CN109111508B

CN109111508B - Avian influenza virus hemagglutinin antigen, CHO cell strain expressing avian influenza virus hemagglutinin antigen, preparation method and vaccine

Info

Publication number: CN109111508B
Application number: CN201811104957.5A
Authority: CN
Inventors: 贺笋; 李俊辉; 于新茂; 李延涛; 寇春; 张伟
Original assignee: Tecon Biological Co ltd
Current assignee: Tiankang biopharmaceutical Co.,Ltd.
Priority date: 2018-09-19
Filing date: 2018-09-19
Publication date: 2021-03-19
Anticipated expiration: 2038-09-19
Also published as: CN109111508A

Abstract

The invention provides an avian influenza virus hemagglutinin antigen, a CHO cell strain expressing the avian influenza virus hemagglutinin antigen, a preparation method and a vaccine, and relates to the technical field of biology. The preparation method of the avian influenza virus hemagglutinin antigen and the cell strain expressing the avian influenza virus hemagglutinin antigen can obtain the hemagglutinin protein with broad spectrum and good antigen immunogenicity. The vaccine for the avian influenza virus hemagglutinin antigen is low in production cost and high in immune effect, and after SPF chickens of 3-4 weeks old are immunized, the Geometric Mean Titer (GMT) of HI antibodies detected by H5 subtype antigen in 14 days is not lower than 1:64, so that the vaccine is superior to the conventional vaccine.

Description

Avian influenza virus hemagglutinin antigen, CHO cell strain expressing avian influenza virus hemagglutinin antigen, preparation method and vaccine

Technical Field

The invention relates to the technical field of biology, in particular to an avian influenza virus hemagglutinin antigen, a CHO cell strain expressing the avian influenza virus hemagglutinin antigen, a preparation method and a vaccine.

Background

Avian Influenza Virus (AIV) is a mononegavirale virus, spherical or filamentous, with a spherical diameter of 80-120 nm. The genome is divided into 8 segments and encodes 10 proteins, among which HA, NA and M are located on the envelope surface of the virus and are the main protective antigens of AIV. Located under the viral bilayer lipid envelope is matrix protein M, the most abundant protein in the viral particle, which constitutes the framework of the viral envelope. Two glycoprotein spikes, namely Hemagglutinin (HA) and Neuraminidase (NA), encoded by viral genes are embedded in the lipid bilayer membrane, and are the basis for classifying influenza virus subtypes, and the antigenicity of the two glycoprotein spikes is very easy to change, so that the avian influenza outbreak is very easy to cause once a new subtype is formed.

HA is the main component constituting the viral envelope fiber and belongs to type I glycoprotein. HA exists as a trimer. HA plays a key role in virus adsorption, model penetration, and determination of virus and cell fusion. Whether it can be split into two parts, HA1 and HA2, is an important factor for the high or low virulence of the virus. AIV infection can produce anti-HA antibodies, which are the primary protective antibodies and play an important role in preventing viral infection. The HA protein is one of the main protective antigens of avian influenza virus. It can not only stimulate organism to produce protective antibody, but also induce organism to produce cytotoxicity, and has excellent protection effect on the same subtype virus.

To date in 2003, highly pathogenic avian influenza of the H5N1 subtype occurred in an oversize pandemic and caused significant economic losses in birds in europe, africa, america and asia. At present, avian influenza inactivated vaccines are produced by using chick embryos or MDCK full-suspension cells, the process for producing the vaccines by using the chick embryos is complex, the used chick embryos are possibly polluted by exogenous viruses, the chick embryos need to be inoculated with venom, allantoic fluid needs to be collected and inactivated, the chick embryo waste needs to be treated under high pressure after the allantoin fluid is collected, more production waste is generated, and pressure is brought to environmental protection. The process of domesticating the virus adaptive cells is needed in the production of the avian influenza vaccine by MDCK full suspension cells, the period is long, the expected virus price can not be achieved sometimes, and the culture cost and the production process are complex. Thus, an improved avian influenza vaccine is currently in demand in the marketplace.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide an avian influenza virus hemagglutinin antigen which has broad spectrum and good immunogenicity, can generate high-titer antibodies after being immunized to poultry and can prevent avian influenza of various subtypes.

The second object of the present invention is to provide a cell line expressing the avian influenza virus hemagglutinin antigen, which can express the influenza virus hemagglutinin antigen with broad spectrum.

The third purpose of the invention is to provide a preparation method of the avian influenza virus hemagglutinin antigen and the cell strain, which has simple operation and good immunogenicity of the obtained antigen.

The fourth purpose of the invention is to provide a vaccine containing the avian influenza virus hemagglutinin antigen, which has low production cost and high immune efficacy.

In order to solve the technical problems, the invention adopts the following technical scheme:

an avian influenza virus hemagglutinin antigen, which is a hemagglutinin protein expressed by a sequence shown as SEQ ID NO. 1; the hemagglutinin protein is a protein expressed by eukaryotic cells.

Preferably, the eukaryotic cell is a CHO cell.

A cell strain expressing the avian influenza virus hemagglutinin antigen;

preferably, the cell line is a CHO cell line.

The preparation method of the avian influenza virus antigen or the cell strain comprises the steps of providing a recombinant vector comprising a sequence shown in SEQ ID NO.1, and then expressing the recombinant vector in host cells to obtain the avian influenza virus hemagglutinin antigen and the cell strain expressing the avian influenza virus hemagglutinin antigen;

preferably, the production method comprises the step of subjecting the host cell containing the recombinant vector to pressure screening.

Preferably, the screening reagent for the recombinant vector comprises G418; the host cell is a CHO cell; the pressure screening of the host cell comprises the following steps:

a) the CHO cells containing the recombinant vector are subjected to pressure screening for 3-5 generations by using a culture medium containing G418 until the cell survival rate is 10% -50%; preferably 20% -40%; more preferably 30%;

b) culturing the CHO cells selected in step a) in a conventional medium to a survival rate of at least 90%;

c) repeating steps a) and b) once;

preferably, the concentration of G418 in the G418-containing medium is 400-1000. mu.g/ml, preferably 600-900. mu.g/ml, more preferably 800. mu.g/ml.

Preferably, the recombinant vector is an expression vector obtained by recombining the sequence shown in SEQ ID NO.1 with at least one vector selected from the following vectors: pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13.

Preferably, the CHO cells after pressure screening are subjected to monoclonal screening.

Further, the screened CHO cells are placed at 36-38 ℃ and 5% CO₂Shaking the flask in a shaking table for 48-72 hours, and inoculating the cells at a density of 0.3 × 10⁶～0.5×10⁶VC/ml；

Preferably, the temperature is reduced to 31-33 ℃ and the pH is adjusted to 7.4-7.6 after the culture is carried out at 36-38 ℃ to the 5 th day;

preferably, 10% of initial working volume of effective Feed is added on the 4 th day and the 9 th day respectively, the glucose concentration is detected every day, and when the glucose concentration is lower than 2.5g/L, the glucose is supplemented to 3-4 g/L;

preferably, the avian influenza virus antigen is obtained by harvesting the supernatant when the cell viability is less than 80%.

An avian influenza vaccine, which comprises the avian influenza virus hemagglutinin antigen;

preferably, the dosage of the avian influenza virus hemagglutinin antigen in the avian influenza vaccine is 1-10 mug/feather, preferably 2-8 mug/feather, and more preferably 3-6 mug/feather.

Preferably, the avian influenza vaccine further comprises an adjuvant;

preferably, the adjuvant is selected from one or more of aluminium hydroxide Gel, mineral oil, carbomer, Gel01, propolis, ISA206 or ISA760 VG;

preferably, the avian influenza vaccine comprises an antigen and an adjuvant in a mass ratio of 1:1, preferably water-in-oil-in-water.

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

the avian influenza virus hemagglutinin antigen provided by the invention is hemagglutinin protein expressed by a sequence shown as SEQ ID NO. 1; the sequence shown in SEQ ID NO.1 is obtained by selecting avian influenza hemagglutinin gene which is popular in 5 years for comparison and analysis, and the sequence is subjected to codon optimization and modification, so that the aims of further improving the broad spectrum of the antigen and improving the expression quantity of the antigen are fulfilled. And the hemagglutinin protein is a protein expressed by eukaryotic cells, so that the loss of conformation epitope of the antigen is avoided. The cell strain for expressing the avian influenza virus hemagglutinin antigen provided by the invention has the beneficial effects of the avian influenza virus hemagglutinin antigen.

The preparation method of the avian influenza virus hemagglutinin antigen can obtain the hemagglutinin protein with broad spectrum and good antigen immunogenicity, and the method is simple to operate and suitable for large-scale production.

The vaccine containing the avian influenza virus hemagglutinin antigen is low in production cost and high in immune efficiency, and after SPF (specific pathogen free) chickens of 3-4 weeks are immunized, the Geometric Mean Titer (GMT) of HI antibodies in H5 subtype antigen determination is not lower than 1:64 within 14 days, and the Geometric Mean Titer (GMT) of HI antibodies in H5 subtype antigen determination is not lower than 1:256 within 21 days; the HI antibody Geometric Mean Titer (GMT) is not less than 1:64 at 21 days after the immunization of the conventional inactivated vaccine, so that the vaccine is superior to the conventional vaccine.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a diagram showing the results of restriction enzyme identification of recombinant plasmids provided in example 3 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The invention provides an avian influenza virus hemagglutinin antigen which is hemagglutinin protein expressed by a sequence shown as SEQ ID NO. 1; the sequence shown in SEQ ID NO.1 is obtained by selecting avian influenza HA genes which are popular in 5 years for comparison and analysis, and codon optimization and modification are carried out on the sequence so as to achieve the purposes of further improving the broad spectrum of the antigen and improving the expression quantity of the antigen. And the hemagglutinin protein is a protein expressed by eukaryotic cells, preferably a protein expressed by CHO cells, so as to avoid losing conformational epitope of the antigen. The invention also provides a cell strain expressing the avian influenza virus hemagglutinin antigen, so that the avian influenza virus hemagglutinin antigen has the beneficial effect of the avian influenza virus hemagglutinin antigen.

Among them, typical CHO cells may be CHO-K1 cells.

The invention also provides a preparation method of the avian influenza virus hemagglutinin antigen and the cell strain, which comprises the steps of providing a recombinant vector comprising a sequence shown in SEQ ID NO.1 and then expressing the recombinant vector in host cells. In a preferred embodiment, the antigen is prepared using CHO cells and the sequence shown in SEQ ID NO.1 is recombined with at least one of the following vectors: pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13; then the recombinant vector is transfected into a CHO cell for expression, and pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13 all use G418 as a reporter gene, so that after the recombinant vector is transfected into the CHO cell, a culture medium containing G418 is adopted to carry out pressurized screening on the CHO cell expressing the hemagglutinin protein, the pressurized screened cell strain is subjected to monoclonal screening of the cell, and the cell strain with relatively high expression level is obtained through screening. Then, a cell strain with higher hemagglutinin protein is finally obtained by optimizing the culture condition, the feeding time and the feeding amount of the cell strain, and the cell strain is used for expressing the hemagglutinin protein with high and stable expression level. After the cell strain with high hemagglutinin protein expression is screened out, the cell strain is cultured under the optimized culture condition, protein with high expression quantity can be obtained, and hemagglutinin protein can be obtained after the protein is collected and cell fragments are removed, namely the avian influenza virus antigen of the vaccine can be prepared.

In a preferred embodiment, the pressure screening is performed using the following steps:

a) CHO cells containing the recombinant vector are pressure screened with G418 containing medium for 3-5 passages to a cell viability of 10% -50%, which may be, for example, but not limited to, 10%, 20%, 30%, 40% or 50%; preferably 20% -40%; more preferably 30%;

c) repeating steps a) and b) once;

in some preferred embodiments, the concentration of G418 in the G418-containing medium is 400-1000. mu.g/ml, such as, but not limited to, 400. mu.g/ml, 500. mu.g/ml, 600. mu.g/ml, 700. mu.g/ml, 800. mu.g/ml, 900. mu.g/ml or 1000. mu.g/ml, preferably 600-900. mu.g/ml, more preferably 800. mu.g/ml, for the purpose of optimizing the pressure screening effect.

In some preferred embodiments, the selected CHO cells are subjected to a temperature of 36-38 ℃ and 5% CO₂Shaking the flask in a shaker for 48-72 hours, for example, but not limited to 48 hours, 60 hoursHour or 72 hours; the cell seeding density is 0.3X 10⁶～0.5×10⁶VC/ml may be, for example, but is not limited to, 0.3X 10⁶VC/ml、0.4×10⁶VC/ml or 0.5X 10⁶VC/ml。

Preferably, the temperature is reduced to 31-33 ℃ and the pH is adjusted to 7.4-7.6 at the culture temperature of 36-38 ℃ to the 5 th day.

Preferably, 10% of the initial working volume of the effective Feed is added on the 4 th day and the 9 th day respectively, the glucose concentration is detected every day, and when the glucose concentration is lower than 2.5g/L, the glucose is supplemented to 3-4 g/L.

The method can obtain the stable and high-expression CHO cells of the hemagglutinin protein, the expression quantity of the hemagglutinin protein can reach 5mg/ml, the production cycle of the hemagglutinin protein produced by the screened CHO cells as the avian influenza hemagglutinin antigen is short, the protein expression quantity is high, and the produced antigen has good immunogenicity.

The invention also provides an avian influenza vaccine, which comprises the avian influenza virus hemagglutinin antigen, wherein the dosage of the antigen in the vaccine is preferably 1-10 mu g/feather, and can be, but not limited to, 1 mu g/feather, 2 mu g/feather, 3 mu g/feather, 4 mu g/feather, 5 mu g/feather, 6 mu g/feather, 7 mu g/feather, 8 mu g/feather, 9 mu g/feather or 10 mu g/feather, preferably 2-8 mu g/feather, and more preferably 3-6 mu g/feather.

Preferably, the avian influenza vaccine further comprises an adjuvant; for example, but not limited to, aluminum hydroxide Gel, mineral oil, carbomer, Gel01, propolis, ISA206 or ISA760VG, the avian influenza vaccine preferably uses ISA206VG with the mass fraction of 10% -60%, and the antigen slow-release effect and the immunogenicity of the vaccine can be effectively improved.

The advantageous effects of the present invention will be further described with reference to preferred embodiments.

Example 1A sequence expressing hemagglutinin protein

The current and recent 5-year epidemic avian influenza H5 subtype HA gene sequences are downloaded from Genebank for comparison and analysis, dominant epitopes are selected as components of vaccine antigens, and codon optimization and modification are carried out on the sequence of the avian influenza virus HA gene according to the bias of CHO cell codons so as to improve the level of target protein expression HA protein. Meanwhile, in order to enable the HA gene to have broad-spectrum antigenicity, screening a relatively conserved antigen sequence of the dominant epitope of HA1, connecting matrix protein M2e (containing 23 amino acids) at the 3' end of HA2 in a flexible amino acid form to form a base sequence of an artificial polypeptide, searching a protein module in a PDB library by utilizing the homology of the structure and the sequence through protein homology modeling, simulating the space three-dimensional conformation of different connection methods of each epitope, analyzing the antigenicity, and finally screening out a combination mode with optimal antigenicity as a gene sequence of the HA target protein.

Example 2A recombinant vector expressing hemagglutinin protein

The above synthesized fragment encoding the amino acid sequence of the HA protein and having a His tag at the C-terminus was inserted and cloned into the eukaryotic transfer vector pcDNA3.1 via Sal I and Xho I sites. Connecting by T4DNA ligase at 16 ℃ overnight to obtain a connecting product, transforming by Escherichia coli competent DH5a, coating on an LB plate containing the ampicillin, culturing at 37 ℃ overnight, picking out positive colonies, culturing in an LB culture medium containing the ampicillin, and extracting plasmids.

EXAMPLE 3 enzymatic identification of recombinant vectors expressing hemagglutinin proteins

Plasmid DNA was prepared, restriction enzyme digestion was performed using Sal I and Xho I sites, and the digestion products were subjected to 1% agarose gel electrophoresis, and the results are shown in FIG. 1. Wherein, lane M: DNA Marker, lanes 1-3: post-digestion plasmid, lane 4: plasmid before enzyme digestion. From the figure, it can be seen that the target of the expected size appears, indicating that the target gene was successfully inserted into the vector.

The positive result plasmid is sent to Shanghai engineering sequencing verification to finally obtain a positive recombinant expression plasmid, the sequence of which is shown as SEQ ID NO.1, wherein flexible amino acids coded by CCTCCCAGC are connected among the antigen epitopes 1402-1431, 1441-1476 and 1486-1512.

Example 4 recombinant cell pressure screening of recombinant vectors expressing hemagglutinin proteins

The plasmid is transfected into CHO cells which grow well and are suspended completely, the cells are passaged 3 days later, G418 with the concentration of 800 mu G/ml is added into the culture medium, and the pressurization is stopped when the activity rate is about 30 percent. Culturing with conventional culture medium, replacing with new adherent culture medium, culturing for 7 days, and pressure screening once again when cell survival rate reaches above 90%, and similarly cell survival rate reaches 90%.

EXAMPLE 5 monoclonal screening of recombinant vector cells expressing hemagglutinin proteins

And (3) positive clone selection and detection, namely selecting cells growing in an adherent manner into a 96-well plate in a flat plate after 7 days of culture, adding 100gL of suspension culture medium MSX to express and culture for 2 days after 7 days of culture by utilizing the adherent culture medium, using the culture medium in an empty plate for dot hybridization detection, transferring high-expression clones into a 24-well plate, using the adherent culture medium to culture for 2 days, then switching to MSX culture, using the culture medium for Western blotting detection, and finally obtaining the high-expression clone strain according to an experimental result. All cells were cultured at 37 ℃ in 5% CO₂。

EXAMPLE 6 culture of recombinant vector cells expressing hemagglutinin protein

The selected CHO cells were resuspended in CHO medium (containing 25. mu.M MSX) or other suitable medium at a cell seeding density of 0.3X 10⁶～0.5×10⁶VC/ml, inoculating cells into suitable volume of shake flask, placing at 37 + -1 deg.C, and containing 5% CO₂And (5) carrying out shake flask culture in a shaking table for 48-72 hours, and continuously carrying out subculture amplification on the cells.

Performing culture fermentation by using a bioreactor step by step according to needs, generally performing 5-8 times amplification, performing antigen expression when the amplification is carried out to a specific volume, performing culture at 37 +/-1 ℃ to 5 days, reducing the temperature to 32 +/-1 ℃, adjusting the pH to 7.5 +/-0.1, performing culture at a proper rotating speed, adding 10% of initial working volume of effective Feed at 4 days and 9 days, detecting the glucose concentration every day, and supplementing glucose to 3-4 g/L when the glucose concentration is lower than 2.5 g/L. When the egg cell survival rate is lower than 80%, the supernatant is the required antigen.

By optimizing the culture condition and the feeding time of the cell strain, the HA protein expression of 1.5mg/ml is finally obtained by the feeding amount.

Example 7 an avian influenza vaccine

And (3) after cell debris is removed, quantifying the expression, mixing the cell debris with an adjuvant to prepare a vaccine, and immunizing SPF chickens of 3-4 weeks old, wherein the result shows that the Geometric Mean Titer (GMT) of the HI antibody in H5 subtype antigen determination is not lower than 1:64 in 14 days, and the Geometric Mean Titer (GMT) of the HI antibody in H5 subtype antigen determination is not lower than 1:256 in 21 days.

Comparative example 1

Inoculating the seed virus into 10-11 days old chick embryo allantoin liquid, culturing for about 72 hours, collecting chick embryo allantoin liquid, inactivating, emulsifying, and simultaneously performing autoclaving treatment on chick embryos absorbing allantoin liquid. HI antibody Geometric Mean Titer (GMT) was not less than 1:64 on day 21 post immunization.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Tiankang biological products Ltd

<120> avian influenza virus hemagglutinin antigen, CHO cell strain expressing same, preparation method and vaccine

<160> 1

<170> PatentIn version 3.5

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<211> 1524

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atggagaaga tcgtgctgct gttcgccatc gtgagcctgg tgaagagcga ccagatctgc 60

atcggctacc acgccaacaa cagcaccgag caggtggaca ccatcatgga gaagaacgtg 120

accgtgaccc acgcccagga catcctggag aagacccaca acggcaagct gtgcgaccac 180

gacggcgtga agcccctgat cctgagggac tgcagcgtgg ccggctggct gctgggcaac 240

cccatgtgcg acgagcccat caacgtgccc gagtggagct acatcgtgga gaaggccaac 300

cccgccaacg acctgtgcta ccccggcgac ttcaacgact acgaggagct gaagcacctg 360

ctgagcagga tcaaccactt cgagaagatc cagatcatcc ccaagaacag ctggagcagc 420

cacgagaccc ccctgggcgt gagcagcgcc tgcccctacc agggcaagag cagcttcttc 480

aggaacgtgg tgtggctgat caagaagaac aacgcctacc ccaccatcaa gaggagctac 540

aacaacacca accaggagga cctgctggtg ctgtggggca tccaccaccc caacgacgcc 600

gccgagcaga ccaggctgta ccagaacccc accacctaca tcagcgtggg caccagcacc 660

ctgaaccaga ggcctgtgcc caagatcgcc accaggagca agatcaacgg ccagagcggc 720

aggatcgact tcttctggac catcctgaag cccaacgacg ccatccactt cgagagcaac 780

ggcaacttca tcgcccccga gtacgcctac aagatcgtga agaagggcga cagcaccatc 840

atgaggagcg aggtggagta cggcaactgc aacaccaggt gccagacccc cgtgggcgcc 900

accaggggat ccttcggcgc catcgccggc ttcatcgagg gcggctggca gggcatggtg 960

gacggctggt acggctacca ccacagcaac gagcagggca gcggctacgc cgccgacaag 1020

gagagcaccc agaaggccat ggacggcgtg accaacaagg tgaacagcat catcgacaag 1080

atgaacaccc agttcgaggc cgtgggcagg gagttcaaca acctggagag gaggatcgag 1140

aacctgaaca agaagatgga ggacggcttc ctggacgtgt ggacctacaa cgccgagctg 1200

ctggtgctga tggagaacga gaggaccctg gacttccacg acagcaacgt gaagaacctg 1260

tacgacaagg tgaggctgca gctgaaggac aacgccaagg agctgggcaa cggctgcttc 1320

gagttctacc acaagtgcaa caacgagtgc atggagagcg tgaggaacgg cacctacgac 1380

tacccccagt accctcccag catcctgggc ttcgtgttca cgctgaccgt gcctcccagc 1440

ggccccctga aggccgagat cgcgcagagg ctggagcctc ccagcggcat cctgggcttc 1500

gtgttcaccc tccctcccag ctga 1524

Claims

1. An avian influenza virus hemagglutinin antigen, which is characterized in that the avian influenza virus hemagglutinin antigen is a hemagglutinin protein expressed by a sequence shown as SEQ ID NO. 1; the hemagglutinin protein is a protein expressed by eukaryotic cells.

2. The avian influenza virus hemagglutinin antigen of claim 1, wherein the eukaryotic cell is a CHO cell.

3. A cell strain expressing the avian influenza virus hemagglutinin antigen of claim 1 or 2.

4. The cell strain of avian influenza virus hemagglutinin antigen of claim 3, wherein the cell strain is a CHO cell strain.

5. A method for preparing the avian influenza virus hemagglutinin antigen of claim 1 or 2 or the cell strain of claim 3 or 4, wherein the avian influenza virus hemagglutinin antigen and the cell strain expressing the avian influenza virus hemagglutinin antigen are obtained by providing a recombinant vector comprising the sequence shown in SEQ ID No.1 and expressing the recombinant vector in a host cell;

the host cell is a CHO cell.

6. The method according to claim 5, wherein the method comprises the step of subjecting the host cell containing the recombinant vector to pressure screening.

7. The method according to claim 6, wherein the screening reagent for the recombinant vector comprises G418; the host cell is a CHO cell; the pressure screening of the host cell comprises the following steps:

a) the CHO cells containing the recombinant vector are subjected to pressure screening for 3-5 generations by using a culture medium containing G418 until the cell survival rate is 10% -50%;

c) repeating steps a) and b) once.

8. The method according to claim 7, wherein in the step a, the CHO cells containing the recombinant vector are subjected to pressure screening using a G418-containing medium for 3 to 5 passages until the survival rate of the CHO cells is 20 to 40%.

9. The method according to claim 8, wherein the CHO cell containing the recombinant vector is pressure-screened with the medium containing G418 for 3 to 5 passages until the cell survival rate is 30% in step a.

10. The method according to claim 7, wherein the concentration of G418 in the G418-containing medium is 400-1000. mu.g/ml.

11. The method as set forth in claim 10, wherein the concentration of G418 in the G418-containing medium is 600-900. mu.g/ml.

12. The method according to claim 11, wherein the concentration of G418 in the medium containing G418 is 800. mu.g/ml.

13. The method according to claim 7, wherein the recombinant vector is an expression vector obtained by recombining the sequence shown in SEQ ID No.1 with at least one of the following vectors: pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13.

14. The method according to claim 7, wherein the CHO cells subjected to the pressure screening are subjected to monoclonal screening.

15. The method according to claim 7, wherein the selected CHO cells are exposed to a temperature of 36 to 38 ℃ and 5% CO₂Shaking the flask in a shaking table for 48-72 hours, and inoculating the cells at a density of 0.3 × 10⁶~0.5×10⁶ VC/ml。

16. The method according to claim 15, wherein the temperature is decreased to 31 to 33 ℃ and the pH is adjusted to 7.4 to 7.6 at the day of culturing at 36 to 38 ℃ to 5 days.

17. The method of claim 15, wherein the glucose concentration is measured daily by adding 10% of the initial working volume of effective Feed on day 4 and day 9, and the glucose is supplemented to 3-4 g/L when the glucose concentration is less than 2.5 g/L.

18. The method of claim 15, wherein the avian influenza virus antigen is obtained by harvesting the supernatant when the cell viability is less than 80%.

19. An avian influenza vaccine comprising the avian influenza virus hemagglutinin antigen of claim 1 or 2.

20. The avian influenza vaccine of claim 19, wherein the amount of avian influenza virus hemagglutinin antigen in the avian influenza vaccine is 1-10 μ g per plume.

21. The avian influenza vaccine of claim 20, wherein the amount of avian influenza virus hemagglutinin antigen in the avian influenza vaccine is 2-8 μ g per plume.

22. The avian influenza vaccine of claim 21, wherein the amount of avian influenza virus hemagglutinin antigen in the avian influenza vaccine is 3-6 μ g per plume.

23. The avian influenza vaccine of claim 19, further comprising an adjuvant.

24. The avian influenza vaccine of claim 23, wherein the adjuvant is selected from one or more of aluminium hydroxide Gel, mineral oil, carbomer, Gel01, propolis, ISA206, ISA201 or ISA760 VG.

25. The avian influenza vaccine of claim 23, comprising the antigen and the adjuvant in a mass ratio of 1: 1.

26. The avian influenza vaccine of claim 25, wherein the avian influenza vaccine is of the water-in-oil-in-water type.