CN102174090B

CN102174090B - A kind of porcine type A influenza virus head protein and its preparation method and application

Info

Publication number: CN102174090B
Application number: CN201110069590.XA
Authority: CN
Inventors: 高福; 宣春玲
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2011-03-23
Filing date: 2011-03-23
Publication date: 2014-01-08
Anticipated expiration: 2031-03-23
Also published as: CN102174090A

Abstract

The invention discloses a porcine type A influenza virus head protein and its preparation method and application, the amino acid sequence of which is shown in SEQ ID NO.1. The method provided by the invention can improve the yield of influenza virus HA, and the purified HA protein can be more than 200 mg per two liters of culture medium. It only takes 5-6 days to get a batch of purified protein, shortening the production time. In addition, since the influenza virus is not used, the biological safety of the vaccine can be guaranteed, and there is no danger of virus shedding, and the virus does not need to be amplified in chicken embryos, which greatly reduces the production cost and has a good application prospect.

Description

One boar source A type influenza virus head protein and its preparation method and application

Technical field

The present invention relates to a kind of influenza virus protein and its preparation method and application, particularly a kind of functional pig source A type influenza head protein of high expression level amount.

Background technology

Influenza (abbreviation influenza) is (influenza) acute infectious disease that a kind of people, fowl, the poultry that are caused by influenza virus suffer from altogether.Influenza virus belongs to orthomyxoviridae family on virus taxis.According to the feature of virus nucleoprotein (nucleocapside protein, NP) and stromatin (matrix protein), influenza virus is divided into first (A), second (B) and 3 types of third (C).According to the difference of virion surface hemagglutinin (hamagglutinin, HA) and neuraminidase (neuraminidase, NA) protein antigenicity, influenza A virus can be further divided into different hypotypes again.The influenza A virus of finding at present has 16 HA hypotypes, 9 NA hypotypes.Influenza A virus was found in 1901, yet the influenza virus A hominis until just be separated in 1933, and it often exists with popular form, can cause worldwide flu outbreak.Since the human influenza virus is found, the influenza virus A hominis once occurred that the hypotype strain was the H2N2 hypotype of nineteen fifty-seven for several times, also claimed Asia influenza; The H3N2 hypotype of nineteen sixty-eight, also claim Mao flu; The H1N1 hypotype of 1977, also claim Russian influenza, and they are all starting in China.Within 1997 and 1998, in China Hong Kong Special Administrative Region and interior ground, fowl H5N1 and H9N2 flu cases have also been found.Simultaneously, in the influenza virus vaccine strain that since 1998, WHO announces every year, great majority are from China.Therefore, China by universally acknowledged be the multiple ground of influenza.

The coded product of influenza virus has 10 kinds of albumen, and wherein RNA polymerase has 3 kinds, is respectively PB1, PB2 and PA.Nucleoprotein (NP), M1 and M2 albumen, NS1 and NS2 albumen and hemagglutinin (HA) and neuraminidase (NA).Removing NS1 and NS2 is that non-structural protein is ultrawhite, and all the other are structural protein.There are 3 kinds of structural protein, i.e. HA, NA and M2 in influenza A virus grain surface.At first HA is 76kD at the endocytoplasmic reticulum synthetic molecular weight, contain 562-566 amino acid whose HA amyloid protein precursor (HA0), the HA0 molecule is hydrolyzed into HA1(47kD subsequently) and two polypeptide of HA2 (29kD), the former contains 319-328 amino acid, the latter is contained 221-222 amino acid, is connected to form the HA molecule with typical I type Membrane protein conformation by disulfide linkage between the two.The major part of HA albumen is positioned at outside film, and the hydrophobic region of nearly HA2 C-terminal, through BLM, is embedded in the HA molecule on film.Utilize the X-ray diffraction technology to confirm, the HA albumen of influenza virus is present on BLM with trimerical form, and the HA protein monomer by 3 non-covalent combinations is formed.HA albumen tripolymer can be divided into the basis pontis that is shaft-like and be spherical head.The receptor binding site of virus is shallow pocket-like, is positioned at the head of HA albumen, and the amino acid of receptor binding site forms at different subtype virus HA albumen camber conservative.HA plays an important role in virus replication, and its is identified and, in conjunction with the acceptor on host cell surface, can make virus envelope and host cell membrane merge, and can stimulate body to produce the protectiveness neutralizing antibody.NA plays an important role in new synthetic virion dispose procedure, and it can prevent from newly synthesizing virion and mutually be gathered in host cell surface, can't discharge and remove to infect new host cell again.Its antibody capable reduces viral excretion and plaque formation amount, but can not in and viral infection.M2 plays the ionic channel effect, can make the M1 film open, and viral RNP (ribosomal protein) enters endochylema.All the other structural protein, all in virion inside, can't stimulate body to produce protection antibody.HA albumen is one of major antigen of influenza virus, and the infection of its antibody capable neutralized stream Influenza Virus is main protection antibody.The result of molecule epidemic disease-ology research proves, influenza virus HA gene is mainly that the HA1 coding region is continuously, undergo mutation fast.Cause the fast-changing reason of HA gene to comprise quick change and two kinds of factors of crowd's natural selection pressure of its rna gene itself, the former is because the viral RNA polymerase lacks the function of checking that the DNA polymerase has, can not correct the mistake in rna replicon, be the incomplete faithful to parental virus of gene of progeny virus.The latter refers to the effect that crowd's specific immunity is evolved to HA albumen.

The effective means of current flu-prevention is still the inoculation of influenza virus vaccine, for the research of influenza vaccines, since the thirties in last century, just carries out.Nineteen thirty-seven chicken embryo culture influenza virus succeeds, and making to produce in a large number vaccine becomes possibility.Inactivated influenza virus vaccine goes through to use in the U.S. in nineteen forty-one first.Nineteen forty-three, the U.S. started to use in army, and confirmed effectively.1945 start in U.S.'s widespread use.This early stage rough vaccine, used the chick embryo allantoic liquid containing the influenza virus grain, through red corpuscle absorption and release, carries out limited purifying, then strengthens deactivation.After vaccination, reaction local and whole body is all very strong.The sixties in 20th century, the application of ultracentrifuge and thin layer chromatography technology, improve the virion purification process greatly, made purifying totivirus grain vaccine.Yet, when children use still untoward reaction can appear.As the purified vaccine with cracking, untoward reaction just greatly reduces.Nineteen sixty-eight, the U.S. ratified to use split vaccine first.20th century 70 and the eighties, on the basis of split vaccine, developed again virion subunit (HA and NA) vaccine.Use and confirmed that immune effect is identical with the cracking seedling in Britain, and can be used for children.Britain in 1980 ratifies to use first, then expands to other countries.In order further to improve vaccine output, reduce costs, worked out the reprovision vaccine strain of high yield.As (PR8) strain and street strain carry out the vaccine strain of reprovision to be used in the long-term A/PR/8/34 (H1N1) adapted in laboratory.The vaccine strain of reprovision not only possesses the surface antigen (HA and NA) of street strain like this, also obtains the replication of PR8 strain in the chicken embryo simultaneously.Recently go back the applying gene clone technology, obtain the influenza virion HA subunit of a large amount of high purities, high-titer in baculovirus silk mori system (baculovirus silk worm system).Along with molecular biological develop rapidly, promoted the foundation of genetically engineered and protein engineering.Molecular biological develop rapidly, promoted the foundation of genetically engineered and protein engineering.Use this type of technology to carry out many-sided research to vaccine.Along this direction, influenza vaccines have following development work.Someone develops NP, M1 and M2 vaccine; because the specificity of type is guarded and had to their antigenicities, therefore the antibody that stimulates body to produce has cross protection, the tool broad spectrum between the different subtype strain; needn't change with the epidemic isolates antigenic variation, but they are still under test at present.The micro-virion vaccine of MDP-utilizes adjuvant to improve immune effect, and Japan has started to use in the volunteer.The antibody male rotary rate of this vaccine is comparatively desirable, but untoward reaction is arranged.A kind of influenza dna vaccine of development in recent years, it not only can make body produce protection antibody and also produce strong cell response.But still more unanimously think that so far DNA vaccination is still in cradle, many problems still have to be solved.

It is mainly by chicken embryo culture amplicon virus that China produces influenza vaccines, and the virus of then collecting in allantoic fluid is carried out inactivation treatment, then with immunological adjuvant, is mixed with into inactivated virus vaccine.It faces the high problem of production cycle long production cost in process of production, and needs in process of production a large amount of SPF chicken embryos.

Summary of the invention

Technical problem to be solved by this invention is to provide a boar source A type influenza virus protein.

Described influenza proteins is (a) or protein (b):

(a) protein formed by the amino acid shown in SEQ ID NO.1,

(b) aminoacid sequence in (a) is through replacing, lacking or add an amino acid or several amino acid and have the antigenic protein derivative by (a) of influenza proteins.

Another technical problem that the present invention will solve is to provide a kind of genetic engineering bacterium that produces described influenza proteins.

Described genetic engineering bacterium is e. coli bl21.

Described genetic engineering bacterium is used secreted expression carrier, and described expression vector is pET series.

Another technical problem that the present invention will solve is to provide a kind of method for preparing described influenza proteins.

For solving the problems of the technologies described above, concrete technical scheme of the present invention is:

(1) according to the GenBank:FJ966082.1 sequence, the nucleotide sequence SEQ ID NO.4 of the described protein of synthetic coding SEQ ID NO.1;

(2) nucleotide fragments obtained in step (1) is cloned into to expression vector pET21a;

(3) expression vector step 2 obtained transforms e. coli bl21;

(4) the described protein of abduction delivering claim 1;

(5) protein that purification step (4) obtains, obtain a large amount of inclusion bodys;

(6) inclusion body step (5) obtained carries out the renaturation processing, prepares activated viral protein.

Described renaturation condition is: 1mL dissolution buffer (6M Guanidine Hydrochloride; 10% glycerine; 100mM NaCl; 10mM EDTA; 10mM DTT), 4 ℃ of stirring and dissolving; Preparation refolding buffer(100mM Tris pH8.0; 400mM L-Arginine monohydrochloride; 2mM EDTA; 5mM GSH; 0.5mM GSSG) on ice or 4 ℃ of precoolings; With the 5mL syringe by the about 3-5mL of inclusion body after dissolving at twice every minor tick within 8 hours, add syringe, make it drop by drop in refolding buffer, to drip, slowly stir 8-10 hour.

Inventive principle: vaccination is still the most effective a kind of means of current flu-prevention, yet the easy variation of influenza antigen causes vaccine inoculation invalid, is influenza morbidity and the popular one of the main reasons that can't control fully so far.The HA header area is main variation position, can be separated to by epidemiology survey the influenza virus strain of advantage serotype, and its HA header area gene clone is tested to this in designed expression vector, utilize colibacillary expression system with a large amount of HA of production of the fastest time header area albumen, thus the variation of reply influenza antigen.The present invention is on the early-stage Study basis, and the first fragment of clone HA header area subunit's immunity, utilize escherichia expression system with a large amount of HA of production of the fastest time header area protein subunit.

The present invention can improve the output of influenza virus HA, and the HA albumen that every two liters of substratum can obtain purifying is greater than 200 milligrams.Only need can obtain in 5-6 days the albumen of a collection of purifying, shorten the production time.Owing to can not using influenza virus therefore can guarantee the biological safety of vaccine, not there will be loose malicious danger, and do not need chicken embryo amplicon virus to greatly reduce production cost.The HA albumen that experimental results show that the purifying acquisition by western blotting can be combined (Fig. 1) with the polyclonal antibody of influenza virus, shows that the HA albumen of prokaryotic expression has immunogenicity.The present invention has built the expression vector of influenza virus HA header area albumen, thereby can constantly change virogene along with the influenza antigen variation as required and can obtain a large amount of albumen for the preparation of vaccine.

The accompanying drawing explanation

Fig. 1 H1HA head protein detection of antigenicity

Fig. 2 H1HA header area protein purification and SDS-PAGE

A:H1HA header area protein purification B:H1HA header area protein SDS-PAGE detects

Fig. 3 H1HA header area is attacked malicious Protection.

Embodiment

embodiment 1 influenza virus protein

Described influenza proteins amino acid forms as shown in SEQ ID NO.1.

SEQ ID NO.1：APLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERN

Described influenza virus protein amino acid forms: the aminoacid sequence shown in SEQ ID NO.1 in albumen is through replacing, lacking or add an amino acid or several amino acid and have the antigenic protein of influenza proteins, as SEQ ID NO.2:

APLQLGKCNIAGWLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFIDYEELRE QLSSVSSFEKFEIFPKTSSWPNHETTKGVTAACSYAGASSFYRNLLWLTKKGSSYP KLSKSYVNNKGKEVLVLWGVHHPPTGTDQQSLYQNADAYVSVGSSKYNRRFTPEIA ARPKVRDQAGRMNYYWTLLEPGDTITFEATGNLIAPWYAFALN; Or SEQ ID NO.3:

Shown in APLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELRE QLSSVSSFERFEIFPKESSWPNHNTTKGVTAACSHAGKSSFYRNLLWLTEKEGSYP KLKNSYVNKKGKEVLVLWGIHHPSNSKDQQNIYQNENAYVSVVTSNYNRRFTPEIA ERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPRYAFALS, the preparation method of SEQ ID NO.2 and SEQ IN NO.3 albumen is with reference to SEQ ID NO.1 albumen.

the preparation of embodiment 2 influenza virus proteins

1, the acquisition of goal gene

This tests selected influenza virus A/California/04/2009 (H1N1), GenBank:FJ966082.1, according to artificial complete synthesis (this synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) the nucleotide sequence SEQ ID NO.4 of GenBank:FJ966082.1 sequence

gccccatt gcatttgggt aaatgtaaca ttgctggctg gatcctggga aatccagagt gtgaatcact ctccacagca agctcatggt cctacattgt ggaaacacct agttcagaca atggaacgtg ttacccagga gatttcatcg attatgagga gctaagagag caattgagct cagtgtcatc atttgaaagg tttgagatat tccccaagac aagttcatgg cccaatcatg actcgaacaa aggtgtaacg gcagcatgtc ctcatgctgg agcaaaaagc ttctacaaaa atttaatatg gctagttaaa aaaggaaatt catacccaaa gctcagcaaa tcctacatta atgataaagg gaaagaagtc ctcgtgctat ggggcattca ccatccatct actagtgctg accaacaaag tctctatcag aatgcagata catatgtttt tgtggggtca tcaagataca gcaagaagtt caagccggaa atagcaataa gacccaaagt gagggatcaa gaagggagaa tgaactatta ctggacacta gtagagccgg gagacaaaat aacattcgaa gcaactggaa atctagtggt accgagatat gcattcgcaa tggaaagaaat。

2, the preparation of carrier

PCR product and expression vector pET21a are cut with restriction enzyme, then carry out the DNA electrophoresis, reclaiming test kit by DNA gel reclaims out by carrier and DNA fragmentation respectively, pass through the T4DNA ligase enzyme at the carrier by reclaiming and fragment, 16 ℃ of connections of spending the night, finally will connect product and be transformed in competent escherichia coli cell DH5 α.The recon obtained is identified by the method for bacterium liquid PCR and plasmid enzyme restriction respectively, shown that the clone is correct.Then the positive colony of having identified is extracted to Plasmid Transformation in competent escherichia coli cell BL21.

The restriction enzyme that this experiment is used is all Dalian precious biotechnology company limited products.The operation instruction of using Takara company to provide according to the restriction enzyme used is selected suitable damping fluid and enzyme Qie Wendu, and the enzyme system of cutting is 20 μ L.

10×H Buffer 2μL

EcoR1 1μL

Xho1 1μL

DNA 16μL

37 ℃ of enzymes are cut 4 hours.During recovery, use DNA gel to reclaim test kit.

Linked system is as follows:

Vector DNA 100ng

Insert DNA Variable

10×T4 DNA Ligation Buffer 1μL

T4 DNA Ligase 1μL

Total system 10 μ L, connect mixture and be placed in 16 ℃ of connection 2h, and then 4 ℃ are spent the night.The mole number of carrier in each linked system: the mole number ratio of inserting DNA is about 1:1 or 1:3.

Connect the conversion of product and plasmid: get-70 ℃ of frozen competent cells on ice after melting, add and connect product 5 μ L, mix gently ice bath 30 minutes.42 ℃ of water-bath heat shocks 90 seconds, then put back to rapidly 2min on ice.Add 400 μ L LB liquid nutrient mediums, after 37 ℃ of 200r/min-220r/min shaking culture 45min, to recover the resistance of plasmid.4 ℃ of centrifugal 5min of 4000r/min, supernatant discarded 400 μ L, mix rear painting ammonia benzyl flat board by remaining 100 μ L bacterium liquid, cultivates the 30min(plate for 37 ℃ and just put), treat the absorption of bacterium liquid fully, then plate is inverted and is cultivated about 12h-16h, to single bacterium colony appearance.

Recon is identified: PCR Rapid identification: with aseptic toothpick picking bacterium transformant, be applied in aseptic EP pipe bottom, add the aseptic distilled water of 20 μ L, vortex mixes, and boils 5 min, in quenching on ice, centrifugal 1 min of 12,000 r/min, get the template of supernatant as pcr amplification, carry out the PCR evaluation, result is indicated in recon the plasmid contained after conversion.

The recombinant plasmid enzyme is cut evaluation: the recon that the picking Rapid identification is positive is inoculated in the liquid LB substratum containing corresponding resistant 37 ℃ of 250-300 r/min overnight incubation, extract plasmid, by suitable digestion with restriction enzyme, electrophoresis on 1% sepharose, observations under ultraviolet lamp.

3, clone gene is expressed

Express in a small amount: the e. coli bl21 list bacterium colony that picking contains the correction plasmid, be inoculated in the LB substratum that 5mL contains penbritin, 37 ℃ of 0.1mM IPTG induce 4 hours, and the blank that does not add IPTG is set simultaneously.SDS-PAGE verifies the solubility of this albumen.

Extensive expression and purification: the single colony inoculation that contains the correction plasmid on the fresh flat board of picking contains the LB liquid nutrient medium of penbritin in 2mL, and then 37 ℃ of concussion overnight incubation are transferred to same overnight incubation in 20mL LB liquid nutrient medium.20mL bacterium liquid all is transferred to 2L containing in the LB liquid nutrient medium of penbritin, and about 3-5 hour is cultivated in 37 ℃ of concussions.When OD600 reaches 0.4 left and right, add IPTG to final concentration 0.1mM, induce for 37 ℃ and lead 4 hours, the centrifugal results thalline of 6000 rpm.After thalline is resuspended with PBS under 4 ℃, is positioned over and uses Ultrasonic Cell Disruptor cracking bacterium on ice.Centrifugal 30 minutes of 12,000rpm, abandoning supernatant is peeled bacterial debris off with glass stick, and precipitation is inclusion body.

4, purifying expressing protein from inclusion body: the high level expression of protein in intestinal bacteria usually causes forming visible cytoplasmic granule or inclusion body under phase microscope.These inclusion bodys that are gathered into by expressing protein are easy to separate with embrane-associated protein with soluble proteins.The bacterium of high level expression foreign protein is after centrifugal concentrating, and the method that can add stain remover by mechanical process, Ultrasonic treatment or N,O-Diacetylmuramidase is carried out cracking.Inclusion body available Triton X-100 and EDTA or wash with urea after centrifugation.The purpose of washing is to remove as far as possible bacterioprotein soluble, that adhere to from the foreign protein of assembling.For obtaining the activated protein of solubility, must, by washed solubilization of inclusion bodies, then carry out refolding.

The method of HA header area albumen renaturing inclusion bodies:

With washing buffer(0.5% TritonX-100; 50mM Tris pH8.0; 300mM NaCl; 10mM EDTA; 10mM DTT) inclusion body is hanged.Can see after centrifugal that bacterium has or not complete cracking, if can not be again ultrasonic (4 seconds, 10 seconds, 43 times), ultrasound condition can be adjusted voluntarily.The centrifugal 10-20 minute of 12,000rpm after ultrasonic removes bacterial debris with washing buffer washing as far as possible.With resuspension buffer (50mM Tris pH8.0; 100mM NaCl; 10mM EDTA; 10mM DTT) resuspended inclusion body 12, the centrifugal 10-20 of 000rpm minute supernatant discarded, and inclusion body is weighed.Add 1mL dissolution buffer (6M Guanidine Hydrochloride according to the 30mg inclusion body; 10% glycerine; 100mM NaCl; 10mM EDTA; 10mM DTT), 4 ℃ of stirring and dissolving.

Renaturing inclusion bodies: preparation refolding buffer(100mM Tris pH8.0; 400mM L-Arginine monohydrochloride; 2mM EDTA; 5mM GSH; 0.5mM GSSG) on ice or 4 ℃ of precoolings.With the 5mL syringe by the about 3-5mL of inclusion body after dissolving at twice every minor tick within 8 hours, add syringe, make it drop by drop in refolding buffer, to drip, slowly stir 8-10 hour.After renaturation, available concentrated cup concentrating sample, then change the damping fluid that is suitable for this albumen into, then it is interior concentrated to forward evaporating pipe to, if the renaturation system is little, can directly with evaporating pipe, change liquid.By the Superdex 200(Amersham Biosciences for albumen obtained) gel-filtration column is further purified (Fig. 2), collects the elution peak containing target protein, with super filter tube, albumen is concentrated into to approximately 30 mg.mL ^-1.

embodiment 3 protein animal experiments

The experiment of H1HA header area protein immunization originality:

Laboratory animal: 24 of SPF chickens, every 6 are divided into one group totally 4 groups, are respectively: blank group, adjuvant control group, 50 μ g antigen amount groups, 100 μ g antigen amount groups

Reagent: Freund's complete adjuvant, incomplete Freund's adjuvant are purchased from sigma company

Method: mix with Freund's complete adjuvant and carry out the intramuscular injection immunity with the H1HA header area recombinant protein 50 μ g of purifying and 100 μ g respectively, booster immunization after mixing with incomplete Freund's adjuvant with corresponding antigen after 3 weeks.Establish blank group and adjuvant control group simultaneously.Reach the last immunity before immunity and within latter 10 days, collect chicken serum.After the last immunity, the 21st day via intranasal application inoculated 50 μ L containing 10 ⁶eID ₅₀the diluent of A/California/04/2009 (H1N1) virus quantity, and observe the mean body weight variation of monitoring chicken death situation and survival in 20 days, result shows the SPF chicken death rate low (Fig. 3) after H1HA header area protein immunization.

the application of embodiment 4 H1HA header area albumen in the subunit vaccine preparation

The H1HA header area albumen that purifying is obtained, be mixed and made into adjuvant the subunit vaccine that contains influenza virus hemagglutinin, and the intramuscular injection immune animal makes animal obtain the initiative immunity, thus the infection of opposing influenza virus.Application in subunit vaccine production can be with reference to following document.

[1] Oriol Manuel, Manuel Pascual, Katja Hoschler. Humoral Response to the Influenza A H1N1/09 Monovalent AS03-Adjuvanted Vaccine in Immunocompromised Patients. Clin Infect Dis. 2011 Jan;52(2):248-56

[2] Robert B. Belshe, Sharon E. Frey, Irene Graham. Safety and immunogenicity of influenza A H5 subunit vaccines: effect of vaccine schedule and antigenic variant. J Infect Dis. 2011 Mar;203(5):666-73

Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.

<110 > Institute of Microorganism, Academia Sinica

<120 > boar source A type influenza virus head protein and its preparation method and application

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<213 > pig source A type influenza virus H1N1 (Swine-origin A influenza virus H1N1)

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gataaaggga aagaagtcct cgtgctatgg ggcattcacc atccatctac tagtgctgac 420

caacaaagtc tctatcagaa tgcagataca tatgtttttg tggggtcatc aagatacagc 480

aagaagttca agccggaaat agcaataaga cccaaagtga gggatcaaga agggagaatg 540

aactattact ggacactagt agagccggga gacaaaataa cattcgaagc aactggaaat 600

ctagtggtac cgagatatgc attcgcaatg gaaagaaat 639

Claims

1. a boar source A type influenza virus head protein, its aminoacid sequence is as shown in SEQ ID NO.1.

2. a property right profit requires the genetic engineering bacterium of 1 described pig source A type influenza virus head protein, it is characterized in that described genetic engineering bacterium is for expressing protein colon bacillus BL21 shown in SEQ ID NO.1.

3. genetic engineering bacterium according to claim 2, is characterized in that described engineering bacteria is used secreted expression carrier.

4. genetic engineering bacterium according to claim 3, is characterized in that described expression vector is pET series.

5. a method for preparing the described pig of claim 1 source A type influenza virus head protein, is characterized in that comprising the steps:

(3) expression vector step (2) obtained transforms e. coli bl21;

(4) the described protein of abduction delivering claim 1;

6. method according to claim 5, it is characterized in that described inductive condition is: thalline OD600 adds IPTG to final concentration 0.1mM while reaching 0.4 left and right, induces 4 hours the centrifugal results thalline of 6000rpm for 37 ℃.

7. the application of the described influenza virus head protein of claim 1 in preparing subunit vaccine.