CN1936009A - Avian influenza virus matrix protein and nucleoprotein fusion antigen protein expression preparation - Google Patents

Abstract

The invention relates to an expression making method for Avian Influenza virus matrix protein. It supplies a confluence antigen protein compounded by two AIV antigen proteins, and could effectively detect A type Avian Influenza virus matrix protein and make the expression. It includes the following steps: constructing pPIC9K-M-Np, expressing and making M-Np protein Pichia pastoris eukaryon expression. It has the feature of high expression quantity, strong specificity, easy to purify and full space conformation.

Description

The expression preparation of avian influenza virus stromatin and nucleoprotein fusion antigen protein

Technical field:

The invention belongs to biological technical field, particularly relate to a kind of AIV fusion antigen protein and preparation.

Background technology:

(Avian Influenza AI) is a kind of poultry that caused by A type influenza virus and infection and/or the disease syndrome of wild fowl in bird flu.AIV have the height variability, based on hemagglutinin (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA) the antigenicity difference of two kinds of surface antigens can be divided into different hypotypes, has found 15 of HA hypotypes, 9 of NA hypotypes at present.According to influenza virus nucleoprotein (Nucleoprotein, NP) and stromatin (Matrix proteins, M) antigenic difference, influenza virus is divided into three serotypes, be A, B, C type (or claiming first, second, third type), can test by AGP between various, complement fixation test (CFT) etc. detected and distinguished.All AIV are A type influenza virus.

From Perroncito in 1878 reported first since bird flu, nineteen fifty-nine take place high pathogenic avian influenza take place in Italy, the whole world has at least breaks out on a large scale with popular for 23 times, all causes heavy economic losses at every turn.All there is breaking out with popular of this disease in world many countries and area at present.This disease is defined as the category-A transmissible disease by OIE (OIE).China also classifies it as a class animal epidemic.In Hong Kong bird flu incidents in 1997, obstacle direct infection people and cause people's death between H5N1 AIV breaks through kind first, broken H1, H2 are only arranged under the natural condition, routine that H3 subtype influenza virus can infected person, given AIV brand-new public health meaning thus.At the beginning of the year ends to 2,004 2003, successively broken out bird flu in countries and regions such as Korea S, Japan, Thailand, Vietnam, China's Mainland, TaiWan, Chinas, strain is the H5N1 hypotype.The infection of H5N1 has taken place again in China Qinghai in 2005, causes the extensive concern of the whole world to AI once more.

Bird flu is the viral deadly infectious disease of serious threat aviculture development.China is that the first in the world is supported fowl big country, support the fowl total amount and reach 10,000,000,000 plumages, in recent years, the generation of bird flu and popular, enormous economic loss and serious social influence have been caused in China, especially occurred avian influenza virus (AIV) direct infection people's incident in 1997 and 1999, at the beginning of the year ends to 2,004 2003, successively broken out bird flu in Southeast Asian countries and regions.The Asia had 53 people to infect avian influenza virus in 2004,41 people's death, and bird flu causes global extensive concern and even fear.The public health meaning of bird flu more causes common people's shock and concern, and its anti-system has been directly connected to the sustainable and stable development of China's aviculture and the people's health health.Thereby early stage quick diagnosis and immune antibody monitoring be the key of the anti-system of bird flu, and be significant.

At present, domestic and international research to AIV early diagnosis technology and detection technique has all obtained progress in various degree.Bird flu H5, H7, H9 hypotype multiplex real-time reverse transcriptase PCR detection kit are succeeded in developing in Shenzhen; The research and development of Taiwan Chung Hsing University detect ferment immunosorption reaction (ELISA) quick test cover group method of H5 and H7 antibody, can be applicable to the screening of an a large amount of serum corpse or other object for laboratory examination and chemical testing; Medical College of Shantou University, medical college of Hong Kong University associating influenza research centre and Xiamen University's cooperation research and development go out the world's first avian influenza virus H 5 N 1 rapid diagnosis kit test kit; Korea S develops a kind of simple diagnosis box that can make a definite diagnosis bird flu rapidly.Simultaneously, the development of avian influenza vaccine is also among extensively carrying out.The early diagnosis of this explanation AIV, the research that detects and prevent and treat method have caused worldwide concern.

To the diagnosis of bird flu, mainly contain methods such as checking chicken serum antibody, avian influenza virus isolation identification, detection of nucleic acids at present.And directly detect avian influenza virus antigen method relevant report seldom.In view of the present domestic AIV deactivation vaccine that begun to be extensive use of, detect serum antibody and diagnose this disease impossible; Virus is separated needs responsive animal of inoculation or culturing cell, has shortcomings such as length consuming time, complex operation; Immunofluorescence needs equipment such as fluorescent microscope, and there is subjectivity in result's judgement; The RT-PCR method requires to extract organizes RNA, and technical requirements is higher, is unsuitable for basic unit's laboratory operation.In addition, aforesaid method all is not easy to carry out the detection of batch samples, is difficult to satisfy the needs of current bird flu epidemic situation development.Enzyme linked immunosorbent assay (ELISA) has sensitivity, the incomparable advantage of other method such as accurate, quick, simple, and the bottleneck that this method is set up be whether to have a large amount of biologically actives, specific antigen protein examines and determine.Stromatin (M) is the maximum a kind of albumen of content in the viral protein of influenza virus, can guarantee the integrity of virus particle, regulates the activity of polysaccharase, is also playing an important role aspect the assembling of progeny virus particle.

Technology contents:

The invention provides a kind of fusion antigen protein of forming by two kinds of AIV antigen proteins, can effectively detect the expression preparation of the avian influenza virus stromatin of antibody and the nucleoprotein fusion antigen protein of very high A type avian influenza virus stromatin of pathogenicity rate and nucleoprotein.

The present invention at first makes up plasmid pPIC9K-M-Np: the pMD18-T-M plasmid is also reclaimed by EcoR I, EcoRV double digestion; The pMD18-T-Np plasmid is by EcoR I, EcoRV double digestion and recovery; Interstitial granules pMD18-T-M-Np during above recovery product promptly obtained after the T4 ligase enzyme connects; PMD18-T-M-Np is through EcoR I, Not I double digestion and recovery; The pPIC9K plasmid is by EcoR I, Not I double digestion and recovery; Above recovery product after connecting, the T4 ligase enzyme is promptly obtained purpose plasmid pPIC9K-M-Np;

M-Np albumen pichia spp eukaryotic expression and preparation then: with Bpu1102I linearizing pPIC9K-M-Np recombinant plasmid; After the linearizing its electricity is converted in the GS115 competence, in the MD flat board, cultivate, then the single bacterium colony of picking is recovered in the YPD substratum, put forward the recombinant gene group and carry out the PCR evaluation, the positive strain that filters out shakes bacterium in the BMGY substratum, go in the BMMY substratum after centrifugal to begin methanol induction, gets supernatant after product is centrifugal, add saturated ammonium sulphate albumen, centrifugal collection promptly gets the M-Np antigen protein.

The present invention is by avian influenza virus stromatin and nucleoprotein fusion antigen protein that aforesaid method obtained, and its gene order is SEQ ID NO:1.

Nucleoprotein (NP) is the major antigen of cytotoxic lymphocyte identification.Therefore, the present invention has carried out the research of the M-Np fusion antigen protein that utilizes pichia spp eukaryotic expression system expression preparation.Its expression amount height, high specificity, be easy to purifying, have the evaluation requirement that complete characteristics such as space conformation meet the ELISA quick detection kit fully.

1, invents the antigen protein that obtains, can detect AIV antibody in the body on the one hand; Can assist the development of AIV testing tool on the other hand and lay a good foundation for the development of AIV novel gene engineered vaccine.

2, related antigen protein in the invention, at A type AIV stromatin and nucleoprotein, popularity is strong, has very important practice significance.

3, related antigen protein in the invention utilizes the pichia spp eukaryotic expression system, and except that having good biological activity, but also great expression and purifying are easy to industrialization.

4, therefore the domestic so far amalgamation and expression that does not also utilize yeast expression system to carry out the AIV viral protein, originally delivers high novelty.

Description of drawings:

Fig. 1 is pPIC9K-M-Np plasmid construction figure of the present invention;

Fig. 2 is M-Np antigen protein eukaryotic expression of the present invention and preparation mode figure.

Embodiment:

The present invention at first makes up plasmid pPIC9K-M-Np: the pMD18-T-M plasmid is also reclaimed by EcoR I, EcoRV double digestion; The pMD18-T-Np plasmid is by EcoR I, EcoRV double digestion and recovery; Interstitial granules pMD18-T-M-Np during above recovery product promptly obtained after the T4 ligase enzyme connects; PMD18-T-M-Np is through EcoR I, Not I double digestion and recovery; The pPIC9K plasmid is by EcoR I, Not I double digestion and recovery; Above recovery product after connecting, the T4 ligase enzyme is promptly obtained purpose plasmid pPIC9K-M-Np;

M-Np albumen pichia spp eukaryotic expression and preparation then: with Bpu1102I linearizing pPIC9K-M-Np recombinant plasmid.After the linearizing its electricity is converted in the GS115 competence, cultivated 2～3 days in the MD flat board under 28 ℃ of conditions, then the single bacterium colony of picking is recovered in the YPD substratum, put forward the recombinant gene group and carry out the PCR evaluation, the positive strain that filters out shakes bacterium 16～18h under 28 ℃ of conditions in the BMGY substratum, go to after centrifugal in the BMMY substratum and begin methanol induction, get supernatant after product is centrifugal, add an amount of saturated ammonium sulphate albumen, make the ammonium sulfate final concentration reach 70%, centrifugal collection promptly gets the M-Np antigen protein.

The present invention is by avian influenza virus stromatin and nucleoprotein fusion antigen protein that aforesaid method obtained, and its gene order is SEQ ID NO:1.

Narrate implementation method of the present invention below:

1, method

1.1 the genetic manipulation of molecular biology routine

The extraction of the preparation of competent escherichia coli cell and conversion, plasmid and digestion with restriction enzyme, DNA

The connection of segmental recovery, linear DNA fragment, the screening of recombinant plasmid and evaluation, pcr amplification reaction, SDS-polyacrylamide gel electrophoresis, protein blot experiment are translated " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., and Science Press (1992) related Sections carries out.

1.2 the structure of recombinant expression plasmid pPIC9K-M-Np

The structure of middle interstitial granules pMD18-T-M-Np: the pMD18-T-M plasmid is by EcoR I, EcoRV double digestion and recovery; The pMD18-T-Np plasmid is by EcoR I, EcoRV double digestion and recovery.Interstitial granules pMD18-T-M-Np during above recovery product promptly obtained after the T4 ligase enzyme connects.

The structure of purpose plasmid pPIC9K-M-Np: pMD18-T-M-Np is through EcoR I, Not I double digestion and recovery; The pPIC9K plasmid is by EcoR I, Not I double digestion and recovery; Above recovery product after connecting, the T4 ligase enzyme is promptly obtained purpose plasmid pPIC9K-M-Np.

1.3 the preparation of yeast competent cell

The single bacterium colony of picking pichia pastoris phaff bacterium GS115 is to 5ml YPD liquid nutrient medium, and 30 ℃ of joltings are spent the night.Be transferred to next day in the fresh YPD substratum of 500ml, 30 ℃ of joltings are cultured to OD ₆₀₀=1.3～1.5.In centrifugal 5 minutes of 4 ℃ of 2500rpm, collect thalline.Ice-cold aseptic deionized water suspends, and washs 2 times, and the washing of the sorbyl alcohol of ice-cold aseptic 1M is once got the sorbyl alcohol suspension of precipitation with the aseptic 1M of 1/500 volume ice precooling at last again, is prepared into the yeast competent cell, promptly can be used for transforming.

1.4 the preparation of recombinant bacterial strain

With the recombinant expression plasmid pPIC9K-M-Np Bpu1102I linearizing that makes up, getting linearizing recombinant expression plasmid of 5～20 μ g and freshly prepd GS115 yeast competent cell mixes, with sample injector sample drop is added between 0.2cm electricity revolving cup two electrodes, ice bath 5min, with the ECM830 electroporation in voltage 300V, electric capacity 25 μ F, electricity transforms under the resistance 200 Ω conditions.After the electric shock, add the cold sorbyl alcohol of 1ml 1M ice bath immediately, mixing is got 200～600 μ l bacterium liquid and is coated on the MD selection flat board, hatches 2～3 days for 30 ℃, YPD extracts the yeast genes group with glass bead method after expanding bacterium, with carried genome is the template pcr amplification, selects exogenous origin gene integrator and goes into the interior positive bacteria of yeast genes group, then these bacterium is carried out methanol induction and expresses, select the high expression level bacterial strain, condition such as oxygen saturation is optimized to substratum and when expressing simultaneously.

Upstream primer: 5 '-TACTATTGCCAGCATTGCTGC-3;

Downstream primer: 5 '-GCAAATGGCATTCTGACATCC-3 '.

1.5 the methanol induction secreting, expressing of Yeast engineering bacterium strain

Inoculate single positive strain or glycerol stock (20 ℃ of preservations) in the 100mlYPD substratum, 28～30 ℃, the about 16～18h of 250rpm shaking culture, the centrifugal 10min of 5000rpm receives bacterium, and is resuspended with 100ml BMGY substratum, 28～30 ℃, the 250rpm shake-flask culture makes OD ₆₀₀Reach 2～6 (about 16～18h), centrifugal collection thalline, precipitate resuspended with the BMMY substratum of 5～10 times of volumes, 28～30 ℃, 250rpm continues shaking culture 2～6d, and after abduction delivering is initial, added methyl alcohol to 0.5～1.0% of cumulative volume every 24 hours, take out the 1ml sample every 24h and be used to test expression amount.

1.6 the SDS-PAGE of exogenous gene expression product analyzes

The engineering yeast cultivated certain hour in the BMMY substratum after, the centrifugal 10min of 5000rpm collects thalline and substratum supernatant, thalline adopts direct boiling lysis cracking tropina in Loadingbuffer, expressing protein in the substratum supernatant is packed in the dialysis tubing, elder generation is 4 ℃ of dialysis 24h in PBS, with PEG20000 carry out 10 times concentrate after, 12h again dialyses in PBS, take out enriched product and 2 times of Loading buffer balanced mix, carry out SDS-PAGE with cracked yeast tropina, the proteic expression of analysis purposes.

1.7 indirect enzyme-linked immunosorbent assay (indirect ELISA)

According to State Standard of the People's Republic of China (GB/T18936-2003) " high pathogenic avian influenza diagnostic techniques " indirect enzyme-linked immunologic adsorption test method, expressed antigen is measured.Process is as follows: remove A1, B1, C1 and D1 hole and do not add sample, stay and do blank zeroing, pPIC9K empty carrier expression product and A type AIV standard antigen are respectively as outside negative control and the positive control, all the other holes get 1: 400 the dilution by Reichl's test, every hole 100 μ l bag is carried out record by plate with the application of sample position.Outwell hole endoperidium liquid, it is inferior to give a baby a bath on the third day after its birth with PBS.Add A type AIV standard serum, Sptting plate is built the rearmounted 37 ℃ of environment of lid effect 30min down.Outwell liquid in the hole, do in that thieving paper is overhead, washing lotion is filled it up with in every hole, outwells after leaving standstill 1～2min, and empty doing repeats to wash 2 times again.Except that A1, B1, C1 and D1 hole, the anti-chicken two anti-100 μ l that other every holes add alkali phosphatase enzyme mark build the rearmounted 37 ℃ of environment of lid effect 30min down.Wash again three times with above-mentioned same method, add substrate and use liquid (adding the BCIP/NBT mixed solution in the alkaline phosphatase damping fluid), every hole 90 μ l, put room temperature lucifuge colour developing 2～3min, add and end liquid (vitriol oil 11.1ml+ distilled water 88.9ml), every hole 90 μ l make its termination reaction.Measuring each hole with microplate reader (is OD in the optical density value of 490nm wavelength ₄₉₀Value), OD 〉=0.2 are judged to be the positive, and 0.18≤OD＜0.2 needs repeated test 1 time, if still then be judged to the positive in this scope, OD＜0.18 are judged to be feminine gender.

1.8 the optimization of expression condition

Characteristics during according to the pichia spp abduction delivering are come the optimization expression condition by different dissolved oxygen (95%, 85%, 75%, 65%, 55%), incubation time (2d, 3d, 4d, 5d), methanol concentration (0.5%, 1.0%, 1.5%, 2.0%, 2.5.%), rolling bottle revolution (100,150,200,250) and pH value (3.0,4.0,5.0,6.0,7.0,8.0).

1.9 the yeast transformant genetic stability is analyzed

Respectively positive yeast transformant is carried out the thalline amplification and the abduction delivering in 10 cycles, carry out PCR and identify and detection of expression, analyze foreign gene and whether lose.

2, interpretation of result

2.1 the structure of recombinant expression plasmid pPIC9K-M-Np

Enzyme is cut the evaluation collection of illustrative plates and is seen Figure of description 1, and recombinant plasmid pPIC9K-M-Np size can occur and be the linearizing band of 11300bp behind EcoR I single endonuclease digestion as seen from the figure; Behind the EcoRV single endonuclease digestion, 10100bp and 1200bp two bands can appear; Behind EcoR I, Not I double digestion, 9300bp and 2000bp two bands can appear, prove that this plasmid construction is correct.

2.2 the PCR of yeast recon identifies

Pcr amplification the results are shown in Figure of description 2, as seen from the figure, by universal primer PCR amplification GS115/pPIC9K-M-Np transformant, amplify the band of 2.1kb size, the sizableness that just in time adds secretion signal with goal gene M-Np proves that our goal gene has been integrated in the yeast genes group.

2.3 the SDS-PAGE of expression product

To express supernatant 70% ammonium sulfate precipitation behind the transformant inoculation BMGY and BMMY substratum abduction delivering of screening, and get precipitation and be dissolved among the PBS (PH7.4) of former 1/10 volume, SDS-PAGE is carried out in sampling, and it the results are shown in Figure of description 3.From Figure of description 3 as can be seen, the GS115/pPIC9K-M-Np transformant induced product band that occurs at the 74kDa place conforming to the expectation size.

2.4 expression condition optimum result

Through series optimization, finally determine target protein optimum expression condition be greater than 80% dissolved oxygen, 1% methanol induction, to cultivate 4d, shake bottle rotating speed be 6.0 greater than 200rpm, medium pH value.High expression level amount reaches 18.6% of soluble proteins in the nutrient solution supernatant, is about 52.9mg/L.

2.5 genetic stability analysis

The Yeast engineering bacteria GS115/pPIC9K-M-Np that makes up is seeded in goes down to posterity in YPD liquid nutrient medium and the BMMY substratum after 10 times, carry out the detection of the pcr amplification and the expression product of goal gene.The result shows that Yeast engineering bacteria has good genetic stability, can amplify target gene fragment in the Yeast engineering bacteria karyomit(e) after repeatedly going down to posterity, with the expression that can detect target protein behind the methanol induction in the nutrient solution supernatant.

2.6 indirect ELISA experimental result

Detect GS115/pPIC9K-M-Np expression product measured value OD through ELISA ₄₉₀〉=0.2, decidable is positive; GS115/pPIC9K expression product measured value OD ₄₉₀＜0.18, decidable is negative.Prove prepared antigen protein tool good biological activity, can be specifically in and M antibody and Np antibody.

3, conclusion

Implement by above method, successfully obtain recombinant plasmid pPIC9K-M-Np, yeast recon GS115/pPIC9K-M-Np, and behind methanol induction, can obtain purpose antigen protein M-Np, the tool biologic activity, can be specifically in and the antibody of M and Np.In the methanol induction process, its expression condition is continued to optimize the output that can increase expression product.Find that simultaneously the yeast transformant genetic stability is stronger.In sum, the present invention successfully obtains the M-Np antigen protein of biologically active, it will all embody important value of exploiting and utilizing in the development of the development of antibody test, AIV recombinant vaccine and AIV Detection of antigen goods, its high efficiency and stability also provide good prospect for the industrialization preparation.

Sequence table

＜110〉Jin Ningyi

＜120〉expression of avian influenza virus stromatin and nucleoprotein fusion antigen protein preparation

<160>2

<170>PatentIn?version?3.2

<210>1

<211>1974

<212>DNA

<213>Avian

<221>CDS

<222>(1)..(1974)

<400>1

atg?gaa?ttc?gag?ctc?ggt?acc?cgg?gga?tcc?tct?aga?gat?tgc?gga?tcc 48

att?tgg?cgc?caa?gcg?aac?aat?gga?gaa?gat?gca?act?gct?ggt?ctc?act 96

cat?ctg?atg?atc?tgg?cat?tcc?aat?cta?aat?gat?gcc?aca?tac?cag?agg 144

aca?aga?gct?ctc?gtg?cgt?act?ggg?atg?gac?ccc?aga?atg?tgt?tct?cta 192

atg?caa?gga?tca?act?ctc?ccg?agg?aga?tct?gga?gct?gca?ggt?gca?gca 240

gta?aag?gga?gtc?gga?acg?atg?gtg?atg?gag?cta?att?cgg?atg?ata?aag 288

cga?ggg?att?aat?gat?cgg?aat?ttc?tgg?aga?ggt?gaa?aat?ggg?cga?aga 336

acg?agg?att?gca?tat?gag?aga?atg?tgc?aac?atc?ctc?aaa?ggg?aaa?ttc 384

caa?aca?gca?gca?caa?aga?gca?atg?atg?gac?cag?gta?cgg?gaa?agc?agg 432

aat?cct?ggg?aat?gcc?gaa?att?gaa?gat?ctc?atc?ttt?ctg?gca?cgg?tct 480

gca?ctc?atc?ctg?aga?gga?tca?gtg?gct?cac?aag?tcc?tgc?ttg?cct?gct 528

tgt?gtg?tac?ggg?ctt?gct?gta?gcc?agt?gga?tat?gac?ttt?gag?aga?gaa 576

ggg?tat?tct?ctg?gtc?ggg?att?gat?ccc?ttt?cgt?ctg?ctg?caa?aac?agc 624

caa?gtc?ttc?agt?cta?att?aga?cca?aat?gag?aac?cca?gca?cat?aaa?agt 672

caa?ctg?gta?tgg?atg?gca?tgc?cat?tct?gca?gca?ttt?gaa?gat?ctg?agg 720

gtc?tca?agt?ttc?atc?aga?ggg?aca?aga?gta?gtt?cca?agg?gga?caa?cta 768

tct?act?aga?gga?gtt?caa?att?gct?tca?aat?gag?aac?atg?gat?aca?atg 816

gac?tcc?aac?act?cta?gaa?ctg?aga?agc?aga?tat?tgg?gct?ata?aga?acc 864

agg?agt?gga?gga?aac?acc?aat?cag?cag?aga?gca?tct?gca?gga?caa?atc 912

agt?gta?cag?cct?act?ttt?tcg?gta?cag?aga?aat?ctt?ccc?ttc?gag?aga 960

gcg?acc?att?atg?gcg?gca?ttt?aca?ggg?aat?aca?gag?ggc?aga?aca?tct 1008

gac?atg?agg?act?gaa?ata?ata?aga?atg?atg?gaa?agc?gcc?aga?cca?gaa 1056

gat?gtg?tct?ttc?cag?ggg?cgg?gga?gtc?ttc?gag?ctc?tcg?gac?gaa?aag 1104

gca?acg?aac?ccg?atc?ttg?cct?tcc?ttt?gac?atg?agt?aat?gaa?gga?tct 1152

tac?ttc?ttc?gga?gac?aat?gca?gag?gag?tac?gac?aat?ggt?gga?ggc?gga 1200

agc?gga?tat?cga?aag?atg?agt?ctt?cta?acc?gag?gtc?gaa?acg?tac?gtt 1248

ctc?tct?atc?aac?ccg?tca?ggc?ccc?ctc?aaa?gcc?gag?atc?gcg?cag?aga 1296

ctt?gaa?gat?gtc?tct?gca?ggg?aag?aac?aca?gat?cct?gag?gct?ctc?atg 1344

gaa?tgg?cta?aag?aca?aga?cca?atc?ctg?tca?cct?ctg?act?aag?ggg?att 1392

tta?ggg?ttt?gtg?ttt?acg?ctc?acc?gtg?ccc?agt?gag?cga?gga?ctg?cag 1440

cgt?aga?cga?ttt?gtc?caa?aat?gcc?cta?aat?ggg?aat?gga?gac?cca?aac 1488

aac?atg?gac?agg?gca?gtc?aaa?cta?tac?aag?aag?ctt?aag?agg?gaa?atg 1536

acg?ttc?cat?gga?gca?aag?gaa?gtc?gca?ctc?agt?tac?tca?act?ggt?gcg 1584

ctt?gcc?agt?tgc?atg?ggt?ctc?ata?tac?aac?cga?atg?gga?acg?gta?acc 1632

aca?gaa?gtg?gct?ctt?ggc?cta?gta?tgt?gcc?act?tgt?gag?cag?att?gct 1680

gat?tca?cac?cat?agg?tct?cac?aga?cag?atg?gcg?act?acc?acc?aac?cca 1728

cta?atc?agg?cat?gag?aac?aga?atg?gta?cta?gcc?agc?act?aca?gct?aag 1776

gcc?atg?gag?caa?atg?gct?gga?tcg?agt?gag?cag?gca?gcg?gaa?gcc?atg 1824

gag?gtt?gca?agt?cag?gct?agg?cag?atg?gtg?cag?gcg?atg?agg?aca?att 1872

ggg?act?caa?cct?agc?tcc?agt?gca?ggt?ctg?aaa?gat?gat?ctt?att?gaa 1920

aat?ttg?cag?gca?tac?cag?aaa?cgg?atg?ggg?gtg?cag?atg?cag?cga?ttc 1968

aag?tga 1974

Claims (2)

1, the expression preparation method of a kind of avian influenza virus stromatin and nucleoprotein fusion antigen protein is characterized in that:

At first make up plasmid pPIC9K-M-Np: the pMD18-T-M plasmid is also reclaimed by EcoR I, EcoRV double digestion; The pMD18-T-Np plasmid is by EcoR I, EcoRV double digestion and recovery; Interstitial granules pMD18-T-M-Np during above recovery product promptly obtained after the T4 ligase enzyme connects; PMD18-T-M-Np is through EcoR I, Not I double digestion and recovery; The pPIC9K plasmid is by EcoR I, Not I double digestion and recovery; Above recovery product after connecting, the T4 ligase enzyme is promptly obtained purpose plasmid pPIC9K-M-Np;

M-Np albumen pichia spp eukaryotic expression and preparation then: with Bpu1102I linearizing pPIC9K-M-Np recombinant plasmid; After the linearizing its electricity is converted in the GS115 competence, in the MD flat board, cultivate, then the single bacterium colony of picking is recovered in the YPD substratum, put forward the recombinant gene group and carry out the PCR evaluation, the positive strain that filters out shakes bacterium in the BMGY substratum, go in the BMMY substratum after centrifugal to begin methanol induction, gets supernatant after product is centrifugal, add saturated ammonium sulphate albumen, centrifugal collection promptly gets the M-Np antigen protein.

2, the avian influenza virus stromatin and the nucleoprotein fusion antigen protein that are obtained of the described method of claim 1, its gene order is SEQ ID NO:1.

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