and deposit 6-12 month.The interferon that do not have by this Experiment Preparation is that the C type foot-and-mouth disease gene engineering subunit vaccine of adjuvant is to 100LD ₅₀C type FMDV strain AV104 strong virus attack can not provide protection, and is that the C type foot-and-mouth disease gene engineering subunit vaccine of adjuvant can be to 50LD with the interferon ₅₀C type FMDV strain AV104 strong virus attack reaches 100% protection, to 100LD ₅₀C type FMDV strain AV104 strong virus attack also can provide 83.3% protective rate; Simultaneously, be that the pig of adjuvant is not with the common pig of porcine alpha-interferon 2.2 times with C type foot-and-mouth disease gene engineering vaccine with C type foot-and-mouth disease gene engineering vaccine immunity pig antibody horizontal with the porcine alpha-interferon.

Description of drawings

Fig. 1 .PCR amplification a-interferon gene, wherein: M 1DL2000Marker, swimming lane 1RT-PCR amplification porcine alpha-interferon product.

Fig. 2 .PCR and double digestion are identified the interferon recombinant vector; Wherein: M DL10000Marker; The porcine alpha-interferon product that swimming lane 1PCR increases from cloning vehicle; The porcine alpha-interferon product that swimming lane 2PCR increases from expression vector, swimming lane 3EcoR V, EcoR I double digestion are identified recombinant expression carrier.

Fig. 3. pig α-IFN expression relatively, wherein: pig α-IFN gene Fusion albumen that swimming lane 1 is involved in the present invention, swimming lane 2 the present invention be used for the expression contrast the full gene Fusion albumen of pig α-IFN.

Fig. 4. porcine alpha-interferon fusion rotein renaturing inclusion bodies and purification SDS-PAGE analyze, wherein: the purified product of swimming lane 1 pig α-IFN fusion rotein, the full bacterium product of swimming lane 2 pigs α-IFN fusion rotein.

Fig. 5. pig interferon fusion rotein Western-blotting analyzes after the renaturation, wherein: M albumen Marker, the reaction of swimming lane 1 pig α-IFN fusion rotein and ELIAS secondary antibody IgG.

Fig. 6 .ELISA detects the antibody horizontal behind the vaccine immunity pig

The specific embodiment

The embodiment of the invention is divided into two parts to be accomplished, and promptly embodiment one: the prokaryotic expression of porcine alpha-interferon and renaturation; Embodiment two: recombinant IFN-alpha is as the potency test at C type hoof-and-mouth disease subunit recombinant vaccine (comprising immunity and the counteracting toxic substances measuring of Cavia porcellus) of vaccine adjuvant

Embodiment one: the prokaryotic expression of porcine alpha-interferon and renaturation:

Obtain with the fresh spleen sample of the pig of butchering in 7 days behind the foot and mouth disease virus counteracting toxic substances; Extract the total RNA of spleen with the precious biological test kit in Dalian; From the total RNA of spleen, obtain 181 amino acid whose pig α-IFN genes through the RT-PCR method; Its aminoacid sequence is seen SEQ.NO.3, and its nucleotide sequence coding is seen SEQ.NO.4.With said gene cloning vehicle pMD18-T, transformed into escherichia coli DH5 α, picking positive plasmid order-checking and with the porcine alpha-interferon of Genbank relatively, obtain correct gene; The specific expressed primer of design porcine alpha-interferon; Its sequence is SEQ.NO.2, adds EcoR V and EcoR I restriction enzyme site, inserts pET-30a; Transformed into escherichia coli DH5 α; The positive plasmid called after pET-SIFN that obtains with the LB culture medium culturing, carries out purification, renaturation and also analyzes pig interferon fusion rotein characteristic with SDS-PAGE and Western-blotting.Concrete steps are following:

The RT-PCR amplification of 1 porcine alpha-interferon:

Genomic templates 3ul, 1 * step Enzyme mix, 0.8 μ l, 2 * buffer, 10 μ l, positive each 50pmol of anti-primer, aseptic ultra-pure water to the cumulative volume of benefit are 50 μ l, mix homogeneously.Amplification condition: 50 ℃ of 30min, 94 ℃ of 10min, carry out 30 circulations in following condition then: 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 40s, last 72 ℃ are extended 8min.

The present invention's porcine alpha-interferon Auele Specific Primer that is used to increase, its sequence is respectively SEQ.NO.1 and SEQ.NO.2, that is:

The IFN-forward primer: 5 '-GACCTGGAAGCCTGTGTCAT-3 '

The IFN-reverse primer: 5 '-CTGTCTTGCAGGTTTGTGGA-3 ';

The concrete steps that the 2PCR product is connected with plasmid are:

(1) target dna fragment (20ng), pMD18-T carrier (50ng), T4DNALigase 5U, 10 * T4DNA Ligase Buffer, 1 μ l mend aseptic ultra-pure water to cumulative volume 10 μ l.

(2) 16 ℃ connect 10～12 hours and are prepared into the gene that connects carrier, called after pMD-SIFN.

(3) sample that step (2) is connected carries out PCR and detects; Connect product 1ul, dNTP (10mM) 5 μ l, 10 * pfu buffer, 5 μ l, pfu DNA polymerase 5U is 50 μ l with positive each 50pmol of anti-primer, aseptic ultra-pure water to the cumulative volume of benefit, mix homogeneously; Amplification condition: 94 ℃ of 10min, carry out 30 circulations in following condition then: 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 40s, last 72 ℃ are extended 8min.

Wherein used forward and reverse primer is seen SEQ.NO.3 and SEQ.NO.4, for:

The IFN-forward primer: 5 '-GACCTGGAAGCCTGTGTCAT-3 '

The IFN-reverse primer: 5 '-CTGTCTTGCAGGTTTGTGGA-3 ';

(4) the 1%TBE agarose gel electrophoresis detects, and uv analyzer is observed the DNA band down.

The prokaryotic expression construction of recombinant vector method of 3 pig alpha-interferon genes sequences:

(1) from target dna fragment (20ng), pET-30a carrier (50ng), T4DNA Ligase 5U, 10 * T4DNA Ligase Buffer, the 1 μ l of pMD-SIFN amplification, mends aseptic ultra-pure water to cumulative volume 10 μ l.

(2) 16 ℃ connect 10～12 hours and are prepared into the gene that connects carrier.

Wherein this experiment sees that from the target dna fragment and the used forward and reverse primer of PCR order-checking of pMD-SIFN amplification SEQ.NO.5 and SEQ.NO.6 are:

IFN-ES:5-GCGATATCGCCCCAACCTCAGCCTTCCT-3 (underscore is labeled as the EcoRV restriction enzyme site)

IFN-EAs:5-GCGAATTCCTCCTTCTTCCTGAGTCTGTCTTGC-3 (underscore is labeled as EcoR I restriction enzyme site)

(4) the pET-SIFN recombination 10ul that connects, 10 * H buffer 2ul, EcoR I (8-20U/ μ l) and each 1.5ul of Xho I (8-20U/ μ l), ddH2O is settled to 20ul.

(5) the 1%TBE agarose gel electrophoresis detects, and uv analyzer is observed the DNA band down.

The prokaryotic expression of 4 pig alpha-interferon genes sequences, renaturation and detection:

To be accredited as male recombinant vector bacterium liquid 20 μ L and be seeded in the 6mL LB culture medium, 37 ℃, 200rpm jolting spend the night.Get overnight culture 20 μ L and be inoculated in the 6mL LB culture medium, when 37 ℃ of joltings are cultured to OD600 and are 0.6-1.0, add IPTG to final concentration be 0.5mM, induce 8h for 37 ℃, SDS-PAGE observes the bacterium expression of respectively recombinating.

Concrete grammar is: with getting bacterium liquid appearance 12, the centrifugal 3min of 000rpm abandons supernatant.Residual with the resuspended thalline of deionized water to there not being big antibacterial piece, the centrifugal 3min of 12.000rpm, supernatant discarded, the resuspended thalline of deionized water.Multigelation 3 times, 12, the centrifugal 3min of 000rpm draws supernatant, and thalline is resuspended with deionized water.Get each 20 μ L of cleer and peaceful resuspended thalline and add 5 μ L, 5 * Loading Buffer, boil 7min behind the mixing.Protein Marker boils 7min equally.Draw sample 5 μ L to be analyzed by predefined procedure with micro sample adding appliance, slowly add well.Whenever add a sample, should in electrode buffer, thoroughly clean micro sample adding appliance.Add protein molecular weight standard Marker and do reference.About 200 volts of voltage electrophoresis 40min, after electrophoresis finished, dyeing and decolouring were waited to take pictures, the record result.

The purification of the expression product of porcine alpha-interferon recombination, specific as follows:

(1) proteic preparation

PET-30VP1 and pET-30INF BL21 (DE3) bacterium overnight culture 500 μ L are added respectively in the 200mL culture medium, and 37 ℃, 0.5mM IPTG induces 8h.Induce after the end bacterium liquid 3800rpm, 4 ℃ centrifugal 15 minutes, supernatant discarded.Deionized water is resuspended, and the same terms is centrifugal, supernatant discarded.With the resuspended thalline of 50mL TE buffer, the ultrasonic 3s of 200w stops 3s, and is ultrasonic to bacterium liquid no longer till the thickness.12,4 ℃ of centrifugal 10min of 000rpm, supernatant discarded.

(2) inclusion body washing and degeneration

Use inclusion body cleaning mixture BufferA, B, C, D to prepared inclusion body washing successively, wherein use Buffer B washing 5 times, all the other 1 time.Use 8M urea liquid dissolving washing back inclusion body at last, 4 ℃ are spent the night.12,000rpm, 4 ℃ of centrifugal 10min.Collect supernatant, get a little supernatant, use SDS-PAGE to observe clean result.

(3) renaturing inclusion bodies

Renaturing inclusion bodies is handled the method for the formic acid of the carbamide can take variable concentrations respectively, guanidine hydrochloride, guanidine hydrochloride (containing DDT and EDTA) and 70% and carried out renaturation.The inclusion body that renaturation is successful uses the SDS-PAGE observed result.

(4)Western-blotting

The Western blotting of above-mentioned porcine alpha-interferon recombination analyzes:

(1) electrophoresis: get purified various albumen sample preparations and carry out SDS-PAGE.

(2) transfer printing: after the gel excision that electrophoresis is intact concentrates glue, washed with de-ionized water surface, balance 30min in the film transfering buffering liquid then.Cut 1 with the pvdf membrane of gel phase with size, in 100% methanol, soak about 3min back and gel balance in buffer and finish to 30min.8 of clips and the identical filter paper of getting PVDF size again, and in transfering buffering liquid, soak 10min.Order by four metafiltration paper, pvdf membrane, gel, four metafiltration paper stacks neatly on positive plate then, pushes gently with Glass rod, drives each interlayer bubble away, guarantees that each layer contacts negative plate on the bonnet fully, and 120mA shifts 150min.

(3) sealing: after transfer printing finishes; Take off pvdf membrane, cut protein molecular weight standard Marker location pvdf membrane, place the amino black dyeing liquor to contaminate 2-3min according to labelling in advance; Take out the back with rinsing liquid (150mM NaCl; 50mM Tris-HCL, pH 7.5) decolouring, until background Lan Setuo to the greatest extent.All the other pvdf membranes wash with PBS, add the 10-20mL confining liquid, and room temperature is jolting 2h gently, or 4 ℃ spend the night (to guarantee that defatted milk powder fully dissolves during the preparation confining liquid, so as not to undissolved milk powder particles adhere to film on, influence experiment effect).

(4) with anti-combining: after sealing finishes, clean pvdf membrane 3 times with PBST, jolting 10min on shaking table at every turn, add with PBST suitably dilution respectively detect antibody 10mL, room temperature jolting 1.5h is with PBST flushing 3 times, at every turn at the slow shake 10min of shaking table.

(5) with two anti-reactions: add suitably dilution horseradish peroxidase-labeled with one anti-corresponding two anti-, jog is hatched 1h under the room temperature, takes out with PBST flushing 3 times, at every turn at the slow shake 10min of shaking table.

(6) colour developing: pvdf membrane is transferred in the 10-20mL substrate solution, and room temperature lucifuge jog 3min observes the colour developing situation, when waiting band to occur, changes cessation reaction in the PBS buffer immediately over to, and room temperature preservation is treated IMAQ.

5 aforementioned product order-checking and experimental results

5.1 through order-checking, see the SEQ.NO.7 of gene order table by aforementioned gained porcine alpha-interferon recombination sequence, its aminoacid sequence is seen the SEQ.NO.8 of gene order table.

5.2 the foregoing description one experimental result 1

Use the RT-PCR technology, the pig alpha-interferon genes that increased, the target DNA fragment that the expection size is identical can be seen two specific bands (accompanying drawing 1) about a 550bp in electrophoretogram.Will be from agarose gel the porcine alpha-IFN gene dna fragmentation and the expression vector pET-30a recombinant vector of purification, identify that through PCR, enzyme action size conforms to expected results.(accompanying drawing 2).Through order-checking, the present invention has obtained 8 amino acid whose pig α-IFN gene orders of rejecting leading portion, sees sequence table SEQ .NO.7 and SEQ.NO.8.

5.3 the foregoing description one experimental result 2

E. coli bl21 (DE3) genetic engineering bacterium that will contain recombiant plasmid is expressed under variable concentrations IPTG induces, and gets the bacterium liquid pyrolysis product of each concentration under inducing and is SDS-PAGE and analyzes, and the result is illustrated in 28kDa and specific band occurs.In IPTG concentration is that expression is maximum under the 0.5mmol/ml; Expression product is fusion rotein and exists with the inclusion body form; And weed out 8 amino acid whose pig α-IFN expression of gene amounts of leading portion and be higher than and do not reject 8 amino acid whose pig α-IFN genes of signal peptide leading portion and be about 20%, see accompanying drawing 3 disastrously.It is consistent with the destination protein size to use Model 422Electro-Eluter to cut glue recover recovery albumen; And it is high by preferred refolding method of the present invention (promptly adopt and contain DDT and EDTA and guanidine hydrochloride renaturation) products therefrom purity; Ultraviolet spectrophotometry detects and shows; Its purity can reach 85%, referring to accompanying drawing 4.

The renaturation of the foregoing description one fusion rotein inclusion body is used carbamide, guanidine hydrochloride, guanidine hydrochloride respectively, and ((wherein the final concentration of DDT is 1mM to contain DDT and EDTA; The final concentration of EDTA is 10mM) and the method for 70% formic acid carry out the renaturation result and see table 1; Wherein guanidine hydrochloride (containing DDT and EDTA) renaturation yield can reach more than 85%, and comparing result sees the following form 1.

The experimental result of the different renaturation material of table 1 renaturation yield

Can find out from result of the test; Adopt different recombinant protein reagent by this experiment; The renaturation yield that its protein renaturation result is respectively variable concentrations carbamide is 44.6%; 70% formic acid renaturation yield is 34.4%, and the renaturation yield of guanidine hydrochloride is 60.3%, and the renaturation yield of guanidine hydrochloride (containing DDT and EDTA) is 85.7%.70% formic acid renaturation yield is the poorest; The renaturation yield of guanidine hydrochloride (containing DDT and EDTA) is best, is because this experiment purpose gene contains cysteine (SEQ.NO.7), adds Reducing agent DTT and possibly open all disulfide bond in the fusion rotein as much as possible; Simultaneously, but EDTA chelating Cu ²⁺, Fe ³⁺Deng the sulfydryl generation oxidation reaction of metal ion and reducing condition, thereby make that its renaturation yield is the highest.

3. purified product is transferred on the nitrocellulose filter, uses C type FMD positive serum that the VP3 fusion rotein is carried out reactionogenicity and detect, specific band occurs, prove that cutting the glue purification afterproduct has good reactionogenicity, referring to accompanying drawing 5 at the 28kDa place.

Embodiment two: recombinant IFN-alpha is as the potency test at C type hoof-and-mouth disease subunit recombinant vaccine of vaccine adjuvant

2.1 materials and methods

2.1.1 supplying the present invention to contrast, uses by vaccine by the interferon of prokaryotic expression C type foot-and-mouth disease gene engineering subunit vaccine, lot number: 20110501,20110515,20110605 as adjuvant.C type foot and mouth disease inactivated vaccine is by this Experiment Preparation, lot number: 20110410.Not having interferon is the C type foot-and-mouth disease gene engineering subunit vaccine of adjuvant, lot number: 20110502,20110512,20110531.The interferon vaccine of prokaryotic expression, lot number: 20110508,20110512,20110516.

2.1.2 experimental animal and strain body weight are about the Cavia porcellus of 350～450g available from Lanzhou institute of Biological Products Experimental Animal Center.C type FMDV strain AV104 preserves by the BHK-21 passage and by this chamber.

Female Cavia porcellus divides into groups at random 2.1.3 immune animal will grow up, and 6 every group, 4 kinds of immunogens that prepare are altogether carried out subcutaneous injection in 0d, 14d, and immunizing dose is 200 μ l/.Immunity is all laboratory animals employing 100LD50 counteracting toxic substances after 28 days.

Immune group 100% protection when 2.1.4 the result judges 80% morbidity of virus control group; Or during matched group 80% morbidity the above protection of immune group 80% protection can be judged to vaccine qualified.

2.2 result of the test and discussion

2.2.1 result of the test use respectively 50 with 100LD50/ type FMDV strain AV104 counteracting toxic substances 14 days, the contrast Cavia porcellus all falls ill, and the protective rate of C type foot-and-mouth disease gene engineering subunit vaccine immune group is 83.3%, sees table 2, table 3 for details.

Table 2.C type foot-and-mouth disease gene engineering subunit vaccine and inactivated vaccine immunity comparative test result

2.2.2 result

Can find out from result of the test, be that the C type foot-and-mouth disease gene engineering subunit vaccine of adjuvant is to 100LD by the interferon that do not have of this Experiment Preparation ₅₀C type FMDV strain AV104 strong virus attack only can provide 50% protective rate, with the animal of interferon immunity to 100LD ₅₀C type FMDV strain AV104 strong virus attack can not provide protection.And be that the C type foot-and-mouth disease gene engineering subunit vaccine of adjuvant can be to 50LD with the interferon ₅₀C type FMDV strain AV104 strong virus attack reaches 100% protection, to 100LD ₅₀C type FMDV strain AV104 strong virus attack also can provide 83.3% protective rate; Hence one can see that with the interferon is the immune effect that vaccine adjuvant can significantly improve vaccine; Improve the protection of immune animal, thereby the C type foot-and-mouth disease gene engineering subunit vaccine that makes us study reaches more satisfactory protection effect.

The C type foot-and-mouth disease gene engineering subunit vaccine pig immuning effect test that embodiment three is an adjuvant with the above-mentioned porcine alpha-interferon that makes, specific as follows:

3.1 materials and methods

3.1.1 of the present inventionly tried VP1 that vaccine is a C type foot and mouth disease and VP3 mixes by protein content at 1: 1; The porcine alpha-interferon that adds previous embodiment preparation of the present invention more therein is an adjuvant; The adjuvant volume that is added is 1: 1 (V/V) with the ratio of the cumulative volume of VP1 and VP3; Being tried vaccine name lot number is: 20110501,20110515,20110605.

Comparative Examples and vaccine are that the VP1 and the VP3 of C type foot and mouth disease mixes by protein content at 1: 1, add isopyknic 206 oily adjuvants more therein, experiment lot number: 20110502,20110512,20110531; The pig of this prepared in laboratory-interferon expression purified product, lot number: 20110508,20110512,20110516.

3.1.2 experimental animal and strain body weight are about the feeder pig of body weight 40kg, purchase in the pig farm, Dingxi.

3.1.3 5 of every group of pigs of immune animal (1) are divided into and are: PBS group, interferon group, recombinant vaccine (adding interferon) group, recombinant vaccine (not adding the interferon group).The immunogen for preparing is carried out subcutaneous injection in 0d, 14d, the 1ml/ head.(2) all the other control vaccines carry out immunity in a manner described, and negative control is set simultaneously.Above experimental animal 7d after immunity, 14d, 28d, 35d, 42d blood sampling also detect.

3.1.4 immune animal TPPA

3.1.4.1 indirect ELISA detects the anti-C type FMDV antibody horizontal in the serum sample, concrete steps are following:

(1) antigen coated: encapsulate buffer with ELISA C type FMDV cell toxicant inactivation antigen is carried out dilution in 1: 2, every then hole 50 μ L add in the ELISA micro-reaction plate, and 4 ℃ are spent the night.

(2) sealing: the ELISA Sptting plate of envelope antigen is taken out, discard in the hole behind the liquid,, clap dried with washing buffer washing reaction plate 3 times.Add confining liquid then, every hole 50 μ L, 37 ℃ of effect 2.5h.

(3) add serum to be checked: after sealing finished, (1 * PBST, pH7.4) the washing reaction plate was 3 times, claps the dry reaction plate with lavation buffer solution.Serum to be checked is carried out 1: 200 dilution back with the PBST buffer add in the Sptting plate, every hole 50 μ L, each sample repeats three holes.Simultaneously standard FMDV feminine gender and positive serum are diluted back adding Sptting plate equally, as feminine gender and positive control.37 ℃ of effect 1h.

(4) add ELIAS secondary antibody: take out the ELISA Sptting plate, discard in the hole behind the liquid, with washing buffer washing reaction plate 3 times, bat dry reaction plate.Add ELIAS secondary antibody, every hole 50 μ L, 37 ℃ of effect 45min.

(5) colour developing: take out the ELISA Sptting plate, discard in the hole behind the liquid, with washing buffer washing reaction plate 3 times, clap the every hole of dry reaction plate and add OPD substrate colour developing liquid 100 μ L, 37 ℃ act on 20min.

(6) stop: after chromogenic reaction finishes, add 100 μ L stop buffer (1.25M sulphuric acid) cessation reactions in every hole.Measure the OD value at the 490nm place with ELIASA.

3.2 experimental result

The antibody titer that is directed against C type FMDV in the pig body is measured in the immunity back through indirect ELSA method; The result shows: back 14 days to 42 days of immunity; With the porcine alpha-interferon be the pig of adjuvant with C type foot-and-mouth disease gene engineering vaccine immunity pig antibody horizontal apparently higher than the pig that does not add the interferon adjuvant with C type foot-and-mouth disease gene engineering vaccine, its porcine alpha-interferon is that the antibody horizontal of the subunit genetic engineering vaccine of adjuvant is common pig 2.2 times with C type foot-and-mouth disease gene engineering vaccine.Experimental result shows, with the porcine alpha-interferon be raising pig that adjuvant can highly significant with C type foot-and-mouth disease gene engineering vaccine antibody level, and safety is good, referring to accompanying drawing 6.

Claims

1. a boar is characterized in that with C type hoof-and-mouth disease subunit vaccine adjuvant this adjuvant is a recombinant IFN-alpha.

2. pig according to claim 1 is characterized in that with C type hoof-and-mouth disease subunit vaccine adjuvant used recombinant IFN-alpha has weeded out 8 signal peptides of front end of interferon, and its nucleotides sequence is classified SEQ ID No3 as.

3. claim 1 or 2 described pigs are used for the C type hoof-and-mouth disease subunit vaccine that VP1 and VP3 protein mixture by C type foot and mouth disease constitute with C type hoof-and-mouth disease subunit vaccine adjuvant.

4. the described pig of claim 1 is with C type foot-and-mouth disease gene engineering vaccine adjuvant method for preparing; It is characterized in that: use the fresh spleen of pig of behind foot and mouth disease virus counteracting toxic substances, butchering to extract the total RNA of spleen; Obtain pig α-IFN gene through reverse transcription method again; Resulting pig α-IFN gene is connected in constitutes plasmid on the prokaryotic expression carrier, change plasmid over to prokaryotic expression system again and express, expression product obtains desired adjuvant after the renaturation purification process.

5. the described pig of claim 2 is with C type foot-and-mouth disease gene engineering vaccine adjuvant method for preparing; It is characterized in that: use the fresh spleen of pig of behind foot and mouth disease virus counteracting toxic substances, butchering to extract the total RNA of spleen; Obtain pig α-IFN gene through reverse transcription method again; Resulting pig α-IFN gene is connected in constitutes plasmid on the prokaryotic expression carrier; Change prokaryotic expression system again over to and express, expression product obtains desired adjuvant after the renaturation purification process, and used a pair of pig alpha-interferon genes uses the RT-PCR primer to be:

The IFN-forward primer: 5 '-GACCTGGAAGCCTGTGTCAT-3 '

The IFN-reverse primer: 5 '-CTGTCTTGCAGGTTTGTGGA-3 ';

6. pig according to claim 5 is with C type foot-and-mouth disease gene engineering vaccine adjuvant method for preparing; It is characterized in that the product renaturation has adopted the guanidine hydrochloride that contains DDT and EDTA when handling; Wherein the final concentration of guanidine hydrochloride is 1.5M, and the final concentration of DDT is 1mM, and the final concentration of EDTA is 10mM.