CN117946981A

CN117946981A - H1N1 influenza virus cold-adaptation vaccine skeleton strain CV2-PR8, construction method and application thereof

Info

Publication number: CN117946981A
Application number: CN202310662031.2A
Authority: CN
Inventors: 毕玉海; 杨静茹; 孙举; 张宁; 杨婧
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2022-12-30
Filing date: 2023-06-06
Publication date: 2024-04-30

Abstract

The application discloses a cold-adaptive vaccine skeleton strain CV2-PR8 of H1N1 influenza virus and a construction method and application thereof, belonging to the technical field of medical preparations. The application aims to solve the technical problems that: how to obtain influenza strains which can be used as influenza vaccine skeletons. The present application provides a biological material for constructing a recombinant influenza a virus, the biological material comprising a protein composition comprising a PB2 mutein, a PB1 mutein, a PA mutein, an NP mutein, an M1 mutein and an M2 mutein. The application also provides the recombinant influenza virus prepared from the biological material and application of the recombinant influenza virus in preparation of influenza vaccines. The influenza cold-adaptive vaccine skeleton strain obtained by the application can be used as a cold-adaptive vaccine skeleton strain, and can be reconstructed with the HA and NA genes of epidemic influenza viruses to prepare the influenza vaccine. Provides more choices for cold adaptation of influenza virus to attenuated vaccine frameworks.

Description

H1N1 influenza virus cold-adaptation vaccine skeleton strain CV2-PR8, construction method and application thereof

Technical Field

The application belongs to the technical field of medical preparations, and particularly relates to a cold-adaptive vaccine skeleton strain CV2-PR8 of H1N1 influenza virus, and a construction method and application thereof.

Background

Influenza virus is a segmented single-stranded negative-strand RNA virus, divided into A, B, C and D types, and currently, the main influenza viruses in the population are the type A H1N1 and H3N2 subtypes and the type B Yamagata and Victoria seasonal influenza viruses. Up to now, influenza viruses have caused four pandemics, respectively the H1N1 subtype in 1918, the H2N2 subtype in 1957, the H3N2 subtype in 1968 and the H1N1 subtype in 2009, each time bringing great harm to the life and health of people worldwide. Since influenza appeared, seasonal influenza was epidemic and occurs annually worldwide, bringing serious disease burden to society.

Influenza vaccines are the most effective way to prevent influenza virus infection and epidemic at present, and the influenza vaccines mainly comprise inactivated influenza vaccines, attenuated live vaccines and the like. The inactivated vaccine is a vaccine which is used in a large number at present, the inactivated influenza vaccine strain is mainly prepared from 6 internal gene segments of chicken embryo-adapted A/Puerto Rico/8/1934 (PR 8) (H1N 1) skeleton strains, and is constructed by reconfiguration with HA and NA gene segments of popular strains recommended by WHO, and the vaccine mainly plays a role by inducing organisms to generate humoral immunity, and HAs the characteristics of short protection time and poor cross protection effect.

The main donor skeletons of the current attenuated live vaccines of influenza A viruses are two types, namely A/Leningrad/17/57H2N2LAIV (LenLAIV) and A/Ann Arbor/6/60H2N2 (AA/60), and the attenuated live vaccines are constructed by reassortment of 6 internal genes of donor strains and epidemic virus HA and NA genes recommended by WHO. The attenuated live vaccine is inoculated mainly by a nasal spray mode, imitates a natural infection way, can induce a host to generate humoral immunity, cellular immunity and immune response at the same time, and the cellular immune response plays an important role in cross immune protection against different subtype viruses.

However, studies have now shown that the effectiveness of live attenuated vaccines in the resulting lot has been reduced. This decrease in immunogenicity is most likely caused by the large differences in the vaccine backbone strains used from the currently prevalent strains. H2N2 subtype influenza virus has disappeared early in the human population, and currently the main pandemic is H1N1 and H3N2 subtype influenza virus. In addition, the phenomenon of mutation of vaccine strains is found in the production process of attenuated live vaccines and in children vaccinated with attenuated live vaccines, which also raise a question about the safety of attenuated live vaccines used at present. Various phenomena suggest that a new influenza attenuated live vaccine framework needs to be constructed.

Disclosure of Invention

The invention aims to solve the technical problems that: how to prepare cold-adapted influenza strains, and the cold-adapted influenza strains are used as cold-adapted vaccine skeleton strains for preparing influenza vaccines.

To solve the above technical problem, the first aspect of the present invention provides a biological material for constructing recombinant influenza a virus, characterized in that: the biological material is selected from any one of the following B1) -B7):

B1 Protein composition comprising PB2 mutein, PB1 mutein, PA mutein, NP mutein, M1 mutein and M2 mutein,

The PB2 mutein is selected from A1) or A2):

a1 A protein having an amino acid sequence of SEQ ID No. 1;

A2 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A1);

the PB1 mutein is selected from A3) or A4):

A3 A protein having an amino acid sequence of SEQ ID No. 2;

a4 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A3);

The PA mutein is selected from A5) or A6):

a5 A protein having an amino acid sequence of SEQ ID No. 3;

a6 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A5);

The NP mutein is selected from A7) or A8):

A7 A protein having an amino acid sequence of SEQ ID No. 4;

A8 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A7);

the M1 mutein is selected from A9) or a 10):

A9 A protein having an amino acid sequence of SEQ ID No. 7;

A10 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A11);

the M2 mutein is selected from a 11) or a 12):

A11 A protein having an amino acid sequence of SEQ ID No. 8;

A12 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A13);

b2 A set of DNA molecules (DNA molecule composition) encoding the protein composition of B1) comprising a DNA molecule encoding the PB2 mutein, a DNA molecule encoding the PB1 mutein, a DNA molecule encoding the PA mutein, a DNA molecule encoding the NP mutein and a DNA molecule encoding the M1 mutein and the M2 mutein,

B3A kit of vectors (vector composition) comprising the following B3 a), B3B), B3 c), B3 d) and B3 e), B3 a), a recombinant vector comprising a DNA molecule encoding said PB2 mutein;

B3B), a recombinant vector comprising a DNA molecule encoding said PB1 mutein;

b3 c), a recombinant vector comprising a DNA molecule encoding said PA mutein;

b3 d), a recombinant vector containing a DNA molecule encoding said NP mutein;

b3 e), a recombinant vector containing DNA molecules encoding said M1 mutein and M2 mutein;

b4 A microorganism comprising B2) said set of DNA molecules or B3) said set of vectors;

B5 An animal cell line comprising B2) said set of DNA molecules or B3) said set of vectors;

B6 Animal tissue containing B2) said set of DNA molecules or B3) said set of vectors;

b7 An animal organ containing B2) said set of DNA molecules or B3) said set of vectors.

Further, in the biological material, the protein composition of B1) further comprises HA protein, NA protein, NS1 protein and NS2 protein of influenza A virus,

The NS1 protein is selected from a 13) or a 14), and the NS2 protein is selected from a 15) or a 16):

a13 A protein having an amino acid sequence of SEQ ID No. 5;

A14 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A13);

A15 A protein having an amino acid sequence of SEQ ID No. 6;

A16 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A15);

The HA protein is selected from a 17) or a 18):

a17 A protein having an amino acid sequence of SEQ ID No. 9;

A18 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A17);

The NA protein is selected from a 19) or a 20):

a19 A protein having an amino acid sequence of SEQ ID No. 9;

a20 A fusion protein obtained by ligating a tag to the N-terminal and/or C-terminal of A19).

In the invention, the protein tag (protein-tag) refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag, and/or a SUMO protein tag, etc.

Further, in the biological material, B2) the set of DNA molecules further includes a DNA molecule encoding HA protein of the influenza a virus, a DNA molecule encoding NA protein of the influenza a virus, and a DNA molecule encoding NS1 protein and NS2 protein of the influenza a virus.

Further, in the biological material, the nucleotide sequence of the DNA molecule encoding the PB2 mutein is SEQ ID No.31; the nucleotide sequence of the DNA molecule encoding the PB1 mutein is SEQ ID No.32; the nucleotide sequence of the DNA molecule encoding the PA mutein is SEQ ID No.33; the nucleotide sequence of the DNA molecule encoding the NP mutein is SEQ ID No.34; the nucleotide sequence of the DNA molecule encoding the M1 mutein and the M2 mutein is SEQ ID No.36.

Further, in the biological material, the complete carrier of B3) also comprises B3 f) -B3 h),

B3 f), a recombinant vector comprising DNA molecules encoding the NS1 and NS2 proteins of influenza a virus;

B3 g), a recombinant vector comprising a DNA molecule encoding an HA protein of an influenza A virus;

b3 h), recombinant vector containing DNA molecules encoding NA protein of influenza A virus.

Further, in the biological material, the nucleotide sequence of the DNA molecule encoding the NS1 protein and the NS2 protein of the influenza A virus is SEQ ID No.35; the nucleotide sequence of the DNA molecule encoding the HA protein of the influenza A virus is SEQ ID No.37; the DNA molecule nucleotide sequence of NA protein of the coded influenza A virus is SEQ ID No.38.

Further, in the biological material, the DNA molecule encoding HA protein of influenza a virus and the DNA molecule encoding NA protein of influenza a virus can be adaptively adjusted according to the type of epidemic influenza a virus, so as to ensure that HA and NA in the recombinant flow virus are the same as those of the epidemic influenza a virus.

In the present invention, the vector may be a reverse genetics rescue system plasmid of influenza virus, for example, a virus rescue system plasmid. In one embodiment of the invention, the virus rescue system plasmid is pHW2000. Constructing cDNA of genome of each segment of influenza virus to virus rescue system plasmid to obtain recombinant vector, co-transfecting cell with all segments of plasmid, and the recombinant vector can generate genome RNA of influenza virus or influenza virus protein to obtain recombinant influenza virus.

In a second aspect, the invention provides a recombinant influenza a virus that expresses or comprises a protein composition as described above.

Further, the genome of the recombinant influenza A virus is single-strand negative-strand segmented RNA, the single-strand negative-strand segmented RNA is transcribed to obtain complete positive-strand RNA complementary to the single-strand negative-strand segmented RNA, the complete positive-strand RNA comprises PB2-RNA, PB1-RNA, PA-RNA, NP-RNA and M-RNA, and the PB2-RNA is an RNA molecule for encoding the PB2 mutant protein; the PB1-RNA is an RNA molecule for encoding the PB2 mutant protein; the PA-RNA is an RNA molecule for encoding the PA mutein; the NP-RNA is an RNA molecule encoding the NP mutein; the M-RNA is an RNA molecule for encoding the M1 protein and the M2 protein.

Further, the positive strand RNA set in the recombinant influenza A virus also comprises NS-RNA, HA-RNA and NA-RNA, wherein the NS-RNA is an RNA molecule for encoding the NS mutant protein; the HA-RNA is an RNA molecule for encoding the HA protein; the NA-RNA is an RNA molecule encoding the NA protein.

Further, in the recombinant influenza A virus, the PB2-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 11; the PB1-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 12; the PA-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 13; the NP-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 14; the M-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 16.

Further, in the recombinant influenza A virus, the NS-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 15; the HA-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 17; the NA-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 18.

Furthermore, in the recombinant influenza A virus, the HA-RNA and the NA-RNA can be adaptively adjusted according to the type of the epidemic influenza A virus so as to ensure that the HA-RNA and the NA-RNA in the recombinant influenza A virus have the same sequence as the epidemic influenza A virus.

Further, the strain number of the recombinant influenza A virus is CV2-A/PuertoRico/8/1934 (PR 8) (H1N 1), and the preservation number of the recombinant influenza A virus in China general microbiological culture Collection center is CGMCC No.45374.

In a third aspect, the invention provides a method of constructing a recombinant influenza A virus, said method comprising M1) or/and M2),

M1), introducing the complete set of vectors, a recombinant vector containing an NS protein gene of the influenza A virus, a recombinant vector containing an HA protein gene of the influenza A virus and a recombinant vector containing an NA protein gene of the influenza A virus into cells to obtain recombinant influenza A virus containing the influenza A virus HA and NA;

M2), mixing and culturing the recombinant influenza a virus and the epidemic influenza a virus to obtain the recombinant influenza a virus containing the epidemic influenza a viruses HA and NA.

Further, in the method, the cell may be a packaging cell. In one embodiment of the invention, the method of M1) comprises introducing the vector into MDCK and 293T cells, transfecting chick embryos with a mixed cell culture, and collecting chick embryo allantoic fluid to obtain EggP1 containing a recombinant influenza virus CV-PR8 backbone strain.

In a fourth aspect, the present invention provides the use of the recombinant influenza virus described above or the biological material described above in the preparation of a medicament for the prophylaxis or/and treatment of influenza virus.

In a fifth aspect, the invention provides the use of a recombinant influenza virus as described above or a biological material as described above in the preparation of an influenza vaccine.

In a sixth aspect, the present invention provides an influenza vaccine having the recombinant influenza a virus described above as a backbone strain or as an active ingredient.

In the present invention, the backbone strain may be an influenza virus comprising the above-described PB2 mutein, PB1 mutein, PA mutein, NP mutein and M1 mutein, M2 mutein and corresponding genomes. The HA protein and NA protein and the corresponding genome sequence thereof can be adaptively adjusted according to the type of the epidemic influenza A virus so as to ensure that the influenza vaccine generates an immune effect on the target epidemic influenza A virus strain.

The invention has the beneficial technical effects that

The influenza cold-adaptive vaccine skeleton strain obtained by the invention can be used as a cold-adaptive vaccine skeleton strain, and can be reconstructed with the HA and NA genes of epidemic influenza viruses to prepare the influenza vaccine. At present, the intellectual property and the products of the influenza virus cold adaptation attenuated vaccine framework in China are seriously deficient, and the invention can make up for the deficiency. Meanwhile, the immunity effect of the currently internationally approved influenza virus cold-adaptive attenuated vaccine is reduced, and the safety of the vaccine is questioned by research.

Preservation description

Strain name: influenza virus A

Latin name: influenza A virus A

Strain number: CV2-A/PuertoRico/8/1934 (PR 8) (H1N 1)

Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

The preservation organization is abbreviated as: CGMCC

Address: beijing, chaoyang district North Star, west Lu No. 1, 3

Preservation date: 2022, 12, 19

Accession numbers of the preservation center: CGMCC No.45374.

Drawings

FIG. 1 shows replication ability of constructed CV-PR8 backbone strains at different temperatures.

FIG. 2 shows replication ability of constructed CV2-PR8 cold-adapted vaccine backbone strains under different temperature conditions.

FIG. 3 is a gel electrophoresis chart of PCR amplification products.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

MDCK cells are given benefit from the group of high-quality subjects of the institute of microbiology, academy of China, and document "Song H,Gao GF.Evaluation of the Glycan-Binding and Esterase Activities of Hemagglutinin-Esterase-Fusion Glycoprotein from Influenza D Virus.Methods Mol Biol.2022;2556:187-203.doi:10.1007/978-1-0716-2635-1_15.PMID:36175636." discloses that the above biological materials are available to the public from the applicant, and the obtained biological materials are only used for repeated experiments of the present invention and are not used for other purposes.

293T cells were purchased from basic medical institute of China medical sciences-basic college of Beijing synergetic medical sciences, cell number 1101HUM-PUMC000091, order number: 2021010716891.

MDCK and 293T cells were cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco) and double antibodies (100U/mL penicillin+100. Mu.g/mL streptomycin) at 37℃in an incubator with 5% CO ₂. SPF grade chick embryos 9-11 days old were from Beijing Bolin invagination Hantong Biotechnology Co. The pHW2000 vector in the following examples was constructed from Erich Hoffmann and was disclosed as having the nucleotide sequence of SEQ ID No.19.

The A/Anhui/1/2005 (H5N 1) and A/Hong Kong/2108/2003 (H9N 2) strains in the examples described below synthesized corresponding cDNAs based on genomic information provided by the GISAID accession numbers of the strains, and the corresponding strains were obtained by virus rescue plasmids using reverse genetics techniques with reference to the method of example 1 (both transfection temperature and chick embryo culture temperature were changed to 37 ℃) in example 1.

A/Guangdong-Maonan/SWL1536/2019 (H1N 1) and A/Hong Kong/2671/2019 (H3N 2) -in the examples below are strains provided by the Chinese disease control center.

A/Anhui/1/2013 (H7N 9) in the examples below was stored in this laboratory and was disclosed in paper "Bi Y,Xie Q,Zhang S,Li Y,Xiao H,Jin T,Zheng W,Li J,Jia X,Sun L,Liu J,Qin C,Gao GF,Liu W.Assessment of the internal genes of influenza A(H7N9)virus contributing to high pathogenicity in mice.J Virol.2015Jan;89(1):2-13.doi:10.1128/JVI.02390-14.Epub 2014Oct 15..", which was made available to the public from the applicant in accordance with national biosafety regulations, and was used only for repeated experiments of the present invention and not as other uses.

A/Shenzhen/TH002/2016 (H5N 6) in the examples described below was stored in this laboratory and was disclosed in paper "Bi Y,Tan S,Yang Y,Wong G,Zhao M,Zhang Q,Wang Q,Zhao X,Li L,Yuan J,Li H,Li H,Xu W,Shi W,Quan C,Zou R,Li J,Zheng H,Yang L,Liu WJ,Liu D,Wang H,Qin Y,Liu L,Jiang C,Liu W,Lu L,Gao GF,Liu Y.Clinical and Immunological Characteristics of Human Infections With H5N6 Avian Influenza Virus.Clin Infect Dis.2019Mar 19;68(7):1100-1109." as being available to the public from the applicant in accordance with national biosafety regulations, and was used only in duplicate experiments of the invention and not as a further use.

Example 1 construction and culture of influenza virus CV-PR8 backbone Strain

The CV-PR8 backbone strain was constructed by artificially introducing mutant gene sites PB2 (N265S), PB1 (K391E, E581G, A T), NP (M239L) on the basis of the A/Puerto Rico/8/1934 (PR 8) (H1N 1) vaccine backbone strain (also called WT-PR8, genome publication number GenBank: AB 671295.1) by reverse genetics manipulation system.

1) Acquisition of CV-PR8 plasmid: recombinant plasmids pHW2000-CV-PB2, pPHW-CV-PB 1, pHW2000-CV-NP, pHW2000-CV-PA, pHW2000-CV-M, pHW2000-CV-NS, pHW2000-CV-HA and pHW2000-CV-NA are respectively constructed by taking PR8 virus rescue system gene plasmid pHW2000 (the complete vector sequence of which is SEQ ID No. 19) as a skeleton vector. Wherein CV-PB2 contains a mutation of N268S (CV-PB 2), CV-PB1 contains a mutation of K391E, E581G, A I (CV-PB 1) and CV-NP contains a mutation of M239L (CV-NP), which are all accomplished by the company of Kirschner Biotechnology, inc.

The recombinant plasmid pHW2000-CV-PB2 is a recombinant expression plasmid obtained by replacing fragments between cleavage sites of the backbone plasmid pHW2000 BsmBI with a DNA molecule (cDNA of CV-PB 2) having a nucleotide sequence of SEQ ID No.20, and keeping other nucleotide sequences of the backbone plasmid pHW2000 unchanged. The cDNA of the recombinant plasmid pHW2000-CV-PB2 transferred into cells generates CV-PB2 single-strand negative strand RNA of influenza virus, and the single-strand negative strand RNA is translated to obtain influenza virus protein CV-PB2, wherein the amino acid sequence of the influenza virus protein CV-PB2 is SEQ ID No.25.

The recombinant plasmid pHW2000-CV-PB1 is a recombinant expression plasmid obtained by replacing fragments between cleavage sites of the backbone plasmid pHW2000 BsmBI with a DNA molecule (cDNA of CV-PB 1) having a nucleotide sequence of SEQ ID No.21, and keeping other nucleotide sequences of the backbone plasmid pHW2000 unchanged. The cDNA of the recombinant plasmid pHW2000-CV-PB1 transferred into cells generates CV-PB1 single-strand negative strand RNA of influenza virus, and the single-strand negative strand RNA is translated to obtain influenza virus protein CV-PB1, wherein the amino acid sequence of the influenza virus protein CV-PB1 is SEQ ID No.26.

The recombinant plasmid pHW2000-CV-PA is a recombinant expression plasmid obtained by replacing fragments between cleavage sites of the backbone plasmid pHW2000 BsmBI with a DNA molecule (cDNA of CV-PA) having a nucleotide sequence of SEQ ID No.22, and keeping other nucleotide sequences of the backbone plasmid pHW2000 unchanged. The cDNA of the recombinant plasmid pHW2000-CV-PA transferred into cells generates CV-PA single-strand negative strand RNA of influenza virus, and the single-strand negative strand RNA is translated to obtain influenza virus protein CV-PA, and the amino acid sequence of the influenza virus protein CV-PA is SEQ ID No.27.

The recombinant plasmid pHW2000-CV-NP is a recombinant expression plasmid obtained by replacing a fragment between cleavage sites of the backbone plasmid pHW2000 BsmBI with a DNA molecule (cDNA of CV-NP) having a nucleotide sequence of SEQ ID No.23, and keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. The cDNA of the recombinant plasmid pHW2000-CV-NP is transferred into cells to generate CV-NP single-strand negative strand RNA of influenza virus, and the single-strand negative strand RNA is translated to obtain influenza virus protein CV-NP, and the amino acid sequence of the influenza virus protein CV-NP is SEQ ID No.28.

The recombinant plasmid pHW2000-CV-NS is a recombinant expression plasmid obtained by replacing the fragment between the cleavage sites of the backbone plasmid pHW2000 BsmBI with a DNA molecule (cDNA of CV-NS) having the nucleotide sequence of SEQ ID No.35, and keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. cDNA of the CV-NS after the recombinant plasmid pHW2000-CV-NS is transferred into cells generates CV-NS single-strand negative-strand RNA of influenza virus, and the single-strand negative-strand RNA is translated to obtain influenza virus proteins CV-NS1 and CV-NS2, wherein the amino acid sequence of the influenza virus protein CV-NS1 is SEQ ID No.5, and the amino acid sequence of the influenza virus protein CV-NS2 is SEQ ID No.6.

The recombinant plasmid pHW2000-CV-M is a recombinant expression plasmid obtained by replacing a fragment between cleavage sites of the backbone plasmid pHW2000 BsmBI with a DNA molecule (cDNA of CV-M) having a nucleotide sequence of SEQ ID No.24, and keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. cDNA of the recombinant plasmid pHW2000-CV-M is transferred into cells to generate CV-M single-strand negative strand RNA of influenza virus, and the single-strand negative strand RNA is translated to obtain influenza virus proteins CV-M1 and CV-M2, wherein the amino acid sequences of the influenza virus proteins CV-M1 and CV-M2 are SEQ ID No.29 and SEQ ID No.30 respectively.

The recombinant plasmid pHW2000-CV-HA is a recombinant expression plasmid obtained by replacing fragments between cleavage sites of the backbone plasmid pHW2000 BsmBI with a DNA molecule (CV-HA cDNA) having a nucleotide sequence of SEQ ID No.37, and keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. After the recombinant plasmid pHW2000-CV-HA is transferred into cells, the cDNA of CV-HA is transcribed under the action of pol I to obtain CV-HA single-strand negative strand RNA of influenza virus, the single-strand negative strand RNA is transcribed under the action of pol II to obtain CV-HA positive strand RNA (mRNA) of influenza virus, the CV-HA positive strand RNA of influenza virus is further translated into influenza virus protein CV-HA, and the amino acid sequence of the influenza virus protein CV-HA is SEQ ID No.9.

The recombinant plasmid pHW2000-CV-NA is a recombinant expression plasmid obtained by replacing a fragment between cleavage sites of the backbone plasmid pHW2000 BsmBI with a DNA molecule (cDNA of CV-NA) having a nucleotide sequence of SEQ ID No.38, and keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. After the recombinant plasmid pHW2000-CV-NA is transferred into cells, the cDNA of CV-NA is transcribed under the action of pol I to obtain CV-NA single-strand negative-strand RNA of influenza virus, the single-strand negative-strand RNA is transcribed under the action of pol II to obtain CV-NA positive-strand RNA (mRNA) of influenza virus, the CV-NA positive-strand RNA of influenza virus is further translated into influenza virus protein CV-NA, and the amino acid sequence of the influenza virus protein CV-NA is SEQ ID No.10.

2) Transfection method: MDCK:293T was plated in six-well plates at a ratio of 1:5-10, and transfection experiments were performed after incubation with DMEM containing 10% fetal bovine serum and diabody in an incubator at 37℃for 16-20h with 5% CO ₂. During transfection, recombinant plasmids pHW2000-CV-PB2, pHW2000-CV-PB1, pHW2000-CV-NP, pHW2000-CV-PA, pHW2000-CV-M, pHW-CV-NS, pHW2000-CV-HA, pHW2000-CV-NA and a transfection reagent Lipofectamine 2000 (500 ng of each recombinant plasmid and 10 mu L of the transfection reagent are added into each hole in a six-hole plate) are respectively added into an EP tube containing MEM-Opti (Gibco) and mixed uniformly, standing is carried out for 5min, the mixture is mixed, then the mixture is uniformly added into a six-hole plate and gently shaken to uniformly disperse the mixture, then the mixture is placed at 33 ℃ for 6-8h and then is changed, each hole is added into Opti-MEM containing 1 mu g/mL of TPCK-pancreatin (Sigma-Aldrich), after being placed into a 5% CO ₂ incubator for 72h, two SPF-stage chickens of 9-11 days old are inoculated, each chicken embryo is placed into a 60 ℃ incubator, and then the chicken embryo is placed into a 50-11 th medium for 1% of Peking cage for 1 mu L of biological embryo, namely, after the chicken embryo strain is placed into a limited biological embryo strain for 1-96 h, and the chicken strain is placed into a cage for 1% of biological embryo strain for 1-Peking, and the strain is placed for 1% of biological embryo, after the strain is placed into a cage for 1h, and the cage is placed for the cage is subjected to a limited, and the cage is placed for the cage is cultured.

3) Measurement of growth curves: the growth curve of the strain is measured by using chick embryos, the virus is diluted by 0.1HAU times, the inoculation amount of each chick embryo is 0.1HAU/100 mu L, 9 SPF grade chick embryos of 9-11 days old are inoculated together, 3 chick embryos are respectively placed at 37 ℃, 33 ℃ and 25 ℃, then 300 mu L of chick embryo allantoic fluid is respectively extracted at 24 hours, 48 hours, 72 hours, 96 hours and 120 hours, the blood coagulation titer of the obtained allantoic fluid is respectively measured, the CT value is measured by using a qRT-PCR kit (Vazyme, Q223-01), and the copies (copy number) of the chick embryos are calculated through the CT value (the conversion formula is that copies= -0.2654 x CT value +11.842, if no CT value is directly recorded as 0).

Kit model Vazyme Q-01, nucleotide sequence of primer probe used is as follows (5 '-3'):

IFA-BF：TGGITAAAGACAAGACCAATCYTG；

IFA-BR：TCTACGYTGCWGTCCTCGCTCA；

IFA-BP：TTGTRTTYACGCTCACCGTGCCCAG。

wherein I represents inosine, Y represents T or C, W represents A or T, and R represents G or A.

The results are shown in FIG. 1, wherein the left graph in FIG. 1 shows the hemagglutination titers of allantoic fluids obtained by culturing chick embryos at 37 ℃, 33 ℃ and 25 ℃, and the ordinate shows HA titer and the abscissa shows the culture time of chick embryos; the right panel in FIG. 1 shows the results of measurement of influenza virus copy number of allantoic fluid obtained by culturing chick embryos at 37 ℃, 33 ℃ and 25 ℃, wherein the ordinate indicates the logarithmic value (base 2) of influenza virus copy number per unit volume (mL) and the abscissa indicates the culture time of chick embryos. The results of fig. 1 show that: the constructed CV-PR8 skeleton strain does not have the replication capacity at 37 ℃, can replicate well at 33 ℃, but has poor replication capacity at 25 ℃.

Example 2 Cold-adapted influenza Virus vaccine Strain CV2-PR8 acquisition

It was found that CV-PR8 backbone strains could replicate at 33℃but not at 37℃and 25℃as shown in FIG. 1. Thus, in subsequent experiments, further cold-adapted acclimation passaging was performed in the following manner: the vaccine backbone strain CV-PR8 was serially passaged at 25℃and during passaging we found that the strain gradually gained replicative capacity at 25℃and finally a cold adapted strain CV2-PR8 was obtained that had good replicative capacity at 25 ℃.

During passage, changes in gene loci before, during and after cold adaptation were analyzed by first and second generation sequencing, and as a result, it was found that adaptive mutations occurred in multiple genes with virus adaptation to 25 ℃. Therefore, the genetic locus mutations are determined to endow the vaccine skeleton strain CV2-PR8 with the growth capacity at 25 ℃, an H1N1 subtype influenza virus cold adaptation vaccine skeleton strain Egg P50 suitable for chick embryo propagation is also obtained, and the Egg P50 strain is numbered CV2-A/PuertoRico/8/1934 (PR 8) (H1N 1) and is called CV2-PR8 hereinafter. Furthermore, CV2-PR8 was continuously passaged at 37℃for 5 passages and still could not be grown at 37 ℃. The constructed H1N1 subtype influenza virus cold-adaptation vaccine skeleton strain HAs good genetic stability, can be used as a skeleton strain for constructing a type A cold-adaptation vaccine strain, and can be used for inserting epidemic influenza virus HA and NA genes into the vaccine skeleton strain so as to construct the cold-adaptation vaccine strain aiming at the epidemic influenza virus.

The method comprises the following specific steps:

1) Passaging method obtained by Egg P50: directly carrying out cold adaptation passage at 25 ℃, if the previous generation has no hemagglutination titer, directly inoculating the next generation with stock solution for subculture, wherein the inoculation amount of each chick embryo (9-11 days old) is 100 mu L

The inoculated chick embryo is placed in a incubator at 25 ℃ for culturing for 120 hours, placed in a refrigerator at 4 ℃ for overnight, and then chick embryo allantoic fluid is collected and the hemagglutination titer and CT value of the chick embryo allantoic fluid are measured. If the blood coagulation titer is available, 1HAU/100 mu L and 0.1HAU/100 mu L are inoculated for subculture, the chick embryo allantoic fluid is placed at 4 ℃ overnight after being cultured for 96 hours or 120 hours at 25 ℃, then the blood coagulation titer is measured by 1% chicken blood, and the CT value is measured by a kit (Vazyme Q-01).

2) Measurement of growth curves: diluting virus 0.1HAU times with chick embryo strain growth curve, inoculating 9 chick embryos together at 37 deg.C, 33 deg.C and 25 deg.C, respectively placing 3 chick embryos, extracting chick embryo allantoic fluid 300 μl at 24 hr, 48 hr, 72 hr, 96 hr and 120 hr, and measuring blood coagulation titer of allantoic fluid obtained at each time

3) Measurement of hemagglutination titre, HA titer: samples were diluted 2-fold in 96-well V-bottom plates with 1 x PBS, then an equal volume of 1% chicken suspension red blood cells was added, left to stand at room temperature for 20min, and then the hemagglutination titer of the samples was read by observing whether the chicken red blood cells settled.

The results of measurement of the hemagglutination titers are shown in FIG. 2, in which the HA titers are logarithmic in the coordinates in FIG. 2 and the chick embryo incubation times are on the abscissa. The results of fig. 2 show that: the strain has good replication ability at 33 ℃ and 25 ℃ and poor replication ability at 37 ℃.

4) Mutation site detection of CV2-PR8

The chicken embryo allantoic fluid RNA obtained by inoculating the CV2-PR8 is used as a template (ddH ₂ O is used as a negative control), MBTuni-12 and MBTuni-13 are used as primers, a PCR kit (Vazyme P-01) is used for PCR amplification, a PCR amplification product (an electrophoresis gel diagram is shown below) is obtained, and the amplification product is sent for measurement, and second generation sequencing is carried out. Sequencing results showed that: the CV2-PR8 strain showed mutations as shown in Table 1, compared to the backbone strain CV-PR 8.

PCR amplification primer information:

MBTuni-12 ACg CgT gAT CAg CAA AAg CAg g；

MBTuni-13ACg CgT gAT CAg TAg AAA CAA gg。

the gel diagram of the PCR amplification product is shown in FIG. 3.

Table 1: the adaptive mutation site found in CV2-PR8 (compared to CV-PR 8)

That is, compared to existing strains, the CV2-PR8 strain has the following mutations:

C1 The influenza virus PB2 protein contains mutations of N268S (backbone strain CV-PR 8), Q439H and V731M;

C2 The influenza PB1 protein contains mutations of K391E, E581G, A I (backbone strain CV-PR 8), R187K and M645I;

C3 Influenza virus PA protein contains a mutation of E243V, R269K, E K;

c4 Influenza virus Nucleoprotein (NP) contains mutations E18G, T M and M374I;

c5 Influenza virus M1 protein contains mutations of a 137D;

C6 Influenza virus M2 protein contains a86S mutations.

That is, sequencing results indicate that CV2-PR8 strains express PB2 muteins, PB1 muteins, PA muteins, NP muteins, NS1 proteins, NS2 proteins, M1 muteins, M2 muteins, HA proteins and NA proteins. The PB2 mutein is the protein with the amino acid sequence of SEQ ID No.1, the PB1 mutein is the protein with the amino acid sequence of SEQ ID No.2, the PA mutein is the protein with the amino acid sequence of SEQ ID No.3, the NP mutein is the protein with the amino acid sequence of SEQ ID No.4, the NS1 protein is the protein with the amino acid sequence of SEQ ID No.5, the NS2 protein is the protein with the amino acid sequence of SEQ ID No.6, the M1 mutein is the protein with the amino acid sequence of SEQ ID No.7, the M2 mutein is the protein with the amino acid sequence of SEQ ID No.8, the HA protein is the protein with the amino acid sequence of SEQ ID No.9, and the NA protein is the protein with the amino acid sequence of SEQ ID No. 10.

The genome of CV2-PR8 influenza virus is single-strand negative-strand segmented RNA, the single-strand negative-strand segmented RNA is transcribed to obtain complete set of positive-strand RNA complementary to the single-strand negative-strand segmented RNA, and the complete set of positive-strand RNA comprises PB2-RNA, PB1-RNA, PA-RNA, NP-RNA, NS-RNA, M-RNA, HA-RNA and NA-RNA. The PB2-RNA is an RNA molecule for encoding the PB2 mutant protein, the PB1-RNA is an RNA molecule for encoding the PB1 mutant protein, the PA-RNA is an RNA molecule for encoding the PA mutant protein, and the NP-RNA is an RNA molecule for encoding the NP mutant protein; the NS-RNA is an RNA molecule for encoding the NS1 protein and the NS2 protein, the M-RNA is an RNA molecule for encoding the M1 mutant protein and the M2 mutant protein, the HA-RNA is an RNA molecule for encoding the HA protein, and the NA-RNA is an RNA molecule for encoding the NA protein. That is, the PB2-RNA is an RNA molecule having a nucleotide sequence of SEQ ID No.11, the PB1-RNA is an RNA molecule having a nucleotide sequence of SEQ ID No.12, the PA-RNA is an RNA molecule having a nucleotide sequence of SEQ ID No.13, the NP-RNA is an RNA molecule having a nucleotide sequence of SEQ ID No.14, the NS-RNA is an RNA molecule having a nucleotide sequence of SEQ ID No.15, the M-RNA is an RNA molecule having a nucleotide sequence of SEQ ID No.16, the HA-RNA is an RNA molecule having a nucleotide sequence of SEQ ID No.17, and the NA-RNA is an RNA molecule having a nucleotide sequence of SEQ ID No. 18.

And (3) preserving the CV2-PR8 strain in a common microorganism center of China Committee for culture Collection of microorganisms by a patent program, wherein the preservation date is 2022, 12 and 19 days, and the registration number of the preservation center is CGMCC No.45374.

CV2-PR8 was obtained by rescue according to the method of example 1. Wherein the nucleotide sequence of the cDNA molecule of CV1-PB2 is SEQ ID No.31; the nucleotide sequence of the cDNA molecule of CV1-PB1 is SEQ ID No.32; the nucleotide sequence of the cDNA molecule of CV1-NP is SEQ ID No.33; the nucleotide sequence of the cDNA molecule of CV1-PA is SEQ ID No.34; the nucleotide sequence of the cDNA molecule of CV1-NS is SEQ ID No.35; the nucleotide sequence of the cDNA molecule of CV1-M is SEQ ID No.36; the nucleotide sequence of the cDNA molecule of CV1-HA is SEQ ID No.37; the nucleotide sequence of the DNA molecule of CV1-NA is SEQ ID No.38.

Example 3 CV2-PR8 animal experiments and the formation of novel strains as a backbone

3.1, Test animals:

SPF-grade BALB/c mice, female, 6-7 weeks old, weighing 16-18 g, were purchased from Beijing Fukang Biotech Co.

3.2 Test procedure

Test mice were divided into three groups of 5 mice, the first group was treated with 25. Mu.L of 1 XPBS as a control, the second group was infected with 25. Mu.L of 10 ⁶EID₅₀/. Mu.L of PR8 wild-type influenza strain (WT-PR 8 in example 1), the third group was infected with 25. Mu.L of 10 ⁶EID₅₀/. Mu.L of CV2-PR8 influenza virus, survival was monitored (mice were judged to die and weight loss 25% and above), blood was taken and serum was isolated 14 days after immunization, and HI antibody titer was measured. The 14-day survival of the mice and the HI antibody are shown in the table. The mice in the PBS control group and the CV1-PB1 group have no obvious weight loss within 14 days, the mortality rate is 0 percent, and the mortality rate of the mice in the WT-PR8 group with equal doses reaches 100 percent, which proves that CV2-PR8 achieves the purpose of attenuation and has certain safety. HI antibodies were all higher than 1 after 14 days of immunization: 40, demonstrating the protective effect of antibodies raised after nasal drops of CV2-PR8 (Table 2).

The preparation method of 10 ⁶EID₅₀/50 mu L CV2-PR8 influenza virus comprises the following steps: CV2-PR8 was diluted to 10 ⁶EID₅₀/50. Mu.L with 1 XPBS.

10 ⁶EID₅₀/50. Mu.L of PR8 wild-type influenza strain was prepared with reference to CV2-PR8 influenza virus.

3.3, HI antibody titre determination method

Taking 20 mu L of separated serum in a 1.5mL EP tube, adding 80 mu L of RBD, uniformly mixing, performing the action at 37 ℃ for 16-18 h, performing the inactivation at 56 ℃ for 30min, adding 20% of erythrocyte mud, uniformly mixing, and repeatedly reversing and uniformly mixing at 37 ℃ for 1 h; centrifuging at 1000 Xg to obtain supernatant, and standing at 4deg.C. mu.L of 1 XPBS was added to each well of a 96-well V-type coagulum using an eight-way pipette. Then, 25 mu L of treated serum is respectively added into the 1 st hole of the 96-hole V-shaped blood coagulation plate by a single-channel pipette, the mixture is blown and uniformly mixed, then 25 mu L of the serum is sucked out of the 1 st hole to the 2 nd hole by an eight-channel pipette, the mixture is blown and uniformly mixed, the mixture is diluted to the 11 th hole in turn by a multiple ratio, and 25 mu L of serum is discarded. Wells 12 were negative controlled by adding 25 μl of saline or PBS. Sequentially adding 25 μL of 4HAU virus solution by eight pipettes, standing at room temperature for 15-20 min, and observing the result

And (3) result judgment: the reaction plate is inclined to 45 degrees, and the red blood cells which are deposited at the bottom of the hole flow linearly downwards along the inclined plane are deposited, so that the red blood cells are not or incompletely aggregated by viruses; if the red blood cells at the bottom of the well are spread out to form a uniform thin layer, the red blood cells do not flow after tilting, indicating that the red blood cells are aggregated by viruses or the platelets are placed. The highest dilution that resulted in complete inhibition of agglutination of erythrocytes was used as the endpoint of the assay, i.e., HI titer.

Table 2: results of animal experiments

Strain name	Mortality rate of mice	Weight loss	14Dpi HI antibody titre
				1×PBS	0％	No significant drop was observed	0
Wt	100％	>25％	Total death was undetected for 14 days
				CV1-PR8	0％	No significant drop was observed	5 Mice all >1:40

Example 4 viral construction

4.1 Reverse genetics rescue Virus

MDCK:293T was plated in six-well plates at a ratio of 1:5-10, and transfection experiments were performed after incubation with DMEM containing 10% fetal bovine serum and diabody in an incubator at 37℃for 16-20h with 5% CO ₂. At the time of transfection, recombinant plasmids pHW2000-CV2-PB2、pHW2000-CV2-PB1、pHW2000-CV2-NP、pHW2000-CV2-PA、pHW2000-CV2-M、pHW2000-CV2-NS、pHW2000-HA(, HA, pHW2000-NA (NA of the strain shown in Table 3) and a transfection reagent Lipofectamine 2000 (500 ng of each recombinant plasmid and 10. Mu.L of the transfection reagent were added to each well of a six-well plate) were added to an EP tube containing MEM-Opti (Gibco) and mixed uniformly, and left to stand for 5 minutes, then the two were mixed, the mixture was left to stand for 20 minutes, the mixture was uniformly added to the six-well plate and gently shaken to disperse the mixture uniformly, then left to stand for 6-8 hours at 33℃to change the liquid, each well was added to Opti-MEM containing 1. Mu.g/mL of TPCK-pancreatin (Sigma-Aldrich), two SPF-grade chick embryos 9-11 days old were inoculated after being placed in a culture box at 33℃for 5% CO ₂, each chick embryo was inoculated at 500. Mu.L, and left to stand for 96 hours in a box at 4℃and then the chick embryo was collected and allowed to stand overnight for 1% chick blood (Beijing blood titer limited blood titer).

Preparation of recombinant plasmids pHW2000-CV2-PB2、pHW2000-CV2-PB1、pHW2000-CV2-NP、pHW2000-CV2-PA、pHW2000-CV2-M、pHW2000-CV2-NS、pHW2000-HA( HA, pHW2000-NA (NA of the strains shown in Table 3) of the strains shown in Table 3 reference example 1, except that the cDNA was replaced correspondingly. Among them, cDNA molecules of HA and NA can be directly synthesized with reference to genomic information of the strains shown in Table 3.

4.2 Obtaining reassortant Virus from Natural infection

CV2-PR8 is diluted to 10 ^-3, and is respectively mixed with strains in equal volume, chick embryos are inoculated, each chick embryo is inoculated with 200 mu L, and the chick embryos are placed in a temperature box at 37 ℃ for culturing for 72 hours, and chick embryo allantoic fluid is collected. The harvested chick embryo allantoic fluid was mixed with equal volumes of serum against PR8, neutralized at 37℃for 2 hours, then inoculated with chick embryos (100. Mu.L/piece), incubated at 25℃for 96 hours, then chick embryo allantoic fluid was harvested, continuously neutralized with antisera and then inoculated with chick embryos, and the hemagglutination titer (1% chick blood cells) was measured for a total of 4 antisera cycles.

The preparation method of the PR 8-resistant serum comprises the following steps: after the beta-propiolactone is inactivated by WT-PR8, the mixture is uniformly mixed with an aluminum adjuvant according to the method carried by instructions, and emulsified, and rabbits (New Zealand white rabbits and 70 days old) are immunized by intramuscular injection, and blood is taken after multiple immunization to separate serum. Specifically, the injection volume of each rabbit is 500 mu L-1mL, and the immune dose for inactivating WT-PR8 in primary immunization is 100 mu g/rabbit. Two weeks after the first immunization, the inactivated WT-PR8 was mixed with an aluminum adjuvant to boost the immunization at an immunization dose of 100. Mu.g/dose of inactivated WT-PR 8. Boost was performed every two weeks for 4 total boosts. And (3) taking blood from rabbits at an immunization interval, taking about 5mL each time, centrifuging at 2000rpm for 5min, and taking supernatant to obtain the immune serum of the WT-PR8 immune group. H I the potency is more than or equal to 1 and 2 and 80, and blood can be completely collected.

Table 3: influenza a strain information

In the invention, the transformation is carried out on the existing inactivated vaccine skeleton strain A/Puerto Rico/8/34 (H1N 1) adapted to the chick embryo as a basic virus strain, and the H1N1 subtype cold-adapted vaccine skeleton strain suitable for chick embryo propagation is constructed by a technical method of combining reverse genetics technology with cold-adapted passage, so that technical support is provided for producing cold-adapted vaccine on the basis of not changing chick embryo vaccine production line, and the possibility of actual production of vaccine conversion is improved. The cold adaptation sites discovered in the invention can greatly weaken the replication capacity of strains at 37 ℃, and can ensure that the strains replicate well at low temperature (33 ℃ and 25 ℃), and the replication capacity under the three temperature conditions meets the requirement of the cold adaptation attenuated influenza vaccine on the replication capacity at different temperatures, so that the strains containing the sites have the potential of becoming attenuated live vaccine skeletons.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims

1. A biomaterial for use in constructing a recombinant influenza a virus, characterized in that: the biological material is selected from any one of the following B1) -B7):

The PB2 mutein is selected from A1) or A2):

a1 A protein having an amino acid sequence of SEQ ID No. 1;

the PB1 mutein is selected from A3) or A4):

A3 A protein having an amino acid sequence of SEQ ID No. 2;

The PA mutein is selected from A5) or A6):

a5 A protein having an amino acid sequence of SEQ ID No. 3;

The NP mutein is selected from A7) or A8):

A7 A protein having an amino acid sequence of SEQ ID No. 4;

the M1 mutein is selected from A9) or a 10):

A9 A protein having an amino acid sequence of SEQ ID No. 7;

the M2 mutein is selected from a 11) or a 12):

A11 A protein having an amino acid sequence of SEQ ID No. 8;

b2 A set of DNA molecules encoding the protein composition of B1), said set of DNA molecules comprising a DNA molecule encoding the PB2 mutein, a DNA molecule encoding the PB1 mutein, a DNA molecule encoding the PA mutein, a DNA molecule encoding the NP mutein and a DNA molecule encoding the M1 mutein and M2 mutein,

B3A kit of vectors comprising B3 a), B3B), B3 c), B3 d) and B3 e) described below,

B3 a), a recombinant vector comprising a DNA molecule encoding said PB2 mutein;

B3B), a recombinant vector comprising a DNA molecule encoding said PB1 mutein;

b3 c), a recombinant vector comprising a DNA molecule encoding said PA mutein;

b3 d), a recombinant vector containing a DNA molecule encoding said NP mutein;

2. The biomaterial according to claim 1, characterized in that:

the nucleotide sequence of the DNA molecule encoding the PB2 mutein is SEQ ID No.31;

the nucleotide sequence of the DNA molecule encoding the PB1 mutein is SEQ ID No.32;

the nucleotide sequence of the DNA molecule encoding the PA mutein is SEQ ID No.33;

the nucleotide sequence of the DNA molecule encoding the NP mutein is SEQ ID No.34;

The nucleotide sequence of the DNA molecule encoding the M1 mutein and the M2 mutein is SEQ ID No.36.

3. A recombinant influenza a virus characterized by: the recombinant influenza a virus expresses or contains the protein composition of claim 1.

4. A recombinant influenza a virus according to claim 3, characterized in that: the genome of the recombinant influenza A virus is single-strand negative-strand segmented RNA, the single-strand negative-strand segmented RNA is transcribed to obtain complete positive-strand RNA complementary to the single-strand negative-strand segmented RNA, the complete positive-strand RNA comprises PB2-RNA, PB1-RNA, PA-RNA, NP-RNA and M-RNA,

The PB2-RNA is an RNA molecule encoding the PB2 mutein of claim 1;

the PB1-RNA is an RNA molecule encoding the PB1 mutein of claim 1;

The PA-RNA is an RNA molecule encoding the PA mutein of claim 1;

the NP-RNA is an RNA molecule encoding the NP mutein of claim 1;

the M-RNA is an RNA molecule encoding the M1 mutein and the M2 mutein of claim 1.

5. The recombinant influenza a virus of claim 4, wherein:

The PB2-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 11;

The PB1-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 12;

The PA-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 13;

the NP-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 14;

The M-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 16.

6. The recombinant influenza a virus of any one of claims 3-5, wherein: the strain number of the recombinant influenza A virus is CV2-A/PuertoRico/8/1934 (PR 8) (H1N 1), and the preservation number of the recombinant influenza A virus in China general microbiological culture Collection center is CGMCC No.45374.

7. The construction method of the recombinant influenza A virus is characterized by comprising the following steps: the method comprises M1) or/and M2),

M1), introducing the set of vectors of claim 1, a recombinant vector comprising a gene encoding an NS protein of influenza a virus, a recombinant vector comprising a gene encoding an HA protein of influenza a virus, and a recombinant vector comprising a gene encoding an NA protein of influenza a virus into cells to obtain a recombinant influenza a virus comprising HA and NA of influenza a virus;

M2), mixing and culturing the recombinant influenza a virus according to any one of claims 3-6 with an epidemic influenza a virus to obtain a recombinant influenza a virus comprising said epidemic influenza a viruses HA and NA.

8. Use of the biomaterial of claim 1 or 2 or the recombinant influenza virus of any one of claims 3-6 in the manufacture of a medicament for the prevention or/and treatment of influenza virus.

9. Use of the biomaterial of claim 1 or 2 or the recombinant influenza virus of any one of claims 3-6 in the preparation of an influenza vaccine.

10. An influenza vaccine characterized in that: the influenza vaccine has the recombinant influenza virus of any one of claims 3 to 6 as a backbone strain or as an active ingredient.