CN104372013B - A kind of duck hepatitis A virus gene engineering subunit vaccine - Google Patents
A kind of duck hepatitis A virus gene engineering subunit vaccine Download PDFInfo
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Abstract
The object of this invention is to provide a kind of duck hepatitis A virus gene engineering subunit vaccine, namely obtain novel duck hepatitis A virus (HAV) Structural protein VP1 by screening, and prepare duck hepatitis A virus gene engineering subunit vaccine using this Structural protein VP1 as antigen.Duck hepatitis A virus (HAV) Structural protein VP1 in the present invention, the aminoacid sequence of its proteins encoded is SEQ ID NO:1; Its a kind of nucleotide sequence is SEQ ID NO:2.The present invention utilizes prokaryotic expression vector construction can express e. coli bl21 (DE3) Host Strains of duck hepatitis A virus (HAV) Structural protein VP1.Analyze through SDS-PAGE, given expression to 28kD restructuring target protein.Be prepared into genetic engineering subunit vaccine by after recombinant protein purification, immune 4 monthly age sheldrakes, the duckling adaptive immune of hatching after immunity, can be made to protect.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of duck hepatitis A virus gene engineering subunit vaccine.
Background technology
Duck viral hepatitis is by duck hepatitis A virus (HAV) (Duck hepatitis A viral, DHAV) and the one that causes of duck Astrovirus (Duck astrovirus) highly cause a disease, propagate virus disease rapidly, this disease mainly appears on the duckling in 1-3 age in week, and mortality ratio is up to more than 90%.And duck hepatitis A virus (HAV) is world wide distribution, provisions duck industrial belt is lost greatly.
Duck hepatitis A virus (HAV) is the unique member of Picornaviridae fowl hepatovirus, is mainly divided into 3 genotype: A type, Type B and C type, corresponding to three serotype DHAV-1, DHAV-2 and DHAV-3.Wherein DHAV-1 obtains from U.S.'s separation in nineteen fifty the earliest, is classical strains, is distributed in all over the world at present, is also Chinese popular at present main strain.DHAV-2 is separated the novel strain from Taiwan, mainly popular in Taiwan.DHAV-3 is separated the novel strain from Korea S the earliest, also have in China's Mainland now increasing this serotype strain is separated obtains, the mixed infection of itself and DHAV-1 often causes the immuning failure of duck hepatitis vaccine.Research shows between these three kinds of serotypes without cross protection or only limited cross protection.
The VP1 gene of DHAV is by 714 based compositions; coding VP1 albumen size is 26.4kD; containing multiple DHAV epitope; it is the main protective antigen of duck hepatitis virus; body can be induced to produce stronger antibody response and adaptive immune protection, be the preferred object gene of design DHAV new generation vaccine.Fu Yuzhi etc. (2012) are by inserting in alphavirus replicon pSCA1 carrier by duck hepatitis virus VP1 gene; obtain recombinant expression plasmid pSCA1/VP1; build the Suicidal DNA of I type duck hepatitis virus VP1 gene; and to its expression in cell with study the immune protective effect of duckling; result shows; pSCA1/VP1 not only can obviously induce duckling lymphopoiesis; and duckling can be induced to produce the DHAV specific antibody of higher titre, and there is good immanoprotection action to duckling.The duck plague virus TK Deleted Transfer set up is transformed by Zhang Jing etc., I type duck hepatitis virus VP1 gene is inserted in its green fluorescence expression cassette, build the restructuring duck plague virus containing I type duck hepatitis virus VP1 gene, and this recombinant virus of tentative confirmation can in eukaryotic cell the fusion rotein of correction VP1 gene and green fluorescent protein, for the research building duck plague and duck viral hepatitis bivalent genetic engineering vaccine is laid a good foundation.
But the duck viral hepatitis that China DHAV-3 causes in recent years frequently breaks out, and common popular in some areas with DHAV-1, show that existing vaccine has not had effective provide protection.And the domestic duck virus hepatitis vaccine (duck viral hepatitis low virulent strain A66 strain, CH60 strain) having ratified to go on the market is DHAV-1 strain at present, yet there are no DHAV-3 vaccine strain and occur.The sound development of new generation vaccine strain to foster duck industry of therefore developing DHAV-3 is significant undoubtedly.
Summary of the invention
The object of this invention is to provide a kind of duck hepatitis A virus gene engineering subunit vaccine, namely obtain novel duck hepatitis A virus (HAV) Structural protein VP1 by screening, and prepare duck hepatitis A virus gene engineering subunit vaccine using this Structural protein VP1 as antigen.
First the present invention provides a kind of duck hepatitis A virus (HAV) Structural protein VP1, and the aminoacid sequence of its proteins encoded is SEQ ID NO:1;
A kind of nucleotides sequence of above-mentioned albumen is classified as SEQ ID NO:2;
The present invention also provides a kind of duck hepatitis A virus gene engineering recombinant subunit vaccine, and antigen is wherein above-mentioned duck hepatitis A virus (HAV) Structural protein VP1, and its concentration is between 0.1-1mg/ml;
Duck hepatitis A virus gene engineering subunit vaccine of the present invention, its preparation process is as follows:
1) by VP1 expression vector, recombinant expression is built into;
2) recombinant expression built is transformed Host Strains, construct the recombination engineering bacteria expressing duck hepatitis A virus (HAV) Structural protein VP1; Duck hepatitis A virus (HAV) Structural protein VP1 is gone out by this recombination engineering bacterium expression;
3), after purifying being carried out to recombinant expressed VP1 albumen, add white-oil adjuvant and make vaccine.
E. coli bl21 (DE3) Host Strains that the present invention utilizes pET28a (+) Expressing vector to construct to express duck hepatitis A virus (HAV) Structural protein VP1.Analyze through SDS-PAGE, given expression to 28kD restructuring target protein.Be prepared into genetic engineering subunit vaccine by after recombinant protein purification, immune 4 monthly age sheldrakes (kind of laying eggs), the duckling adaptive immune of hatching after immunity, can be made to protect.
Embodiment
Applicant has on the basis of new duck hepatitis A virus gene of autoimmune properties acquisition one, gone out e. coli bl21 (DE3) the recombination engineering bacteria expressing duck hepatitis A virus (HAV) Structural protein VP1 by pET28a (+) Prokaryotic expression vector construction, by the induction to engineering bacteria, ultrasonication, protein purification, quantitatively and the preparation such as adjuvant proportioning and production method prepared duck hepatitis A virus gene engineering recombinant subunit vaccine.
The present invention is further described below in conjunction with embodiment; but what those skilled in the art should understand that is; can modify to the details of technical scheme of the present invention and form when not departing from technical scheme of the present invention or replace, these amendments and replacement all fall in scope.
The screening of the VP1 gene of embodiment 1DHAV-3
1, in May, 2012, duck viral hepatitis was broken out in Qingdao duckery, caused a lot of duck in duck field dead.But the duck group of outburst duck viral hepatitis had injected commercially available duck virus hepatitis vaccine, therefore inferred that pathogenic cause of disease there occurs the variation in heredity, therefore, selected above-mentioned morbidity duck to carry out the separation of Disease-causing gene as sample.
First the liver pathological material of disease stroke-physiological saline solution of morbidity duck ground, multigelation 3 times, adds dual anti-(mycillin, 1000U/ml), the SPF duck embryo of allantoic cavity inoculation 11-13 age in days, 0.2ml/ piece.Observe according to egg, in 24h, died discards.Get allantoic fluid, the idiosome of the duck embryo of peak mortality phase death in 24-96 hour respectively ,-20 DEG C frozen, and observe dead idiosome and liver has atypism duck hepatitis symptom.Connect biography 3 generation, be separated and obtain strain called after VF-3.
2, get the allantoic fluid of the obvious strain VF-3 of pathology, extract total serum IgE by the method for Trizol test kit, and carry out reverse transcription synthesis cDNA, carry out the VP1 gene of pcr amplification DHAV-3 using the cDNA synthesized as template, primer sequence is as follows:
P1:5′-cgcggatcctccaatcagcttggt-3′
P2：5′-aagctttta ttcaatctccagatg-3′
The condition of amplification is as follows: 94 DEG C of 5min sex change, 94 DEG C of 30S, 65 DEG C of 30S, 72 DEG C of 1min, 30 circulations, and 72 DEG C extend 10min.
Amplified production size is about 714bp, by the Blast comparison on NCBI of VP1 gene order after checking order, found that with the homology of DHAV-3FX strain (accession number: KF826745) the highest, be 95%, the protein sequence homologies of deriving is up to 92%, shows that strain VF-3 is the DHAV-3 virus strain morphed.The aminoacid sequence of its VP1 gene translation is SEQ ID NO:1.
The structure of embodiment 2 engineered protein expression vector and the acquisition of engineering bacteria
1, be connected into the corresponding restriction enzyme site of pET28a carrier after the VP1 gene protein gene BamH I that obtains of above-mentioned amplification and Hind III double digestion, build pET28a/VP1 expression vector.
2, CaCl is used
2pET28a/VP1 expression vector is transformed into e. coli bl21 (DE3) by method, coats the agar plate containing 50 μ g/ml kantlex, 37 DEG C of incubated overnight.Choose 10 single bacterium colonies and extract plasmids, BamH I and the positive bacterium colony of Hind III double digestion checking check order qualification further.Positive colony after sequence verification is added in LB substratum 0.3mMIPTG induction 4 ~ 5 hours during fermentation culture to 0.6 ~ 0.8, collected by centrifugation thalline runs SDS-PAGE electrophoresis, sets up simultaneously and does not induce thalline in contrast.After result induction, positive colony has more a protein band than contrast bacterium at 28kD place, and consistent with recombinant protein theoretical molecular, expression amount is about more than 35%.Through the calibrating of DHAV-3 antibody immunoblotting, display positive reaction.Prove that the positive colony obtained is the engineering bacteria of high expression engineered protein, called after YG strain.
Embodiment 3 is fermented, the preparation of purifying and duck hepatitis A virus gene engineering subunit vaccine
1, zymotechnique
1) LB substratum is as seed culture medium, containing 5g/L glucose and 5g/L MgSO
47H
2the LB substratum of O is as fermention medium, and feed supplement is glucose 400g/L, peptone 24g/L, yeast extract 10g/L, NaCl 5g/L, MgSO
47H
2o 5g/L.
2) fermenting process
The engineering bacteria that picking the was identified concentration be inoculated in containing kantlex is the LB substratum of 50 μ g/ml, and 37 DEG C of shaking culture 8 hours, as seed liquor.Be inoculated in fermentor tank by seed liquor by 2% inoculum size, regulate each parameter, 37 DEG C, 200 turns, dissolved oxygen controls more than 20%.Ferment and start flow feeding after 4 hours, fermenting adds 0.3mmol/L IPTG after 6 hours and carries out abduction delivering, and after expressing, 6 hours fermentation terminate.
3) ni-sepharose purification, desalination
4) affinity chromatography
Adopt nickel ion metal chelate chromatography post, recombinant protein can be washed with the elutriant containing 300mmol/L imidazoles, and purity reaches more than 90%
5) desalination
Dialysis tubing put into by the recombinant protein elutriant collected, and PBS liquid, as extracellular fluid dialysis, surveys protein concentration with after formalin-inactivated after dialysis desalting, is diluted to 0.5mg/ml with PBS liquid, and 0.22 μm of filtration sterilization, obtains recombinant protein liquid.
2, the preparation of duck hepatitis A virus gene engineering subunit vaccine
The recombinant protein 96 parts of preparation and 4 parts of tween-80s of sterilizing are fully uniformly mixed as aqueous phase.Injection white oil 94 parts simultaneously, adds department's basis 80, the aluminum stearate 2 parts of 6 parts, mixes, as oil phase after autoclaving.Duck hepatitis A virus gene engineering subunit vaccine can be prepared in aqueous phase and oil phase 1:3 ratio mixing and emulsifying.The vaccine of preparation is carried out proterties, steriling test, safety verification by the method for inspection of regulations, and detected result shows that vaccine indices is all qualified.
Concrete operations are as follows:
1 bacterium kind
1.1 manufacture bacterial classifications are that bacterial strain YG strain produced by duck hepatitis A virus gene engineering subunit vaccine; The virulent strain detected is DHAV-3 virulent strain VF-3.
1.2 production bacterial standards
1.2.1 form and biochemical characteristic
Incubated overnight on LB agar plate containing kantlex, culture plate presents circle, neat in edge, projection, the glossiness smooth colony of oyster white, after gramstaining, is shown as Gram-negative tyrothricin under mirror; Biochemical results be glucose fermentation+, indole test+, methyl red test+, VP-, Citric Acid utilize test-.
1.2.2 cultural characters can grow in containing the substratum of kantlex.
1.2.3 diagnostic test
1.2.3.1PCR detect and the LB liquid culture 3 μ l of this bacterium is done template, carry out pcr amplification by following PCR primer, the fragment of about 714bp should be able to be amplified.
P1:5′-cgcggatcctccaatcagcttggt-3′
P2：5′-aagctttta ttcaatctccagatg-3′
The condition of amplification is as follows: 94 DEG C of 5min sex change, 94 DEG C of 30S, 65 DEG C of 30S, 72 DEG C of 1min, 30 circulations, and 72 DEG C extend 10min.
1.2.3.2Western-blot detect and the recombinant bacterium list colony inoculation on LB solid culture flat board is contained in the LB liquid nutrient medium of kantlex in 5ml, be cultured to OD
600add the IPTG induction of 0.8mM when value is between 0.6 ~ 1.0, continue cultivation 4 hours, collect thalline, broken with the resuspended rear high pressure homogenization of PBS, 8000r/min is centrifugal goes precipitation, and supernatant liquor and anti-duck hepatitis A virus (HAV) 3 type positive serum carry out Western-blot test, should occur specific band.
1.2.4 pure appropriate media inspection, should be pure.
1.2.6 basic bacteria generation F4 ~ F10 generation.
1.2.7 preserve freeze-drying lactobacillus ,-20 DEG C, preservation period is 2 years.
2 vaccine manufacture and the inspections of semifinished product
2.1 production Spawn preparation
2.1.1 freeze-drying lactobacillus is inoculated in the LB liquid nutrient medium containing kantlex by first order seed breeding and qualification respectively, 37 DEG C of shaking culture 8 ~ 10 hours, then streak inoculation in containing kantlex LB solid medium on 37 DEG C cultivate 16 ~ 18 hours, as first order seed.2 ~ 8 DEG C of preservations, be no more than 14 days; Substratum goes down to posterity, should be no more than for 2 generations.
2.1.2 secondary seed breeding is chosen the colonies typical 10 meeting 1.2.1 item standard and is mixed in a small amount of LB nutrient solution in first order seed, is inoculated in the LB nutrient solution containing kantlex, cultivates 8 ~ 10 hours, purely checks for 37 DEG C, should be pure.Put 2 ~ 8 DEG C of preservations, 3 days should be no more than.
2.2 seedling substratum are improvement LB substratum, containing Tryptones 10g in every 1000ml substratum, and yeast leaching powder 5g, sodium-chlor 10g, glucose 5g, MgSO
47H
2o 5g.
2.3 prepared by antigen for vaccine liquid
2.3.1 bacterium liquid is cultivated and is used culture tank aerated culture, loads appropriate substratum (about 70%) and defoamer by culture tank volume, and by the 2% inoculation secondary seed bacterium liquid cultivating base unit weight after sterilizing, 37 DEG C of aerated culture, treat the OD of bacterium liquid
600when value reaches 7.0, add the alpha-lactose induction of 8g/L, then continue cultivation 6 ~ 8 hours.Use 20%NaOH to regulate pH 7.0, control dissolved oxygen more than 20% by rotating speed association.When dissolved oxygen rises rapidly, flow feeding.
2.3.2 after broken bacterium is cultivated and terminates, collected by centrifugation thalline.The thalline PBS collected cleans 2 times, by the suspension that the thalline PBS of collection makes 10%, uses high pressure homogenizer disrupt bacteria at 4 DEG C.Bacterium liquid after fragmentation, 8000r/min, centrifugal 15 minutes, collects supernatant liquor.
2.3.3 dialysis tubing put into by the recombinant protein elutriant that nickel column chromatography purification is collected, and PBS liquid, as extracellular fluid dialysis, namely obtains recombinant protein liquid after dialysis desalting.
2.3.4 the deactivation formaldehyde solution that will add 10% in the supernatant liquor of purifying in proportion, the ultimate density of formaldehyde solution is 0.2%, 37 DEG C of deactivations 12 hours, with the intestinal bacteria that deactivation is remaining.The sample that takes a morsel carries out the inspection of semifinished product.2 ~ 8 DEG C of preservations, are no more than 7 days; Less than-15 DEG C preservations, are no more than 60 days.
2.4 the inspection of semifinished product
2.4.1VP1 content detection BCA method detects Supernatant protein concentration, is diluted to final concentration 0.5mg/ml, sterile filtration, stand-by.
2.4.2 steriling test is tested by existing " Chinese veterinary pharmacopoeia ", answers asepsis growth.
2.4.3 endotoxin content detects and carries out intracellular toxin detection by tachypleus amebocyte lysate method, and endotoxin content can not be used for seedling higher than 1000EU/ml person.
2.5 vaccine preparations
2.5.1 oil phase preparation gets high-quality injection white oil 94 parts, aluminum stearate 2 parts.Mix in oil phase tank, heating and melting is to translucent, then Jia Siben-806 parts, maintains 30 minutes, be cooled to room temperature for subsequent use when temperature reaches 125 ~ 130 DEG C.
2.5.2 the tween-80 4 parts of sterilizing is got in aqueous phase preparation, adds the work in-process 96 parts be up to the standards, is stirred well to tween-80 and dissolves completely.
2.5.3 emulsification is got oil phase 3 parts and is placed in high-speed shearing machine, starts motor stirring at low speed, slowly adds aqueous phase 1 part simultaneously, then with 3600r/min emulsification 40 minutes, before termination is stirred, add 1% Thiomersalate solution, final concentration reached 0.01%.After emulsification, sampling 10ml adds centrifuge tube, with 3000r/min centrifugal 15 minutes, separates out aqueous phase and should be no more than 0.5ml at the bottom of pipe.
2.5.4 packing quantitative separating, sealing bottleneck.
3 inspection after constructions
3.1 physical behavior
Outward appearance is oyster white emulsion.
Formulation is water-in-oil-type.Get a clean suction pipe, draw a small amount of vaccine and drip in cold water, except the 1st, all should indiffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, through 3000r/min centrifugal 15 minutes, and the aqueous phase of separating out at the bottom of pipe should be no more than 0.5ml.
Viscosity is undertaken by existing " Chinese veterinary pharmacopoeia ", conforms with the regulations.
Loading quantity inspection is undertaken by existing " Chinese veterinary pharmacopoeia ", conforms with the regulations.
3.2 steriling tests are undertaken by existing " Chinese veterinary pharmacopoeia ", asepsis growth.
3.3 safety verifications get 1 age in days duckling 10, and muscle or neck subcutaneous injection vaccine 0.5ml/ only, observe 14 days, do not occur the local that caused by vaccine and systemic adverse reactions.
The following method of 3.4 efficacy test is appointed and is selected one.
3.4.1 4 monthly age sheldrakes (kind of laying eggs) 10 are equally divided into two groups by antibody test, first group is immune group, (second time immunity is carried out in first time immunity after 21 days to carry out second immunisation with the duck hepatitis A virus gene engineering subunit vaccine of preparation, every 0.5ml at every turn, subcutaneous injection), the serum NAT to DHAV-3 is surveyed in second time immunity for 14 days afterwards.Second group is control group, the stroke-physiological saline solution of injection equal volume.The serum NAT of immune group all should >=1:152, and control group NAT all should≤1:4.
3.4.2 attack poison protection and 4 monthly age sheldrakes (kind of laying eggs) 10 are equally divided into two groups, often organize 5.First group is immune group, the duck hepatitis A virus gene engineering subunit vaccine prepared with the present invention carries out second immunisation, and (second time immunity is carried out in first time immunity after 21 days, every 0.5ml at every turn, subcutaneous injection), second group is saline control group, the stroke-physiological saline solution of injection equal volume.Within 21-28 days, collect the duck's egg that each group of duck produce after second time immunity to hatch, go out with duckling respectively and respectively get 10 at random, with DHAV-3 virulent strain VF-3 (100LD in 14 days after shell
50) inject and attack poison, attack to add up afterwards for malicious 14 days and attack malicious protection ratio (not dead number of elements accounts for the percentage of total number of elements).Immune group should at least protect 8, and control group is all dead.
3.5 formaldehyde, the antiseptic mercurials determination of residual amount are undertaken by existing " Chinese veterinary pharmacopoeia ", all conform with the regulations.
Antibody titer after the immunity of embodiment 4 duck hepatitis A virus gene engineering subunit vaccine
4 monthly age sheldrakes (kind of laying eggs) 10 are equally divided into two groups, first group is immune group, (second time immunity is carried out in first time immunity after 21 days to carry out second immunisation with the duck hepatitis A virus gene engineering subunit vaccine of preparation, every 0.5ml at every turn, subcutaneous injection), the serum NAT to DHAV-3 is surveyed in second time immunity for 14 days afterwards.Second group is control group, the stroke-physiological saline solution of injection equal volume.Result show the serum NAT of immune group all >=1:128, and control group NAT is feminine gender.
Table 1: the antibody titer after the immunity of duck hepatitis A virus gene engineering subunit vaccine
Protest test after the immunity of embodiment 5 duck hepatitis A virus gene engineering subunit vaccine
4 monthly age sheldrakes (kind of laying eggs) 60 are equally divided into two groups, often organize 30.First group is immune group, the duck hepatitis A virus gene engineering subunit vaccine prepared with the present invention carries out second immunisation, and (second time immunity is carried out in first time immunity after 21 days, every 0.5ml at every turn, subcutaneous injection), second group is saline control group, the stroke-physiological saline solution of injection equal volume.Within 21-28 days, collect the duck's egg that each group of duck produce after second time immunity to hatch, within 14 days after duckling goes out shell, respectively get 10 at random, with DHAV-3 virulent strain VF-3 (100LD
50) inject and attack poison, attack to add up afterwards for malicious 14 days and attack malicious protection ratio (not dead number of elements accounts for the percentage of total number of elements).Set up vaccine control group simultaneously, its immune programme for children by specification carries out: get saline control group duck's egg and hatch, get rear 1 age in days duckling, 30 the subcutaneous inoculation duck hepatitis attenuated vaccines (A66 strain) of hatching, 0.5ml/ only, 10 are got at random, with DHAV-3 virulent strain VF-3 (100LD in latter 14 days of immunity is each
50) inject and attack poison, attack to add up afterwards for malicious 14 days and attack malicious protection ratio (not dead number of elements accounts for the percentage of total number of elements).
Table 2: the protest test after the immunity of duck hepatitis A virus gene engineering subunit vaccine
| Group | Attack poison strain | Attack malicious age in days | Attack protection ratio after malicious 14 days |
| Immune group | DHAV-3 strain VF-3 | 14 age in days ducklings | 8/10 |
| Vaccine control group | DHAV-3 strain VF-3 | 14 age in days ducklings | 4/10 |
| Saline control group | DHAV-3 strain VF-3 | 14 age in days ducklings | 0/10 |
Result show prepare duck hepatitis A virus gene engineering subunit vaccine to the passive protection phase of duckling more than 21 days; duckling after immunity 14 days attack malicious protection ratio more than 80%; and the immune protective rate of duck hepatitis attenuated vaccine strain (A66 strain) is 40%, the malicious protection ratio of attacking of saline control group is 0%.Prove that existing vaccine strain can not provide DHAV-3 virulent strain VF-3 to protect completely, vaccine of the present invention then protection ratio is good.
Claims (7)
1. a duck hepatitis A virus (HAV) Structural protein VP1 gene, is characterized in that, the aminoacid sequence of the proteins encoded of described gene is SEQ ID NO:1.
2. gene as claimed in claim 1, it is characterized in that, the nucleotides sequence of described gene is classified as SEQID NO:2.
3. a duck hepatitis A virus (HAV) Structural protein VP1 albumen, is characterized in that, the aminoacid sequence of described albumen is SEQ ID NO:1.
4. duck hepatitis A virus (HAV) Structural protein VP1 gene according to claim 1 is preparing the application in vaccine.
5. a duck hepatitis A virus (HAV) subunit vaccine, is characterized in that, the antigen of described vaccine is duck hepatitis A virus (HAV) Structural protein VP1 albumen according to claim 3.
6. vaccine as claimed in claim 5, it is characterized in that, in described vaccine, the concentration of duck hepatitis A virus (HAV) Structural protein VP1 albumen is 0.1-1mg/ml.
7. the preparation method of vaccine according to claim 5, is characterized in that, described preparation method includes following step:
1) VP1 gene is connected in expression vector, be built into recombinant expression;
2) recombinant expression built is transformed Host Strains, build the recombination engineering bacteria expressing duck hepatitis A virus (HAV) Structural protein VP1; Duck hepatitis A virus (HAV) Structural protein VP1 is gone out by this recombination engineering bacterium expression;
3), after purifying being carried out to recombinant expressed VP1 albumen, add white-oil adjuvant and make vaccine.
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