CN109880835B

CN109880835B - Recombinant H9N2 avian influenza virus strain, preparation method thereof, avian influenza vaccine and application thereof

Info

Publication number: CN109880835B
Application number: CN201910248018.6A
Authority: CN
Inventors: 石火英; 苏海龙
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2019-03-28
Filing date: 2019-03-28
Publication date: 2022-03-25
Anticipated expiration: 2039-03-28
Also published as: CN109880835A

Abstract

The invention discloses a PB2 mutant gene, a mutant protein, a recombinant vector and a recombinant cell. The invention also discloses a recombinant virus and its preparation method and application. The invention also discloses an H9N2 avian influenza virus vaccine and its application. In the present invention, mutations of 1185K and R355K amino acids are respectively introduced into the PB2 gene of the H9N2 subtype avian influenza virus F/98 strain, so that the replication ability of the F/98 strain in chicken embryos can be significantly enhanced, and the virus replication titer can be significantly increased. It provides an effective technical method to improve vaccine production efficiency and reduce production cost. The invention has great market applicability and creates significant economic benefits.

Description

Recombinant H9N2 avian influenza virus strain, preparation method thereof, avian influenza vaccine and application thereof

Technical Field

The invention belongs to the field of vaccine production, and particularly relates to a recombinant H9N2 avian influenza virus strain, a preparation method thereof, an avian influenza vaccine and application thereof.

Background

Avian influenza is a highly contagious disease of poultry and wild birds infection caused by influenza a virus, and is classified into high pathogenicity and low pathogenicity. The H9N2 subtype belongs to low pathogenic influenza virus, can cause serious reduction of the laying rate of poultry, and is easy to cause concurrent infection, causing serious economic loss. The H9N2 influenza virus epidemic outbreak in China since 1998, and the vaccine plays an important role in the prevention and control of the disease, and particularly the immunization effect of the avian influenza virus inactivated oil emulsion vaccine is extremely obvious. The inactivated vaccine produced by the H9N2 subtype avian influenza virus has better immune protection effect on poultry for preventing H9N2 subtype avian influenza. But the problems of high early mortality rate and final mortality rate of chick embryos, small average single embryo yield, unstable titer and the like exist in the production process. The conventional immunization dose cannot contain enough multiple antigens, so that the immunization efficacy is influenced; in order to obtain sufficient avian influenza virus antigen for the immunized animal, the injection dosage must be increased, thereby affecting the application of the vaccine. The effect of the vaccine is positively correlated with the antigen amount, and a sufficient amount of antigen is indispensable to obtain a high immune protection rate. Increasing the antigen content per unit volume is key to the production of influenza vaccines. The polymerase of avian influenza virus consists of 3 heterotrimeric complex subunits, PB2, PB1 and PA proteins, respectively. PB2 and PB1 are basic proteins and PA is an acidic protein. Studies have shown that mutations in the amino acids of the polymerase affect the virulence and replication capacity of the virus.

The reverse genetic manipulation technique of RNA virus is a technique for assembling a new RNA virus from virus genome cDNA and various accessory proteins by constructing infectious molecular clone of RNA virus in vitro, i.e., by reverse transcribing virus genome RNA into cDNA and performing various in vitro manual manipulations on the DNA molecular level. Since the finally 'rescued' RNA virus is derived from cDNA clone, various in vitro manual operations such as gene mutation, gene knockout (deletion), gene insertion, gene replacement, gene complementation and the like can be carried out on the RNA virus genome at the DNA level through the artificially added DNA link in the intermediate process to construct a new virus strain.

At present, the avian influenza vaccine is mainly a inactivated vaccine which is mainly derived from chick embryo amplification. But the problems of high early mortality rate and final mortality rate of chick embryos, small average single embryo harvest, unstable titer and the like exist in the production process. In order to obtain sufficient avian influenza virus antigen for the immunized animal, the injection dosage must be increased, thereby affecting the application of the vaccine. Increasing the antigen content per unit volume is the key to the production of avian influenza virus vaccines.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a PB2 mutant gene.

The technical problem to be solved by the invention is to provide a recombinant protein, a recombinant vector, a recombinant cell, a recombinant virus and a preparation method thereof.

The invention finally solves the technical problem of an H9N2 avian influenza virus vaccine and application thereof.

The technical scheme is as follows: aiming at the problems, in order to improve the titer of the avian influenza virus in chick embryo allantoic fluid, the invention utilizes a reverse genetic manipulation technology, 8 genes of a low-pathogenicity avian influenza virus chick embryo highly-adaptive strain A/Chciken/Shanghai/F/98(H9N2) separated from China mainland in 1998 are utilized to construct a transcription/expression plasmid of 8 genes of an H9N2 subtype avian influenza virus F/98 strain, and a point mutation I185K or R355K is introduced into a PB2 gene, so that recombinant viruses rFPB2-I185K and rFPB2-R355K strains are successfully rescued, and the virulence, pathogenicity and immunity efficacy of the strains are identified.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a PB2 mutant gene is mutated from T to A at the 554 th nucleotide and/or from G to A at the 1064 th nucleotide of PB2 gene sequence of H9N2 avian influenza virus.

Wherein, the nucleotide sequence of the mutated PB2 gene sequence is shown as SEQ ID No: 1 or SEQ ID No: 3, respectively.

The invention also comprises a PB2 mutant protein and a PB2 mutant gene coded by the same, wherein the sequence of the recombinant protein is shown as SEQ ID No: 2 or SEQ ID No: 4, respectively.

The invention also comprises a recombinant vector containing the PB2 mutant gene and the mutant protein.

The invention also comprises a recombinant cell which contains the PB2 mutant gene, the PB2 mutant protein and the recombinant vector.

The invention also comprises a recombinant virus rFPB2-I185K, which contains the PB2 mutant gene, the PB2 mutant protein, the recombinant vector and the recombinant cell.

The invention also provides a preparation method of the recombinant virus, which comprises the following steps:

1) the PB2 gene of H9N2 avian influenza virus F/98 strain is subjected to site-directed mutagenesis, the 554 th nucleotide is mutated from T to A, or the 1064 th nucleotide is mutated from G to A to obtain a PB2 mutant gene;

2) inserting the PB2 mutant gene into a transcription or expression vector to obtain a PB2 mutant gene-containing plasmid;

3) 7 gene plasmids of F/98 strain and PB2 mutant gene plasmids are mixed and cotransfected to 393T cells to obtain the recombinant virus.

Wherein, the 7 gene plasmids of the F/98 strain in the step 3) are 7 expression vector plasmids of pHW202-PB1, pHW203-PA, pHW204-HA, pHW205-NP, pHW206-NA, pHW207-M and pHW208-NS respectively.

The invention further provides an H9N2 avian influenza virus vaccine, wherein the H9N2 avian influenza virus vaccine comprises an immunizing amount of the recombinant virus antigen.

Wherein, the H9N2 avian influenza virus EID₅₀Is 10^-6.670.2mL or 10^-6.67/0.2mL。

The invention also comprises the application of the H9N2 avian influenza virus vaccine in the preparation of medicaments for preventing or treating H9N2 avian influenza.

The invention takes F/98 strain (A/Chiken/Shanghai/F/98, H9N2) as a framework, adopts a reverse genetic technology method, respectively introduces mutation of I185K or R355K amino acid into PB2 gene of the F/98 strain, and rescues recombinant virus strain with PB2 gene containing point mutation.

The method comprises the following specific steps:

(1) firstly, constructing transcription/expression plasmids of 8 genes of an H9N2 subtype avian influenza virus F/98 strain, namely respectively inserting 8 gene cDNAs of the F/98 strain into a transcription/expression vector pHW2000 to respectively form 8 transcription/expression vector plasmids: 201(PB2), 202(PB1), 203(PA), 204(HA), 205(NP), 206(NA), 207(M), 208 (NS);

(2) then, a PB2 gene of the F/98 strain is taken as a template, a mutation (I185K) of a T-to-A base is introduced into the position 554 of a PB2 fragment of the F/98 strain by a molecular cloning method such as overlap PCR and the like, and the fragment is inserted into a transcription/expression vector pHW 2000; 2 transcription/expression vectors containing PB2 amino acid mutation were obtained separately: pHWPB 2-I185K;

(3) the recombinant virus rFPB2-I185K strain is rescued by utilizing a reverse genetic technology.

(4) The recombinant virus was serially diluted 10-fold with tetra-anti-PBS from 10^-4To 10^-115 SPF chick embryos of 10 days old are inoculated per dilution via the allantoic cavity, 0.2 mL/embryo, and incubated at 37 ℃. Discarding dead embryos within 24h, aseptically collecting allantoic fluid of dead and non-dead chick embryos for 24h to 120h, and determining HA titer. Meanwhile, the death condition of the chick embryo is observed and recorded in time every 12h, and the EID of the virus to be detected is calculated according to the Reed-Muench formula₅₀。

Has the advantages that: the invention introduces mutation of I185K and R355K amino acids on PB2 gene of H9N2 subtype avian influenza virus F/98 strain, which can obviously enhance the replication capacity of F/98 strain in chick embryo and obviously increase virus replication titer, thus providing an effective technical method for improving vaccine production efficiency and reducing production cost. EID of avian influenza virus rFPB2-I185K strain of the present invention₅₀Is 10^-16.250.2mL, and the EID of the parental virus F/98 strain₅₀Is 10^-6.670.2mL, EID of mutant rFPB2-I185K compared to parental virus₅₀Is improved by 9X 10⁹Multiple (P < 0.001). The result shows that the I185K and R355K amino acids in the PB2 gene can obviously improve the replication titer of the H9N2 influenza virus in chick embryo allantoic fluid. The invention has great market applicability and creates remarkable economic benefit.

Drawings

FIG. 1 is a flow chart of the construction of strains of avian influenza virus rFPB2-I185K and rFPB 2-R355K.

FIG. 2 is a diagram of the whole genome PCR amplification of strain F/98; m represents DL2000DNAmarker, lane 1 represents PB2-1 gene, lane 2 represents PB2-2 gene, lane 3 represents PB1-1 gene, lane 4 represents PB1-2 gene, lane 5 represents PA-1 gene, lane 6 represents PA-2 gene, lane 7 represents HA gene, lane 8 represents NP gene, lane 9 represents NA gene, lane 10 represents M gene, lane 11 represents NS gene;

FIG. 3 is PCR amplification electrophoretogram of full PB2 gene of avian influenza virus strains rFPB2-I185K and rFPB 2-R355K; m represents DL2000DNA Marker, lane 1 represents PB2-1 gene of rFPB2-I185K strain, lane 2 represents PB2-2 gene of rFPB2-I185K strain, lane 3 represents PB2-1 gene of rFPB2-R355K strain, and lane 4 represents PB2-2 gene of rFPB2-R355K strain.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the 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 commercially available.

The low-pathogenicity avian influenza virus A/Chiken/Shanghai/F/98 strain (H9N2, abbreviated as F/98 strain) serving as an H9N2 subtype vaccine is selected in the embodiment of the invention, the strain is separated and purified by a livestock and poultry infectious disease focus open laboratory of Yangzhou university department of agriculture, is identified as the H9N2 subtype by the national influenza center, and has a whole gene sequence of AY253750-AY 253756.

8 plasmid virus rescue systems pHW2000 was offered by Robert Webster doctor of St.Jude Children research Hospital, USA.

Example 1: extraction of F/98-strain RNA

Inoculating F/98 strain to SPF chick embryo, collecting allantoic fluid 300ml, centrifuging at 6000rpm (30# rotor) for 15min, and collecting supernatant; 18000rpm was centrifuged at 4 ℃ for 1.5 hours, and the pellet was suspended with 40ml STE (10mM pH8.0Tris-HCl, 100mM NaCl, 5mM pH8.0 EDTA). Bedding with 10% sucrose, carefully adding the suspension, centrifuging at 18000rpm for 1.5h at 4 ℃ to remove protein, discarding the supernatant, suspending the precipitate with 30mL STE, centrifuging at 18000rpm for 1h at 4 ℃ to remove sucrose, suspending the precipitate with 7mL STE, and packaging into finger tubes with 450. mu.L each tube.

Using Shanghai bioengineering technology servicesRNA was extracted using an RNA extraction kit from Co., Ltd. Adding 300 mu L of virus allantoic fluid into 400 mu L of Trizol, and mixing well; adding 100. mu.L chloroform/isoamyl alcohol (24: 1), shaking for 30 seconds, and centrifuging at 12,000rpm for 5 minutes; transferring the supernatant into a sterile 1.5mL RNase-free centrifuge tube, adding 150 mu L of absolute ethyl alcohol, and uniformly mixing; placing in MNIQ-10 column, placing for 2 minutes at room temperature, and centrifuging for 1 minute at 8,000 rpm; discarding the waste liquid, adding 450 mu LSolution RPE into the column, 10,000rpm, and centrifuging for 30 seconds; repeating the previous step once; discarding the waste liquid, 10,000rpm, centrifuging for 15 seconds; transferring the column into a sterile, RNase-free centrifuge tube, and adding 50. mu.L DEPC-H to the center of the column₂O, standing at 55-80 ℃ for 2 minutes at 10,000rpm, centrifuging for 1 minute, and collecting the solution in the tube to obtain the RNA sample.

Example 2 amplification, cloning and sequencing of 8 genes of F/98 Strain

Primers were designed to amplify 8 gene sequences of F/98 strain and sequenced.

The primer sequences for amplifying 8 internal genes of the F/98 strain are as follows:

Bm-HA-1 5’-TATTCGTCTCAGGGAGCAAAAGCAGGGG-3’，

Bm-HA-2 5-ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3’。

Bm-NA-1 5’-TATTCGTCTCAGGGAGCAAAAGCAGGAGT-3’；

Bm-NA-2 5’-ATATCGTCTCGTATTAGTAGAAACAAGGAGTTTTTT-3’，

Bm-PB1-1 5’-TATTCGTCTCAGGGAGCGAAAGCAGGCA-3’；

Bm-PB1-2 5’-ATATCGTCTCGTATTAGTAGAAACAAGGCATTT-3’，

Bm-PA-1 5’-TATTCGTCTCAGGGAGCGAAAGCAGGTACTGAT-3’；

Bm-PA-2 5’-ATATCGTCTCGTATTAGTAGAAACAAGGTACTTTT-3’，

Bm-NP-1 5’-TATTCGTCTCAGGGAGCA AAAGCAGGGTAGATAATC-3’；

Bm-NP-2 5’-ATATCGTCTCGTATTAGTAGAAACAAGGGTATTTTTC-3’；

Bm-PB2-1 5’-TATTCGTCTCAGGGAGCGAAAGCAGGTC-3’；

Bm-PB2-2 5’-ATATCGTCTCGTATTAGTAGAAACAAGGTCGTTT-3’，

Bm-M-1 5’-TATTCGTCTCAGGGAGCAAAAGCAGGTAG-3’；

Bm-M-2 5’-ATATCGTCTCGTATTAGTAGAAACAAGGTAGTT TTT-3’，

Bm-NS-1 5’-TATTCGTCTCAGGGAGCAAAAGCAGGGTG-3’；

Bm-NS-2 5’-ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3’。

the general primer is as follows: 12uni5 '-AGCAAAAGCAGG-3'

In order to facilitate the cloning of the above 8 gene fragments into a transcription/expression vector (BsmBI enzyme cutting sites are arranged on the transcription/expression vector), when primers of PCR amplified gene fragments are designed, the cloned enzyme cutting sites BsmBI sites are added at the 5' ends of the upstream and downstream primers, the BsmBI sites are abbreviated as Bm, and the underlined parts are recognition sites of corresponding enzymes. In addition, 12nt, which is present at the 5' -end of all the fragment cDNAs, was designed as a universal primer (12uni) for Reverse Transcription (RT) and used for reverse transcription. All primers were synthesized by Takara Shuzo (Dalian) Co., Ltd.

Reverse transcription F/98 strain whole genome (25. mu.l reaction system):

15 μ L of the F/98 strain genomic RNA extracted above is taken respectively, 1 μ L of 50 pmol/L12 nt primer is added for denaturation at 70 ℃ for 5min, the mixture is immediately placed at 0 ℃, and then the following reagents are added in sequence:

acting at 42 deg.C for 1 hr, and using immediately or placing at-20 deg.C for use.

Polymerase Chain Reaction (PCR) amplified full-length fragments of 8 genes (50 μ L reaction):

take 10. mu.L of the above RT product (cDNA) to 0.5mL PCR reaction tube, wherein the PB2 fragment was amplified using primers: Bm-PB2-1, Bm-PB 2-2; primers were used for amplification of PB1 fragment: Bm-PB1-1, Bm-PB 1-2; primers were used for amplification of PA fragments: Bm-PA-1, Bm-PA-2; primers were used for amplification of HA fragments: Bm-HA-1, Bm-HA-2; primers were used for amplification of NP fragments: Bm-NP-1, Bm-NP-2; primers were used for amplification of NA fragments: Bm-NA-1, Bm-NA-2; primers were used for amplification of M fragments: Bm-M-1, Bm-M-2; primers were used for amplification of NS fragments: Bm-NS-1, Bm-NS-2; the following reagents were added in sequence:

transient centrifugation, pre-denaturation at 94 ℃ for 5min, and addition of 0.5. mu.L (2.5U) of Expand High Fidelity polymerase at 75-80 ℃. 30 cycles of 94 ℃ for 40s, 58 ℃ for 35s and 72 ℃ for 2 min. Finally, extension was carried out at 72 ℃ for 7 minutes and run at 4 ℃ for 10 min. Taking 5 mu L of PCR product, and carrying out electrophoresis identification by using 0.8% agarose gel. The electrophoretic identification result is shown in FIG. 2.

Cloning, identification and sequencing of the PCR products:

and (3) carrying out electrophoretic identification on the PCR product, carrying out electrophoresis on the rest product, cutting Gel containing target fragments, recovering by using an agarose Gel DNA Extraction Kit, connecting with pGEM-T easy vector, transforming to escherichia coli JM109 competent cells, carrying out enzyme digestion identification on the escherichia coli JM109 competent cells, and simultaneously carrying out sequencing on the PCR product and the product connected with the T vector by bio-engineering (Shanghai) corporation.

RT-PCR amplified fragments were spliced into the full sequence of each gene using DNASTAR 11.0 software, with EditSeq editing sequences, Clustal method in MegAlign. The whole gene sequence of the F/98 strain is registered in GenBank with the registration numbers AY253750-AY 253756.

Example 3 construction of transcription/expression vector plasmid for the 8 genes of strain F/98:

in 8 plasmid transcription/expression vector pHW2000, F/98 strain 8 internal gene (PB2, PB1, PA, HA, NP, M, NS, NA) cDNAs were inserted to form 8 reassortant plasmids: pHW202-PB2, pHW202-PB1, pHW203-PA, pHW204-HA, pHW205-NP, pHW206-NA, pHW207-M, and pHW 208-NS.

The correct sequence was selected for cloning into the 8 plasmid transcription/expression vector pHW2000 by sequence analysis. First, 6 internal genes of F/98 strain, NA gene of D3 strain and 8 plasmid transcription/expression vector pHW2000 were digested with BsmBI, which was digested in the following manner (40. mu.L), and 5. mu.L of plasmid DNA (0.3. mu.g/. mu.L) was taken. The reaction is carried out for 4 hours at 55 ℃.

The enzyme digestion fragment and the vector are respectively subjected to gel electrophoresis and recovery, then are connected by T4DNA ligase, are transformed into escherichia coli JM109 competent cells, colonies are selected for amplification culture, plasmids are extracted in small quantity, corresponding plasmids with standard sizes in an 8 plasmid system are used as a reference, gel electrophoresis is carried out, and the fragments with consistent sizes are preliminarily identified as positive clones, and are frozen and stored at the temperature of minus 20 ℃.

EXAMPLE 4 construction of transcription/expression vector plasmid for PB2 Point mutation Gene

Using pHW202-PB2 of F/98 strain as a template to design a PCR primer pair pHW-PB2-185-1, and pHW-PB2-185-2 to mutate 554 site nucleotide to A on PB2 gene, so that 185 site amino acid of PB2 gene is mutated from I to K (I185K); the primer pairs are as follows:

pHW-PB2-185-1：CAGAATCGCAATTGACAAAAACAAA

pHW-PB2-185-2：TCAATTGCGATTCTGATGTCAATA

the following reagents were added in sequence:

transient centrifugation, pre-denaturation at 94 ℃ for 5min, and addition of 0.5. mu.L (2.5U) of Expand High Fidelity polymerase at 75-80 ℃. 30 cycles of 94 ℃ for 40s, 58 ℃ for 35s and 72 ℃ for 2 min. Finally, extension was carried out at 72 ℃ for 10 minutes and run at 4 ℃ for 10 min.

Taking the PCR product, identifying by 1% agarose gel electrophoresis, and recovering the gel. Homologous recombination was performed according to the instructions of the Novozam homologous recombination kit. Firstly, the following reagents are sequentially added under the ice bath condition according to the following system:

the recombinant plasmid is transformed into competent cells of Escherichia coli JM109, colonies are selected for amplification culture, and a small amount of the plasmid is extracted, amplified by PCR and sequenced (figure 3), so that the plasmid is identified as a positive clone and frozen at-20 ℃.

Example 5 rescue and identification of PB2 Point mutant avian influenza Virus

Preparation of plasmids for transfection and cells for transfection:

plasmids from strain F/98: pHW202-PB1, pHW203-PA, pHW205-NP, pHW207-M, pHW208-NS and PB2 gene plasmid pHW-PB2-185 containing point mutation respectively are enlarged and cultured, plasmid is extracted by a small plasmid-upgrading kit of QIAGEN company, and the content and purity are measured, and high-purity plasmid is prepared for transfection. The 293T cells with good growth status were digested and counted as 10⁶Cell concentrations per well were plated in 24-well cell culture plates overnight.

Virus rescue:

8 plasmids from F/98 strain: pHW201-PB2, pHW202-PB1, pHW203-PA, pHW205-NP and pHW207-M, pHW208-NS are respectively mixed with plasmids pHW-PB2-185 and pHW-PB2-355 containing PB2 point mutation according to the ratio of 1: 1. Transfection according to QIAGEN transfection reagent protocol: the mixed plasmid and transfection reagent are added into 293T cells, and the cells are placed in a medium containing 5% CO₂Culturing in an incubator at 37 ℃ for 24h, scraping transfected cells, and inoculating SPF (specific pathogen free) chick embryos of 10 days old; after culturing for 48h, harvesting allantoic fluid, continuously inoculating SPF chick embryos of 10 days old, observing death and lesion conditions of the chick embryos every day, collecting allantoic fluid of the chick embryos at proper time for virus identification, wherein the rescued virus is called rFPB2-1185 for short, and rFPB2-R355K strain avian influenza virus.

Virus identification:

hemagglutination tests for avian influenza virus were performed according to the OIE standard: the allantoic fluid hemagglutination titer of the inoculated first-generation 10-day-old SPF chick embryo reaches 2⁸(ii) a Diluting allantoic fluid of first-generation 10-day-old SPF chick embryo by 10³～10⁵The maximum blood coagulation titer is 2 after the 10-day-old SPF chick embryos are inoculated in a sterile way and subcultured¹¹。

Sequence identification: the genes PB2 and HA of rFPB2-I185K and rFPB2-R355K strain avian influenza virus obtained by RT-PCR method were identified by Biotechnology engineering (Shanghai) GmbH, and the method is shown in example 2. As a result, 7 genes other than the PB2 gene were F/98-related genes, K was found at position 185 of the PB2 gene of rFPB2-1185K strain, and K was found at position 355 of the PB2 gene of rFPB2-R355K strain.

Example 6 stability assay of mutations in the PB2 Gene of rFPB2-I185K and rFPB2-R355K strains of avian influenza Virus in SPF chick embryos

The stability of the rFPB2-1185K and rFPB2-R355K strains of avian influenza virus in SPF chick embryo passage is tested, and F/98 strains are used as controls:

allantoic fluid of first-generation 10-day-old SPF chick embryos inoculated with transfected 293T cells and having HA hemagglutination titer of 2⁸(ii) a Continuously transferring allantoic fluid of first generation 10 day-old SPF chick embryo for 20 generations, and diluting allantoic fluid of previous generation for 10 generations each time⁵The HA titer is 8-10 in 1-15 generations and reaches 2 in the allantoic fluid of chick embryo in the 20 th generation¹¹(1: 2048) (Table 1).

The PB2 genes of the F/98 strain, the rFPB2-I185K and the rFPB2-R355K strains of the avian influenza virus of the 20 th generation in SPF chick embryos are obtained by an RT-PCR method and are sequenced by a company Limited in Biotechnology engineering (Shanghai), and the result proves that the 554 th nucleotide of the PB2 gene of the F/98 strain is still T and the 1064 th nucleotide is G; the 554 th nucleotide of PB2 gene of rFPB2-I185K is A, and the 1064 th nucleotide of PB2 gene of rFPB2-R355K strain avian influenza virus is A. The results show that the mutation of rFPB2-I185K and rFPB2-R355K strain avian influenza virus is not changed after passage.

TABLE 1 HA Titers of rFPB2-I185K and rFPB2-R355K avian influenza Virus passaged in SPF chick embryos (2)ⁿ)

Example 7F/98 and rFPB2-I185K and rFPB2-R355K avian influenza Virus half the infection in chick Embryos (EID)₅₀Replication titer) assay

Allantoic fluids of test viruses (F/98 and rFPB2-1185K) were made 10 separately with tetra-resistant PBS^-1～10^-20Multiple ofAnd (4) specific dilution. 5 SPF chick embryos of 10 days old are inoculated per dilution, 0.2mL per chick, and incubated at 37 ℃. The dead embryos within 24h are discarded, and allantoic fluid of dead and non-dead chick embryos is aseptically harvested for 24h to 120 h. Detecting the hemagglutination titer (HA) of allantoic fluid of virus subjected to chick embryo half infection quantity measurement, wherein the HA titer of each virus is more than or equal to 2³The test was judged to be positive (Table 2), and calculated according to the Reed-Muench formula. The results showed that EID of rFPB2-1185K strain₅₀Is 10^-16.6250.2mL, and the EID of the parental virus F/98 strain₅₀Is 10^-6.670.2mL, EID of mutant rFPB2-I185K compared to parental virus F/98₅₀Is improved by 9X 10⁹Multiple (P < 0.001). The result shows that the I185K amino acid in the PB2 gene can obviously improve the replication titer of the H9N2 influenza virus in chick embryo allantoic fluid through two amino acid point mutations on PB 2.

Table 2: rFPB2-I185K and rFPB2-R355K avian influenza virus EID₅₀Detecting dilution

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that. Various modifications and alterations of those details may be made in light of the overall teachings of the disclosure, and are within the scope of the invention. The full scope of the invention is given by the appended patent claims and any equivalents thereof.

Sequence listing

<110> Yangzhou university

<120> recombinant H9N2 avian influenza virus strain, preparation method thereof, avian influenza vaccine and application thereof

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2341

<212> DNA

<213> PB2-185 mutant Gene (PB2-185)

<400> 1

agcgaaagca ggtcaaatat attcagtatg gagagaataa aagaattaag agatctaatg 60

tcgcaatccc gcacccgcga gatactaaca aaaaccactg tggaccatat ggccataatc 120

aagaagtaca catcaggaag acaagagaag aaccctgctc tcagaatgaa atggatgatg 180

gctatgaaat atccaatcac agcagacaag agaataatgg agatgattcc cgaaaggaat 240

gagcaagggc aaacgctttg gagcaagaca aatgatgctg gatcggacag ggtgatggtg 300

tctcccctag ctgtaacttg gtggaacagg aatgggccaa caacaagtac agtccattac 360

ccaaaggttt acaaaacata ctttgagaag gttgagaggt taaagcatgg aacctttggt 420

cccgttcact tccgaaatca agttaaaata cgccgccggg ttgatataaa cccgggtcat 480

gcagatctca gtgctaaaga agcacaagat gttatcatgg aggtcgtttt cccaaatgaa 540

gtgggagcta gaaaattgac atcagaatcg caattgacaa aaacaaaaga gaagaaagaa 600

gagctccagg attgtaagat tgctccttta atggtggcat acatgttgga aagagaactg 660

gtccggaaaa ccagattcct accggtagca ggtggaacaa gcagtgtata cattgaagta 720

ttgcatttga ctcaagggac ctgctgggaa cagatgtaca ctccaggcgg agaagtgaga 780

aatgatgatg ttgaccagag tttgatcatt gctgccagaa atattgttag gagatcaacg 840

gtgtcagcag atccattggc atcactatta gagatgtgcc acagcacaca aattggtggg 900

ataaggatgg tggacatcct taggcaaaat ccaactgagg aacaagctgt ggatatatgc 960

aaagcagcaa tgggattgag gatcagttca tcttttagct ttggaggatt cactttcaaa 1020

agaacaagtg ggtcatctgt caagaaggaa gaggaagtgc ttacgggcaa cctccaaaca 1080

ctgaaaataa gagtacatga ggggtatgag gaattcacaa tggttgggcg gagagcaaca 1140

gctatcctga ggaaagcaac cagaaggctg attcagctga tagtaagtgg aagagatgaa 1200

caatcaatcg ctgaagcgat cattgtagca atggtgttct cacaggaaga ttgcatgata 1260

aaagcagtca gaggtgatct gaatttcgta aatagagcaa atcaaaggtt aaaccccatg 1320

catcaactcc ttaggcactt ccaaaaagat gcaaaggtgc tatttcagaa ctggggaatt 1380

gaacctattg acgatgtcat ggggatgatc ggactattac ctgacatgac tccaagcaca 1440

gaaatgtcac tgagaggagt aagagttagt aaaatggggg tggatgaata ttccagcact 1500

gagagagtgg ttgtaagtat tgaccgtttc ttaagggttc gagatcagcg ggggaacgta 1560

ctcttatccc ccgaagaggt cagcgaaaca caggggactg agaaattgac aataacatat 1620

tcatcaccaa tgatgtggga aatcaacggt cctgaatcag tgcttgttaa cacctatcaa 1680

tggatcatca gaaattggga aactgtgaag attcaatggt ctcaagaccc aacaatgctg 1740

tacaataaga tggagtttga accgttccaa tccttggtgc ctaaagctgc cagaggtcaa 1800

tacagtggat ttgtgagaac actattccaa cagatgcgtg acgtattggg aacatttgat 1860

actgtacaga taataaagct gctaccattt gcagcagccc caccggagca gagcagaatg 1920

cagttttctt ctctaactgt gaatgtgaga ggctcgggaa tgaggatact cgtaagggga 1980

aactcccccg tgttcaacta taataaggca accaaaaggc ttaccgttct tggaaaggat 2040

gcaggtgcat taacagaaga tccagatgag ggaacagcag gagtggaatc tgcagtactg 2100

aggggattcc taattctagg caaggaggac aaaaggtatg gaccagcatt gagcatcaat 2160

gaactgagca atcttgcgaa aggggagaaa gctaatgtgc ttatagggca aggagacgtg 2220

gtgttggtaa tgaaacggaa acgggactct agcatactta ctgacagcca gacagcgacc 2280

aaaaggattc ggatggccat caattagtgt cgaattgttt aaaaacgacc ttgtttctac 2340

t 2341

<210> 2

<211> 759

<212> PRT

<213> PB2-185 mutant protein (PB2-185)

<400> 2

Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr

1 5 10 15

Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys

20 25 30

Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys

35 40 45

Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met

50 55 60

Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys

65 70 75 80

Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val

85 90 95

Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Val His Tyr Pro

100 105 110

Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly

115 120 125

Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg

130 135 140

Val Asp Ile Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln

145 150 155 160

Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Lys

165 170 175

Leu Thr Ser Glu Ser Gln Leu Thr Lys Thr Lys Glu Lys Lys Glu Glu

180 185 190

Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu

195 200 205

Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr

210 215 220

Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp

225 230 235 240

Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Val Asp

245 250 255

Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ser Thr Val

260 265 270

Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln

275 280 285

Ile Gly Gly Ile Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu

290 295 300

Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser

305 310 315 320

Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser

325 330 335

Ser Val Lys Lys Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu

340 345 350

Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg

355 360 365

Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu

370 375 380

Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val

385 390 395 400

Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly

405 410 415

Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His

420 425 430

Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn

435 440 445

Trp Gly Ile Glu Pro Ile Asp Asp Val Met Gly Met Ile Gly Leu Leu

450 455 460

Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val

465 470 475 480

Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val

485 490 495

Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Val Leu

500 505 510

Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr

515 520 525

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

530 535 540

Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Thr Val

545 550 555 560

Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Met Glu

565 570 575

Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Ala Arg Gly Gln Tyr

580 585 590

Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly

595 600 605

Thr Phe Asp Thr Val Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala

610 615 620

Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val

625 630 635 640

Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe

645 650 655

Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala

660 665 670

Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser

675 680 685

Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Lys Arg Tyr

690 695 700

Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu

705 710 715 720

Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys

725 730 735

Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys

740 745 750

Arg Ile Arg Met Ala Ile Asn

755

<210> 3

<211> 2341

<212> DNA

<213> PB2-355 mutant Gene (PB2-355)

<400> 3

agcgaaagca ggtcaaatat attcagtatg gagagaataa aagaattaag agatctaatg 60

tcgcaatccc gcacccgcga gatactaaca aaaaccactg tggaccatat ggccataatc 120

aagaagtaca catcaggaag acaagagaag aaccctgctc tcagaatgaa atggatgatg 180

gctatgaaat atccaatcac agcagacaag agaataatgg agatgattcc cgaaaggaat 240

gagcaagggc aaacgctttg gagcaagaca aatgatgctg gatcggacag ggtgatggtg 300

tctcccctag ctgtaacttg gtggaacagg aatgggccaa caacaagtac agtccattac 360

ccaaaggttt acaaaacata ctttgagaag gttgagaggt taaagcatgg aacctttggt 420

cccgttcact tccgaaatca agttaaaata cgccgccggg ttgatataaa cccgggtcat 480

gcagatctca gtgctaaaga agcacaagat gttatcatgg aggtcgtttt cccaaatgaa 540

gtgggagcta gaatattgac atcagaatcg caattgacaa taacaaaaga gaagaaagaa 600

gagctccagg attgtaagat tgctccttta atggtggcat acatgttgga aagagaactg 660

gtccggaaaa ccagattcct accggtagca ggtggaacaa gcagtgtata cattgaagta 720

ttgcatttga ctcaagggac ctgctgggaa cagatgtaca ctccaggcgg agaagtgaga 780

aatgatgatg ttgaccagag tttgatcatt gctgccagaa atattgttag gagatcaacg 840

gtgtcagcag atccattggc atcactatta gagatgtgcc acagcacaca aattggtggg 900

ataaggatgg tggacatcct taggcaaaat ccaactgagg aacaagctgt ggatatatgc 960

aaagcagcaa tgggattgag gatcagttca tcttttagct ttggaggatt cactttcaaa 1020

agaacaagtg ggtcatctgt caagaaggaa gaggaagtgc ttacgggcaa cctccaaaca 1080

ctgaaaataa aagtacatga ggggtatgag gaattcacaa tggttgggcg gagagcaaca 1140

gctatcctga ggaaagcaac cagaaggctg attcagctga tagtaagtgg aagagatgaa 1200

caatcaatcg ctgaagcgat cattgtagca atggtgttct cacaggaaga ttgcatgata 1260

aaagcagtca gaggtgatct gaatttcgta aatagagcaa atcaaaggtt aaaccccatg 1320

catcaactcc ttaggcactt ccaaaaagat gcaaaggtgc tatttcagaa ctggggaatt 1380

gaacctattg acgatgtcat ggggatgatc ggactattac ctgacatgac tccaagcaca 1440

gaaatgtcac tgagaggagt aagagttagt aaaatggggg tggatgaata ttccagcact 1500

gagagagtgg ttgtaagtat tgaccgtttc ttaagggttc gagatcagcg ggggaacgta 1560

ctcttatccc ccgaagaggt cagcgaaaca caggggactg agaaattgac aataacatat 1620

tcatcaccaa tgatgtggga aatcaacggt cctgaatcag tgcttgttaa cacctatcaa 1680

tggatcatca gaaattggga aactgtgaag attcaatggt ctcaagaccc aacaatgctg 1740

tacaataaga tggagtttga accgttccaa tccttggtgc ctaaagctgc cagaggtcaa 1800

tacagtggat ttgtgagaac actattccaa cagatgcgtg acgtattggg aacatttgat 1860

actgtacaga taataaagct gctaccattt gcagcagccc caccggagca gagcagaatg 1920

cagttttctt ctctaactgt gaatgtgaga ggctcgggaa tgaggatact cgtaagggga 1980

aactcccccg tgttcaacta taataaggca accaaaaggc ttaccgttct tggaaaggat 2040

gcaggtgcat taacagaaga tccagatgag ggaacagcag gagtggaatc tgcagtactg 2100

aggggattcc taattctagg caaggaggac aaaaggtatg gaccagcatt gagcatcaat 2160

gaactgagca atcttgcgaa aggggagaaa gctaatgtgc ttatagggca aggagacgtg 2220

gtgttggtaa tgaaacggaa acgggactct agcatactta ctgacagcca gacagcgacc 2280

aaaaggattc ggatggccat caattagtgt cgaattgttt aaaaacgacc ttgtttctac 2340

t 2341

<210> 5

<211> 759

<212> PRT

<213> PB2-355 mutant protein (PB2-355)

<400> 5

Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr

1 5 10 15

Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys

20 25 30

Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys

35 40 45

Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met

50 55 60

Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys

65 70 75 80

Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val

85 90 95

Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Val His Tyr Pro

100 105 110

Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly

115 120 125

Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg

130 135 140

Val Asp Ile Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln

145 150 155 160

Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile

165 170 175

Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu

180 185 190

Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu

195 200 205

Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr

210 215 220

Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp

225 230 235 240

Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Val Asp

245 250 255

Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ser Thr Val

260 265 270

Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln

275 280 285

Ile Gly Gly Ile Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu

290 295 300

Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser

305 310 315 320

Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser

325 330 335

Ser Val Lys Lys Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu

340 345 350

Lys Ile Lys Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg

355 360 365

Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu

370 375 380

Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val

385 390 395 400

Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly

405 410 415

Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His

420 425 430

Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn

435 440 445

Trp Gly Ile Glu Pro Ile Asp Asp Val Met Gly Met Ile Gly Leu Leu

450 455 460

Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val

465 470 475 480

Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val

485 490 495

Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Val Leu

500 505 510

Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr

515 520 525

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

530 535 540

Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Thr Val

545 550 555 560

Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Met Glu

565 570 575

Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Ala Arg Gly Gln Tyr

580 585 590

Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly

595 600 605

Thr Phe Asp Thr Val Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala

610 615 620

Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val

625 630 635 640

Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe

645 650 655

Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala

660 665 670

Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser

675 680 685

Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Lys Arg Tyr

690 695 700

Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu

705 710 715 720

Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys

725 730 735

Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys

740 745 750

Arg Ile Arg Met Ala Ile Asn

755

<210> 5

<211> 28

<212> DNA

<213> upstream primer (Bm-HA-1)

<400> 5

tattcgtctc agggagcaaa agcagggg 28

<210> 6

<211> 35

<212> DNA

<213> downstream primer (Bm-HA-2)

<400> 6

atatcgtctc gtattagtag aaacaagggt gtttt 35

<210> 7

<211> 29

<212> DNA

<213> upstream primer (Bm-NA-1)

<400> 7

tattcgtctc agggagcaaa agcaggagt 29

<210> 8

<211> 36

<212> DNA

<213> downstream primer (Bm-NA-2)

<400> 8

atatcgtctc gtattagtag aaacaaggag tttttt 36

<210> 9

<211> 28

<212> DNA

<213> upstream primer (Bm-PB1-1)

<400> 9

tattcgtctc agggagcgaa agcaggca 28

<210> 10

<211> 35

<212> DNA

<213> downstream primer (Bm-PA-2)

<400> 10

atatcgtctc gtattagtag aaacaaggta ctttt 35

<210> 11

<211> 36

<212> DNA

<213> upstream primer (Bm-NP-1)

<400> 11

tattcgtctc agggagcaaa agcagggtag ataatc 36

<210> 12

<211> 37

<212> DNA

<213> downstream primer (Bm-NP-2)

<400> 12

atatcgtctc gtattagtag aaacaagggt atttttc 37

<210> 13

<211> 28

<212> DNA

<213> upstream primer (Bm-PB2-1)

<400> 13

tattcgtctc agggagcgaa agcaggtc 28

<210> 14

<211> 34

<212> DNA

<213> downstream primer (Bm-PB2-2)

<400> 14

atatcgtctc gtattagtag aaacaaggtc gttt 34

<210> 15

<211> 29

<212> DNA

<213> upstream primer (Bm-M-1)

<400> 15

tattcgtctc agggagcaaa agcaggtag 29

<210> 16

<211> 36

<212> DNA

<213> downstream primer (Bm-M-2)

<400> 16

atatcgtctc gtattagtag aaacaaggta gttttt 36

<210> 17

<211> 29

<212> DNA

<213> upstream primer (Bm-NS-1)

<400> 17

tattcgtctc agggagcaaa agcagggtg 29

<210> 18

<211> 35

<212> DNA

<213> downstream primer (Bm-NS-2)

<400> 18

atatcgtctc gtattagtag aaacaagggt gtttt 35

<210> 19

<211> 12

<212> DNA

<213> random primer (12nt)

<400> 19

agcaaaagca gg 12

<210> 20

<211> 25

<212> DNA

<213> upstream primer (pHW-PB2-185-1)

<400> 20

cagaatcgca attgacaaaa acaaa 25

<210> 21

<211> 24

<212> DNA

<213> downstream primer (pHW-PB2-185-2)

<400> 21

tcaattgcga ttctgatgtc aata 24

Claims

1. a PB2 mutant gene, which is mutated into A by T at the 554th nucleotide of the PB2 gene sequence of the H9N2 subtype avian influenza virus vaccine strain F/98 strain, or at the 1064th nucleotide by G is mutated to A.

2 . A recombinant vector comprising the PB2 mutant gene of claim 1 . 3 .

3 . A recombinant cell comprising the PB2 mutant gene of claim 1 or the recombinant vector of claim 2 . 4 .

4 . A recombinant virus comprising the PB2 mutant gene of claim 1 , the recombinant vector of claim 2 or the recombinant cell of claim 3 . 5 .

5. the preparation method of the described recombinant virus of claim 4, is characterized in that, comprises the following steps:

1) By site-directed mutation of the PB2 gene of H9N2 avian influenza virus F/98 strain, the 554th nucleotide is mutated from T to A, or the 1064th nucleotide is mutated from G to A to obtain PB2 mutation type gene;

2) Insert the PB2 mutant gene into the transcription or expression vector to obtain a plasmid containing the PB2 mutant gene;

3) Mix the 7 gene plasmids of the F/98 strain with the PB2 mutant gene plasmid, and co-transfect 293T cells to obtain a recombinant virus. The 7 gene plasmids are pHW202-PB1, pHW203-PA, pHW204-HA, pHW205- NP, pHW206-NA, pHW207-M, pHW208-NS, the 7 gene plasmids are in the expression vector pHW2000, respectively insert the 7 internal genes PB1, PA, HA, NP, M, NS, NA of the F/98 strain 7 gene plasmids formed from the cDNA.

6. An H9N2 avian influenza virus vaccine, characterized in that the H9N2 avian influenza virus vaccine comprises the recombinant virus of claim 4 in an immunization amount.

7. The application of the H9N2 avian influenza virus vaccine of claim 6 in the preparation of a medicine for preventing or treating H9N2 avian influenza.