CN102174090A - Swine-origin influenza A virus head protein and preparation method and application thereof - Google Patents

Abstract

The invention discloses a swine-origin influenza A virus head protein and a preparation method and application thereof. The protein has an amino acid sequence shown as SEQ ID NO.1. By the method, the yield of hemagglutinin (HA) in influenza virus can be improved, and more than 200 milligrams of purified HA protein can be obtained in each two liters of culture medium; a batch of purified proteins can be obtained only after 5 to 6 days, and production time is shortened; in addition, the influenza virus is not used, so the biological safety of a vaccine can be ensured, and the danger of spreading poison is avoided; a chick embryo is not required to be used for amplifying the virus, so production cost is greatly reduced; and the protein has a good application prospect.

Description

One boar source A type influenza virus head albumen and its production and application

Technical field

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

Background technology

Influenza (abbreviation influenza) is the acute infectious disease of being suffered from altogether by a kind of people, fowl, poultry that influenza virus causes (influenza).Influenza virus belongs to orthomyxoviridae family on virus taxis.(nucleocapside protein, NP) and the feature of stromatin (matrix protein), influenza virus is divided into first (A), second (B) and 3 types of third (C) according to virus nucleoprotein.According to virion surface hemagglutinin (hamagglutinin, HA) and neuraminidase (influenza A virus can be further divided into different hypotypes again for neuraminidase, the NA) difference of protein antigenicity.The influenza A virus of being found at present has 16 HA hypotypes, 9 NA hypotypes.Influenza A virus was found in 1901, yet the influenza virus A hominis just was separated up to 1933, and it often exists with popular form, can cause worldwide flu outbreak.Since the human influenza virus was found, it was the H2N2 hypotype of nineteen fifty-seven that several hypotype strain once appearred in the influenza virus A hominis, also claims Asia influenza; The H3N2 hypotype of nineteen sixty-eight also claims Mao flu; H1N1 hypotype in 1977 also claims Russian influenza, and they are all run initially in China.In reaching, China Hong Kong Special Administrative Region also found fowl H5N1 and H9N2 influenza case in 1997 and 1998.Simultaneously, great majority are from China in the influenza virus vaccine strain that WHO is announced every year since 1998.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.HA is 76kD at the endocytoplasmic reticulum synthetic molecular weight at first, 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, and linking to each other by disulfide linkage between the two forms the HA molecule with typical type I membranin structure.The proteic major part of HA is positioned at outside the film, and the hydrophobic region of nearly HA2 C-terminal passes BLM, and the HA molecule is embedded on the film.Utilize the X-ray diffraction technology to confirm that the HA albumen of influenza virus is present on the BLM with trimerical form, promptly is made up of 3 non-covalent bonded HA protein monomers.HA albumen tripolymer can be divided into the basis pontis that is shaft-like and be the globular head.The Virus receptors combining site is shallow pocket-like, is positioned at the proteic head of HA, and the amino acid of receptor binding site is formed at different subtype virus HA albumen camber conservative.HA plays an important role in virus replication, and its is discerned and in conjunction with the acceptor on the host cell surface, virus envelope and host cell membrane is merged, 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 that newly synthetic virion from accumulating in host cell surface mutually, 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 then plays the ionic channel effect, and the M1 film is opened, and viral RNP (ribosomal protein) enters endochylema.All the other structural protein can't stimulate body to produce protection antibody all in virion inside.HA albumen is one of major antigen of influenza virus, in its antibody capable and the infection of influenza virus, is main protection antibody.The result of molecule epidemic disease-ology research proves, influenza virus HA gene mainly is 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 had, can not correct the mistake in the 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, just carries out since the thirties in last century for the research of influenza vaccines.Nineteen thirty-seven chicken embryo culture influenza virus is succeedd, and makes the mass production vaccine become possibility.Inactivated influenza virus vaccine goes through to use in the U.S. in nineteen forty-one first.Nineteen forty-three, the U.S. began to use in army, and confirmed effectively.Beginning was in U.S.'s widespread use in 1945.This early stage rough vaccine uses the chick embryo allantoic liquid that contains the influenza virus grain, through red corpuscle absorption and release, carries out limited purifying, strengthens deactivation then.After the vaccination, reaction local and whole body is all very strong.The sixties in 20th century, the application of ultracentrifuge and chromatography chromatographic technique improves the virion purification process greatly, has made purifying totivirus grain vaccine.Yet, when children use still untoward reaction can appear.As use the cracked purified vaccine, 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 virion subunit (HA and NA) vaccine again.Use has 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 cost, 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) that adapts 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, the influenza virion HA subunit that a large amount of high purities of acquisition, height are tired 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 has promoted the foundation of genetically engineered and protein engineering.Use this type of technology that vaccine has been carried out many-sided research.Along this direction, influenza vaccines have following development work.Someone develops NP, M1 and M2 vaccine; because of the specificity of type is guarded and had to their antigenicities, so stimulate the antibody that body produced cross protection to be arranged, 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 little virion vaccine of MDP-utilizes adjuvant to improve immune effect, and Japan has begun 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 the intensive cell response.But still more consistently so far think that dna vaccination still is in cradle, many problems still have to be solved.

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

Summary of the invention

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

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

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

(b) aminoacid sequence in (a) is through replacing, lack or adding an amino acid or several amino acid and have influenza proteins antigenic by (a) deutero-protein.

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

Described genetic engineering bacterium is an e. coli bl21.

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

Another technical problem that the present invention will solve provides 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 described proteinic nucleotide sequence SEQ ID NO.4 of synthetic coding SEQ ID NO.1;

(2) nucleotide fragments that obtains in the step (1) is cloned into expression vector pET21a;

(3) the expression vector transformed into escherichia coli BL21 that step 2 is obtained;

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

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

(6) inclusion body that step (5) is 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; The about 3-5mL of inclusion body after will dissolving with the 5mL syringe is each at twice to add syringe in 8 hours at interval, made it drop by drop to drip in refolding buffer, slowly stirred 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 the influenza virus strain of advantage serotype by epidemiology survey, and with its HA header area gene clone in the designed expression vector of this experiment, utilize colibacillary expression system with the fastest time mass production HA 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 utilizes escherichia expression system with the fastest time mass production HA header area protein subunit.

The present invention can improve the output of influenza virus HA, and per two liters of substratum can obtain the HA albumen of purifying greater than 200 milligrams.Only need to obtain in 5-6 days the albumen of a collection of purifying, shorten the production time.Owing to can not use influenza virus therefore can guarantee the biological safety of vaccine, the danger of poison of loosing can not occur, and not need chicken embryo amplicon virus to greatly reduce production cost.Experimental results show that by western blotting the HA albumen that purifying obtains can combine (Fig. 1) with the polyclonal antibody of influenza virus, shows that the HA albumen of prokaryotic expression has immunogenicity.The present invention has made up the proteic expression vector of influenza virus HA header area, thereby can constantly change virogene as required along with the influenza antigen variation and can obtain a large amount of albumen and be used to prepare vaccine.

Description of drawings

Fig. 1 H1HA head protein antigenicity detects

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 poison protection experiment.

Embodiment

Embodiment 1 influenza virus protein

Described influenza proteins amino acid is formed shown in SEQ ID NO.1.

SEQ?ID?NO.1：APLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADTYVFVGSS

Described influenza virus protein amino acid form can also for: the aminoacid sequence shown in the SEQ ID NO.1 in the albumen through replacing, lack 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 the APLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELRE QLSSVSSFERFEIFPKESSWPNHNTTKGVTAACSHAGKSSFYRNLLWLTEKEGSYP KLKNSYVNKKGKEVLVLWGIHHPSNSKDQQNIYQNENAYVSVVTSNYNRRFTPEIA ERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPRYAFALS, SEQ ID NO.2 and the proteic preparation method of SEQ IN NO.3 are 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 GenBank:FJ966082.1 sequence manually complete synthesis (this is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) nucleotide sequence SEQ ID NO.4

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 with carrier and dna fragmentation respectively, pass through the T4DNA ligase enzyme at carrier that will reclaim and fragment, 16 ℃ of connections of spending the night will connect product at last and be transformed among the competent escherichia coli cell DH5 α.The recon that obtains is identified by the method for bacterium liquid PCR and plasmid enzyme restriction respectively, shown that the clone is correct.To identify that then good positive colony extracts plasmid and is transformed among the competent escherichia coli cell BL21.

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

10×H?Buffer 2μL

EcoR1 1μL

Xho1 1μL

DNA 16μL

37 ℃ of enzymes were cut 4 hours.Use dna gel to reclaim test kit during recovery.

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 place 16 ℃ to connect 2h, and 4 ℃ are spent the night then.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, mixing gently, ice bath 30 minutes.2min is on ice put back in 42 ℃ of water-bath heat shocks 90 seconds more rapidly.Add 400 μ L LB liquid nutrient mediums, behind 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 will be coated with ammonia benzyl flat board behind the remaining 100 μ L bacterium liquid mixings, cultivate the 30min(plate for 37 ℃ and just put), treat the absorption of bacterium liquid fully, plate is inverted is cultivated about 12h-16h again, 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, the vortex mixing boils 5 min, in quenching on ice, centrifugal 1 min of 12,000 r/min gets the template of supernatant as pcr amplification, carry out PCR and identify, the result indicates the plasmid that contains in the recon after the conversion.

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

3, clone gene is expressed

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

Extensive expression and purification: the single colony inoculation that contains correct expression plasmid on the fresh flat board of picking contains the LB liquid nutrient medium of penbritin in 2mL, and 37 ℃ of concussion overnight incubation are transferred to same overnight incubation in the 20mL LB liquid nutrient medium then.20mL bacterium liquid all is transferred to 2L contains in the LB liquid nutrient medium of penbritin, 37 ℃ of concussions were cultivated about 3-5 hour.Treat to add when OD600 reaches 0.4 left and right sides IPTG to final concentration 0.1mM, induce for 37 ℃ and lead 4 hours, the centrifugal results thalline of 6000 rpm.Thalline is positioned over and uses Ultrasonic Cell Disruptor cracking bacterium on ice after using PBS resuspended under 4 ℃.12, centrifugal 30 minutes of 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 the 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 can carry out cracking by the method that mechanical process, supersound process method or N,O-Diacetylmuramidase add stain remover after centrifugal concentrate.Inclusion body available Triton X-100 and EDTA or wash after centrifugation with urea.The purpose of washing is to remove soluble, adherent bacterioprotein as far as possible from the accumulative foreign protein.For obtaining the activated protein of solubility, washed solubilization of inclusion bodies must be carried out refolding then.

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 ultrasonic once more (4 seconds, 10 seconds, 43 times), ultrasound condition can be adjusted voluntarily.Bacterial debris the centrifugal 10-20 of 000rpm minute, is removed with washing buffer washing in ultrasonic back 12 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 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.The about 3-5mL of inclusion body after will dissolving with the 5mL syringe is each at twice to add syringe in 8 hours at interval, made it drop by drop to drip in refolding buffer, slowly stirred 8-10 hour.Available concentrated cup concentrating sample after the renaturation changes into then and is suitable for this proteic damping fluid, forwards in the evaporating pipe to concentrate again, and can directly change liquid with evaporating pipe if the renaturation system is little.With the albumen that obtains with Superdex 200(Amersham Biosciences) gel-filtration column is further purified (Fig. 2), collects the elution peak that contains target protein, with the ultrafiltration pipe albumen is concentrated into about 30 mg.mL ^-1

Embodiment 3 protein animal experiments

The experiment of H1HA header area protein immunization originality:

Laboratory animal: 24 of SPF chickens, per 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 available from sigma company

Method: mix with Freund's complete adjuvant with the H1HA header area recombinant protein 50 μ g of purifying and 100 μ g respectively and carry out the intramuscular injection immunity, 3 week the back mix back booster immunization with incomplete Freund's adjuvant with corresponding antigen.Establish blank group and adjuvant control group simultaneously.Reach the last immunity before the immunity and collected chicken serum in back 10 days.The 21st day via intranasal application inoculated 50 μ L and contained 10 after the last immunity 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, the result shows the SPF chicken death rate low (Fig. 3) behind H1HA header area protein immunization.

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

H1HA header area albumen with purifying obtains is mixed and made into the subunit vaccine that contains influenza virus hemagglutinin with adjuvant, and the intramuscular injection immune animal makes animal obtain the initiative immunity, thus the infection of opposing influenza virus.Application during subunit vaccine is produced 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

Though the present invention with preferred embodiment openly as above; but it is not in order to qualification 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 albumen and its production and application

 

<160> 4

 

<170> PatentIn?version?3.3

 

<210> 1

<211> 213

<212> PRT

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

 

<400> 1

 

Ala?Pro?Leu?His?Leu?Gly?Lys?Cys?Asn?Ile?Ala?Gly?Trp?Ile?Leu?Gly

1 5 10 15

 

 

Asn?Pro?Glu?Cys?Glu?Ser?Leu?Ser?Thr?Ala?Ser?Ser?Trp?Ser?Tyr?Ile

20 25 30

 

 

Val?Glu?Thr?Pro?Ser?Ser?Asp?Asn?Gly?Thr?Cys?Tyr?Pro?Gly?Asp?Phe

35 40 45

 

 

Ile?Asp?Tyr?Glu?Glu?Leu?Arg?Glu?Gln?Leu?Ser?Ser?Val?Ser?Ser?Phe

50 55 60

 

 

Glu?Arg?Phe?Glu?Ile?Phe?Pro?Lys?Thr?Ser?Ser?Trp?Pro?Asn?His?Asp

65 70 75 80

 

 

Ser?Asn?Lys?Gly?Val?Thr?Ala?Ala?Cys?Pro?His?Ala?Gly?Ala?Lys?Ser

85 90 95

 

 

Phe?Tyr?Lys?Asn?Leu?Ile?Trp?Leu?Val?Lys?Lys?Gly?Asn?Ser?Tyr?Pro

100 105 110

 

 

Lys?Leu?Ser?Lys?Ser?Tyr?Ile?Asn?Asp?Lys?Gly?Lys?Glu?Val?Leu?Val

115 120 125

 

 

Leu?Trp?Gly?Ile?His?His?Pro?Ser?Thr?Ser?Ala?Asp?Gln?Gln?Ser?Leu

130 135 140

 

 

Tyr?Gln?Asn?Ala?Asp?Thr?Tyr?Val?Phe?Val?Gly?Ser?Ser?Arg?Tyr?Ser

145 150 155 160

 

 

Lys?Lys?Phe?Lys?Pro?Glu?Ile?Ala?Ile?Arg?Pro?Lys?Val?Arg?Asp?Gln

165 170 175

 

 

Glu?Gly?Arg?Met?Asn?Tyr?Tyr?Trp?Thr?Leu?Val?Glu?Pro?Gly?Asp?Lys

180 185 190

 

 

Ile?Thr?Phe?Glu?Ala?Thr?Gly?Asn?Leu?Val?Val?Pro?Arg?Tyr?Ala?Phe

195 200 205

 

 

Ala?Met?Glu?Arg?Asn

210

 

 

<210> 2

<211> 211

<212> PRT

＜213〉artificial synthesized sequence

 

<400> 2

 

Ala?Pro?Leu?Gln?Leu?Gly?Lys?Cys?Asn?Ile?Ala?Gly?Trp?Leu?Leu?Gly

1 5 10 15

 

 

Asn?Pro?Glu?Cys?Asp?Leu?Leu?Leu?Thr?Ala?Ser?Ser?Trp?Ser?Tyr?Ile

20 25 30

 

 

Val?Glu?Thr?Ser?Asn?Ser?Glu?Asn?Gly?Thr?Cys?Tyr?Pro?Gly?Asp?Phe

35 40 45

 

 

Ile?Asp?Tyr?Glu?Glu?Leu?Arg?Glu?Gln?Leu?Ser?Ser?Val?Ser?Ser?Phe

50 55 60

 

 

Glu?Lys?Phe?Glu?Ile?Phe?Pro?Lys?Thr?Ser?Ser?Trp?Pro?Asn?His?Glu

65 70 75 80

 

 

Thr?Thr?Lys?Gly?Val?Thr?Ala?Ala?Cys?Ser?Tyr?Ala?Gly?Ala?Ser?Ser

85 90 95

 

 

Phe?Tyr?Arg?Asn?Leu?Leu?Trp?Leu?Thr?Lys?Lys?Gly?Ser?Ser?Tyr?Pro

100 105 110

 

 

Lys?Leu?Ser?Lys?Ser?Tyr?Val?Asn?Asn?Lys?Gly?Lys?Glu?Val?Leu?Val

115 120 125

 

 

Leu?Trp?Gly?Val?His?His?Pro?Pro?Thr?Gly?Thr?Asp?Gln?Gln?Ser?Leu

130 135 140

 

 

Tyr?Gln?Asn?Ala?Asp?Ala?Tyr?Val?Ser?Val?Gly?Ser?Ser?Lys?Tyr?Asn

145 150 155 160

 

 

Arg?Arg?Phe?Thr?Pro?Glu?Ile?Ala?Ala?Arg?Pro?Lys?Val?Arg?Asp?Gln

165 170 175

 

 

Ala?Gly?Arg?Met?Asn?Tyr?Tyr?Trp?Thr?Leu?Leu?Glu?Pro?Gly?Asp?Thr

180 185 190

 

 

Ile?Thr?Phe?Glu?Ala?Thr?Gly?Asn?Leu?Ile?Ala?Pro?Trp?Tyr?Ala?Phe

195 200 205

 

 

Ala?Leu?Asn

210

 

 

<210> 3

<211> 211

<212> PRT

＜213〉artificial synthesized sequence

 

<400> 3

 

Ala?Pro?Leu?Gln?Leu?Gly?Lys?Cys?Asn?Ile?Ala?Gly?Trp?Leu?Leu?Gly

1 5 10 15

 

 

Asn?Pro?Glu?Cys?Asp?Pro?Leu?Leu?Pro?Val?Arg?Ser?Trp?Ser?Tyr?Ile

20 25 30

 

 

Val?Glu?Thr?Pro?Asn?Ser?Glu?Asn?Gly?Ile?Cys?Tyr?Pro?Gly?Asp?Phe

35 40 45

 

 

Ile?Asp?Tyr?Glu?Glu?Leu?Arg?Glu?Gln?Leu?Ser?Ser?Val?Ser?Ser?Phe

50 55 60

 

 

Glu?Arg?Phe?Glu?Ile?Phe?Pro?Lys?Glu?Ser?Ser?Trp?Pro?Asn?His?Asn

65 70 75 80

 

 

Thr?Thr?Lys?Gly?Val?Thr?Ala?Ala?Cys?Ser?His?Ala?Gly?Lys?Ser?Ser

85 90 95

 

 

Phe?Tyr?Arg?Asn?Leu?Leu?Trp?Leu?Thr?Glu?Lys?Glu?Gly?Ser?Tyr?Pro

100 105 110

 

 

Lys?Leu?Lys?Asn?Ser?Tyr?Val?Asn?Lys?Lys?Gly?Lys?Glu?Val?Leu?Val

115 120 125

 

 

Leu?Trp?Gly?Ile?His?His?Pro?Ser?Asn?Ser?Lys?Asp?Gln?Gln?Asn?Ile

130 135 140

 

 

Tyr?Gln?Asn?Glu?Asn?Ala?Tyr?Val?Ser?Val?Val?Thr?Ser?Asn?Tyr?Asn

145 150 155 160

 

 

Arg?Arg?Phe?Thr?Pro?Glu?Ile?Ala?Glu?Arg?Pro?Lys?Val?Arg?Asp?Gln

165 170 175

 

 

Ala?Gly?Arg?Met?Asn?Tyr?Tyr?Trp?Thr?Leu?Leu?Lys?Pro?Gly?Asp?Thr

180 185 190

 

 

Ile?Ile?Phe?Glu?Ala?Asn?Gly?Asn?Leu?Ile?Ala?Pro?Arg?Tyr?Ala?Phe

195 200 205

 

 

Ala?Leu?Ser

210

 

 

<210> 4

<211> 639

<212> DNA

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

 

<400> 4

gccccattgc?atttgggtaa?atgtaacatt?gctggctgga?tcctgggaaa?tccagagtgt 60

 

gaatcactct?ccacagcaag?ctcatggtcc?tacattgtgg?aaacacctag?ttcagacaat 120

 

ggaacgtgtt?acccaggaga?tttcatcgat?tatgaggagc?taagagagca?attgagctca 180

 

gtgtcatcat?ttgaaaggtt?tgagatattc?cccaagacaa?gttcatggcc?caatcatgac 240

 

tcgaacaaag?gtgtaacggc?agcatgtcct?catgctggag?caaaaagctt?ctacaaaaat 300

 

ttaatatggc?tagttaaaaa?aggaaattca?tacccaaagc?tcagcaaatc?ctacattaat 360

 

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 (8)

1. a boar source A type influenza virus head albumen is characterized in that as (a) or protein (b):

(a) protein of forming by the amino acid shown in the SEQ ID NO.1;

(b) aminoacid sequence in (a) is through replacing, lack or adding an amino acid or several amino acid and have influenza proteins antigenic by (a) deutero-protein.

2. a property right profit requires the genetic engineering bacterium of 1 described influenza proteins, and its spy is being that described genetic engineering bacterium is for expressing protein colon bacillus BL21 shown in the SEQ ID NO.1.

3. genetic engineering bacterium according to claim 2, its spy is being that described engineering bacteria uses secreted expression carrier.

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

5. a method for preparing the described viral protein of claim 1 is characterized in that comprising the steps:

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

(2) nucleotide fragments that obtains in the step (1) is cloned into expression vector pET21a;

(3) the expression vector transformed into escherichia coli BL21 that step (2) is obtained;

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

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

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

6. method according to claim 5 is characterized in that described inductive condition is: add IPTG when thalline OD600 reaches 0.4 left and right sides to final concentration 0.1mM, induce for 37 ℃ and lead 4 hours, the centrifugal results thalline of 6000 rpm.

7. method according to claim 5 is characterized in that described renaturation part is: 1mL solubilization of inclusion bodies damping fluid (6M Guanidine Hydrochloride; 10% glycerine; 100mM NaCl; 10mM EDTA; 10mM DTT), 4 ℃ of stirring and dissolving; Preparation renaturing inclusion bodies damping fluid (100mM Tris pH8.0; 400mM L-Arginine monohydrochloride; 2mM EDTA; 5mM GSH; 0.5mM GSSG) on ice or 4 ℃ of precoolings; The about 3-5mL of inclusion body after will dissolving with the 5mL syringe is each at twice to add syringe in 8 hours at interval, made it drop by drop to drip in refolding buffer, slowly stirred 8-10 hour.

8. the described influenza virus head of claim 1 albumen is applied to the subunit vaccine preparation.

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