CN109180820A

CN109180820A - Fusion protein of equine influenza virus H3N8 hypotype and preparation method thereof, application and vaccine

Info

Publication number: CN109180820A
Application number: CN201811095817.6A
Authority: CN
Inventors: 贺笋; 任立松; 李延涛; 陈泽良; 任郭子君
Original assignee: Tiankang Biological Ltd By Share Ltd
Current assignee: Tiankang biopharmaceutical Co.,Ltd.
Priority date: 2018-09-19
Filing date: 2018-09-19
Publication date: 2019-01-11
Anticipated expiration: 2038-09-19
Also published as: CN109180820B

Abstract

The present invention provides fusion protein of a kind of equine influenza virus H3N8 hypotype and preparation method thereof, application and vaccines, are related to field of biotechnology.The fusion protein connects equine influenza virus H3N8 hypotype HA gene and NA gene order, and the fusion protein of expression has antigenic good, the high advantage of expression quantity.The fusion protein can be applied to prepare vaccine, antibody and the kit for detecting equine influenza virus.Vaccine comprising the fusion protein has preferable immunogenicity, can produce the antibody titer compared with high titre after immune animal, animal is made to obtain preferable protecting effect.

Description

Fusion protein of equine influenza virus H3N8 hypotype and preparation method thereof, application and vaccine

Technical field

The present invention relates to field of biotechnology, a kind of fusion protein more particularly, to equine influenza virus H3N8 hypotype and its Preparation method, application and vaccine.

Background technique

Epizootic catarrhalfever (Equine Influenza, EI) is by equine influenza virus (Equine Influenza Virus, EIV) caused by a kind of acute infectious disease, it has the characteristics that acute, high degree in contact is communicable.H3N8 hypotype is main Epidemic strain is one of horse respiratory infectious disease.

Equine influenza virus belongs to orthomyxoviridae family (Orthomyxoviridae), influenza A category.It is a kind of segmented The sub-thread strand RNA to differ in size have the virus of cyst membrane, genome is about 13.6kb.There are many viral particle morphology, most of Shape spherical in shape, diameter are about 80-120nm, and Filamentous physical efficiency is up to a few micrometers.Vims particle structures are broadly divided into three layers, outermost The cyst membrane of layer is made of bilayer lipid matter, and the different important albumen protrusion of two kinds of forms is inlayed in cyst membrane, a kind of picture Mushroom sample is neuraminidase (Neuraminidase, NA), another as rodlike structure is hemagglutinin (Hemagglutinin, HA), the ratio of NA and HA is about 5:1-4:1 on cyst membrane.Middle layer is that stromatin (M1) is formed Globular protein shell, there are also a small amount of ionophorous proteins (Ionchannelprotein) in double lipid membrane, referred to as M2 egg It is white.

Epizootic catarrhalfever can occur throughout the year, and propagation is extremely rapid, be in outbreak of epidemic more.Contaminate equine influenza virus Afterwards, spread speed is very fast, and with fever, mental depression, conjunctiva flush, cough and stream slurry property nose liquid, expiratory dyspnea, appetite are big Subtract, and mostly causes aggravation or death, pregnant mare that miscarriage, horse racing forfeiture sports energy may occur because of secondary infection Power.The major source of infection of influenza is disease horse with malicious horse, and the horse of each age and each kind has a possibility that generation, spring and summer Autumn and winter can all occur, and illness rate can achieve 100%, cause larger economic loss to horse keeping industry.The country there is no prevention horse at present The subunit vaccine of influenza.

In view of this, the present invention is specifically proposed.

Summary of the invention

The first object of the present invention is to provide a kind of fusion protein of equine influenza virus H3N8 hypotype, fusion protein general The HA albumen and NA albumen of equine influenza virus H3N8 hypotype carry out expressing in series.

The second object of the present invention is to provide a kind of preparation method of above-mentioned fusion protein, which can give expression to Above-mentioned fusion protein.

The third object of the present invention is the albumen for providing a kind of above-mentioned fusion protein or above-mentioned preparation method is prepared Application, can be applied to prepare many aspects such as vaccine, antibody and viral diagnosis.

The fourth object of the present invention is to provide a kind of vaccine comprising above-mentioned fusion protein, and the vaccine immunogenicity is good, Vaccine effectiveness is high.

In order to solve the above technical problems, spy of the present invention adopts the following technical scheme that

A kind of fusion protein of equine influenza virus H3N8 hypotype, including HA section and NA section；The HA section mainly by The expression of nucleotide sequence shown in SEQ ID NO.1, the NA section mainly nucleotides sequence list as shown in SEQ ID NO.2 It reaches.

Preferably, HA section and NA section put in order as HA-NA, are connected by Linker；

Preferably, the Linker has the nucleotide sequence as shown in SEQ ID NO.3.

Preferably, the fusion protein has the amino acid sequence as shown in SEQ ID NO.4.

The present invention also provides a kind of preparation method of above-mentioned fusion protein, the preparation method is by by the fusion protein Gene express in host；

Preferably, the gene of the fusion protein is expressed using mammalian expression systems；

Preferably, using the gene of fusion protein described in expressing cho cell system expression.

Preferably, the expression vector of the gene comprising expressing the fusion protein is provided, the expression vector is imported into CHO In cell, pressurization screening then is carried out to Chinese hamster ovary celI, then the Chinese hamster ovary celI domestication that pressurization is filtered out is at the cell for the culture that suspends Strain makes the cell strain of the culture that suspends express the fusion protein.

Preferably, the Chinese hamster ovary celI strain of the fusion protein is expressed using glutamine synthelase screening amplification system screening；

Preferably, expression vector used in the glutamine synthelase screening amplification system is pcDNA3, pEE6.4 Or pEE12.4.

Preferably, the expression fusion protein for the culture that suspends is obtained using method passage at least 6 generations gradually tamed Chinese hamster ovary celI strain.

The present invention also provides a kind of applications for the albumen that above-mentioned fusion protein or above-mentioned preparation method are prepared, including At least one of (a)-(d) as follows:

(a) equine influenza virus vaccine is prepared；

(b) antibody of equine influenza virus is prepared；

(c) kit of preparation detection equine influenza virus antibody；

(d) equine influenza virus diagnostic antigen is prepared.

The vaccine for the horse bird flu H3N8 hypotype comprising above-mentioned fusion protein that the present invention also provides a kind of.

Preferably, the concentration of fusion protein described in the vaccine be 20-100 μ g/ml, preferably 30-80 μ g/ml, it is more excellent Select 50 μ g/ml；

Preferably, the vaccine further includes auxiliary material, and the auxiliary material includes one of vaccine adjuvant, stabilizer and antibiotic Or it is a variety of；

Preferably, the vaccine adjuvant includes aluminium hydroxide gel, Freund's complete adjuvant, incomplete Freund's adjuvant, white oil assistant Agent, MF59 adjuvant or ISA201VG are, it is preferable to use ISA201VG.

Compared with prior art, the invention has the following beneficial effects:

The fusion protein of equine influenza virus H3N8 hypotype provided by the invention, by equine influenza virus H3N8 hypotype HA gene and NA gene order is connected, and for the present invention by analyzing HA gene and NA gene, it is antigenic good to have chosen, easily high expression Region connect, obtained fusion protein has antigenic good, the high advantage of expression quantity.

The preparation method of above-mentioned fusion protein provided by the invention, by by the gene of above-mentioned fusion protein table in host It reaches.

The application range for the albumen that above-mentioned fusion protein provided by the invention and above-mentioned preparation method are prepared is very wide It is general, it can be to prepare the antibody of equine influenza virus vaccine and equine influenza virus.The antibody of equine influenza virus and above-mentioned fusion egg It is white to can be applied to prepare a variety of detection reagents and kit, with for detecting equine influenza virus antibody, such as using containing horse The ELISA kit of the antibody of influenza virus is exempted to detect equine influenza virus, or using the colloidal gold containing the fusion protein Epidemic disease chromatographic test paper detects the equine influenza virus antibody content in test serum sample.

Vaccine provided by the invention comprising above-mentioned fusion protein has preferable immunogenicity, and being immunized after animal can be with The antibody titer compared with high titre is generated, animal is made to obtain preferable protecting effect.

Detailed description of the invention

It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor It puts, is also possible to obtain other drawings based on these drawings.

Fig. 1 is the PEE6.4-HA-NA plasmid map that the embodiment of the present invention 2 provides；

Fig. 2 is the digestion qualification result that the embodiment of the present invention 2 provides；

Fig. 3 is the Werstern-Blotting detection that the cell that the embodiment of the present invention 6 provides expresses albumen；

Fig. 4 is BALB/c mouse antibody after the immune equine influenza virus HA-NA subunit vaccine that the embodiment of the present invention 8 provides Level variation.

Specific embodiment

Technical solution of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention. The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same or instrument are not Production firm person is indicated, is the conventional products that can be obtained by commercially available purchase.

The present invention provides a kind of fusion protein of equine influenza virus H3N8 hypotype, which is that equine influenza virus melts Hop protein, the fusion protein include HA section and NA section；The main nucleotides sequence as shown in SEQ ID NO.1 of the HA section List reaches, and the NA section mainly express by the nucleotide sequence as shown in SEQ ID NO.2.

Hemagglutinin (hemagglutinin, HA) is the major surface antigen of equine influenza virus, is responsible for virion, host The connection of cell, HA gene nucleotide length are 1742-1778 base, only one encoder block, coding is containing 562-566 ammonia The protein peptides of base acid, size 70KD, for carboxyl in cyst membrane, aminoterminal is typical I type glycoprotein outside cyst membrane.

Neuraminidase (Neuraminidase, NA) is another major surface antigen of equine influenza virus, and ingredient is whole Body membrane glycoprotein contains an open reading frame, there is 1443-1492 nucleotide, encodes about 459 amino acid, there is 3-5 glycosyl Change site.The catalytic center of NA enzymatic activity is located at apical head, and each NA fibre is prominent, and there are four antigen sites, and each site is contained again Multiple antigenic determinants.

Fusion protein provided by the invention connects equine influenza virus H3N8 hypotype HA gene and NA gene order, The present invention has chosen antigenic height, and be easier to highly expressed region and gone here and there by analyzing HA gene and NA gene Connection, obtained fusion protein have antigenic good, the high advantage of expression quantity.

In some optionally embodiments, putting in order as HA-NA for HA section and NA section is connected by Linker It connects, fusion protein can be separated from each other by Linker, not influence the formation of the respective higher structure of HA and NA albumen, and the present invention is preferred Use the Linker with the nucleotide sequence as shown in SEQ ID NO.3.In one preferred embodiment, the fusion Albumen with the amino acid sequence as shown in SEQ ID NO.4 and has the nucleotide sequence as shown in SEQ ID NO.5.

The present invention also provides a kind of preparation method of above-mentioned fusion protein, the preparation method is by by above-mentioned fusion protein Gene express in host, such as can be but be not limited in escherichia expression system, yeast expression system, elder brother Worm expression system, plant expression system or mammalian expression systems, since the albumen of mammalian cell expression passes through After translation process, structure and biological characteristics are closer to native protein, therefore present invention preferably uses mammal expression The gene of fusion protein described in system expression, more preferably using the gene of fusion protein described in expressing cho cell system expression. Chinese hamster ovary celI, that is, Chinese hamster ovary cell (Chinese hamster ovary), expressing cho cell system has the advantages that

(1) there is folding and rhetorical function after accurately translating, the albumen of expression is in molecular structure, physicochemical property and biology Function aspect is learned closest to native protein molecule.

(2) have the function of product exocytosis, isolated and purified convenient for downstream product.

(3) with the efficient amplification and ability to express of recombination.

(4) there is adherent growth characteristic, tolerance shearing force and osmotic pressure ability with higher.It can also carry out suspension training It supports, expression is higher.

(5) CHO belongs to fibroblast, seldom secretes the endogenous protein of itself, conducive to the separation of foreign protein.

In some preferred embodiments, the present invention carries out pressurization screening to CHO expression system, is melted with obtaining high expression The Chinese hamster ovary celI strain of hop protein.It is alternatively possible to select glutamine synthetase gene screening amplification system or dihydrofoilic acid also Nitroreductase gene screening system carries out pressurization screening, both systems can be used alone, and can also be used in combination, the present invention is to this With no restrictions.

Selected marker and gene magnification expressing cho cell carrier there are two main classes selected marker.One kind is non-amplification gene, It does not influence the copy number of target gene, for constructing transient expression vector.It is another kind of to have the function of gene magnification, Claim coamplification gene, such as dihydrofolate reductase gene (dihydrofolatereductase, dhfr), glutamine synthelase Gene (glutaminesynthetase, GS).It is available to select after carrying the expression plasmid transfection CHO cell of GS gene The cell clone of culture basal growth is selected, GS can be inhibited by methionine Asia maple (MSX), thin in the resistance that only a few survives In born of the same parents, GS gene is expanded, and as a result will lead to the coamplification of the foreign gene together with GS gene tandem, and copy number can Increase several hundred to thousands of times, to make target gene high level expression, to offset the depression effect of MSX.

When screening amplification system using glutamine synthetase gene, first the gene cloning for expressing the fusion protein is arrived Recombinant vector is obtained on expression vector with GS selection markers, then imports recombinant vector in Chinese hamster ovary celI, then by making It is pressurizeed with the culture medium culture Chinese hamster ovary celI containing MSX to Chinese hamster ovary celI, to filter out high expression target gene.Some excellent In the embodiment of choosing, the expression vector with GS selection markers uses pcDNA3, pEE6.4 or pEE12.4.

Its culture of the cell of suspension growth and passage are all very easy.Redisperse is not needed when passage, is only needed dilute in proportion It can continue to cultivate after releasing.This method cell Proliferation is fast, yield is high, incubation is simple, is the reason of large-scale culture zooblast Think mode.Both the characteristic of suspension culture can also be carried out, therefore the present invention is preferred since Chinese hamster ovary celI has with adherent growth The Chinese hamster ovary celI for expressing the fusion protein is tamed, can suspend culture.And it is preferred that using the side gradually tamed Method passes on the cell strain for obtaining the expression fusion protein for the culture that suspends at least 6 generations, so that suspension cell strain cell expression quantity Height, and pass on and stablize.

In one preferred embodiment, the preparation method refers to following scheme:

First by after the equine influenza HA-NA gene magnification of optimization by digestion be connected to carrier for expression of eukaryon pcDNA3, PEE6.4 or pEE12.4 obtains the positive plasmid containing HA-NA gene by identification.Then it by expanding culture, extracts positive Plasmid is transfected to Chinese hamster ovary celI.

Pressurization screening, transfection at least start to pressurize afterwards for 24 hours, pressurize 7 days；Be forced into negative control cell dead 90% with On, start monoclonal screening.

Cell strain domestication is cultivated at suspending, every counting cell and vigor for 24 hours, when first generation Cell viability reaches 94% left side When right, second generation culture is carried out；The second generation, the third generation to the 6th are commissioned to train support after obtained Cell viability reach 95%, 7 weeks after it is thin Born of the same parents bred for 3 generations after being inoculated with 3 days, and density reaches Cell viability and reaches 1 × 10⁶A/ml, it is believed that adapt to the culture that suspends.

Cell culture fluid, centrifuging and taking supernatant are collected, then filter membrane purifies, measure protein concentration, concentration using BCA method It about can achieve between 0.7-1.0g/L, purity detected using HPLC method, purity can achieve 95% or more.

The present invention also provides a kind of applications for the albumen that above-mentioned fusion protein and above-mentioned preparation method are prepared, including At least one of (a)-(d) as follows: equine influenza virus vaccine (a) is prepared；(b) antibody of equine influenza virus is prepared；(c) it prepares Detect the kit of equine influenza virus antibody；(d) equine influenza virus diagnostic antigen is prepared.

It, can be with since equine influenza virus H3N8 hypotype fusion protein provided by the invention has preferable immunogenicity To prepare equine influenza virus vaccine, HA-NA subunit vaccine is preferably prepared, can produce the drop of higher antibody after immune animal Degree.The antibody of the equine influenza virus prepared using the fusion protein and the fusion protein can be applied to prepare a variety of detection examinations Agent and kit, for detecting equine influenza virus antibody, such as the ELISA kit using the antibody containing equine influenza virus To detect equine influenza virus, or using in the colloidal gold immune chromatography test detection test serum sample containing the fusion protein Equine influenza virus antibody content.

The vaccine for the horse bird flu H3N8 hypotype comprising above-mentioned fusion protein that the present invention also provides a kind of, the vaccine have Preferable immunogenicity can produce the antibody titer compared with high titre after immune animal, animal made to obtain preferable protecting effect.

In some preferred embodiments, the concentration of fusion protein described in vaccine is 20-100 μ g/ml, preferably 30- 80 μ g/ml, more preferable 50 μ g/ml.Preferably, the vaccine further includes auxiliary material, for example, can be but be not limited to vaccine adjuvant, Stabilizer or antibiotic.Preferably include vaccine adjuvant, the vaccine adjuvant for example can be but be not limited to aluminium hydroxide gel, not Family name's Freund's complete adjuvant, incomplete Freund's adjuvant, white-oil adjuvant, MF59 adjuvant or ISA201VG are, it is preferable to use ISA201VG.

Beneficial effects of the present invention are further illustrated below with reference to preferred embodiment, wherein reagent of the present invention and medicine source As follows: Chinese hamster ovary cell (CHO) is purchased from U.S. ATCC company；Cell culture medium and serum are purchased from U.S. gibcom public affairs Department；Carrier for expression of eukaryon PEE6.4 is purchased from U.S. ThermoFisher company；Lipofectamine LTX is purchased from Beijing Suo Laibao Science and Technology Ltd.；Aminomethyl petrin (mnethotrexate MTX) is purchased from Sigma company；Methionine sulfoxide imonium (L- Methioninesulfoximine MSX) it is purchased from Sigma company；BCA quantification of protein kit is purchased from the U.S. ThermoFisher company；For ISA201VG purchased from match BIC Corp of France, other reagents, kit and consumptive material are conventional commercial Product.

It should be noted that the reaction system and reaction condition in embodiment is only a kind of citing.Each step in embodiment In reaction system and reaction condition can be adjusted within the acceptable range, to optimize reaction condition, the present invention couple This is with no restrictions.

The design of embodiment 1:HA-NA gene and synthesis

The design of HA-NA gene and synthesis: after HA gene (GenBank accession number KX815377.1) is optimized, design length 810bp has the sequence as shown in SEQ ID NO.1；Design length after NA gene (GenBank accession number MG132049.1) is optimized 900bp is spent, there is the sequence as shown in SEQ ID NO.2.It is connected between HA gene and NA gene using linker sequence, linker sequence Column have the sequence as shown in SEQ ID NO.3.HA-NA full length gene 1740bp has the amino acid sequence as shown in SEQ ID NO.4 Column and the nucleotide sequence as shown in SEQ ID NO.5.HA-NA gene chemical synthesis is completed by Shanghai Sheng Gong biotech firm.

Embodiment 2:PEE6.4-HA-NA construction of recombinant plasmid

2.1 addition restriction enzyme sites: the upstream and downstream by PCR amplification in HA-NA gene order adds digestion position respectively Point: Hind III, SmaI, PCR amplification upstream primer is as shown in SEQ ID NO.6, PCR amplification downstream primer such as SEQ ID NO.7 It is shown.PEE6.4-HA-NA plasmid map is as shown in Figure 1.

2.2HA-NA gene and carrier double enzyme digestion reaction

2.2.1 50 μ L reaction systems are constructed, according to following table by each component 37 DEG C of water-bath 2h after mixing.

2.2.2 target DNA fragment recycles: DNA gel QIAquick Gel Extraction Kit (being purchased from Beijing Lai Bao Science and Technology Ltd.) is used, Digestion target fragment is recycled, steps are as follows:

(1) after agarose gel electrophoresis, target DNA band is carefully cut from Ago-Gel with blade, is put into In 1.5mL EP pipe, weight is weighed.

(2) 3 times of volume sol solutions are added into EP pipe, during which 50-55 DEG C of water-bath 10min is carefully turned over centrifuge tube, it is ensured that Blob of viscose sufficiently dissolves.

(3) previous step acquired solution is added in adsorption column (adsorption column is put into collecting pipe), 12000rpm is centrifuged 30- 60s outwells the waste liquid in collecting pipe, adsorption column is reentered into collecting pipe.

(4) 600 μ L rinsing liquids are added into adsorption column, 12000rpm is centrifuged 1min, abandons waste liquid, adsorption column is put into collection Guan Zhong.

(5) 600 μ L rinsing liquids are added into adsorption column, 12000rpm is centrifuged 1min, abandons waste liquid, adsorption column is put into collection Guan Zhong.

(6) 12000rpm is centrifuged 2min, as far as possible removing rinsing liquid.Adsorption column opening is placed in room temperature or 50 DEG C of incubators are placed 2min。

(7) adsorption column is put into 1.5mL EP pipe, is preheated in right amount through 65 DEG C of water-baths to the hanging dropwise addition in adsorbed film center Eluent, is placed at room temperature for 2min, and 12000rpm is centrifuged 1min.

2.3HA-NA gene and carrier connection reaction, construct 10 μ L reaction systems, then mix coupled reaction system equal It is even to be placed on 10-16h in 16 DEG C of cold baths, after be put into 65 DEG C of water-bath 15min, last 4 DEG C of preservations.

10×T4buffer	1μL
		DNA fragmentation	6μL
Carrier	2μL
		T4 ligase	1μL

2.4 conversion reaction

10 μ L connection reaction solutions are added in 100 μ L DH5 α competent cells, ice bath 30min after mixing, 42 DEG C of water-baths 100s, then ice bath 2min.EP pipe is then taken out, 600 μ L LB culture solutions are added, is placed in 37 DEG C of constant-temperature tables, 240rpm cultivates 1h EP pipe is taken out afterwards, and room temperature is centrifuged 8000rpm, 2min, absorbs 500 μ l supernatants, and resuspension blows even thallus, the bacterium solution of resuspension is dripped to It converts on plate, is uniformly spread out bacterium solution with bacteria stick is applied.Then conversion plate is placed in constant incubator, 37 DEG C of culture 1h Afterwards, conversion plate inversion is subjected to culture 15h, observes conversion results after culture.

2.5 plasmid extractions and double digestion are identified

2.5.1 plasmid extracts, and using U.S.'s OMEGA plasmid extraction kit, extracting method is according to kit operation instruction Book extracts.

(1) single bacterium colony is into the LB liquid medium of the 5ml resistance of benzyl containing ammonia in picking reformer plate, and 37 DEG C, 220rpm training Support 8h.

(2) it takes bacterium solution 1.5ml into EP pipe, is centrifuged 10000rpm1min under room temperature, abandon supernatant, 250 μ are then added L solution I, oscillation mix；250 μ L solution IIs are added, carefully reverse EP pipe 4-6 times, are stored at room temperature 2min, until clarification, then 350 μ L solution IIIs are added, carefully reverse centrifuge tube 4-6 times, until there is white flock precipitate, the centrifugation of 10000rpm room temperature 10min。

(3) supernatant solution is carefully drawn, and moves to adsorption column center, 10000rpm room temperature is centrifuged 1min, outwells collecting pipe 500 μ L Buffer HB, 10000rpm are added after middle liquid and are centrifuged 1min, filtrate are abandoned, then plus such as 700 μ L Wash Buffer, 10000rpm are centrifuged 1min, abandon filtrate；It is repeated 1 times.

(4) room temperature is centrifuged suction attached column, and adsorption column is put into clean 1.5ml EP pipe, adds 30 by 10000rpm, 2min μ L deionized water is stored at room temperature 5min, 10000rpm, 2min on filter membrane.Save DNA solution in pipe.

2.5.2 double digestion is identified, constructs 20 μ L reaction systems:

10×buffer	2μL
		DNA sample	1μg
HindⅢ	1μL
		SmaI	1μL

Mend ddH₂It is uniformly mixed after 0 to 20 μ L, detected through gel electrophoresis is carried out after 37 DEG C of water-bath 2h, and DNA fragmentation will be inserted into Company is sent to be sequenced, double digestion qualification result is as shown in Figure 2.

2.6 go endotoxin plasmid to mention greatly, and using a large amount of extracts kits of endotoxin plasmid are gone, (Beijing Suo Laibao science and technology is limited Company)

(1) by sequencing, correctly clone is seeded in the culture medium of the 100ml resistance of benzyl containing ammonia, 240rpm, 37 DEG C of constant temperature trainings After supporting 15h, take 50ml bacterial cultures into 50ml centrifuge tube, 11000rpm is centrifuged 1min, absorbs supernatant.

(2) add 4ml solution P1, oscillator suspended bacterial cell precipitation, then plus 4ml solution P2, mild overturn 6-8 times make Thallus sufficiently cracks, and finally adds 4ml solution P3 again, and temperature is 6-8 times reverse immediately, mixes well, until there is white flock precipitate, After 11000rpm is centrifuged 10min, supernatant is moved on in another clean centrifuge tube.

(3) the Endotoxin removal agent of clear 1/5 volume being pre-chilled on ice is added, oscillation mixes, and ice bath 2min to solution becomes clear Bright, then 37 DEG C of water-bath 5min, vibrate frequently.

(4) 11000rpm room temperature is centrifuged 5min, and solution is divided into two-phase, and upper strata aqueous phase contains Plasmid DNA, and lower layer's oil mutually contains endogenous toxic material Upper strata aqueous phase containing Plasmid DNA is transferred to new pipe, abandons lower layer's oil phase by element；In triplicate.

(5) the combination liquid of 12ml is added, is added in adsorption column after mixing well, is placed at room temperature for 2min, 11000rpm centrifugation 1min outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.

(6) 8ml rinsing liquid is then added, 11000rpm is centrifuged 1min, and waste liquid is abandoned, adsorption column is put into collecting pipe, then 6ml rinsing liquid is added, 11000rpm is centrifuged 1min, abandons waste liquid, adsorption column is put into collecting pipe.Then 11000rpm is centrifuged Adsorption column opening is placed in room temperature or 50 DEG C of incubators places 4-5min by 3min.

(7) finally adsorption column is put into a clean centrifuge tube, is washed to adsorbed film drop 2ml through what 65 DEG C of water-baths preheated De- liquid, is placed at room temperature for 5min, and 11000rpm is centrifuged 2min, -20 DEG C of preservations.

Embodiment 3:PEE6.4-HA-NA Transfected Recombinant Plasmid CHO-K1 cell

(1) cell is taken out, culture medium is discarded supernatant, is washed once with the 8ml PBS of pre-temperature, discard PBS, then each culture 2ml 0.25%trypsin-EDTA is added in ware, and room temperature digests 2min, and microscopic observation cell rounding is in individual cells.4ml is added DMEM/F12 (containing 10% serum, 1% ammonia benzyl-streptomysin is dual anti-) terminates digestion reaction, shifts after being dispelled cell with pipettor Into 15ml centrifuge tube, 200rpm is centrifuged 5min.

(2) DMEM/F12 (contain 10% serum, 1% ammonia benzyl-streptomysin dual anti-) suspension cell again, after counting, dilution is thin Born of the same parents are to 2 × 10⁵A/ml, the cell for taking 2ml to mix are added to six hole culture dishes, set 37 DEG C, 5%CO₂It is incubated in cell incubator Overnight.

(3) it observes cell state: starting to transfect when cell degree of crossing reaches 80%-90%, change culture medium before transfection DMEM/F12 (serum-free is without double antibody), the hole 2mL/.

(4) plasmid is diluted with OPTI-MEM, 2.5 μ g plasmids is added in 125 μ l OPTI-MEM, 2.5 μ l are then added Plus is stored at room temperature 5min after mixing.

(5) it dilutes in Lipofectamine LTX:125 μ l OPTI-MEM and 9 μ l Lipofectamine LTX is added, so After 2.5 μ l plus are added, mix gently, be stored at room temperature 5min.

(6) plasmid will be diluted and dilution Lipofectamine LTX is mixed and mixed, 5min is placed at room temperature for, then adds dropwise Enter and is uniformly distributed in six hole culture dishes.

(7) six hole culture dishes are placed in 37 DEG C, 5%CO₂Liquid is changed after cultivating 4-6h in cell incubator: discarding supernatant culture Base is added 2ml DMEM/F12 (dual anti-containing 10% serum, 1% ammonia benzyl-streptomysin), six orifice plates is placed in 37 DEG C, 5%CO₂Cell It is cultivated in incubator.

Embodiment 4: monoclonal cell strain screening

(1) start to pressurize for 24 hours after transfecting: taking out six hole culture dishes from 37 DEG C of incubators, discard supernatant, 2ml is added DMEM/F12 (+25 μM of MSX containing 10% serum), pressurize 7d, and during which under the microscope, dead cell changes liquid more.

(2) when pressurization screening to negative control cell death at least 90% or more, start monoclonal screening；

(3) six hole culture dishes are taken out, culture medium is abandoned, is washed once with PBS, 300 μ l 0.25%trypsin-EDTA are added, Room temperature digests 2min, adds 2ml DMEM/F12 (+25 μM of MSX containing 10% serum) to terminate digestion reaction, is blown cell with pipettor It dissipates, then cell is transferred in 15ml centrifuge tube, 200rpm is centrifuged 5min.

(4) again then cell is diluted to 5 to DMEM/F12 (+25 μM of MSX containing 10% serum) by suspension cell, counting A/ml takes 200 μ L to be added in 96 orifice plates, is placed into 37 DEG C, 5%CO₂It is marked individually after being incubated for 4-6h in cell incubator The hole of cell.

When the hole of individual cells covers in (5) 96 orifice plates, culture medium is abandoned, PBS is washed once, and 100 μ l 0.25% are added Trypsin-EDTA, room temperature digest 2min, and 2ml DMEM/F12 (+25 μM of MSX containing 10% serum) are added and terminate reaction, with shifting Liquid device dispels cell, and cell liquid is then transferred to 12 orifice plates, when cell covers with, takes supernatant, ELISA detection, high efficient expression Continue to cultivate, freeze.

(6) by screening, 2 plants of cell strains are harvested altogether, number is 11 plants, 36 plants.

Embodiment 5:CHO-K1 cell strain is tamed into suspended culture cell strain

(1) Tissue Culture Dish is taken out from 37 DEG C of incubators, discarded supernatant, it is primary to wash cell with 8ml PBS, and discards Then 2ml 0.25%trypsin-EDTA is added to culture dish in PBS, room temperature digests 2min, and microscopic observation cell is rounded by wrinkling, In individual cells.Then 4ml DMEM/F12 (containing 10% serum, 25 μM of MSX) is added and terminates digestion reaction, it will be thin with pipettor Born of the same parents dispel.Then cell liquid is transferred in 15ml centrifuge tube, 200rpm is centrifuged 5min.

(2) using 100%DMEM/F12 (contain 10% serum, 25 μM of MSX) suspension cell, after counting diluting cells to 5 × 10⁵A/ml, inoculation 30ml culture set 37 DEG C, 5%CO based in 125ml shaking flask₂On rail mounted oscillator in cell incubator 130rpm is incubated overnight, and counts 1 time every for 24 hours, observes cell density and vigor.

(3) when Cell viability reaches 94%-97% after first generation cell culture once, second generation culture is carried out, by the Generation cell is transferred in 50ml centrifuge tube, and 200rpm is centrifuged 5min, then by DMEM/F12 (containing 10% serum, 25 μM of MSX) It is mixed with EX-CELL302 by 1:1 mixing while after corresponding concentration puromycin is added, suspension cell, dilutes thin after counting again Born of the same parents are to 5 × 10⁵A/ml, inoculation 30ml culture set 37 DEG C, 5%CO based in a 125ml shaking flask₂Rail in cell incubator 120rpm is incubated overnight on road formula oscillator, and counts 1 time every for 24 hours, observes cell density and vigor.

(4) when second generation culture twice after obtained cell survival rate be greater than 95%, third to six, which is commissioned to train, supports three times afterwards The cell survival rate arrived is greater than 95%.After 7 weeks, cell inoculation breeds three generations after 3 days, and density reaches 1 × 10⁶A/ml, at the same it is thin Born of the same parents' survival rate reaches 95%, which is considered being already adapted to the culture that suspends, and inoculum density is reduced to 3 × 10⁵A/ml.By Domestication, the cell line that 11 plants of number meet the requirements, show to tame successfully.

Embodiment 6: cell fermentation

(1) prepare culture medium: the Ex-cell302 of 60% CD-CHO+40% is placed in 37 DEG C of water-baths and preheats；

(2) from CO₂Constant-temperature table takes out shaking flask cell, and diluting cells are to 3.0 × 10 after counting⁵A/ml, inoculation 30ml training It supports based in 125ml shaking flask, sets 37 DEG C, 5%CO₂In constant-temperature table, 100rpm is incubated overnight.

(3) every cell count for 24 hours, density and vigor are observed, monitors concentration of glucose, when concentration of glucose is lower than 2g/L When, addition glucose to 4g/L；1ml Sample supernatants are taken daily, detect protein expression situation.

(4) feed supplement: the about the 4th day, 70g/L CB5 added the 10% of basal medium；5th day, by CO₂Incubator temperature It adjusts to 32 DEG C；9th day, 70g/L CB5 is supplemented, the 10% of basal medium is added；12nd day, harvest cell.

(5) Werstern-Blotting is detected, as a result as shown in Figure 3.

Embodiment 7: protein purification

(1) cell culture fluid is collected, 4 DEG C, 10000rpm is centrifuged 30min, filter membrane (0.45 μm) after supernatant is taken, in preparation Sample.

(2) column equilibration: with 3 column volumes of ultrapure water balance, ethyl alcohol is discharged and saves liquid；BufferA (20mM is added NaH₂PO₄, 500mM NaCl) and 4-8ml/min, balance 3 column volumes.

(3) loading: using 5ml prepacked column, and 1ml/min carries out loading and (loading flow velocity adjusted according to prepackage column volume, when reservation Between 5min), collect Flow through (FT).

(4) it washs: 4%bufferB (20mM NaH₂PO₄, 500mM NaCl, 100mM imidazole) wash column, flow velocity is 4ml/min, until OD280nm baseline is steady.

(5) it elutes: 50%bufferB (20mM NaH₂PO₄, 500mM NaCl, 100mM imidazole) elution purpose egg It is white, until baseline washes flat, 2ml/min, collect 5ml/ pipe.

(6) it washs: 100%bufferB (20mM NaH₂PO₄, 500mM NaCl, 500mM imidazole) 4ml/min, 2-3 column volume is rinsed, until UV baseline washes flat, ultrapure 3 column volume of water balance.

(7) dialyse: 4 DEG C of Millipore 10KD PBS (pH7.4) dialysis, the secondary liquid imidazoles extension rate that changes is 2, changes liquid Number is 10 times.

(8) aseptic filtration: being filtered with 0.22 μm of filter, and -80 DEG C of refrigerators of protein sample solution save.

(9) protein concentration and purity testing: measuring protein concentration using BCA method, wherein 112 plants of albumen yield are 800mg/ L, other are in 500mg/L or so；Purity is detected using HPLC method, purity is attained by 95% or more.

Embodiment 8: vaccine preparation and immunity inoculation

8.1 equine influenza virus HA-NA subunit vaccines preparation: by the recombinant protein HA-NA of expression and ISA201VG adjuvant 45:55 is mixed by volume, emulsification, and final concentration of protein is 100 μ g/ml.

8.2 vaccine immunities and antibody test: choosing 6 week old BALB/c mouses and carry out immunity inoculation, every time immune CHO expression 50 μ g of HA-NA recombinant protein (note: injection 0.5ml), after head exempts from 2 weeks, carry out second it is immune；Head exempt from before, two exempt from before, two exempt from Blood examination is taken within 14 days to survey antibody level afterwards, as a result as shown in Figure 4.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution The range of scheme.

Sequence table

<110>Tian Kang Biological Co., Ltd.

<120>fusion protein of equine influenza virus H3N8 hypotype and preparation method thereof, application and vaccine

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 810

<212> DNA

<213>equine influenza virus (Equine Influenza Virus)

<400> 1

agtttcttta gccgactgaa ttggctaaca aaatccggaa gctcttaccc cacattgaat 60

gtgacaatgc ctaacaataa aaatttcgac aagctataca tctgggggat ccatcacccg 120

agctcaaatc aagagcagac aaaattgtac atccaagaat cagggcgagt aacagtctca 180

acaaaaagaa gtcaacaaac aataatccct aacatcgggt ctagaccatg gatcagaggt 240

caatcaggta ggataagcat atactggacc attgtaaaac ctggagatat cctaatgata 300

aacagtaatg gcaacttagt tgcaccgcgg ggatatttta aattgaaaac agggaaaagc 360

tctgtaatga gatcagatgt acccatagac atttgtgtgt ctgagtgtat tacaccaaat 420

ggaagcatct ccaacgacaa gccattccaa aatgtgaaca aagttacata tggaaaatgc 480

cccaagtata tcaggcaaaa cactttaaag ctggccactg ggatgaggaa tgtaccagaa 540

aagcaaatca gaggaatctt tggggcaata gcgggattca tcgaaaacgg ctgggaagga 600

atggttgatg gatggtatgg gttccgatac caaaactctg aaggaacagg gcaagctgca 660

gatctaaaga gcactcaagc agccatcgac cagatcaatg gaaagttaaa cagagtgatt 720

gaaagaacca atgagaaatt ccatcaaata gagaaggaat tctcagaagt agaaggaaga 780

attcaggact tggagaaata tgtagaagac 810

<210> 2

<211> 900

<212> DNA

<213>equine influenza virus (Equine Influenza Virus)

<400> 2

tactacatga acaacaccga accactttgt gatgcccaag gctttgcacc attttccaaa 60

gataatggaa tacgaattgg gtcgagaggc catgtttttg tgataagaga accttttgta 120

tcatgttcgc cctcagaatg tagaaccttt ttcctcacac agggctcatt actcaatgac 180

aaacattcta acggcacagt aaaggatcga agtccatata ggactttgat gagtgtcaaa 240

atagggcaat cacctaatgt gtatcaagct aggtttgaat cagtggcatg gtcagcaaca 300

gcatgccacg atggaaaaaa atggatgaca attggagtca cagggcccga caatcaagca 360

accgcaatag tgaactatgg gggcattcca gttgatatta ttaattcatg ggaaggggac 420

atattaagaa cccaagaatc atcatgcacc tgcattaaag gaaactgtta ttgggtaatg 480

actgatggac cagcaaatag gcaagccaaa tacaggatat tcaaagcaaa agatggaaga 540

gtaattggac agaccgatat aagtttcaat gggggacaca tagaggagtg ttcttgttac 600

cccaatgaag ggaaggtgga atgcatatgc agggacaatt ggactggaac aaatagacca 660

attctggtaa tatcttctga tttatcgtac agagttggat atttgtgtgc tggcattccc 720

actgacactc ctaggggaga ggatagtcaa ttcacaggct catgtacaag tcctttggga 780

aataaagggt acggtgtaaa aggttttggg tttcgacaag gaactgacgt atgggccgga 840

aggacaatta gtaggacttc gagatcagga ttcgaaataa taaaaatcag gaatggttgg 900

<210> 3

<211> 30

<212> DNA

<213>artificial sequence ()

<400> 3

ggaggaggag gatctggagg aggaggatct 30

<210> 4

<211> 580

<212> PRT

<213>artificial sequence ()

<400> 4

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

1 5 10 15

Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu

20 25 30

Tyr Ile Trp Gly Ile His His Pro Ser Ser Asn Gln Glu Gln Thr Lys

35 40 45

Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser

50 55 60

Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Ile Arg Gly

65 70 75 80

Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp

85 90 95

Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr

100 105 110

Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Val Pro

115 120 125

Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Ser

130 135 140

Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys

145 150 155 160

Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg

165 170 175

Asn Val Pro Glu Lys Gln Ile Arg Gly Ile Phe Gly Ala Ile Ala Gly

180 185 190

Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe

195 200 205

Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser

210 215 220

Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile

225 230 235 240

Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu

245 250 255

Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Gly Gly

260 265 270

Gly Gly Ser Gly Gly Gly Gly Ser Tyr Tyr Met Asn Asn Thr Glu Pro

275 280 285

Leu Cys Asp Ala Gln Gly Phe Ala Pro Phe Ser Lys Asp Asn Gly Ile

290 295 300

Arg Ile Gly Ser Arg Gly His Val Phe Val Ile Arg Glu Pro Phe Val

305 310 315 320

Ser Cys Ser Pro Ser Glu Cys Arg Thr Phe Phe Leu Thr Gln Gly Ser

325 330 335

Leu Leu Asn Asp Lys His Ser Asn Gly Thr Val Lys Asp Arg Ser Pro

340 345 350

Tyr Arg Thr Leu Met Ser Val Lys Ile Gly Gln Ser Pro Asn Val Tyr

355 360 365

Gln Ala Arg Phe Glu Ser Val Ala Trp Ser Ala Thr Ala Cys His Asp

370 375 380

Gly Lys Lys Trp Met Thr Ile Gly Val Thr Gly Pro Asp Asn Gln Ala

385 390 395 400

Thr Ala Ile Val Asn Tyr Gly Gly Ile Pro Val Asp Ile Ile Asn Ser

405 410 415

Trp Glu Gly Asp Ile Leu Arg Thr Gln Glu Ser Ser Cys Thr Cys Ile

420 425 430

Lys Gly Asn Cys Tyr Trp Val Met Thr Asp Gly Pro Ala Asn Arg Gln

435 440 445

Ala Lys Tyr Arg Ile Phe Lys Ala Lys Asp Gly Arg Val Ile Gly Gln

450 455 460

Thr Asp Ile Ser Phe Asn Gly Gly His Ile Glu Glu Cys Ser Cys Tyr

465 470 475 480

Pro Asn Glu Gly Lys Val Glu Cys Ile Cys Arg Asp Asn Trp Thr Gly

485 490 495

Thr Asn Arg Pro Ile Leu Val Ile Ser Ser Asp Leu Ser Tyr Arg Val

500 505 510

Gly Tyr Leu Cys Ala Gly Ile Pro Thr Asp Thr Pro Arg Gly Glu Asp

515 520 525

Ser Gln Phe Thr Gly Ser Cys Thr Ser Pro Leu Gly Asn Lys Gly Tyr

530 535 540

Gly Val Lys Gly Phe Gly Phe Arg Gln Gly Thr Asp Val Trp Ala Gly

545 550 555 560

Arg Thr Ile Ser Arg Thr Ser Arg Ser Gly Phe Glu Ile Ile Lys Ile

565 570 575

Arg Asn Gly Trp

580

<210> 5

<211> 1740

<212> DNA

<213>artificial sequence ()

<400> 5

agtttcttta gccgactgaa ttggctaaca aaatccggaa gctcttaccc cacattgaat 60

gtgacaatgc ctaacaataa aaatttcgac aagctataca tctgggggat ccatcacccg 120

agctcaaatc aagagcagac aaaattgtac atccaagaat cagggcgagt aacagtctca 180

acaaaaagaa gtcaacaaac aataatccct aacatcgggt ctagaccatg gatcagaggt 240

caatcaggta ggataagcat atactggacc attgtaaaac ctggagatat cctaatgata 300

aacagtaatg gcaacttagt tgcaccgcgg ggatatttta aattgaaaac agggaaaagc 360

tctgtaatga gatcagatgt acccatagac atttgtgtgt ctgagtgtat tacaccaaat 420

ggaagcatct ccaacgacaa gccattccaa aatgtgaaca aagttacata tggaaaatgc 480

cccaagtata tcaggcaaaa cactttaaag ctggccactg ggatgaggaa tgtaccagaa 540

aagcaaatca gaggaatctt tggggcaata gcgggattca tcgaaaacgg ctgggaagga 600

atggttgatg gatggtatgg gttccgatac caaaactctg aaggaacagg gcaagctgca 660

gatctaaaga gcactcaagc agccatcgac cagatcaatg gaaagttaaa cagagtgatt 720

gaaagaacca atgagaaatt ccatcaaata gagaaggaat tctcagaagt agaaggaaga 780

attcaggact tggagaaata tgtagaagac ggaggaggag gatctggagg aggaggatct 840

tactacatga acaacaccga accactttgt gatgcccaag gctttgcacc attttccaaa 900

gataatggaa tacgaattgg gtcgagaggc catgtttttg tgataagaga accttttgta 960

tcatgttcgc cctcagaatg tagaaccttt ttcctcacac agggctcatt actcaatgac 1020

aaacattcta acggcacagt aaaggatcga agtccatata ggactttgat gagtgtcaaa 1080

atagggcaat cacctaatgt gtatcaagct aggtttgaat cagtggcatg gtcagcaaca 1140

gcatgccacg atggaaaaaa atggatgaca attggagtca cagggcccga caatcaagca 1200

accgcaatag tgaactatgg gggcattcca gttgatatta ttaattcatg ggaaggggac 1260

atattaagaa cccaagaatc atcatgcacc tgcattaaag gaaactgtta ttgggtaatg 1320

actgatggac cagcaaatag gcaagccaaa tacaggatat tcaaagcaaa agatggaaga 1380

gtaattggac agaccgatat aagtttcaat gggggacaca tagaggagtg ttcttgttac 1440

cccaatgaag ggaaggtgga atgcatatgc agggacaatt ggactggaac aaatagacca 1500

attctggtaa tatcttctga tttatcgtac agagttggat atttgtgtgc tggcattccc 1560

actgacactc ctaggggaga ggatagtcaa ttcacaggct catgtacaag tcctttggga 1620

aataaagggt acggtgtaaa aggttttggg tttcgacaag gaactgacgt atgggccgga 1680

aggacaatta gtaggacttc gagatcagga ttcgaaataa taaaaatcag gaatggttgg 1740

<210> 6

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 6

ccaagcttag tttctttagc cgac 24

<210> 7

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 7

cccccgggcc aaccattcct gatt 24

Claims

1. a kind of fusion protein of equine influenza virus H3N8 hypotype, which is characterized in that including HA section and NA section；The area HA Section mainly express by the nucleotide sequence as shown in SEQ ID NO.1, the main nucleosides as shown in SEQ ID NO.2 of the NA section Acid sequence expression.

2. fusion protein according to claim 1, which is characterized in that HA section and NA section put in order as HA-NA, It is connected by Linker；

Preferably, the Linker has the nucleotide sequence as shown in SEQ ID NO.3.

3. fusion protein according to claim 1 or 2, which is characterized in that the fusion protein has such as SEQ ID NO.4 Shown in amino acid sequence.

4. a kind of preparation method of fusion protein of any of claims 1-3, which is characterized in that by the fusion egg White gene is expressed in host；

5. the preparation method according to claim 4, which is characterized in that provide the gene comprising the expression fusion protein The expression vector is imported in Chinese hamster ovary celI, then carries out pressurization screening to Chinese hamster ovary celI, then pressurization is filtered out by expression vector Chinese hamster ovary celI domestication at the cell strain for the culture that suspends, so that the cell strain of the culture that suspends is expressed the fusion protein.

6. preparation method according to claim 5, which is characterized in that sieved using glutamine synthelase screening amplification system The Chinese hamster ovary celI strain of the fusion protein is expressed in choosing；

Preferably, expression vector used in glutamine synthelase screening amplification system be pcDNA3, pEE6.4 or pEE12.4。

7. preparation method according to claim 5, which is characterized in that obtained using method passage at least 6 generations gradually tamed Must suspend the Chinese hamster ovary celI strain for expressing the fusion protein cultivated.

8. preparation method system described in any one of fusion protein of any of claims 1-3 or claim 4-7 The application of standby obtained albumen, which is characterized in that including at least one of following (a)-(d):

(a) equine influenza virus vaccine is prepared；

(b) antibody of equine influenza virus is prepared；

(c) kit of preparation detection equine influenza virus antibody；

(d) equine influenza virus diagnostic antigen is prepared.

9. a kind of vaccine of the horse bird flu H3N8 hypotype comprising fusion protein of any of claims 1-3.

10. vaccine according to claim 9, which is characterized in that the concentration of fusion protein described in the vaccine is 20- 100 μ g/ml, preferably 30-80 μ g/ml, more preferable 50 μ g/ml；

Preferably, the vaccine further includes auxiliary material, and the auxiliary material includes one of vaccine adjuvant, stabilizer and antibiotic or more Kind；

Preferably, the vaccine adjuvant include aluminium hydroxide gel, Freund's complete adjuvant, incomplete Freund's adjuvant, white-oil adjuvant, MF59 adjuvant or ISA201VG are, it is preferable to use ISA201VG.