Cold-adapted vaccine backbone strain CV1-PR8 of H1N1 influenza virus, its construction method and application

Abstract

The present application discloses a cold-adapted vaccine backbone strain CV1-PR8 of H1N1 influenza virus, its construction method and application, belonging to the technical field of medical preparations. The technical problem to be solved by the present invention is: how to prepare a cold-adapted influenza strain for the preparation of influenza vaccines. To solve the above technical problem, the present invention provides a recombinant H1N1 influenza virus, characterized in that: the recombinant H1N1 influenza virus expresses or contains PB2 mutant protein, PB1 mutant protein, PA mutant protein, NP mutant protein and NS mutant protein. Its preservation number for patent procedures at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 45373. The influenza cold-adapted vaccine backbone strain CV1-PR8 obtained by the present invention can be used as a cold-adapted vaccine backbone strain to reconstruct influenza vaccines with the HA and NA genes of epidemic influenza viruses.

Description

Technical Field

[0001] This application belongs to the technical field of medical preparations, and specifically relates to the cold-adapted vaccine backbone strain CV1-PR8 of H1N1 influenza virus, its construction method and application. Background Art

[0002] Influenza virus is a segmented single-stranded negative-strand RNA virus, divided into four types: A, B, C, and D. Currently, the main influenza viruses prevalent in the population are the H1N1 and H3N2 subtypes of type A and the Yamagata and Victoria seasonal influenza viruses of type B. As of now, influenza viruses have caused four pandemics, namely the H1N1 subtype in 1918, the H2N2 subtype in 1957, the H3N2 subtype in 1968, and the H1N1 subtype influenza in 2009, each bringing great harm to the lives and health of people all over the world. Since the emergence of influenza, seasonal influenza has prevailed and occurred every year globally, bringing a serious disease burden to society.

[0003] Influenza vaccine is currently the most effective way to prevent influenza virus infection and prevalence. There are mainly several types of influenza vaccines, such as inactivated influenza vaccines and live attenuated vaccines. Inactivated vaccines are the vaccines we currently use more. Currently, the inactivated influenza vaccine strains mainly use the internal 6 gene segments of the chicken embryo-adapted A / Puerto Rico / 8 / 1934 (PR8) (H1N1) backbone strain, and the HA and NA gene segments of the epidemic strains recommended by WHO, which are constructed by reassortment. This type of vaccine mainly exerts its effect by inducing humoral immunity in the body, showing the characteristics of short protection time and poor cross-protection effect.

[0004] Currently, there are two main donor backbones for the live attenuated vaccines of influenza A virus that have been approved, namely A / Leningrad / 17 / 57 H2N2 LAIV (Len LAIV) and A / Ann Arbor / 6 / 60 H2N2 (AA / 60). The live attenuated vaccine is constructed by reassorting the 6 internal genes of the donor strain with the HA and NA genes of the epidemic virus recommended by WHO. The live attenuated vaccine is mainly inoculated by nasal spray, mimicking the natural infection route, and can simultaneously induce the host to produce humoral immunity, cellular immunity, and immune response. Moreover, the cellular immune response plays an important role in cross-immune protection against different subtype viruses.

[0005] However, current research shows that the effectiveness of the approved live attenuated vaccines has declined. This decline in immunogenicity is likely caused by a large difference between the vaccine backbone strains used and the currently prevalent strains. The H2N2 subtype influenza virus has disappeared from the population, and the currently prevalent ones are mainly the H1N1 and H3N2 subtype influenza viruses. In addition, the phenomenon of vaccine strain mutation has been found during the production process of the live attenuated vaccine and in children vaccinated with the live attenuated vaccine, which also raises questions about the safety of the currently used live attenuated vaccine. All these phenomena suggest that a new backbone for the live attenuated influenza vaccine needs to be constructed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is: how to prepare cold-adapted influenza strains as cold-adapted vaccine backbone strains for the preparation of influenza vaccines.

[0007] To solve the above technical problem, in the first aspect, the present invention provides a recombinant influenza A virus, characterized in that: the recombinant influenza A virus expresses or contains PB2 mutant protein, PB1 mutant protein, PA mutant protein, NP mutant protein and NS mutant protein,

[0008] The PB2 mutant protein is selected from A1) or A2):

[0009] A1), a protein with the amino acid sequence of SEQ ID No.1;

[0010] A2), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1);

[0011] The PB1 mutant protein is selected from A3) or A4):

[0012] A3), a protein with the amino acid sequence of SEQ ID No.2;

[0013] A4), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A3);

[0014] The PA mutant protein is selected from A5) or A6):

[0015] A5), a protein with the amino acid sequence of SEQ ID No.3;

[0016] A6), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A5);

[0017] The NP mutant protein is selected from A7) or A8):

[0018] A7), a protein with the amino acid sequence of SEQ ID No.4;

[0019] A8), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A7);

[0020] The NS mutant protein is selected from A9) or A10):

[0021] A9), a protein with an amino acid sequence of SEQ ID No. 5 and / or a protein with an amino acid sequence of SEQ ID No. 6;

[0022] A10), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A9).

[0023] Furthermore, the recombinant influenza A virus further includes an M protein, an HA protein, and an NA protein.

[0024] The M protein includes an M1 protein and an M2 protein. The M1 protein is selected from A11) or A12):

[0025] A11), a protein with an amino acid sequence of SEQ ID No. 7;

[0026] A12), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A11);

[0027] The M2 protein is selected from A13) or A14):

[0028] A13), a protein with an amino acid sequence of SEQ ID No. 8;

[0029] A14), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A13);

[0030] The HA protein is selected from A15) or A16):

[0031] A15), a protein with an amino acid sequence of SEQ ID No. 9;

[0032] A16), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A15);

[0033] The NA protein is selected from A17) or A18):

[0034] A17), a protein with an amino acid sequence of SEQ ID No. 10;

[0035] A18), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A17).

[0036] Furthermore, the HA protein and NA protein in the recombinant influenza A virus can also be adaptively adjusted according to the type of epidemic influenza A virus to ensure that the HA protein and NA protein in the recombinant influenza A virus are the same as those in the epidemic influenza A virus.

[0037] Furthermore, the genome of the recombinant influenza A virus is single-stranded negative-strand, segmented RNA. The single-stranded negative-strand, segmented RNA transcribes to obtain a set of positive-strand RNAs complementary to the single-stranded negative-strand, segmented RNA. The set of positive-strand RNAs includes PB2-RNA, PB1-RNA, PA-RNA, NP-RNA, and NS-RNA. The PB2-RNA is an RNA molecule encoding the above-mentioned PB2 mutant protein; the PB1-RNA is an RNA molecule encoding the above-mentioned PB1 mutant protein; the PA-RNA is an RNA molecule encoding the above-mentioned PA mutant protein; the NP-RNA is an RNA molecule encoding the above-mentioned NP mutant protein; the NS-RNA is an RNA molecule encoding the above-mentioned NS mutant protein.

[0038] Furthermore, in the recombinant influenza A virus, the set of positive-strand RNAs further includes M-RNA, HA-RNA, and NA-RNA. The M-RNA is an RNA molecule encoding the above-mentioned M1 protein and M2 protein; the HA-RNA is an RNA molecule encoding the above-mentioned HA protein; the NA-RNA is an RNA molecule encoding the above-mentioned NA protein.

[0039] Furthermore, 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 NS-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 15.

[0040] Furthermore, in the recombinant influenza A virus, the M-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 16; 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.

[0041] Furthermore, in the recombinant influenza A virus, the HA-RNA and NA-RNA can also be adaptively adjusted according to the variation of influenza virus, for example, being the same as the sequence of epidemic variant strains.

[0042] Furthermore, the recombinant influenza A virus is influenza A virus, with the strain number CV1-A / Puerto Rico / 8 / 1934 (PR8) (H1N1), and the deposit number in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 45373.

[0043] To solve the above technical problems, in a second aspect, the present invention provides biological materials related to the recombinant influenza A virus, and the biological materials are selected from any one of the following:

[0044] E1), a set of DNA molecules encoding the set of positive-strand RNAs of the above recombinant influenza A virus, and the set of DNA molecules includes a DNA molecule encoding PB2-RNA, a DNA molecule encoding PB1-RNA, a DNA molecule encoding PA-RNA, a DNA molecule encoding NP-RNA, and a DNA molecule encoding NS-RNA.

[0045] E2), a set of vectors, including the following E2a), E2b), E2c), E2d) and E2e).

[0046] E2a), a recombinant vector containing a DNA molecule encoding PB2-RNA;

[0047] E2b), a recombinant vector containing a DNA molecule encoding PB1-RNA;

[0048] E2c), a recombinant vector containing a DNA molecule encoding PA-RNA;

[0049] E2d), a recombinant vector containing a DNA molecule encoding NP-RNA;

[0050] E2e), a recombinant vector containing a DNA molecule encoding NS-RNA;

[0051] E3), a microorganism containing the above recombinant influenza A virus;

[0052] E4), an animal cell line containing the above recombinant influenza A virus;

[0053] E5), an animal tissue containing the above recombinant influenza A virus;

[0054] E6), an animal organ containing the above recombinant influenza A virus.

[0055] Furthermore, in the biological materials, in E1), the set of DNA molecules further includes a DNA molecule encoding M-RNA, a DNA molecule encoding HA-RNA, and a DNA molecule encoding NA-RNA.

[0056] Further, in the biological material, E2) the set of vectors further includes: E2f), E2g), and E2h),

[0057] E2f) a recombinant vector containing a DNA molecule encoding the M-RNA;

[0058] E2g) a recombinant vector containing a DNA molecule encoding the HA-RNA;

[0059] E2h) a recombinant vector containing a DNA molecule encoding the NA-RNA.

[0060] Further, in the biological material, E2a) the nucleotide sequence of the DNA molecule encoding PB2-RNA is SEQ ID No. 31; E2b) the nucleotide sequence of the DNA molecule encoding PB1-RNA is SEQ ID No. 32; E2c) the nucleotide sequence of the DNA molecule encoding PA-RNA is SEQ ID No. 33; E2d) the nucleotide sequence of the DNA molecule encoding NP-RNA is SEQ ID No. 34; E2e) the nucleotide sequence of the DNA molecule encoding NS-RNA is SEQ ID No. 35.

[0061] Further, in the biological material, the nucleotide sequence of the DNA molecule encoding M-RNA is SEQ ID No. 36; the nucleotide sequence of the DNA molecule encoding HA-RNA is SEQ ID No. 37; the nucleotide sequence of the DNA molecule encoding NA-RNA is SEQ ID No. 38.

[0062] Further, in the biological material, the DNA molecule encoding HA-RNA and the DNA molecule encoding NA-RNA can also be adaptively adjusted according to the type of influenza A virus, so as to ensure that the recombinant influenza virus HA and NA obtained by preparing the biological material are the same as the influenza A virus.

[0063] To solve the above technical problems, in the third aspect, the present invention provides a method for constructing the recombinant influenza A virus, and the method includes M1) or / and M2):

[0064] M1) introducing the set of vectors described in claim 5, a recombinant vector containing a gene encoding the M protein of influenza A virus, a recombinant vector containing a gene encoding the HA protein of influenza A virus, and a recombinant vector containing a gene encoding the NA protein of influenza A virus into cells to obtain a recombinant influenza A virus containing the HA and NA of influenza A virus;

[0065] M2) Mix and culture the recombinant influenza A virus described in any one of claims 1-4 with an epidemic influenza A virus to obtain a recombinant influenza A virus containing the HA and NA of the epidemic influenza A virus.

[0066] Further, in the method of M1), the cell may be a packaging cell. In an embodiment of the present invention, the method of M1) includes introducing the above vector into MDCK and 293T cells, transfecting the cell mixed culture into chicken embryos, and then collecting the allantoic fluid of the chicken embryos to obtain Egg P1 containing the recombinant influenza virus CV-PR8 backbone strain.

[0067] In the present invention, the vector may be a plasmid of an influenza virus reverse genetics rescue system, such as a virus rescue system plasmid. In an embodiment of the present invention, the virus rescue system plasmid is pHW2000. The cDNA of each segment genome of the influenza virus is constructed into the virus rescue system plasmid to obtain a recombinant vector. When all the segment plasmids are co-transfected into cells, the recombinant vector can generate the genomic RNA of the influenza virus and also generate the influenza virus protein, thereby obtaining a recombinant influenza virus.

[0068] In the recombinant influenza virus, the DNA molecule encoding HA-RNA and the DNA molecule encoding NA-RNA can be adaptively adjusted according to the type of the epidemic influenza A virus to ensure that the HA protein and NA protein in the recombinant influenza A virus are the same as those of the epidemic influenza A virus.

[0069] To solve the above technical problems, in the fourth aspect, the present invention provides the application of the above recombinant influenza virus or the above biological material in the preparation of a drug for treating and / or preventing influenza virus.

[0070] To solve the above technical problems, in the fifth aspect, the present invention provides the application of the above recombinant influenza virus or the above biological material in the preparation of an influenza vaccine.

[0071] To solve the above technical problems, in the sixth aspect, the present invention provides an influenza vaccine, which uses the above recombinant influenza A virus as a backbone strain or uses the above recombinant influenza A virus as an active ingredient.

[0072] In the present invention, the backbone strain may be an influenza virus containing the above PB2 mutant protein, PB1 mutant protein, PA mutant protein, NP mutant protein, and NS mutant protein and the corresponding genome. The HA protein, NA protein, and their corresponding genomic sequences can be adaptively adjusted according to the variation of the virus to ensure that the influenza vaccine also produces an immune effect on the mutated influenza strains.

[0073] In the present invention, the protein tag refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag, SUMO protein tag, etc.

[0074] Beneficial technical effects achieved by the present invention

[0075] The influenza cold-adapted vaccine backbone strain CV1-A / Puerto Rico / 8 / 1934 (PR8) (H1N1) (abbreviated as CV1-PR8) obtained in the present invention can be used as a cold-adapted vaccine backbone strain to reconstruct influenza vaccines by recombining with the HA and NA genes of epidemic influenza viruses. Currently, there is a serious shortage of intellectual property rights and products for influenza virus cold-adapted attenuated vaccine backbones in China, and the present invention can fill this gap. At the same time, the immune effect of internationally approved influenza virus cold-adapted attenuated vaccines has a downward trend, and some studies have questioned the safety of this vaccine. The present invention provides more options for influenza virus cold-adapted attenuated vaccine backbone strains.

[0076] Collection Instructions

[0077] Species name: Influenza A virus

[0078] Latin name: Influenza A virus

[0079] Strain number: CV1-A / Puerto Rico / 8 / 1934 (PR8) (H1N1)

[0080] Depository institution: General Microbiological Center of China Committee for Culture Collection of Microorganisms

[0081] Abbreviation of depository institution: CGMCC

[0082] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0083] Date of deposit: December 19, 2022

[0084] Registration number of the depository center: CGMCC No. 45373. Description of the drawings

[0085] Figure 1 Replication ability of the constructed CV-PR8 backbone strain at different temperatures.

[0086] Figure 2The replication ability of the constructed CV1-PR8 cold-adapted vaccine backbone strain under different temperature conditions.

[0087] Figure 3 It is the gel electrophoresis diagram of the PCR amplification product. Specific implementation manners

[0088] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0089] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0090] The MDCK cells were kindly provided by the group of Gao Fu at the Institute of Microbiology, Chinese Academy of Sciences, and were disclosed in the literature "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." The public can obtain the above biological materials from the applicant. The obtained above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0091] The 293T cells were purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences - School of Basic Medicine, Peking Union Medical College, with the cell number 1101HUM-PUMC000091 and the order number: 2021010716891.

[0092] The MDCK and 293T cells were cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco) and double antibodies (100 U / mL penicillin + 100 μg / mL streptomycin) at 37°C and 5% CO 2Cultivation was carried out in an incubator. SPF chicken embryos at 9 - 11 days old were from Beijing Boehringer Ingelheim Tong Biotechnology Co., Ltd. In the following examples, the pHW2000 vector was constructed and disclosed by Erich Hoffmann, and its nucleotide sequence is SEQ ID No.19.

[0093] For the A / Anhui / 1 / 2005(H5N1) and A / Hong Kong / 2108 / 2003(H9N2) strains in the following examples, the corresponding cDNA was synthesized according to the genomic information provided by the GISAID accession number of the strains, and the corresponding strains were obtained by virus rescue plasmids using reverse genetics technology with reference to the method in Example 1 (the transfection temperature and chicken embryo culture temperature in Example 1 were both changed to 37°C).

[0094] The A / Guangdong-Maonan / SWL1536 / 2019(H1N1) and A / Hong Kong / 2671 / 2019(H3N2) in the following examples were strains provided by the Chinese Center for Disease Control and Prevention.

[0095] The A / Anhui / 1 / 2013 (H7N9) in the following examples was stored in this laboratory and was disclosed in the 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 G F, Liu W. Assessment of the internal genes of influenza A (H7N9) virus contributing to high pathogenicity in mice. J Virol. 2015 Jan;89(1):2 - 13. doi: 10.1128 / JVI.02390 - 14. Epub 2014 Oct 15..". The public can obtain this biological material from the applicant in accordance with the relevant regulations on national biosafety. The obtained above biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0096] A / Shenzhen / TH002 / 2016 (H5N6) in the following embodiments is stored in this laboratory and is disclosed in the 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. 2019 Mar 19;68(7):1100-1109." The public can obtain this biological material from the applicant in accordance with the relevant regulations of national biosafety. The obtained above-mentioned biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0097] Example 1. Construction and culture of the influenza virus CV-PR8 backbone strain

[0098] Based on the A / Puerto Rico / 8 / 1934 (PR8) (H1N1) vaccine backbone strain (also known as WT-PR8, the public accession number of the genome GenBank: AB671295.1), mutant gene loci PB2 (N265S), PB1 (K391E, E581G, A661T), and NP (M239L) were artificially introduced through a reverse genetics operating system to construct the CV-PR8 backbone strain.

[0099] 1), Obtaining the CV-PR8 plasmid: Using the PR8 virus rescue system gene plasmid pHW2000 (the full vector sequence is SEQ ID No. 19) as the backbone vector, recombinant plasmids pHW2000-CV-PB2, pHW2000-CV-PB1, pHW2000-CV-NP, pHW2000-CV-PA, pHW2000-CV-M, pHW2000-CV-NS, pHW2000-CV-HA, and pHW2000-CV-NA were constructed respectively. Among them, CV-PB2 contains the mutation N265S (CV-PB2), CV-PB1 contains the mutations K391E, E581G, and A661I (CV-PB1), and CV-NP has the mutation M239L (CV-NP). These point mutations were all completed by GenScript Biotech Corporation.

[0100] The recombinant plasmid pHW2000-CV-PB2 was obtained by replacing the fragment between the BsmBI cleavage sites of the backbone plasmid pHW2000 with a DNA molecule with the nucleotide sequence SEQ ID No. 20 (the cDNA of CV-PB2), while keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. After the recombinant plasmid pHW2000-CV-PB2 was transfected into cells, the cDNA of CV-PB2 could be transcribed to obtain the single-stranded negative-strand RNA of the influenza virus CV-PB2, and translated to generate the influenza virus protein CV-PB2. The amino acid sequence of the influenza virus protein CV-PB2 is SEQ ID No. 25.

[0101] The recombinant plasmid pHW2000-CV-PB1 was obtained by replacing the fragment between the BsmBI cleavage sites of the backbone plasmid pHW2000 with a DNA molecule with the nucleotide sequence SEQ ID No. 21 (the cDNA of CV-PB1), while keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. After the recombinant plasmid pHW2000-CV-PB1 was transfected into cells, the cDNA of CV-PB1 was transcribed to obtain the single-stranded negative-strand RNA of the influenza virus CV-PB1, and translated to obtain the influenza virus protein CV-PB1. The amino acid sequence of the influenza virus protein CV-PB1 is SEQ ID No. 26.

[0102] The recombinant plasmid pHW2000-CV-PA is a recombinant expression plasmid obtained by replacing the fragment between the BsmBI restriction enzyme cleavage sites of the backbone plasmid pHW2000 with a DNA molecule (the cDNA of CV-PA) having a nucleotide sequence of SEQ ID No. 22, while keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. After the recombinant plasmid pHW2000-CV-PA is transfected into cells, the cDNA of CV-PA transcribes to obtain the single-stranded negative-sense RNA of influenza virus CV-PA, and translates to generate the influenza virus protein CV-PA, and the amino acid sequence of the influenza virus protein CV-PA is SEQ ID No. 27.

[0103] The recombinant plasmid pHW2000-CV-NP is a recombinant expression plasmid obtained by replacing the fragment between the BsmBI restriction enzyme cleavage sites of the backbone plasmid pHW2000 with a DNA molecule (the cDNA of CV-NP) having a nucleotide sequence of SEQ ID No. 23, while keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. After the recombinant plasmid pHW2000-CV-NP is transfected into cells, the cDNA of CV-NP transcribes to obtain the single-stranded negative-sense RNA of influenza virus CV-NP, and translates to generate the influenza virus protein CV-NP, and the amino acid sequence of the influenza virus protein CV-NP is SEQ ID No. 28.

[0104] The recombinant plasmid pHW2000-CV-NS is a recombinant expression plasmid obtained by replacing the fragment between the BsmBI restriction enzyme cleavage sites of the backbone plasmid pHW2000 with a DNA molecule (the cDNA of CV-NS) having a nucleotide sequence of SEQ ID No. 24, while keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. After the recombinant plasmid pHW2000-CV-NS is transfected into cells, the cDNA of CV-NS transcribes to obtain the single-stranded negative-sense RNA of influenza virus CV-NS, and translates to generate the influenza virus proteins CV-NS1 and CV-NS2, the amino acid sequence of the influenza virus protein CV-NS1 is SEQ ID No. 29, and the amino acid sequence of the influenza virus protein CV-NS2 is SEQ ID No. 30.

[0105] The recombinant plasmid pHW2000-CV-M is a recombinant expression plasmid obtained by replacing the fragment between the BsmBI restriction enzyme sites of the backbone plasmid pHW2000 with a DNA molecule (the cDNA of CV-M) having a nucleotide sequence of SEQ ID No. 36, while keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. After the recombinant plasmid pHW2000-CV-M is transfected into cells, the cDNA of CV-M transcribes to obtain the single-stranded negative-sense RNA of influenza virus CV-M, and translates to generate influenza virus proteins CV-M1 and CV-M2. The amino acid sequences of influenza virus proteins CV-M1 and CV-M2 are SEQ ID No. 7 and SEQ ID No. 8, respectively.

[0106] The recombinant plasmid pHW2000-CV-HA is a recombinant expression plasmid obtained by replacing the fragment between the BsmBI restriction enzyme sites of the backbone plasmid pHW2000 with a DNA molecule (the cDNA of CV-HA) having a nucleotide sequence of SEQ ID No. 37, while keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. After the recombinant plasmid pHW2000-CV-HA is transfected into cells, the cDNA of CV-HA transcribes to obtain the single-stranded negative-sense RNA of influenza virus CV-HA, and translates to generate influenza virus protein CV-HA. The amino acid sequence of influenza virus protein CV-HA is SEQ ID No. 9.

[0107] The recombinant plasmid pHW2000-CV-NA is a recombinant expression plasmid obtained by replacing the fragment between the BsmBI restriction enzyme sites of the backbone plasmid pHW2000 with a DNA molecule (the cDNA of CV-NA) having a nucleotide sequence of SEQ ID No. 38, while keeping the other nucleotide sequences of the backbone plasmid pHW2000 unchanged. After the recombinant plasmid pHW2000-CV-NA is transfected into cells, the cDNA of CV-NA transcribes to obtain the single-stranded negative-sense RNA of influenza virus CV-NA, and translates to generate influenza virus protein CV-NA. The amino acid sequence of influenza virus protein CV-NA is SEQ ID No. 10.

[0108] 2). Transfection method: MDCK:293T are seeded in a six-well plate at a ratio of 1:5 - 10, and cultured in DMEM containing 10% fetal bovine serum and double antibiotics at 37 °C and 5% CO 2After culturing in an incubator at a certain temperature for 16 - 20 h, transfection experiments were carried out. During transfection, the recombinant plasmids pHW2000 - CV - PB2, pHW2000 - CV - PB1, pHW2000 - CV - NP, pHW2000 - CV - PA, pHW2000 - CV - M, pHW2000 - CV - NS, pHW2000 - CV - HA and pHW2000 - CV - NA and the transfection reagent Lipofectamine 2000 (500 ng of each recombinant plasmid and 10 μL of the transfection reagent were added to each well of a six - well plate) were respectively added to an EP tube containing MEM - opti (Gibco) and mixed evenly. After standing for 5 min, the two were mixed. After standing the mixture for 20 min, the mixture was evenly added to the six - well plate and gently shaken to disperse the mixture evenly. Then, after placing it at 33°C for 6 - 8 h, the medium was changed. 1 μg / mL of TPCK - trypsin (Sigma - Aldrich) in Opti - MEM was added to each well, and it was placed at 33°C and 5% CO 2 After culturing in an incubator for 72 h, two 9 - 11 - day - old SPF - grade chicken embryos were inoculated, 500 μL was inoculated into each chicken embryo, and they were placed in an incubator at 33°C for 96 h and then placed at 4°C overnight. Then, the allantoic fluid of the chicken embryos was collected and its hemagglutination titer was measured using 1% chicken blood (Beijing Hute Biotechnology Co., Ltd.). If there was a hemagglutination titer, Egg P1 containing the recombinant influenza virus CV - PR8 backbone strain was obtained.

[0109] 3) Determination of the growth curve: The growth curve of the virus strain was measured using chicken embryos. The virus was diluted 0.1 HAU times, and the inoculation amount for each chicken embryo was 0.1 HAU / 100 μL. A total of 9 9 - 11 - day - old SPF - grade chicken embryos were inoculated, and 3 chicken embryos were placed at 37°C, 33°C, and 25°C respectively. Then, 300 μL of the allantoic fluid of the chicken embryos was extracted at 24 h, 48 h, 72 h, 96 h, and 120 h respectively. The hemagglutination titer of the extracted allantoic fluid was measured, and the CT value was measured using a qRT - PCR kit (Vazyme, Q223 - 01), and its copies (copy number) was calculated through the CT value (conversion formula: copies=-0.2654*CT value + 11.842. If there was no CT value, it was directly recorded as 0).

[0110] Kit model: Vazyme Q223 - 01

[0111] The nucleotide sequences of the primers and probes used are as follows (5’ - 3’):

[0112] IFA - BF: TGGITAAAGACAAGACCAATCYTG;

[0113] IFA-BR: TCTACGYTGCWGTCCTCGCTCA;

[0114] IFA-BP: TTGTRTTYACGCTCACCGTGCCCAG.

[0115] Wherein I represents inosine, Y represents T or C, W represents A or T, and R represents G or A.

[0116] The results are as Figure 1 shown, Figure 1 in the left attached figure, the hemagglutination titers of allantoic fluid taken from chicken embryos cultured at 37 °C, 33 °C and 25 °C are shown, where the vertical coordinate is the HA titer and the horizontal coordinate is the culture time of the chicken embryos; Figure 1 in the right attached figure, the results of the determination of the copy number of influenza virus in allantoic fluid taken from chicken embryos cultured at 37 °C, 33 °C and 25 °C are shown, where the vertical coordinate is the logarithm (base 2) of the copy number of influenza virus per unit volume (mL) and the horizontal coordinate is the culture time of the chicken embryos. Figure 1 The results show that the constructed CV-PR8 backbone strain does not have the replication ability at 37 °C, can replicate well at 33 °C, but has a very poor replication ability at 25 °C.

[0117] Example 2. Obtaining of cold-adapted influenza virus vaccine strain CV1-PR8

[0118] It was found in the study that the CV-PR8 backbone strain can replicate at 33 °C, but cannot replicate at 37 °C and 25 °C ( Figure 1 ). Therefore, in the subsequent experiments, the following method was used for further cold adaptation and serial passage: the vaccine backbone strain CV-PR8 was serially passaged at 25 °C. During the passage process, it was found that the strain gradually obtained the replication ability at 25 °C, and finally the cold-adapted strain CV1-PR8 with good replication ability at 25 °C was obtained ( Figure 2 ).

[0119] During the passage process, the changes in gene loci before, during, and after cold adaptation were analyzed by first-generation and second-generation sequencing. The results showed that with the adaptation of the virus to 25°C, multiple genes underwent adaptive mutations (Table 1). Therefore, it was determined that these gene locus mutations conferred the growth ability of the vaccine backbone strain CV1-PR8 at 25°C, and a cold-adapted vaccine backbone strain CV1-Egg P45 of H1N1 subtype influenza virus suitable for propagation in chicken embryos was obtained. CV1-Egg P45 was numbered as CV1-A / Puerto Rico / 8 / 1934 (PR8)(H1N1), hereinafter referred to as CV1-PR8 for short. Moreover, CV1-PR8 was passaged continuously for 5 generations at 37°C and still could not grow under the condition of 37°C. This indicated that the constructed cold-adapted vaccine backbone strain of H1N1 subtype influenza virus had good genetic stability and could be used as the backbone strain for constructing cold-adapted vaccine strains of influenza A. The HA and NA genes of epidemic influenza viruses were inserted into the vaccine backbone strain to construct cold-adapted vaccine strains against epidemic influenza viruses.

[0120] The specific steps are as follows:

[0121] 1). Passage method for obtaining CV1-Egg P45: Directly perform cold adaptation passage at 25°C. If there is no hemagglutination titer in the previous generation, directly inoculate the next generation with the original solution for subculture. The inoculation amount for each chicken embryo (9 - 11 days old) is 100 μL. After inoculating the chicken embryos, place them in an incubator at 25°C for 120 h, then place them in a refrigerator at 4°C overnight, and then collect the allantoic fluid of the chicken embryos and measure its hemagglutination titer and CT value. If there is a hemagglutination titer, inoculate 1 HAU / 100 μL and 0.1 HAU / 100 μL for subculture. After culturing at 25°C for 96 h or 120 h, place them at 4°C overnight, and then collect the allantoic fluid of the chicken embryos and measure its hemagglutination titer with 1% chicken blood and measure its CT value with a kit (Vazyme Q223-01).

[0122] 2). Determination of the growth curve: Use chicken embryos to measure the growth curve of the virus strain. Dilute the virus 0.1 HAU times, and the inoculation amount for each chicken embryo is 0.1 HAU / 100 μL. A total of 9 chicken embryos are inoculated, with 3 chicken embryos placed at 37°C, 33°C, and 25°C respectively. Then, 300 μL of allantoic fluid of the chicken embryos is extracted at 24 h, 48 h, 72 h, 96 h, and 120 h respectively, and then the hemagglutination titer of the allantoic fluid taken at each time is measured.

[0123] 3). Determination of the hemagglutination titer, i.e., HA titer: Dilute the samples (allantoic fluid of chicken embryos extracted in step 2) at each time with 1×PBS in a 96-well V-bottom plate by 2-fold serial dilution, then add an equal volume of 1% chicken suspended red blood cells, let it stand at room temperature for 20 min, and then read the hemagglutination titer of the sample by observing whether the chicken red blood cells sediment.

[0124] The results of the determination of the hemagglutination titer are as Figure 2 shown, Figure 2 where the ordinate is the logarithm (base 2) of the HA titer and the abscissa is the chicken embryo culture time. Figure 2 The results show that: This strain has good replication ability at both 33 °C and 25 °C, and poor replication ability at 37 °C.

[0125] 4), Detection of mutation sites of CV1-PR8

[0126] Extract the RNA of the allantoic fluid of chicken embryos obtained by inoculating CV1-PR8 as a template (using ddH 2 O as a negative control), use MBTuni-12 and MBTuni-13 as primers, and use a PCR kit (Vazyme, P611-01) for PCR amplification to obtain PCR amplification products. The electrophoresis gel image is as Figure 3 , Send the amplification product for sequencing and perform second-generation sequencing. The sequencing results show that: Compared with the backbone strain CV-PR8, the CV1-PR8 strain has the mutations shown in Table 1.

[0127] PCR amplification primer information:

[0128] MBTuni-12 ACg CgT gAT CAg CAA AAg CAg g;

[0129] MBTuni-13 ACg CgT gAT CAg TAg AAA CAA gg.

[0130] Table 1: Adaptive mutation sites in CV1-PR8 (compared with CV-PR8)

[0131]

[0132] Compared with the existing strains, the CV1-PR8 strain has the following mutations:

[0133] C1), The influenza virus PB2 protein contains mutations of N265S (backbone strain CV1-PR8) and S470N;

[0134] C2), The influenza virus PB1 protein contains mutations of K391E, E581G, A661I (backbone strain CV-PR8) and S678N;

[0135] C3), The influenza virus PA protein contains a mutation of L336M;

[0136] C4), The nucleoprotein (NP) of the influenza virus contains a mutation of E18G;

[0137] C5), the influenza virus NS1 protein contains mutations W102C and M119I.

[0138] That is, the sequencing results show that the CV1-PR8 strain expresses PB2 mutant protein, PB1 mutant protein, PA mutant protein, NP mutant protein, NS1 mutant protein, NS2 mutant protein, M1 protein, M2 protein, HA protein and NA protein. The PB2 mutant protein is a protein with the amino acid sequence of SEQ ID No.1, the PB1 mutant protein is a protein with the amino acid sequence of SEQ ID No.2, the PA mutant protein is a protein with the amino acid sequence of SEQ ID No.3, the NP mutant protein is a protein with the amino acid sequence of SEQ ID No.4, the NS1 mutant protein is a protein with the amino acid sequence of SEQ ID No.5, the NS2 mutant protein is a protein with the amino acid sequence of SEQ ID No.6, the M1 protein is a protein with the amino acid sequence of SEQ ID No.7, the M2 protein is a protein with the amino acid sequence of SEQ ID No.8, the HA protein is a protein with the amino acid sequence of SEQ ID No.9, and the NA protein is a protein with the amino acid sequence of SEQ ID No.10.

[0139] The genome of the CV1-PR8 influenza virus is single-stranded negative-stranded and segmented RNA. The single-stranded negative-stranded and segmented RNA is transcribed to obtain a set of positive-stranded RNAs complementary to the single-stranded negative-stranded and segmented RNA. The set of positive-stranded RNAs includes PB2-RNA, PB1-RNA, PA-RNA, NP-RNA, NS-RNA, M-RNA, HA-RNA and NA-RNA. The PB2-RNA is an RNA molecule encoding the above PB2 mutant protein, the PB1-RNA is an RNA molecule encoding the above PB1 mutant protein, the PA-RNA is an RNA molecule encoding the above PA mutant protein, the NP-RNA is an RNA molecule encoding the above NP mutant protein; the NS-RNA is an RNA molecule encoding the above NS mutant protein, the M-RNA is an RNA molecule encoding the above M1 protein and M2 protein, the HA-RNA is an RNA molecule encoding the above HA protein, and the NA-RNA is an RNA molecule encoding the above NA protein.

[0140] That is, 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 NS-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 15, the M-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 16, the HA-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 17, and the NA-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No. 18.

[0141] The obtained CV1-PR8 strain was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms for patent procedures on December 19, 2022, and the accession number registered in the deposit center is CGMCC No. 45373.

[0142] CV1-PR8 can be rescued and obtained by referring to the method of Example 1. Among them, 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-PA is SEQ ID No. 33; the nucleotide sequence of the cDNA molecule of CV1-NP 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.

[0143] In this invention, the existing chicken embryo-adapted inactivated vaccine backbone strain A / Puerto Rico / 8 / 34 (H1N1) was selected as the basic virus strain for modification. The reverse genetics technology was combined with the cold adaptation passage technology method to construct an H1N1 subtype cold-adapted vaccine backbone strain suitable for chicken embryo propagation, providing technical support for the production of cold-adapted vaccines without changing the chicken embryo vaccine production line, and increasing the possibility of transforming this vaccine into actual production. The cold adaptation sites discovered in this invention can greatly weaken the replication ability of the virus strain at 37 °C and enable the virus strain to replicate well at low temperatures (33 °C and 25 °C). The replication ability under these three temperature conditions meets the requirements of cold-adapted attenuated influenza vaccines for replication ability at different temperatures, indicating that the virus strain containing these sites has the potential to become the backbone of live attenuated vaccines.

[0144] Example 3: CV1-PR8 Animal Experiment and Formation of a New Virus Strain as the Backbone

[0145] 3.1 Test Animals:

[0146] SPF-level BALB / c mice, female, 6 - 7 weeks old, weighing 16 - 18 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0147] 3.2 Test Procedures

[0148] The test mice were divided into three groups, with 5 mice in each group. The first group was instilled with 25 μL of 1×PBS as a control. The second group was infected by instillation with 25 μL of 10 6 EID 50 / 50 μL of the wild influenza virus strain of PR8 (WT-PR8 in Example 1). The third group was infected by instillation with 25 μL of 10 6 EID 50 / 50 μL of the CV1-PR8 influenza virus. Their survival was monitored (a mouse was determined to be dead if it died or its weight decreased by 25% or more), and blood was collected 14 days after immunization and serum was separated to measure the HI antibody titer. The survival and HI antibody conditions of the mice on the 14th day are shown in Table 2. There was no significant weight loss in the mice in the PBS control group and the CV1-PB1 group within 14 days, and the lethality rate was 0%. However, the lethality rate of the mice in the equal-dose WT-PR8 group reached 100%, indicating that CV1-PR8 achieved the purpose of attenuation and had a certain degree of safety. The HI antibody after 14 days of immunization was higher than 1:40, indicating that the antibody produced after instillation of CV1-PR8 had a protective effect.

[0149] Among them, the preparation method of 10 6 EID 50 / 50 μL of the CV1-PR8 influenza virus was as follows: CV1-PR8 was diluted to 10 with 1×PBS6 EID 50 / 50 μL.

[0150] 10 6 EID 50 The preparation method of the wild influenza strain of PR8 with / 50 μL refers to CV2-PR8 influenza virus.

[0151] 3.3, HI antibody titer determination method

[0152] Take 20 μL of the separated serum and place it in a 1.5 mL EP tube, add 80 μL of RBD and mix well. Incubate at 37 °C for 16 - 18 h, inactivate at 56 °C for 30 min, add 20% red blood cell mud and mix well, then incubate at 37 °C for 1 h, and invert and mix repeatedly during this period; centrifuge at 1000×g and aspirate the supernatant, which is the well-treated serum, and store it at 4 °C for later use. Use an eight-channel pipette to add 25 μL of 1×PBS to each well of a 96-well V-shaped hemagglutination plate. Then use a single-channel pipette to add 25 μL of the treated serum to the first well of the 96-well V-shaped hemagglutination plate, pipette and mix well, then use an eight-channel pipette to aspirate 25 μL from the first well to the second well, pipette and mix well, and serially dilute it to the 11th well in turn, and discard 25 μL. Add 25 μL of normal saline or PBS to the 12th well as a negative control. Use an eight-channel pipette to sequentially add 25 μl of 4 HAU virus solution, incubate at 37 °C (room temperature) for 30 min, add 1% chicken red blood cell suspension, and let it stand at room temperature for 15 - 20 min, and observe the results.

[0153] Result determination: Tilt the reaction plate at 45°. If the red blood cells sinking to the bottom of the well flow downward in a linear shape along the inclined surface, it is precipitation, indicating that the red blood cells are not or incompletely agglutinated by the virus; if the red blood cells at the bottom of the well spread flat on the bottom of the well, form a uniform thin layer, and do not flow after tilting, it indicates that the red blood cells are agglutinated by the virus. Place the hemagglutination plate. The highest dilution at which the red blood cell agglutination is completely inhibited is used as the determination end point, that is, the HI titer.

[0154] Table 2: Animal experiment results

[0155]

[0156] Example 4, Virus construction

[0157] 4.1, Reverse genetic rescue virus

[0158] MDCK:293T are seeded in a six-well plate at a ratio of 1:5 - 10, and DMEM containing 10% fetal bovine serum and double antibiotics is used at 37 °C, 5% CO 2After culturing in an incubator at a certain temperature for 16 - 20 h, transfection experiments were carried out. During transfection, the recombinant plasmids pHW2000 - CV1 - PB2, pHW2000 - CV1 - PB1, pHW2000 - CV1 - NP, pHW2000 - CV1 - PA, pHW2000 - CV1 - M, pHW2000 - CV1 - NS, pHW2000 - HA (HA of the strain shown in Table 3), pHW2000 - NA (NA of the strain shown in Table 3) and the transfection reagent Lipofectamine 2000 (500 ng of each recombinant plasmid and 10 μL of the transfection reagent were added to each well of a six - well plate) were respectively added to an EP tube containing MEM - opti (Gibco) and mixed evenly. After standing for 5 min, the two were mixed. After standing the mixture for 20 min, the mixture was evenly added to the six - well plate and gently shaken to disperse the mixture evenly. Then, after placing it at 33°C for 6 - 8 h, the medium was changed, and Opti - MEM containing 1 μg / mL TPCK - trypsin (Sigma - Aldrich) was added to each well, and it was placed at 33°C, 5% CO 2 After culturing in an incubator for 72 h, two 9 - 11 - day - old SPF - grade chicken embryos were inoculated, 500 μL was inoculated into each chicken embryo, and they were cultured in an incubator at 33°C for 96 h and then placed at 4°C overnight. Then, the allantoic fluid of the chicken embryos was collected and its hemagglutination titer was measured using 1% chicken blood (Beijing Hute Biotechnology Co., Ltd.). If there was a hemagglutination titer, the target strain was obtained.

[0159] The preparation of the recombinant plasmids pHW2000 - CV1 - PB2, pHW2000 - CV1 - PB1, pHW2000 - CV1 - NP, pHW2000 - CV1 - PA, pHW2000 - CV1 - M, pHW2000 - CV1 - NS, pHW2000 - HA (DNA molecule generating HA of the strain shown in Table 3), pHW2000 - NA (DNA molecule generating NA of the strain shown in Table 3) was referred to Example 1, with the only difference being that the cDNA was replaced correspondingly. Among them, the cDNA molecules generating HA and NA can be directly synthesized with reference to the genomic information of the strains shown in Table 3.

[0160] 4.2, Obtaining reassortant viruses by natural infection

[0161] Dilute CV1 - PR8 to 10 -3, mix them with the virus strains in Table 3 in equal volumes, inoculate chicken embryos, inoculate 200 μL per chicken embryo, place them in an incubator at 37 °C for 72 h, and collect the allantoic fluid of the chicken embryos. Mix the harvested allantoic fluid of the chicken embryos with the anti-PR8 serum in equal volumes, neutralize at 37 °C for 2 h, then inoculate chicken embryos (100 μL / embryo), culture at 25 °C for 96 h, then collect the allantoic fluid of the chicken embryos, continue to neutralize with the anti-PR8 serum and then inoculate chicken embryos, and perform a total of 4 anti-serum cycles, and measure its hemagglutination titer (1% chicken blood cells).

[0162] The preparation method of the anti-PR8 serum is as follows: After inactivating WT-PR8 with β-propiolactone, mix it with aluminum adjuvant according to the method described in the instruction manual and then emulsify it. Immunize rabbits (New Zealand white rabbits and 70-day-old) by intramuscular injection. After multiple immunizations, collect blood to separate the serum. Specifically, the injection volume for each rabbit is 500 μL - 1 mL, and the immunization dose of inactivated WT-PR8 in the first immunization is 100 μg / rabbit. Two weeks after the first immunization, mix inactivated WT-PR8 with aluminum adjuvant according to the immunization dose of 100 μg / rabbit for booster immunization. The booster immunization is carried out once every two weeks for a total of 4 times. Blood is collected from the rabbits during the immunization interval, about 5 mL each time, centrifuged at 2000 rpm for 5 min to obtain the supernatant, that is, the immune serum of the WT-PR8 immunized group. The HI titer ≥ 1280 is required for all blood collections.

[0163] Table 3: Information on influenza A virus strains

[0164]

[0165] The above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. Recombinant influenza A virus, characterized in that: the recombinant influenza A virus expresses or contains PB2 mutant protein, PB1 mutant protein, PA mutant protein, NP mutant protein and NS mutant protein, the PB2 mutant protein is selected from A1) or A2): A1), a protein with an amino acid sequence of SEQ ID No.1; A2), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1); the PB1 mutant protein is selected from A3) or A4): A3), a protein with an amino acid sequence of SEQ ID No.2; A4), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A3); the PA mutant protein is selected from A5) or A6): A5), a protein with an amino acid sequence of SEQ ID No.3; A6), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A5); the NP mutant protein is selected from A7) or A8): A7), a protein with an amino acid sequence of SEQ ID No.4; A8), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A7); the NS mutant protein is selected from A9) or A10): A9), a protein with an amino acid sequence of SEQ ID No.5; A10), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A9).

2. The recombinant influenza A virus according to claim 1, characterized in that: the genome of the recombinant influenza A virus is single-stranded negative-stranded, segmented RNA, and the single-stranded negative-stranded, segmented RNA is transcribed to obtain a set of positive-stranded RNAs complementary to the single-stranded negative-stranded, segmented RNA, and the set of positive-stranded RNAs includes PB2-RNA, PB1-RNA, PA-RNA, NP-RNA and NS-RNA, the PB2-RNA is an RNA molecule encoding the PB2 mutant protein described in claim 1; the PB1-RNA is an RNA molecule encoding the PB1 mutant protein described in claim 1; the PA-RNA is an RNA molecule encoding the PA mutant protein described in claim 1; the NP-RNA is an RNA molecule encoding the NP mutant protein described in claim 1; the NS-RNA is an RNA molecule encoding the NS mutant protein described in claim 1.

3. The recombinant influenza A virus according to claim 2, characterized in that: 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 NS-RNA is an RNA molecule with a nucleotide sequence of SEQ ID No.

15.

4. The recombinant influenza A virus according to any one of claims 1-3, characterized in that: The recombinant influenza A virus is influenza A virus, with the strain number of CV1-A / Puerto Rico / 8 / 1934 (PR8) (H1N1), and the deposit number in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 45373.

5. A biological material related to the recombinant influenza A virus according to any one of claims 1-4, characterized in that: the biological material is selected from any one of the following: E1), a set of DNA molecules encoding the set of positive-strand RNAs of the recombinant influenza A virus according to any one of claims 2-4, the set of DNA molecules including a DNA molecule encoding PB2-RNA, a DNA molecule encoding PB1-RNA, a DNA molecule encoding PA-RNA, a DNA molecule encoding NP-RNA, and a DNA molecule encoding NS-RNA; E2), a set of vectors, including the following E2a), E2b), E2c), E2d) and E2e), E2a), a recombinant vector containing a DNA molecule encoding PB2-RNA; E2b), a recombinant vector containing a DNA molecule encoding PB1-RNA; E2c), a recombinant vector containing a DNA molecule encoding PA-RNA; E2d), a recombinant vector containing a DNA molecule encoding NP-RNA; E2e), a recombinant vector containing a DNA molecule encoding NS-RNA; E3), a microorganism containing the recombinant influenza A virus according to any one of claims 1-4; E4), an animal cell line containing the recombinant influenza A virus according to any one of claims 1-4; E5), an animal tissue containing the recombinant influenza A virus according to any one of claims 1-4; E6), an animal organ containing the recombinant influenza A virus according to any one of claims 1-4.

6. The biological material according to claim 5, characterized in that: the nucleotide sequence of the DNA molecule encoding PB2-RNA in E2a) is SEQ ID No. 31; the nucleotide sequence of the DNA molecule encoding PB1-RNA in E2b) is SEQ ID No. 32; the nucleotide sequence of the DNA molecule encoding PA-RNA in E2c) is SEQ ID No. 33; the nucleotide sequence of the DNA molecule encoding NP-RNA in E2d) is SEQ ID No. 34; the nucleotide sequence of the DNA molecule encoding NS-RNA in E2e) is SEQ ID No.

35.

7. A method for constructing a recombinant influenza A virus, characterized in that: the method includes M1) or / and M2), M1), introducing the set of carriers described in claim 5, the recombinant carrier containing the gene encoding the M protein of influenza A virus, the recombinant carrier containing the gene encoding the HA protein of epidemic influenza A virus, and the recombinant carrier containing the gene encoding the NA protein of the epidemic influenza A virus into cells to obtain a recombinant influenza A virus containing the HA and NA of the epidemic influenza A virus; M2), co-culturing the recombinant influenza A virus described in any one of claims 1-4 with an influenza A virus to obtain a recombinant influenza A virus containing the HA and NA of the influenza A virus.

8. Use of the recombinant influenza A virus described in any one of claims 1-4 or the biological material described in claim 5 or 6 in the preparation of a drug for treating and / or preventing influenza virus.

9. Use of the recombinant influenza virus described in any one of claims 1-4 or the biological material described in claim 5 or 6 in the preparation of an influenza vaccine.

10. An influenza vaccine, characterized in that: the influenza vaccine uses the recombinant influenza A virus described in any one of claims 1-3 as a backbone strain or uses the recombinant influenza A virus described in any one of claims 1-4 as an active ingredient.

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