CN113862284B

CN113862284B - Gene, virus-like particle, vaccine and preparation and application for encoding recombinant avian influenza virus HA protein

Info

Publication number: CN113862284B
Application number: CN202111069024.9A
Authority: CN
Inventors: 樊惠英; 孔德鑫; 廖明; 陈陶然; 胡小龙
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-09-13
Filing date: 2021-09-13
Publication date: 2023-05-26
Anticipated expiration: 2041-09-13
Also published as: CN113862284A

Abstract

The invention belongs to the technical field of genetic engineering vaccines, and in particular relates to a gene for encoding recombinant avian influenza virus HA protein, virus-like particles, a vaccine, preparation and application. The nucleotide sequence of the gene for encoding the recombinant avian influenza virus HA protein is shown as SEQ ID NO. 1. The invention also provides an avian influenza virus-like particle which is assembled by HA protein, NA protein and M1 protein, and the hemagglutination titer can reach 13log2. The invention also provides an avian influenza virus-like particle vaccine containing the virus-like particle, which can provide complete clinical protection and obviously inhibit toxin expelling aiming at lethal attack of homologous and wild H7N9 subtype highly pathogenic avian influenza viruses, and provides a new vaccine choice for prevention and control of H7N9 subtype avian influenza.

Description

Gene, virus-like particle, vaccine and preparation and application for encoding recombinant avian influenza virus HA protein

Technical Field

The invention belongs to the technical field of genetic engineering vaccines, and in particular relates to a gene for encoding recombinant avian influenza virus HA protein, virus-like particles, a vaccine, preparation and application.

Background

Avian influenza virus (Avian Influenza virus, AIV) is a enveloped, segmented, negative-strand RNA virus belonging to the orthomyxoviridae family, the genus influenza, and avian influenza is one of the virulent infectious diseases of birds.

The avian influenza virus encodes a plurality of proteins, the HA (surface antigen hemagglutinin) protein is a membrane protein encoded by the avian influenza virus, plays an important role in the aspect of infection of host cells by the avian influenza virus, and is also a main target antigen for the immune efficacy of the host to the avian influenza virus. NA (neuraminic acid) protein is also a membrane protein, which primarily functions to assist in the release of progeny virus from host cells. The HA and NA proteins are the main antigenic components in the development of avian influenza vaccines.

Vaccination is one of the most effective measures for preventing avian influenza virus infection. The current domestic avian influenza vaccine mainly comprises a whole virus inactivated vaccine, the vaccine is produced by depending on chick embryos, and the defects of insufficient chick embryo supply, large amount of waste, endogenous pollution and the like exist in the influenza epidemic period. Meanwhile, the long-term use of the whole-virus inactivated vaccine accelerates the variation rate of the avian influenza virus, and the long-term immunity selection pressure causes the avian influenza virus to evolve towards the direction of the non-vaccine strain, so that the whole-virus inactivated vaccine needs to continuously update the vaccine strain to cope with the new influenza virus epidemic. Therefore, there is a need to develop a new safe and effective avian influenza vaccine to prevent and control the epidemic of avian influenza virus.

Virus-like particles (VLPs) are viroids assembled from structural proteins of viruses, which are free of viral nucleic acids, are non-infectious, and are a hotspot for the development of novel avian influenza vaccines. Compared with the whole virus inactivated vaccine which mainly uses humoral immunity, the virus-like particle vaccine can induce humoral immunity and cellular immunity at the same time, and the latter is the key of the cross protection of the avian influenza vaccine. The baculovirus expression system has high safety and easy operation, can be used for preparing the avian influenza virus-like particles on a large scale, and is an important tool for developing avian influenza virus-like particle vaccines. Compared with the whole virus inactivated vaccine, the method can rapidly prepare the avian influenza virus-like particles by utilizing the baculovirus expression system only by using the nucleic acid sequence of the avian influenza virus. Therefore, the avian influenza virus-like particle vaccine developed based on the baculovirus expression system has wide prospect.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the primary aim of the invention is to provide a gene for encoding recombinant avian influenza virus HA protein.

It is another object of the present invention to provide an avian influenza virus-like particle.

It is still another object of the present invention to provide a method for producing the above avian influenza virus-like particle.

A fourth object of the present invention is to provide an avian influenza virus-like particle vaccine.

A fifth object of the present invention is to provide the above gene encoding recombinant avian influenza virus HA protein, avian influenza virus-like particle and use of avian influenza virus-like particle vaccine.

The aim of the invention is achieved by the following technical scheme:

a nucleotide sequence of a gene for encoding recombinant avian influenza virus HA protein is shown as SEQ ID NO. 1;

an avian influenza virus-like particle comprising an avian influenza virus HA protein, an avian influenza virus NA protein, and an avian influenza virus M1 (matrix protein) protein; wherein, the gene for encoding the avian influenza virus HA protein is the gene for encoding the recombinant avian influenza virus HA protein;

the nucleotide sequences of the gene for encoding the NA protein of the avian influenza virus and the gene for encoding the M1 protein of the avian influenza virus are respectively shown in SEQ ID NO. 2-3;

the amino acid sequences of the avian influenza virus HA protein, the avian influenza virus NA protein and the avian influenza virus M1 protein are respectively shown in SEQ ID NO. 4-6;

the avian influenza virus-like particles are preferably self-assembled by avian influenza virus HA protein, avian influenza virus NA protein and avian influenza virus M1 protein;

the avian influenza is H7N9 subtype avian influenza;

the preparation method of the avian influenza virus-like particle comprises the following steps:

(1) Species codons of HA, NA and M1 genes of the avian influenza virus are optimized, and the genes are synthesized to obtain genes which code the HA protein of the avian influenza virus, the NA protein of the avian influenza virus and the M1 protein of the avian influenza virus after the codons are optimized, and the nucleotide sequences of the genes are respectively shown as SEQ ID NO. 1-3; through species codon optimization, insect cell expression is facilitated;

(2) Carrying out PCR amplification by taking the gene for encoding the avian influenza virus HA protein, the gene for encoding the avian influenza virus NA protein and the gene for encoding the avian influenza virus M1 protein obtained in the step (1) as templates to obtain HA, NA and M1 gene fragments with enzyme cutting sites, and carrying out enzyme cutting, connection and transformation on the HA, NA and M1 gene fragments with enzyme cutting sites and baculovirus transfer plasmids to respectively obtain HA gene recombination transfer plasmids, NA gene recombination transfer plasmids and M1 gene recombination transfer plasmids;

(3) Transforming and recombining the HA gene recombinant transfer plasmid, the NA gene recombinant transfer plasmid and the M1 gene recombinant transfer plasmid to respectively obtain an HA gene recombinant baculovirus plasmid, an NA gene recombinant baculovirus plasmid and an M1 gene recombinant baculovirus plasmid;

(4) Transfecting the HA gene recombinant baculovirus plasmid, the NA gene recombinant baculovirus plasmid and the M1 gene recombinant baculovirus plasmid into sf9 cells through liposome mediation to obtain the HA gene recombinant baculovirus, the NA gene recombinant baculovirus and the M1 gene recombinant baculovirus respectively;

(5) The HA gene recombinant baculovirus, the NA gene recombinant baculovirus and the M1 gene recombinant baculovirus are used for infecting insect cells together, and extracellular culture supernatant is collected to obtain avian influenza virus-like particles assembled by HA, NA and M1 proteins;

the baculovirus transfer plasmid in step (2) is pACEBac1;

the baculovirus plasmid in the step (3) is Bacmid;

the insect cell in the step (5) is High five;

when the HA gene recombinant baculovirus, the NA gene recombinant baculovirus and the M1 gene recombinant baculovirus are co-infected in the step (5), the MOI is (2-7): (1-4): 2;

when the HA gene recombinant baculovirus, NA gene recombinant baculovirus and M1 gene recombinant baculovirus described in step (5) are co-infected, the MOI is preferably 2:1:2;

an avian influenza virus-like particle vaccine comprising a pharmaceutically acceptable carrier and an immunizing amount of the avian influenza virus-like particle described above;

the pharmaceutically acceptable carrier includes an adjuvant;

the adjuvant is at least one of white oil adjuvant and water-in-oil adjuvant;

the white oil adjuvant is preferably dayer EOLANE 150;

the water-in-oil adjuvant is Montanide ^TM ISA series adjuvants, more preferably Montanide ^TM ISA71VG adjuvant;

the preparation method of the avian influenza virus-like particle vaccine comprises the following steps:

mixing and emulsifying the avian influenza virus-like particles with an adjuvant in an immune dose to obtain an avian influenza virus-like particle vaccine;

when the adjuvant is white oil adjuvant, the volume ratio of the avian influenza virus-like particle to the white oil adjuvant is preferably 1:2;

when the adjuvant is a water-in-oil adjuvant, the volume ratio of the avian influenza virus-like particle to the water-in-oil adjuvant is preferably 3:7, preparing a base material;

the gene for encoding the recombinant avian influenza virus HA protein, the avian influenza virus-like particle and the application of the avian influenza virus-like particle vaccine in preparing medicaments for preventing and/or treating diseases caused by avian influenza virus;

the avian influenza virus comprises H7N9 subtype avian influenza virus;

the administration subjects for preparing the medicine for preventing and/or treating diseases caused by avian influenza virus infection comprise chickens.

Compared with the prior art, the invention has the following advantages and effects:

(1) The HA antigen of the avian influenza virus is a main target antigen for preparing avian influenza subunit vaccine, and the invention optimizes the species codons of the HA, NA and M1 genes of the avian influenza virus, so that the avian influenza virus is not only favorable for insect cell expression, but also HAs strong immunogenicity.

(2) The invention produces H7N9 subtype avian influenza virus-like particle antigen based on an insect-baculovirus expression system, wherein the avian influenza virus-like particle is self-assembled in insect cells by HA, NA and M1 antigens, and is released into extracellular culture supernatant in the form of virus-like particles, and the antigen expression is efficient.

(3) The invention explores the proportion of HA, NA and M1 recombinant baculovirus co-infected insect cells, preferably the HA, NA and M1 recombinant baculovirus HAs MOI=2: 1:2 co-infecting insect cells, wherein the obtained avian influenza virus-like particles have the highest hemagglutination titer, and the hemagglutination titer reaches 13log2.

(4) Compared with the method for producing the avian influenza virus-like particle by adopting the HA, NA and M1 recombinant baculovirus to co-infect insect cells and connecting the HA, NA and M1 genes in series to the same carrier, the method for producing the avian influenza virus-like particle antigen HAs a larger optimization space in the co-infection mode, can controllably increase the content of main target antigens in the avian influenza virus-like particle and regulate and control the proportion of the content of each antigen.

(5) The quantitative H7N9 avian influenza virus-like particles and the white oil adjuvant EOLANE 150 are mixed and emulsified to prepare the vaccine, and the immune efficacy of the vaccine is evaluated. Immunization of 3 week old SPF chickens, 3 weeks after immunization, with average Hemagglutination Inhibition (HI) antibody titers above 6log 2; 3 weeks after immunization, the A/Chicken/Guangdong/16876/2016 (H7N 9) strain was used at 2X 10 ^6.0 ELD ₅₀ Is used for eliminating toxin by collecting throat and cloacal swabs on the 5 th day after eliminating toxin. The results showed that the non-immunized group all died within 2 days after challenge, the vaccine group did not develop clinical symptoms within 14 days after challenge, all survived, and only 1 chicken was detoxified on day 5 after challenge.

(6) The invention uses the quantified H7N9 avian influenza virus-like particles and Montanide ^TM ISA71VG adjuvant is mixed and emulsified to prepare a vaccine, and the immunity efficacy of the vaccine against wild type H7N9 subtype avian influenza virus is evaluated. The results show that the vaccine serum has good cross-reactivity against different wild-type H7N9 avian influenza wild strains. For the A/Chicken/Guangdong/E157/2017 (H7N 9) strain, the average HI titer was 6.875log2 and the average neutralizing antibody titer was 1 at 19 days post immunization: 1706.67; for the a/Chicken/qingayuan/E664/2017 (H7N 9) strain, the average HI titer was 8log2 and the average neutralizing antibody titer was 1 at 19 days post immunization: 3413.33; A/Chicken/Guangdong/E157/2017 (H7N 9) strain was used at 10 ^6.0 EID ₅₀ The dosage of the medicine is 0.2 ml/medicine, and throat and cloacal swabs are collected for detection and detoxification 3, 5, 7 and 9 days after the medicine is detoxified. The results showed that the non-immunized group all died within 3 days after challenge, the vaccine group did not develop clinical symptoms within 14 days after challenge, all survived, and only 1 chicken was detected to expel toxin on day 9 after challenge.

(7) The avian influenza virus-like particle vaccine prepared by the invention has good cross protection. The H7N9 subtype avian influenza virus-like particles prepared by the invention can provide complete clinical protection and obviously inhibit toxin expelling by combining with EOLANE 150 adjuvant and using lethal attack aiming at homologous H7N9 subtype highly pathogenic avian influenza virus; the H7N9 subtype avian influenza virus-like particles prepared by the invention are combined with Montanide ^TM ISA71VG adjuvant induced high levels of HI and neutralizing antibodies against wild-type H7N9 subtype highly pathogenic avian influenza virus; lethal challenge against wild-type H7N9 avian influenza virus provided complete clinical protection, and only one chicken detected detoxification. The H7N9 subtype avian influenza virus-like particle vaccine prepared by the invention provides a new vaccine selection for the prevention and control of H7N9 subtype avian influenza.

Drawings

FIG. 1 is a graph showing analysis of results of restriction enzyme digestion of HA, NA and M1 gene recombinant transfer plasmids, wherein A: pACE-HA, B: pACE-NA, C: pACE-M1.

Fig. 2 is a graph of HA, NA, M1 gene recombinant baculovirus according to moi=2: 1:2 SDS-PAGE and Western blot analysis of avian influenza virus-like particles expressed by insect-infected cells.

FIG. 3 is an electron microscopic view of avian influenza virus-like particles.

FIG. 4 shows avian influenza virus-like particles and Montanide ^TM Serum hemagglutination inhibition antibodies (HI) and neutralizing antibodies results analysis graphs of the ISA71VG mixed emulsion prepared vaccine on days 14 and 19 after immunization of SPF chickens.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

The test materials in the examples described below were all commercially available unless otherwise specified. The test methods described herein are conventional, unless otherwise specified.

EXAMPLE 1 construction of recombinant baculovirus plasmids for HA, NA and M1 genes

Construction of HA, NA, M1 Gene recombination transfer plasmid

(1) In the embodiment, the nucleotide sequences of the HA, NA and M1 genes of the avian influenza virus are subjected to codon optimization, the expression of insect cells is favored, and a 6x his label is added at the C terminal of the HA, NA and M1 genes; the nucleotide sequences of HA, NA and M1 genes with optimized codons are obtained through artificial synthesis and are respectively connected into a PUC57 vector to obtain corresponding recombinant plasmids (Beijing Liuhua large gene technology Co., ltd.); the nucleotide sequence of the HA gene after codon optimization is SEQ ID NO. 1, and the amino acid sequence thereof is SEQ ID NO. 4; the nucleotide sequence of the NA gene after codon optimization is SEQ ID NO. 2, and the amino acid sequence thereof is SEQ ID NO. 5; the nucleotide sequence of the M1 gene after codon optimization is SEQ ID NO. 3, and the amino acid sequence thereof is SEQ ID NO. 6.

Nucleotide sequence of HA gene after codon optimization:

ATGAACACTCAGATCCTGGTCTTCGCTCTGATCGCTATCATCCCCACTAACGCCGACAAGATCTGCCTGGGTCACCACGCTGTGAGCAACGGCACTAAGGTCAACACTCTGACTGAACGTGGTGTCGAGGTCGTGAACGCTACTGAGACTGTGGAACGCACTAACACCCCCCGCATCTGCAGCAAGGGCAAGCGCACCGTCGACCTGGGTCAGTGCGGCCTGCTGGGCACTATCACTGGTCCCCCCCAGTGCGACCAGTTCCTGGAGTTCAGCGCTGACCTGATCATCGAACGCCGCGAGGGTTCCGACGTCTGCTACCCTGGTAAATTCGTCAACGAAGAAGCTCTGCGCCAGATCCTGCGCGAGAGCGGCGGAATCGACAAGGAGCCTATGGGCTTCACTTACAACGGTATCCGCACTAACGGTGTGACTAGCGCTTGCCGCCGCAGCGGTAGCAGCTTCTACGCCGAAATGAAGTGGCTGCTGTCCAACACCGACAACGCTACTTTCCCCCAGATGACCAAGTCCTACAAGAACACTCGCAAGAGCCCCGCCATCATCGTGTGGGGTATCCACCACTCCGTCTCCACTGCTGAACAGACTAAGCTGTACGGTTCCGGTAACAAGCTGGTGACCGTCGGTTCCTCCAACTACCAGCAGTCCTTCGTCCCCAGCCCTGGTGCCCGTCCTCAGGTGAACGGTCAGAGCGGCCGCATCGACTTCCACTGGCTGATCCTGAACCCTAACGACACCGTGACCTTCAGCTTCAACGGTGCTTTCATCGCTCCTGACCGCGCTTCCTTCCTGCGCGGTAAAAGCATGGGTATCCAGTCCGGCGTGCAGGTGGACGCCAACTGCGAAGGCGACTGCTACCACAGCGGCGGTACTATCATCTCCAACCTGCCTTTCCAGAACATCGACAGCCGTGCTGTCGGTAAATGCCCCCGTTACGTCAAGCAGCGCTCCCTGCTGCTGGCTACTGGCATGAAGAACGTCCCTGAGGTTCCTAAGGGCAAGCGTACTGCTCGCGGTCTGTTCGGCGCCATCGCCGGTTTCATCGAGAACGGTTGGGAGGGCCTGATCGACGGCTGGTACGGTTTCCGCCACCAGAACGCCCAGGGCGAGGGCACTGCTGCTGACTACAAGAGCACTCAGTCCGCTATCGACCAGATCACCGGTAAACTGAACCGCCTGATCGCCAAGACCAACCAGCAGTTCAAGCTGATCGACAACGAGTTTAATGAGGTCGAGAAGCAGATCGGCAACGTCATCAACTGGACTCGTGACTCCATCACTGAGGTCTGGAGCTACAACGCCGAGCTGCTGGTGGCTATGGAAAACCAGCACACCATCGACCTCGCTGACTCCGAGATGGACAAGCTGTACGAACGCGTCAAGCGCCAGCTGCGCGAGAACGCTGAAGAAGACGGCACTGGCTGCTTCGAGATCTTCCACAAGTGCGACGACGACTGCATGGCTTCCATCCGTAACAACACCTACGACCACCGTAAGTACCGCGAAGAAGCCATGCAGAACCGTATCCAGATCGACCCCGTCAAGCTGAGCTCCGGCTACAAGGACGTCATCCTGTGGTTCTCCTTCGGTGCCAGCTGCTTCATCCTGCTGGCTATTGTTATGGGTCTGGTCTTCATCTGCGTGAAGAACGGTAACATGCGTTGCACCATCCACCACCACCACCATCACTAA

HA protein amino acid sequence:

MNTQILVFALIAIIPTNADKICLGHHAVSNGTKVNTLTERGVEVVNATETVERTNTPRICSKGKRTVDLGQCGLLGTITGPPQCDQFLEFSADLIIERREGSDVCYPGKFVNEEALRQILRESGGIDKEPMGFTYNGIRTNGVTSACRRSGSSFYAEMKWLLSNTDNATFPQMTKSYKNTRKSPAIIVWGIHHSVSTAEQTKLYGSGNKLVTVGSSNYQQSFVPSPGARPQVNGQSGRIDFHWLILNPNDTVTFSFNGAFIAPDRASFLRGKSMGIQSGVQVDANCEGDCYHSGGTIISNLPFQNIDSRAVGKCPRYVKQRSLLLATGMKNVPEVPKGKRTARGLFGAIAGFIENGWEGLIDGWYGFRHQNAQGEGTAADYKSTQSAIDQITGKLNRLIAKTNQQFKLIDNEFNEVEKQIGNVINWTRDSITEVWSYNAELLVAMENQHTIDLADSEMDKLYERVKRQLRENAEEDGTGCFEIFHKCDDDCMASIRNNTYDHRKYREEAMQNRIQIDPVKLSSGYKDVILWFSFGASCFILLAIVMGLVFICVKNGNMRCTIHHHHHH.

nucleotide sequence of NA gene after codon optimization:

ATGAACCCTAACCAGAAGATCCTGTGCACCTCCGCTACCGCTATCACCATCGGTGCTATCACCGTGCTGATCGGTATCGCTAACCTGGGTCTGAACATCGGTCTGCACCTGAAGTCCGGTTGCAACTGTTCCCGCTCCCAACCTGAGACTACCAACACCTCCCAGACCATCATCAACAACTACTACAACGAGACTAACATCACCAACATCCAGATGGAGGAACGCACCTCCCGCAACTTCAACAACCTGACCAAGGGTCTGTGCACCATCAACTCCTGGCACATCTACGGTAAGGACAACGCTGTGCGCATTGGTGAATCCTCCGACGTTCTGGTGACTCGCGAGCCTTATGTGTCCTGCGACCCTGATGAATGCCGCTTCTACGCTCTGTCCCAGGGTACTACCATTCGCGGTAAGCACTCCAACGGTACTATCCACGACCGTTCCCAATACCGCGCTCTGATCTCTTGGCCTCTGTCCTCTCCTCCTACCGTGTATAACTCCCGCGTGGAGTGTATTGGTTGGTCCTCCACCTCTTGCCACGATGGTAAGTCCCGCATGTCCATCTGCATCTCCGGTCCTAACAACAACGCTTCCGCTGTGATCTGGTACAACCGTCGCCCTGTGGCTGAAATCAACACCTGGGCTCGCAACATCCTGCGTACCCAAGAGTCTGAGTGCGTGTGCCATAACGGTGTGTGCCCTGTGGTGTTCACTGACGGTCCTGCTACTGGTCCTGCTGATACCCGCATCTACTACTTCAAGGAGGGTAAGATCCTGAAGTGGGAGTCCTTGACCGGCACCGCTAAGCACATCGAGGAGTGCTCCTGCTATGGTAAGCGCACCGGTATTACTTGTACCTGCCGCGACAATTGGCAAGGTTCCAACCGCCCTGTGATCCAGATTGACCCTGTGGCTATGACTCACACCTCCCAGTACATCTGCTCCCCTGTGCTGACTGATTCCCCTCGTCCTAACGACCCTAACATCGGTAAGTGCAACGACCCTTACCCTGGTAACAACAACAACGGTGTGAAGGGTTTCTCCTACCTGGACGGTGACAACACTTGGCTGGGTCGTACCATTTCCACCGCTTCCCGTTCCGGTTACGAGATGCTGAAGGTGCCTAACGCTCTGACTGACGACCGCTCCAAGCCTATTCAGGGTCAGACCATCGTGCTGAACGCTGACTGGTCCGGTTACTCCGGTTCCTTCATGGACTACTGGGCTGAGGGTGACTGCTATCGCGCTTGCTTCTACGTTGAGCTGATCCGCGGTAAGCCTAAAGAGGACAAGGTGTGGTGGACCTCCAACTCCATCGTGTCCATGTGCTCCTCCACCGAGTTTCTGGGTCAGTGGAACTGGCCTGACGGTGCTAAGATCGAGTACTTCCTGCACCACCACCACCACCACTAA

NA protein amino acid sequence:

MNPNQKILCTSATAITIGAITVLIGIANLGLNIGLHLKSGCNCSRSQPETTNTSQTIINNYYNETNITNIQMEERTSRNFNNLTKGLCTINSWHIYGKDNAVRIGESSDVLVTREPYVSCDPDECRFYALSQGTTIRGKHSNGTIHDRSQYRALISWPLSSPPTVYNSRVECIGWSSTSCHDGKSRMSICISGPNNNASAVIWYNRRPVAEINTWARNILRTQESECVCHNGVCPVVFTDGPATGPADTRIYYFKEGKILKWESLTGTAKHIEECSCYGKRTGITCTCRDNWQGSNRPVIQIDPVAMTHTSQYICSPVLTDSPRPNDPNIGKCNDPYPGNNNNGVKGFSYLDGDNTWLGRTISTASRSGYEMLKVPNALTDDRSKPIQGQTIVLNADWSGYSGSFMDYWAEGDCYRACFYVELIRGKPKEDKVWWTSNSIVSMCSSTEFLGQWNWPDGAKIEYFLHHHHHH.

nucleotide sequence of the codon-optimized M1 gene:

ATGTCTCTGCTGACCGAGGTGGAGACTTACGTGCTGTCCATCATCCCTTCCGGTCCTCTGAAGGCTGAGATCGCTCAGCGTCTGGAGGATGTGTTCGCTGGTAAGAACGCTGACCTGGAGGCTCTGATGGAGTGGATCAAGACCCGCCCTATCTTGTCCCCTCTGACCAAGGGTATCCTGGGTTTCGTGTTCACCCTGACCGTGCCTTCCGAACGTGGTCTGCAACGTCGTCGTTTCGTGCAGAACGCTCTGAACGGTAACGGTGACCCTAACAACATGGACAAGGCTGTGAAGCTGTACAAGAAGCTGAAGCGCGAGATGACCTTCCACGGTGCTAAGGAGGTGGCTCTGTCCTATTCCACCGGTGCTCTGGCTTCTTGCATGGGTCTGATCTACAACCGCATGGGCACCGTGACTGCTGAAGGTGCTCTGGGTCTGGTTTGTGCTACCTGCGAGCAGATTGCTGACGCTCAGCACCGTTCCCATCGTCAAATGGCTACCACCACCAACCCTCTGATCCGCCACGAAAACCGCATGGTGCTGGCTTCTACCACCGCTAAGGCTATGGAGCAGATGGCTGGTTCCTCCGAGCAAGCTGCTGAGGCTATGGAGGTGGCTTCCCAAGCTCGCCAGATGGTGCAAGCTATGCGCACTGTGGGTACTCACCCTAACTCCTCCACCGGTCTGAAGGACGACCTGATCGAGAACCTGCAGGCTTACCAGAACCGCATGGGTGTTCAACTGCAGCGCTTCAAGCACCATCACCACCACCACTAA

m1 protein amino acid sequence:

MSLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNADLEALMEWIKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYKKLKREMTFHGAKEVALSYSTGALASCMGLIYNRMGTVTAEGALGLVCATCEQIADAQHRSHRQMATTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQARQMVQAMRTVGTHPNSSTGLKDDLIENLQAYQNRMGVQLQRFKHHHHHH.

(2) Designing primers according to the nucleotide sequences of the HA, NA and M1 genes subjected to codon optimization, and carrying out corresponding gene amplification by taking the recombinant plasmid as a template, wherein a PCR reaction system (50 mu L) is as follows: 2X Premix 25. Mu.L, ddH ₂ O22. Mu.L, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, and 1. Mu.L of template; the PCR instrument operation program is as follows: denaturation at 98 ℃,10 s, annealing temperature at 57 ℃, 5s, extension at 72 ℃,2min, 30 cycles; final extension at 72deg.C for 2min; preserving at 4 ℃; electrophoresis is carried out on the PCR product on agarose gel, a target band is cut after the electrophoresis is finished, and a target fragment is recovered by using a DNA gel extraction kit;

TABLE 1 information on HA, NA, M1 Gene amplification primers after codon optimization

Note that: GCCGCCACC (bold) represents a Kozak sequence; the lower straight line is the cleavage site.

(3) The target fragment recovered in step (2) was ligated with pACEBac1 plasmid (Invitrogen) after BamHI and EcoRI double cleavage, and the ligation system (10. Mu.L) was as follows: 1. Mu.L of T4 DNA ligase, 1. Mu.L of 10 XBuffer, 5. Mu.L of target fragment cleavage product and 3. Mu.L of pACEBac1 plasmid cleavage product; the ligation product was transformed into DH 5. Alpha. Competent cells (Invitrogen) as follows: (1) standing in ice bath for 30min, and immediately performing ice bath for 2min after heat shock in water bath at 42 ℃ for 90 s; (2) adding 800. Mu.L of liquid LB medium without resistance into 1.5mL of EP tube under aseptic condition, placing in a constant temperature shaking table, and oscillating at 37 ℃ for 45min (220 rpm); (3) uniformly coating the well-grown bacterial liquid in a biological safety cabinet into a solid LB culture medium containing Gen+ resistance, then pouring a bacterial culture dish into a 37 ℃ incubator, and culturing for 12-16 hours; the plasmid is extracted by using a plasmid small extraction kit, and sequenced after enzyme digestion and electrophoresis identification, positive plasmids with the sequences being fidelity are named pACE-HA, pACE-NA and pACE-M1 respectively, wherein the enzyme digestion identification results of the recombinant transfer plasmids pACE-HA, pACE-NA and pACE-M1 are shown in figure 1.

Construction of recombinant baculovirus plasmids of HA, NA and M1 genes

The correctly sequenced recombinant transfer plasmids pACE-HA, pACE-NA, pACE-M1 were transformed into DH10bac competent cells (Invitrogen) as follows: mixing 1 μL of recombinant transfer plasmid with 100 μL of DH10Bac escherichia coli competent cells, standing on ice for 30min, and immediately carrying out ice bath for 2min after heat shock in a water bath at 42 ℃ for 45 s; adding 900 μl of non-antibiotic LB liquid medium, shaking at 37deg.C and 220rpm for 4 hr, diluting the bacterial liquid 10 times with non-antibiotic LB liquid medium to 10 ^-1 、10 ^-2 、10 ^-3 Uniformly coating 400 mu L of each bacterial liquid into a three-antibody LB plate, and placing the three-antibody LB plate in a 37 ℃ incubator for 48 hours; after culturing for 48h, pickingThe white monoclonal colony is subjected to amplification culture, and plasmids are extracted after the correct identification by PCR, so that recombinant baculovirus plasmids are obtained, which are respectively named as Bacmid-HA, bacmid-NA and Bacmid-M1.

EXAMPLE 2 rescue of recombinant baculoviruses from HA, NA and M1 genes

(1) The recombinant baculovirus plasmids Bacmid-HA, bacmid-NA, bacmid-M1 prepared in example 1 were transfected into sf9 insect cells (Invitrogen) using a conventional liposome-mediated transfection method, respectively, and cultured at 27 ℃; when the cells are cultured for 72 hours, the cells are diseased, and cell culture supernatants are collected, namely, the first generation recombinant baculovirus (P1) BV-HA, BV-NA and BV-M1 are respectively obtained;

(2) Inoculating the P1 generation recombinant baculovirus into sf9 cells, and collecting cell supernatant (namely the P2 generation recombinant baculovirus) when cytopathy is obvious, and continuously obtaining the P3 generation HA, NA and M1 recombinant baculovirus by adopting the sequential method.

EXAMPLE 3 expression, optimization and purification of H7N 9-VLPs in insect cells

(1) P3 generation HA, NA, M1 recombinant baculovirus was used at moi=7: 4:2 inoculating High five cells (Invitrogen company) in suspension culture, inoculating for 96 hours to harvest cells, and centrifuging to obtain extracellular culture supernatant and cells respectively; cell resuspension is followed by disruption, and intracellular disruption supernatant is obtained by centrifugation; measuring the hemagglutination titer of virus-like particles in the extracellular culture supernatant to be 11log2, and measuring the hemagglutination titer of the intracellular disruption supernatant to be 13log2;

(2) P3 generation HA, NA, M1 recombinant baculovirus was used at moi=3: 3:2 inoculating High five cells (Invitrogen company) in suspension culture, inoculating for 96 hours to harvest cells, and centrifuging to obtain extracellular culture supernatant and cells respectively; cell resuspension is followed by disruption, and intracellular disruption supernatant is obtained by centrifugation; measuring the hemagglutination titer of virus-like particles in the extracellular culture supernatant to be 9log2, and crushing the extracellular culture supernatant to be 9log2;

(3) P3 generation HA, NA, M1 recombinant baculovirus was used at moi=2: 1:2 inoculating High five cells (Invitrogen company) in suspension culture, inoculating for 96 hours to harvest cells, and centrifuging to obtain extracellular culture supernatant and cells respectively; cell resuspension is followed by disruption, and intracellular disruption supernatant is obtained by centrifugation; measuring the hemagglutination titer of virus-like particles in the extracellular culture supernatant to be 13log2, and measuring the hemagglutination titer of the intracellular disruption supernatant to be 13log2;

(4) P3 generation HA, NA, M1 recombinant baculovirus was used at moi=2: 1:2 co-transfecting virus-like particle samples in extracellular culture supernatant (prepared in the step (3)) collected from High five cells, performing SDS-PAGE and Western blot analysis and identification, wherein the primary antibody is His-tag monoclonal antibody (His-tag (4C 2) monoclonal antiboby of His protein, bioword TECHNOLOGY company), and the secondary antibody is fluorescence-labeled murine secondary antibody [

800CW gold anti-Mouse IgG (H+L) Secondary Antibody, LI-COR Biosciences Co.

SDS-PAGE and Western blot results are shown in FIG. 2, with the HA protein at about 70kDa, the NA protein at about 53kDa and the M1 protein at about 28kDa.

(5) Virus-like particle purification using sucrose density gradient centrifugation

Preparing sucrose solutions with different concentrations: preparing 20%, 30%, 45%, 60% (m/v) sucrose solution, and filtering with 0.22 μm filter; adding 20%, 30%, 45% and 60% sucrose solutions into a centrifuge tube from top to bottom, adding an avian influenza virus-like particle sample (extracellular culture supernatant collected in the step (3)) at the top, and centrifuging at 4 ℃ for 1h at 100000 Xg; after centrifugation, collecting a white transparent belt between 20% -30% of sucrose layers; 10000 Xg, centrifuging at 4 ℃ for 1.5h to remove sucrose; the avian influenza virus-like particles were resuspended in PBS buffer and stored at 4 ℃. The samples were subjected to subsequent experiments and protein concentration was determined using BCA protein quantification kit, with a protein concentration of about 1.96mg/ml.

EXAMPLE 4 Transmission Electron microscopy of the morphology and structure of H7N 9-VLPs

The sample of avian influenza virus-like particles purified in example 3 (H7N 9-VLP) was added dropwise to a carbon-coated copper mesh for adsorption and incubated at room temperature for 2min. Gently sucking off the excess liquid on the copper mesh with a piece of absorbent paper, drying, then negatively staining the sample with 1wt.% phosphotungstic acid, and incubating for 10min at room temperature; and then slowly absorbing and discarding the excessive phosphotungstic acid on the copper mesh by using water absorbing paper, airing at room temperature, and observing round particles (figure 3) with diameters of about 100nm, capsule membranes and no genetic material inside under a transmission electron microscope, wherein fiber protrusions are visible on the capsule membranes, and the morphological characteristics of the fiber protrusions are highly similar to those of the natural avian influenza virus, so that the recombinant baculovirus co-infection is successfully assembled into the avian influenza virus-like particles (H7N 9-VLP).

EXAMPLE 5 evaluation of vaccine efficacy by H7N9-VLP and white oil adjuvant

(1) Preparation of vaccine

H7N 9-VLPs harvested in example 3 were combined at an immunizing dose with dayer EOLANE 150 adjuvant according to 1:2 (v/v) mixing and emulsifying to prepare the avian influenza virus-like particle vaccine; wherein each 0.3ml of vaccine contains about 30 μ g H N9-VLP antigen;

(2) Evaluation of vaccine immunopotency

30 SPF chickens (purchased from Dahua farm eggs Inc. of New Yoghurt, guangdong, inc. of Ind. With a production license number of SCXK (Guangdong) 2013-0019) were randomly divided into 3 groups of 10 per group. Group 1 avian influenza whole virus inactivated vaccine injected subcutaneously via neck (vaccine source is commercially available, product is produced by agricultural large biological pharmaceutical company in south China, guangzhou, co.) at 0.3 ml/dose; group 2, injected subcutaneously with avian influenza virus-like particle vaccine via the neck, 0.3 ml/dose (0.3 ml vaccine contains about 30 μ g H7N 9-VLP); group 3 was injected with PBS as a blank. All test chickens were bled and serum isolated at week 3 post immunization, the immune serum was subjected to Hemagglutination Inhibition (HI) antibody detection, strain A/Chicken/Guangdong/16876/2016 (H7N 9) (i.e., strain GD16, both supplied by the national institute of medicine and veterinary university of North China, incorporated herein by reference, "She Hejia, enemy reddish, wen Bao, et al H7N9 subtype recombinant avian influenza virus rGD strain inactivated vaccine [ J]Animal medicine progress, 2019,040 (008): 44-48. "as four units of antigen after inactivation. 3 weeks after immunization, challenge was performed using A/Chicken/Guangdong/16876/2016 (H7N 9) strain, inoculated by nasal drip, 0.2 ml/dose (containing 2X 10) ^6.0 ELD ₅₀ ). And observing and timely recording the morbidity or mortality of the test chicken every day after the virus attack, collecting the test chicken throat and cloaca swab at the 5 th day after the virus attack for virus separation, and counting the vaccine protection condition. The results are shown in Table 2.

TABLE 2 vaccine immunopotentiation results

The results showed that the average HI antibody titers of the vaccine groups were above 6log2 at 3 weeks post immunization; after the challenge, the non-immune group died within 2 days after the challenge, the vaccine group did not show clinical symptoms within 14 days after the challenge, and the H7N9 subtype virus-like particle vaccine group only detected 1 chicken throat to expel toxin on day 5 after the challenge.

EXAMPLE 6H 7N9-VLP and Montanide ^TM Evaluation of vaccine efficacy by ISA71VG adjuvant preparation

(1) Preparation of vaccine

The H7N 9-VLPs harvested in example 3 were combined with Montanide at an immunizing dose ^TM ISA71VG adjuvant according to 3:7 (v/v) mixing and emulsifying to prepare the avian influenza virus-like particle vaccine; wherein each 0.3ml of vaccine contains about 30 μ g H N9-VLP antigen.

(2) Evaluation of vaccine immunopotency

20 SPF chickens of 3 weeks of age were randomly divided into 2 groups, 10 per group. 10 chickens were injected intramuscularly with the avian influenza virus-like particle vaccine, 0.3 ml/one (0.3 ml vaccine contained about 30 μ g H7N 9-VLP); another 10 chickens were injected with PBS as a blank, 0.3 ml/chicken.

(1) Antibody level detection

All test chickens were bled and serum isolated at day 14 and 19 post immunization. To evaluate the cross-reactivity of vaccine sera, the immune sera were subjected to cross-reactivity assays with wild-type H7N9 avian influenza strains, respectively, to determine hemagglutination inhibition antibodies (HI) and neutralizing antibody levels. The strains used include: A/Chicken/Guangdong/E157/2017 (H7N 9) (i.e., strain E157, which has been disclosed in application No. "201910117092.4", application name "avian influenza vaccine based on MultiBac baculovirus expression System" and preparation and application ") and A/Chicken/Qiangyuan/E664/2017 (H7N 9) (provided by the national institute of veterinary medicine, university of agricultural, north China). The HI and neutralizing antibodies results are shown in FIG. 4.

The results are shown in fig. 4, and the vaccine serum has good cross reactivity against different wild-type H7N9 avian influenza wild strains. For the A/Chicken/Guangdong/E157/2017 (H7N 9) strain, the average HI titer was 6.875log2 and the average neutralizing antibody titer was 1 at 19 days post immunization: 1706.67; for the a/Chicken/qingayuan/E664/2017 (H7N 9) strain, the average HI titer was 8log2 and the average neutralizing antibody titer was 1 at 19 days post immunization: 3413.33.

(2) toxicity attack protection experiment

3 weeks after immunization, A/Chicken/Guangdong/E157/2017 (H7N 9) strain was used at 10 ^6.0 EID ₅₀ And (3) carrying out toxin eliminating, nasal drip inoculation and the like, wherein the concentration is 0.2 ml/one. And observing and timely recording the morbidity or mortality of the test chicken every day after the virus attack, collecting the test chicken throat and cloaca swab for virus separation in 3, 5, 7 and 9 days after the virus attack, and counting the vaccine protection condition. The results are shown in Table 3.

TABLE 3 vaccine challenge protection results

Note that: dpc: days post challenge.

The results showed that the non-immunized group all died within 3 days after challenge, the vaccine group SPF chickens did not develop clinical symptoms within 14 days after challenge, all survived, and only 1 chicken was detected to expel toxin on day 9. The results show that the H7N9 subtype avian influenza virus-like particle vaccine can provide complete clinical protection and remarkably inhibit toxin expelling against the attack of wild type H7N9 subtype avian influenza virus.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

SEQUENCE LISTING

<110> agricultural university of south China

<120> a gene encoding recombinant avian influenza virus HA protein, virus-like particles, vaccine, preparation and use

<130> 1

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 1707

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of HA Gene after codon optimization

<400> 1

atgaacactc agatcctggt cttcgctctg atcgctatca tccccactaa cgccgacaag 60

atctgcctgg gtcaccacgc tgtgagcaac ggcactaagg tcaacactct gactgaacgt 120

ggtgtcgagg tcgtgaacgc tactgagact gtggaacgca ctaacacccc ccgcatctgc 180

agcaagggca agcgcaccgt cgacctgggt cagtgcggcc tgctgggcac tatcactggt 240

cccccccagt gcgaccagtt cctggagttc agcgctgacc tgatcatcga acgccgcgag 300

ggttccgacg tctgctaccc tggtaaattc gtcaacgaag aagctctgcg ccagatcctg 360

cgcgagagcg gcggaatcga caaggagcct atgggcttca cttacaacgg tatccgcact 420

aacggtgtga ctagcgcttg ccgccgcagc ggtagcagct tctacgccga aatgaagtgg 480

ctgctgtcca acaccgacaa cgctactttc ccccagatga ccaagtccta caagaacact 540

cgcaagagcc ccgccatcat cgtgtggggt atccaccact ccgtctccac tgctgaacag 600

actaagctgt acggttccgg taacaagctg gtgaccgtcg gttcctccaa ctaccagcag 660

tccttcgtcc ccagccctgg tgcccgtcct caggtgaacg gtcagagcgg ccgcatcgac 720

ttccactggc tgatcctgaa ccctaacgac accgtgacct tcagcttcaa cggtgctttc 780

atcgctcctg accgcgcttc cttcctgcgc ggtaaaagca tgggtatcca gtccggcgtg 840

caggtggacg ccaactgcga aggcgactgc taccacagcg gcggtactat catctccaac 900

ctgcctttcc agaacatcga cagccgtgct gtcggtaaat gcccccgtta cgtcaagcag 960

cgctccctgc tgctggctac tggcatgaag aacgtccctg aggttcctaa gggcaagcgt 1020

actgctcgcg gtctgttcgg cgccatcgcc ggtttcatcg agaacggttg ggagggcctg 1080

atcgacggct ggtacggttt ccgccaccag aacgcccagg gcgagggcac tgctgctgac 1140

tacaagagca ctcagtccgc tatcgaccag atcaccggta aactgaaccg cctgatcgcc 1200

aagaccaacc agcagttcaa gctgatcgac aacgagttta atgaggtcga gaagcagatc 1260

ggcaacgtca tcaactggac tcgtgactcc atcactgagg tctggagcta caacgccgag 1320

ctgctggtgg ctatggaaaa ccagcacacc atcgacctcg ctgactccga gatggacaag 1380

ctgtacgaac gcgtcaagcg ccagctgcgc gagaacgctg aagaagacgg cactggctgc 1440

ttcgagatct tccacaagtg cgacgacgac tgcatggctt ccatccgtaa caacacctac 1500

gaccaccgta agtaccgcga agaagccatg cagaaccgta tccagatcga ccccgtcaag 1560

ctgagctccg gctacaagga cgtcatcctg tggttctcct tcggtgccag ctgcttcatc 1620

ctgctggcta ttgttatggg tctggtcttc atctgcgtga agaacggtaa catgcgttgc 1680

accatccacc accaccacca tcactaa 1707

<210> 2

<211> 1416

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of NA Gene after codon optimization

<400> 2

atgaacccta accagaagat cctgtgcacc tccgctaccg ctatcaccat cggtgctatc 60

accgtgctga tcggtatcgc taacctgggt ctgaacatcg gtctgcacct gaagtccggt 120

tgcaactgtt cccgctccca acctgagact accaacacct cccagaccat catcaacaac 180

tactacaacg agactaacat caccaacatc cagatggagg aacgcacctc ccgcaacttc 240

aacaacctga ccaagggtct gtgcaccatc aactcctggc acatctacgg taaggacaac 300

gctgtgcgca ttggtgaatc ctccgacgtt ctggtgactc gcgagcctta tgtgtcctgc 360

gaccctgatg aatgccgctt ctacgctctg tcccagggta ctaccattcg cggtaagcac 420

tccaacggta ctatccacga ccgttcccaa taccgcgctc tgatctcttg gcctctgtcc 480

tctcctccta ccgtgtataa ctcccgcgtg gagtgtattg gttggtcctc cacctcttgc 540

cacgatggta agtcccgcat gtccatctgc atctccggtc ctaacaacaa cgcttccgct 600

gtgatctggt acaaccgtcg ccctgtggct gaaatcaaca cctgggctcg caacatcctg 660

cgtacccaag agtctgagtg cgtgtgccat aacggtgtgt gccctgtggt gttcactgac 720

ggtcctgcta ctggtcctgc tgatacccgc atctactact tcaaggaggg taagatcctg 780

aagtgggagt ccttgaccgg caccgctaag cacatcgagg agtgctcctg ctatggtaag 840

cgcaccggta ttacttgtac ctgccgcgac aattggcaag gttccaaccg ccctgtgatc 900

cagattgacc ctgtggctat gactcacacc tcccagtaca tctgctcccc tgtgctgact 960

gattcccctc gtcctaacga ccctaacatc ggtaagtgca acgaccctta ccctggtaac 1020

aacaacaacg gtgtgaaggg tttctcctac ctggacggtg acaacacttg gctgggtcgt 1080

accatttcca ccgcttcccg ttccggttac gagatgctga aggtgcctaa cgctctgact 1140

gacgaccgct ccaagcctat tcagggtcag accatcgtgc tgaacgctga ctggtccggt 1200

tactccggtt ccttcatgga ctactgggct gagggtgact gctatcgcgc ttgcttctac 1260

gttgagctga tccgcggtaa gcctaaagag gacaaggtgt ggtggacctc caactccatc 1320

gtgtccatgt gctcctccac cgagtttctg ggtcagtgga actggcctga cggtgctaag 1380

atcgagtact tcctgcacca ccaccaccac cactaa 1416

<210> 3

<211> 777

<212> DNA

<213> Artificial

<220>

<223> nucleotide sequence of M1 Gene after codon optimization

<400> 3

atgtctctgc tgaccgaggt ggagacttac gtgctgtcca tcatcccttc cggtcctctg 60

aaggctgaga tcgctcagcg tctggaggat gtgttcgctg gtaagaacgc tgacctggag 120

gctctgatgg agtggatcaa gacccgccct atcttgtccc ctctgaccaa gggtatcctg 180

ggtttcgtgt tcaccctgac cgtgccttcc gaacgtggtc tgcaacgtcg tcgtttcgtg 240

cagaacgctc tgaacggtaa cggtgaccct aacaacatgg acaaggctgt gaagctgtac 300

aagaagctga agcgcgagat gaccttccac ggtgctaagg aggtggctct gtcctattcc 360

accggtgctc tggcttcttg catgggtctg atctacaacc gcatgggcac cgtgactgct 420

gaaggtgctc tgggtctggt ttgtgctacc tgcgagcaga ttgctgacgc tcagcaccgt 480

tcccatcgtc aaatggctac caccaccaac cctctgatcc gccacgaaaa ccgcatggtg 540

ctggcttcta ccaccgctaa ggctatggag cagatggctg gttcctccga gcaagctgct 600

gaggctatgg aggtggcttc ccaagctcgc cagatggtgc aagctatgcg cactgtgggt 660

actcacccta actcctccac cggtctgaag gacgacctga tcgagaacct gcaggcttac 720

cagaaccgca tgggtgttca actgcagcgc ttcaagcacc atcaccacca ccactaa 777

<210> 4

<211> 568

<212> PRT

<213> Artificial

<220>

<223> HA protein amino acid sequence

<400> 4

Met Asn Thr Gln Ile Leu Val Phe Ala Leu Ile Ala Ile Ile Pro Thr

1 5 10 15

Asn Ala Asp Lys Ile Cys Leu Gly His His Ala Val Ser Asn Gly Thr

20 25 30

Lys Val Asn Thr Leu Thr Glu Arg Gly Val Glu Val Val Asn Ala Thr

35 40 45

Glu Thr Val Glu Arg Thr Asn Thr Pro Arg Ile Cys Ser Lys Gly Lys

50 55 60

Arg Thr Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly

65 70 75 80

Pro Pro Gln Cys Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu Ile Ile

85 90 95

Glu Arg Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn

100 105 110

Glu Glu Ala Leu Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp Lys

115 120 125

Glu Pro Met Gly Phe Thr Tyr Asn Gly Ile Arg Thr Asn Gly Val Thr

130 135 140

Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp

145 150 155 160

Leu Leu Ser Asn Thr Asp Asn Ala Thr Phe Pro Gln Met Thr Lys Ser

165 170 175

Tyr Lys Asn Thr Arg Lys Ser Pro Ala Ile Ile Val Trp Gly Ile His

180 185 190

His Ser Val Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn

195 200 205

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

210 215 220

Ser Pro Gly Ala Arg Pro Gln Val Asn Gly Gln Ser Gly Arg Ile Asp

225 230 235 240

Phe His Trp Leu Ile Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe

245 250 255

Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Leu Arg Gly Lys

260 265 270

Ser Met Gly Ile Gln Ser Gly Val Gln Val Asp Ala Asn Cys Glu Gly

275 280 285

Asp Cys Tyr His Ser Gly Gly Thr Ile Ile Ser Asn Leu Pro Phe Gln

290 295 300

Asn Ile Asp Ser Arg Ala Val Gly Lys Cys Pro Arg Tyr Val Lys Gln

305 310 315 320

Arg Ser Leu Leu Leu Ala Thr Gly Met Lys Asn Val Pro Glu Val Pro

325 330 335

Lys Gly Lys Arg Thr Ala Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe

340 345 350

Ile Glu Asn Gly Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe Arg

355 360 365

His Gln Asn Ala Gln Gly Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr

370 375 380

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

385 390 395 400

Lys Thr Asn Gln Gln Phe Lys Leu Ile Asp Asn Glu Phe Asn Glu Val

405 410 415

Glu Lys Gln Ile Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Ile Thr

420 425 430

Glu Val Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln

435 440 445

His Thr Ile Asp Leu Ala Asp Ser Glu Met Asp Lys Leu Tyr Glu Arg

450 455 460

Val Lys Arg Gln Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys

465 470 475 480

Phe Glu Ile Phe His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile Arg

485 490 495

Asn Asn Thr Tyr Asp His Arg Lys Tyr Arg Glu Glu Ala Met Gln Asn

500 505 510

Arg Ile Gln Ile Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Val

515 520 525

Ile Leu Trp Phe Ser Phe Gly Ala Ser Cys Phe Ile Leu Leu Ala Ile

530 535 540

Val Met Gly Leu Val Phe Ile Cys Val Lys Asn Gly Asn Met Arg Cys

545 550 555 560

Thr Ile His His His His His His

565

<210> 5

<211> 471

<212> PRT

<213> Artificial

<220>

<223> NA protein amino acid sequence

<400> 5

Met Asn Pro Asn Gln Lys Ile Leu Cys Thr Ser Ala Thr Ala Ile Thr

1 5 10 15

Ile Gly Ala Ile Thr Val Leu Ile Gly Ile Ala Asn Leu Gly Leu Asn

20 25 30

Ile Gly Leu His Leu Lys Ser Gly Cys Asn Cys Ser Arg Ser Gln Pro

35 40 45

Glu Thr Thr Asn Thr Ser Gln Thr Ile Ile Asn Asn Tyr Tyr Asn Glu

50 55 60

Thr Asn Ile Thr Asn Ile Gln Met Glu Glu Arg Thr Ser Arg Asn Phe

65 70 75 80

Asn Asn Leu Thr Lys Gly Leu Cys Thr Ile Asn Ser Trp His Ile Tyr

85 90 95

Gly Lys Asp Asn Ala Val Arg Ile Gly Glu Ser Ser Asp Val Leu Val

100 105 110

Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Glu Cys Arg Phe Tyr

115 120 125

Ala Leu Ser Gln Gly Thr Thr Ile Arg Gly Lys His Ser Asn Gly Thr

130 135 140

Ile His Asp Arg Ser Gln Tyr Arg Ala Leu Ile Ser Trp Pro Leu Ser

145 150 155 160

Ser Pro Pro Thr Val Tyr Asn Ser Arg Val Glu Cys Ile Gly Trp Ser

165 170 175

Ser Thr Ser Cys His Asp Gly Lys Ser Arg Met Ser Ile Cys Ile Ser

180 185 190

Gly Pro Asn Asn Asn Ala Ser Ala Val Ile Trp Tyr Asn Arg Arg Pro

195 200 205

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

210 215 220

Ser Glu Cys Val Cys His Asn Gly Val Cys Pro Val Val Phe Thr Asp

225 230 235 240

Gly Pro Ala Thr Gly Pro Ala Asp Thr Arg Ile Tyr Tyr Phe Lys Glu

245 250 255

Gly Lys Ile Leu Lys Trp Glu Ser Leu Thr Gly Thr Ala Lys His Ile

260 265 270

Glu Glu Cys Ser Cys Tyr Gly Lys Arg Thr Gly Ile Thr Cys Thr Cys

275 280 285

Arg Asp Asn Trp Gln Gly Ser Asn Arg Pro Val Ile Gln Ile Asp Pro

290 295 300

Val Ala Met Thr His Thr Ser Gln Tyr Ile Cys Ser Pro Val Leu Thr

305 310 315 320

Asp Ser Pro Arg Pro Asn Asp Pro Asn Ile Gly Lys Cys Asn Asp Pro

325 330 335

Tyr Pro Gly Asn Asn Asn Asn Gly Val Lys Gly Phe Ser Tyr Leu Asp

340 345 350

Gly Asp Asn Thr Trp Leu Gly Arg Thr Ile Ser Thr Ala Ser Arg Ser

355 360 365

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

370 375 380

Lys Pro Ile Gln Gly Gln Thr Ile Val Leu Asn Ala Asp Trp Ser Gly

385 390 395 400

Tyr Ser Gly Ser Phe Met Asp Tyr Trp Ala Glu Gly Asp Cys Tyr Arg

405 410 415

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

420 425 430

Val Trp Trp Thr Ser Asn Ser Ile Val Ser Met Cys Ser Ser Thr Glu

435 440 445

Phe Leu Gly Gln Trp Asn Trp Pro Asp Gly Ala Lys Ile Glu Tyr Phe

450 455 460

Leu His His His His His His

465 470

<210> 6

<211> 258

<212> PRT

<213> Artificial

<220>

<223> M1 protein amino acid sequence

<400> 6

Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro

1 5 10 15

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe

20 25 30

Ala Gly Lys Asn Ala Asp Leu Glu Ala Leu Met Glu Trp Ile Lys Thr

35 40 45

Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe

50 55 60

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

65 70 75 80

Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala

85 90 95

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

100 105 110

Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met

115 120 125

Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Ala Glu Gly Ala Leu

130 135 140

Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ala Gln His Arg

145 150 155 160

Ser His Arg Gln Met Ala Thr Thr Thr Asn Pro Leu Ile Arg His Glu

165 170 175

Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met

180 185 190

Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln

195 200 205

Ala Arg Gln Met Val Gln Ala Met Arg Thr Val Gly Thr His Pro Asn

210 215 220

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

225 230 235 240

Gln Asn Arg Met Gly Val Gln Leu Gln Arg Phe Lys His His His His

245 250 255

His His

<210> 7

<211> 43

<212> DNA

<213> Artificial

<220>

<223> HA-BamHI-F

<400> 7

cgggatccgc cgccaccatg aacactcaga tcctggtctt cgc 43

<210> 8

<211> 32

<212> DNA

<213> Artificial

<220>

<223> HA-EcoRI-R

<400> 8

cggaattctt agtgatggtg gtggtggtgg at 32

<210> 9

<211> 42

<212> DNA

<213> Artificial

<220>

<223> NA- BamHI-F

<400> 9

cgggatccgc cgccaccatg aaccctaacc agaagatcct gt 42

<210> 10

<211> 35

<212> DNA

<213> Artificial

<220>

<223> NA-EcoRI-R

<400> 10

cggaattctt agtggtggtg gtggtggtgc aggaa 35

<210> 11

<211> 39

<212> DNA

<213> Artificial

<220>

<223> M1- BamHI-F

<400> 11

cgggatccgc cgccaccatg tctctgctga ccgaggtgg 39

<210> 12

<211> 35

<212> DNA

<213> Artificial

<220>

<223> M1-EcoRI-R

<400> 12

cggaattctt agtggtggtg gtgatggtgc ttgaa 35

Claims

1. An H7N9 avian influenza virus-like particle, which is characterized by comprising an avian influenza virus HA protein, an avian influenza virus NA protein and an avian influenza virus M1 protein; the nucleotide sequences of the gene for encoding the avian influenza virus HA protein, the gene for encoding the avian influenza virus NA protein and the gene for encoding the avian influenza virus M1 protein are respectively shown as SEQ ID NO. 1-3.

2. The H7N9 avian influenza virus like particle of claim 1, wherein:

the avian influenza virus-like particle is formed by self-assembly of avian influenza virus HA protein, avian influenza virus NA protein and avian influenza virus M1 protein.

3. The method for preparing the H7N9 avian influenza virus-like particle as claimed in claim 1 or 2, characterized by comprising the steps of:

(1) Species codons of HA, NA and M1 genes of the avian influenza virus are optimized, and the genes are synthesized to obtain genes which code the HA protein of the avian influenza virus, the NA protein of the avian influenza virus and the M1 protein of the avian influenza virus after the codons are optimized, and the nucleotide sequences of the genes are respectively shown as SEQ ID NO. 1-3;

(5) The HA gene recombinant baculovirus, the NA gene recombinant baculovirus and the M1 gene recombinant baculovirus are used for infecting insect cells together, and extracellular culture supernatant is collected to obtain H7N9 avian influenza virus-like particles assembled by HA, NA and M1 proteins;

when the HA gene recombinant baculovirus, the NA gene recombinant baculovirus and the M1 gene recombinant baculovirus are co-infected in the step (5), the MOI is (2-7): (1-4): 2.

4. the method for preparing H7N9 avian influenza virus like particle according to claim 3, wherein:

when the HA gene recombinant baculovirus, the NA gene recombinant baculovirus and the M1 gene recombinant baculovirus are co-infected in the step (5), the MOI is 2:1:2.

5. an H7N9 avian influenza virus-like particle vaccine comprising a pharmaceutically acceptable carrier and an immunizing amount of the avian influenza virus-like particle of claim 1 or 2.

6. The method for preparing the H7N9 avian influenza virus-like particle vaccine according to claim 5, which is characterized by comprising the following steps:

mixing and emulsifying the avian influenza virus-like particles with an adjuvant according to an immune dose to obtain the H7N9 avian influenza virus-like particle vaccine.

7. Use of the H7N9 avian influenza virus-like particle of claim 1 or 2 or the H7N9 influenza virus-like particle vaccine of claim 6 for the manufacture of a medicament for the prevention and/or treatment of diseases caused by H7N9 avian influenza virus.