Gene for coding recombinant avian influenza virus HA protein, virus-like particle, vaccine, preparation and application
阅读说明：本技术 一种编码重组禽流感病毒ha蛋白的基因、病毒样颗粒、疫苗及制备与应用 (Gene for coding recombinant avian influenza virus HA protein, virus-like particle, vaccine, preparation and application ) 是由 樊惠英 孔德鑫 廖明 陈陶然 胡小龙 于 2021-09-13 设计创作，主要内容包括：本发明属于基因工程疫苗技术领域,具体涉及一种编码重组禽流感病毒HA蛋白的基因、病毒样颗粒、疫苗及制备与应用。所述的编码重组禽流感病毒HA蛋白的基因的核苷酸序列如SEQ ID NO:1。本发明还提供了一种禽流感病毒样颗粒,该禽流感病毒样颗粒由HA蛋白、NA蛋白和M1蛋白组装而成,血凝效价可达13log2。本发明还提供了一种包含上述病毒样颗粒的禽流感病毒样颗粒疫苗,该疫苗针对同源和野生型H7N9亚型高致病性禽流感病毒的致死性攻击能够提供完全的临床保护且显著抑制排毒,为H7N9亚型禽流感的防控提供新的疫苗选择。(The invention belongs to the technical field of genetic engineering vaccines, and particularly relates to a gene for encoding recombinant avian influenza virus HA protein, virus-like particles, a vaccine, and preparation and application thereof. The nucleotide sequence of the gene for coding 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 13log 2. The invention also provides an avian influenza virus-like particle vaccine containing the avian influenza virus-like particle, which can provide complete clinical protection and obviously inhibit toxin expulsion aiming at lethal attack of homologous and wild H7N9 subtype highly pathogenic avian influenza virus, and provides a new vaccine selection for the prevention and control of H7N9 subtype avian influenza.)
1. A gene for coding recombinant avian influenza virus HA protein is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. An avian influenza virus-like particle characterized by comprising an avian influenza virus HA protein, an avian influenza virus NA protein and an avian influenza virus M1 protein; wherein, the gene for coding the avian influenza virus HA protein is the gene for coding the recombinant avian influenza virus HA protein according to claim 1.
3. The avian influenza virus-like particle of claim 2, wherein:
the nucleotide sequences of the gene for coding the NA protein of the avian influenza virus and the gene for coding the M1 protein of the avian influenza virus are respectively shown as SEQ ID NO. 2-3.
4. The avian influenza virus-like particle of claim 3, 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.
5. The method for producing an avian influenza virus-like particle according to any one of claims 2 to 4, characterized by comprising the steps of:
(1) species codon optimization is carried out on HA, NA and M1 genes of the avian influenza virus, and gene synthesis is carried out, so that a gene encoding HA protein of the avian influenza virus, a gene encoding NA protein of the avian influenza virus and a gene encoding M1 protein of the avian influenza virus are obtained after codon optimization, and the nucleotide sequences are respectively shown as SEQ ID NO 1-3;
(2) performing PCR amplification by taking the gene coding the avian influenza virus HA protein, the gene coding the avian influenza virus NA protein and the gene coding the avian influenza virus M1 protein which are obtained in the step (1) after codon optimization as templates to obtain HA, NA and M1 gene segments with enzyme cutting sites, and performing enzyme cutting, connection and transformation on the HA, NA and M1 gene segments with the enzyme cutting sites and the baculovirus transfer plasmid to respectively obtain an HA gene recombination transfer plasmid, an NA gene recombination transfer plasmid and an M1 gene recombination transfer plasmid;
(3) transforming and recombining the HA gene recombination transfer plasmid, the NA gene recombination transfer plasmid and the M1 gene recombination transfer plasmid to respectively obtain an HA gene recombination baculovirus plasmid, an NA gene recombination baculovirus plasmid and an M1 gene recombination baculovirus plasmid;
(4) transfecting an sf9 cell by an HA gene recombinant baculovirus plasmid, an NA gene recombinant baculovirus plasmid and an M1 gene recombinant baculovirus plasmid through liposome mediation to respectively obtain an HA gene recombinant baculovirus, an NA gene recombinant baculovirus and an M1 gene recombinant baculovirus;
(5) the HA gene recombinant baculovirus, the NA gene recombinant baculovirus and the M1 gene recombinant baculovirus infect insect cells together, and the extracellular culture supernatant is collected to obtain the avian influenza virus-like particle assembled by HA, NA and M1 proteins.
6. The method for producing an avian influenza virus-like particle according to claim 5, characterized in that:
when the HA gene recombinant baculovirus, the NA gene recombinant baculovirus and the M1 gene recombinant baculovirus in the step (5) are infected together, the MOI is (2-7): (1-4): 2.
7. the method for producing an avian influenza virus-like particle according to claim 6, characterized in that:
when the HA gene recombinant baculovirus, the NA gene recombinant baculovirus and the M1 gene recombinant baculovirus in the step (5) are infected together, the MOI is 2: 1: 2.
8. an avian influenza virus-like particle vaccine characterized by comprising a pharmaceutically acceptable carrier and an immunizing amount of the avian influenza virus-like particle according to any one of claims 2 to 4.
9. The method for preparing the avian influenza virus-like particle vaccine of claim 8, characterized by comprising the steps of:
mixing and emulsifying the avian influenza virus-like particles with an adjuvant according to the immunizing dose to obtain the avian influenza virus-like particle vaccine.
10. Use of the gene encoding the recombinant avian influenza virus HA protein according to claim 1, the avian influenza virus-like particle according to any one of claims 2 to 4, and the influenza virus-like particle vaccine according to claim 8 in the preparation of a medicament for preventing and/or treating diseases caused by avian influenza virus.
Technical Field
The invention belongs to the technical field of genetic engineering vaccines, and particularly relates to a gene for encoding recombinant avian influenza virus HA protein, virus-like particles, a vaccine, and preparation and application thereof.
Background
Avian Influenza Virus (AIV) is a segmented, negative-strand RNA virus of the capsular type, belonging to the orthomyxoviridae family, the genus Influenza, Avian Influenza being one of the Avian virulent infectious diseases.
The avian influenza virus encodes a plurality of proteins, and HA (surface antigen hemagglutinin) protein is a membrane protein encoded by the avian influenza virus, plays an important role in infecting host cells by the avian influenza virus, and is a main target antigen for generating immune efficacy of the host to the avian influenza virus. The NA (neuraminic acid) protein is also a membrane protein and serves primarily to facilitate the release of progeny virus from the host cell. The HA and NA proteins are the main antigenic components for the development of avian influenza vaccines.
Vaccination is one of the most effective measures to prevent infection by avian influenza virus. At present, the domestic avian influenza vaccine is mainly a whole virus inactivated vaccine, and the vaccine is produced by chicken embryos, so that the defects of insufficient supply of the chicken embryos, generation of a large amount of waste, endogenous pollution and the like during influenza epidemics exist. Meanwhile, the long-term use of the whole virus inactivated vaccine accelerates the variation rate of the avian influenza virus, and the long-term immunoselection pressure enables the avian influenza virus to evolve towards a non-vaccine strain, so that the whole virus inactivated vaccine needs to continuously update the vaccine strain to cope with new influenza virus epidemics. Therefore, there is a need to develop a new, safe and effective avian influenza vaccine to prevent and control the prevalence of avian influenza virus.
Virus-like particles (VLPs) are viroid particles assembled from structural proteins of viruses, which are free from viral nucleic acids and infectious and are a hot spot for the development of novel avian influenza vaccines. Compared with the whole virus inactivated vaccine which mainly takes humoral immunity as a main factor, the virus-like particle vaccine can simultaneously induce humoral immunity and cellular immunity, and the latter is the key of cross protection of the avian influenza vaccine. The baculovirus expression system has high safety and easy operation, can prepare the avian influenza virus-like particles on a large scale, and is an important tool for developing the avian influenza virus-like particle vaccine. Compared with the whole virus inactivated vaccine, the avian influenza virus-like particle can be quickly prepared by using 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 object of the present invention is to provide a gene encoding recombinant avian influenza virus HA protein.
It is another object of the present invention to provide an avian influenza virus-like particle.
Still another object of the present invention is to provide a method for preparing the avian influenza virus-like particle.
The fourth purpose of the invention is to provide an avian influenza virus-like particle vaccine.
The fifth purpose of the invention is to provide 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.
The purpose of the invention is realized by the following technical scheme:
the nucleotide sequence of the gene for coding the 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 coding the avian influenza virus HA protein is the gene for coding the recombinant avian influenza virus HA protein;
the nucleotide sequences of the gene for coding the NA protein of the avian influenza virus and the gene for coding the M1 protein of the avian influenza virus are respectively shown as 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 as SEQ ID NO. 4-6;
the avian influenza virus-like particle is preferably formed by self-assembly of 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 codon optimization is carried out on HA, NA and M1 genes of the avian influenza virus, and gene synthesis is carried out, so that a gene encoding HA protein of the avian influenza virus, a gene encoding NA protein of the avian influenza virus and a gene encoding M1 protein of the avian influenza virus are obtained after codon optimization, and the nucleotide sequences are respectively shown as SEQ ID NO 1-3; through species codon optimization, the expression of insect cells is facilitated;
(2) performing PCR amplification by taking the gene coding the avian influenza virus HA protein, the gene coding the avian influenza virus NA protein and the gene coding the avian influenza virus M1 protein which are obtained in the step (1) after codon optimization as templates to obtain HA, NA and M1 gene segments with enzyme cutting sites, and performing enzyme cutting, connection and transformation on the HA, NA and M1 gene segments with the enzyme cutting sites and the baculovirus transfer plasmid to respectively obtain an HA gene recombination transfer plasmid, an NA gene recombination transfer plasmid and an M1 gene recombination transfer plasmid;
(3) transforming and recombining the HA gene recombination transfer plasmid, the NA gene recombination transfer plasmid and the M1 gene recombination transfer plasmid to respectively obtain an HA gene recombination baculovirus plasmid, an NA gene recombination baculovirus plasmid and an M1 gene recombination baculovirus plasmid;
(4) transfecting an sf9 cell by an HA gene recombinant baculovirus plasmid, an NA gene recombinant baculovirus plasmid and an M1 gene recombinant baculovirus plasmid through liposome mediation to respectively obtain an HA gene recombinant baculovirus, an NA gene recombinant baculovirus and an M1 gene recombinant baculovirus;
(5) co-infecting insect cells by using HA gene recombinant baculovirus, NA gene recombinant baculovirus and M1 gene recombinant baculovirus, collecting extracellular culture supernatant, and obtaining avian influenza virus-like particles assembled by HA, NA and M1 proteins;
the baculovirus transfer plasmid in the step (2) is pACEBac 1;
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 in the step (5) are infected together, the MOI is (2-7): (1-4): 2;
when the HA gene recombinant baculovirus, the NA gene recombinant baculovirus and the M1 gene recombinant baculovirus in the step (5) are infected together, the MOI is preferably 2: 1: 2;
an avian influenza virus-like particle vaccine comprises a pharmaceutically acceptable carrier and an immunizing dose of the avian influenza virus-like particle;
the pharmaceutically acceptable carrier includes an adjuvant;
the adjuvant is at least one of a white oil adjuvant and a water-in-oil adjuvant;
the white oil adjuvant is preferably Doudal EOLANE 150;
the water-in-oil adjuvant is MontanideTMISA series adjuvant, more preferably MontanideTMISA71VG 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 according to the immunizing dose to obtain the avian influenza virus-like particle vaccine;
when the adjuvant is a white oil adjuvant, the volume ratio of the avian influenza virus-like particles 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;
the gene for coding the recombinant avian influenza virus HA protein, the avian influenza virus-like particle and the avian influenza virus-like particle vaccine are applied to the preparation of the medicine for preventing and/or treating diseases caused by the avian influenza virus;
the avian influenza virus comprises H7N9 subtype avian influenza virus;
the administration objects of 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 the avian influenza subunit vaccine, and species codon optimization is carried out on HA, NA and M1 genes of the avian influenza virus, so that the avian influenza virus is not only beneficial to insect cell expression, but also HAs strong immunogenicity.
(2) The invention produces the 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 to the culture supernatant outside the cells in a virus-like particle form, and the antigen expression is high-efficiency.
(3) The invention researches the proportion of HA, NA and M1 recombinant baculovirus co-infected insect cells, and preferably, the HA, NA and M1 recombinant baculovirus HAs the MOI of 2: 1: 2 co-infecting insect cells, the obtained avian influenza virus-like particles have the highest hemagglutination titer, and the hemagglutination titer reaches 13log 2.
(4) The method for producing the avian influenza virus-like particle antigen adopts HA, NA and M1 recombinant baculovirus to co-infect insect cells, and compared with the method for producing the avian influenza virus-like particle by serially connecting HA, NA and M1 genes to the same vector, the co-infection mode HAs larger optimization space, and the content of the main target antigen in the avian influenza virus-like particle can be controllably increased and the proportion of the content of each antigen can be regulated.
(5) The invention mixes and emulsifies the quantified H7N9 avian influenza virus-like particles and white oil adjuvant EOLANE 150 to prepare the vaccine, and evaluates the immune efficacy of the vaccine. Immunizing SPF chickens of 3 weeks of age, wherein the average Hemagglutination Inhibition (HI) antibody titer is more than 6log 23 weeks after immunization; 3 weeks after immunization, 2X 10 using A/Chiken/Guangdong/16876/2016 (H7N9) strain6.0ELD50The dose of the Chinese medicinal composition is used for counteracting toxic substances, and a swab of a throat and a cloaca is collected on the 5 th day after counteracting toxic substances to detect and expel toxic substances. The results showed that the non-immunized group died completely within 2 days after challenge, and the vaccine group did not die within 14 days after challengeClinical symptoms appeared, all survived, and only 1 chicken detoxified on day 5 post challenge.
(6) The invention quantitatively uses the H7N9 avian influenza virus-like particles and MontanideTMISA71VG adjuvant is mixed and emulsified to prepare a vaccine, and the immune efficacy of the vaccine against wild type H7N9 subtype avian influenza virus is evaluated. The results show that the vaccine sera have good cross-reactivity against different wild-type wild strains of avian influenza H7N 9. For the A/Chicken/Guangdong/E157/2017(H7N9) strain, the average HI titer reaches 6.875log2 and the average neutralizing antibody titer reaches 1 at 19 days after immunization: 1706.67, respectively; for the a/Chicken/Qingyuan/E664/2017(H7N9) strain, the mean HI titer reached 8log2, the mean neutralizing antibody titer reached 1: 3413.33, respectively; the A/Chicken/Guangdong/E157/2017(H7N9) strain was used at 106.0EID50The dose of the Chinese herbal medicine is 0.2ml per mouse, and the swabs of the throat and the cloaca are collected 3, 5, 7 and 9 days after toxin attack to detect and expel toxin. The results showed that the non-immunized group died completely within 3 days after challenge, the vaccine group did not show clinical symptoms within 14 days after challenge, all survived, and only 1 chicken was detected to be detoxified 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 particle prepared by the invention is combined with the EOLANE 150 adjuvant, so that complete clinical protection can be provided and detoxification can be obviously inhibited by using lethal attack aiming at homologous H7N9 subtype highly pathogenic avian influenza virus; H7N9 subtype avian influenza virus-like particle combined Montanide prepared by the inventionTMISA71VG adjuvant induces high levels of HI and neutralizing antibodies using highly pathogenic avian influenza virus against wild type subtype H7N 9; lethal challenge against wild-type H7N9 avian influenza virus provided complete clinical protection, and detoxification was detected in only one chicken. 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 an analysis chart of the restriction enzyme analysis result of the recombinant HA, NA and M1 gene transfer plasmid, in which A: pACE-HA, B: pACE-NA, C: pACE-M1.
Fig. 2 shows HA, NA, M1 gene recombinant baculovirus as MOI ═ 2: 1: 2 SDS-PAGE and Western blot analysis of avian influenza virus-like particles expressed by infected insect cells.
FIG. 3 is an electron microscopic view of the avian influenza virus-like particle.
FIG. 4 is a graph of avian influenza virus-like particle and MontanideTMGraph analysis of serum hemagglutination inhibition antibody (HI) and neutralizing antibody results on day 14 and 19 after immunization of SPF chickens with ISA71VG mixed emulsion prepared vaccine.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test materials in the following examples were all commercially available unless otherwise specified. The test methods are conventional test methods unless otherwise specified.
Example 1 construction of recombinant baculovirus plasmids of HA, NA and M1 genes
(I) construction of HA, NA, M1 gene recombination transfer plasmid
(1) In the embodiment, the nucleotide sequence of HA, NA and M1 genes of the avian influenza virus is subjected to codon optimization and is biased to insect cell expression, and 6x his labels are added at the C tail ends of the HA, NA and M1 genes; obtaining nucleotide sequences of HA, NA and M1 genes after codon optimization through artificial synthesis, and respectively connecting the nucleotide sequences into a PUC57 vector to obtain corresponding recombinant plasmids (Beijing Liuhe Huada Gene science and technology Co., Ltd.); wherein, 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
the amino acid sequence of the NA protein:
MNPNQKILCTSATAITIGAITVLIGIANLGLNIGLHLKSGCNCSRSQPETTNTSQTIINNYYNETNITNIQMEERTSRNFNNLTKGLCTINSWHIYGKDNAVRIGESSDVLVTREPYVSCDPDECRFYALSQGTTIRGKHSNGTIHDRSQYRALISWPLSSPPTVYNSRVECIGWSSTSCHDGKSRMSICISGPNNNASAVIWYNRRPVAEINTWARNILRTQESECVCHNGVCPVVFTDGPATGPADTRIYYFKEGKILKWESLTGTAKHIEECSCYGKRTGITCTCRDNWQGSNRPVIQIDPVAMTHTSQYICSPVLTDSPRPNDPNIGKCNDPYPGNNNNGVKGFSYLDGDNTWLGRTISTASRSGYEMLKVPNALTDDRSKPIQGQTIVLNADWSGYSGSFMDYWAEGDCYRACFYVELIRGKPKEDKVWWTSNSIVSMCSSTEFLGQWNWPDGAKIEYFLHHHHHH.
nucleotide sequence of 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 after codon optimization, and carrying out corresponding gene amplification by using the recombinant plasmids as templates, wherein a PCR reaction system (50 mu L) comprises: 2 × Premix 25 μ L, ddH2O22 mu L, upstream primer 1 mu L, downstream primer 1 mu L and template 1 mu L; the running program of the PCR instrument is as follows: denaturation at 98 deg.C for 10s, annealing at 57 deg.C for 5s, extension at 72 deg.C for 2min, and 30 cycles; final extension at 72 deg.C for 2 min; preserving at 4 ℃; carrying out electrophoresis on the PCR product on agarose gel, cutting a target band after the electrophoresis is finished, and recovering a target fragment by using a DNA gel extraction kit;
TABLE 1 codon-optimized HA, NA, M1 Gene amplification primer information
Note: GCCGCCACC (bold) represents Kozak sequence; the lower straight line is the site of restriction enzyme.
(3) The target fragment recovered in step (2) was ligated to pACEBac1 plasmid (Invitrogen) after BamHI and EcoRI 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 enzyme digestion product and 3 mu L of pACEBac1 plasmid enzyme digestion product; the ligation product was transformed into DH 5. alpha. competent cells (Invitrogen) by the following transformation procedure: standing in ice bath for 30min, performing heat shock in water bath at 42 ℃ for 90s, and immediately performing ice bath for 2 min; adding 800 mu L of non-antibiotic liquid LB culture medium into a 1.5mL EP tube under the aseptic condition, placing the tube in a constant temperature shaking table, and oscillating the tube at 37 ℃ (220rpm) for 45 min; uniformly coating the well-grown bacterial liquid in a solid LB culture medium containing Gen + resistance in a biological safety cabinet, and then placing a bacterial culture dish upside down in a 37 ℃ incubator for culture for 12-16 h; plasmids are extracted by using a plasmid miniprep kit, are subjected to enzyme digestion and electrophoretic identification and then are sequenced, and positive plasmids with the maintained sequences are respectively named as pACE-HA, pACE-NA and pACE-M1, wherein the enzyme digestion identification results of recombinant transfer plasmids pACE-HA, pACE-NA and pACE-M1 are shown in figure 1.
(II) construction of HA, NA, M1 gene recombinant baculovirus plasmid
The recombinant transfer plasmids pACE-HA, pACE-NA and pACE-M1, which were correctly sequenced, were transformed into DH10bac competent cells (Invitrogen) according to the following steps: mixing 1 μ L of recombinant transfer plasmid with 100 μ L of DH10Bac Escherichia coli competent cells, standing on ice for 30min, performing heat shock in 42 deg.C water bath for 45s, and immediately performing ice bath for 2 min; adding 900 μ L of nonreactive LB liquid medium, shaking at 37 deg.C and 220rpm, and culturing for 4hDiluting the bacterial liquid by 10 times to 10-1、10-2、10-3Uniformly coating 400 mu L of bacterial liquid in a three-resistant LB flat plate, and placing in an incubator at 37 ℃ for 48 hours; after 48h of culture, selecting white monoclonal colonies for amplification culture, and extracting plasmids after PCR identification to obtain recombinant baculovirus plasmids which are named as Bacmid-HA, Bacmid-NA and Bacmid-M1 respectively.
Example 2 rescue of recombinant baculovirus with HA, NA and M1 genes
(1) The recombinant baculovirus plasmids Bacmid-HA, Bacmid-NA and Bacmid-M1 prepared in example 1 were transfected into sf9 insect cells (Invitrogen) by a conventional liposome-mediated transfection method, respectively, and cultured at 27 ℃; when the culture is carried out 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 sf9 cells with the P1 generation recombinant baculovirus, collecting cell supernatant (namely P2 generation recombinant baculovirus) when cytopathic effect is obvious, and continuously obtaining P3 generation HA, NA and M1 recombinant baculovirus by the sequential method.
Example 3 expression, optimization and purification of H7N9-VLP in insect cells
(1) HA, NA, M1 recombinant baculoviruses passage P3 were combined according to MOI 7: 4: inoculating High five cells (Invitrogen company) in suspension culture, inoculating for 96h, harvesting the cells, and centrifuging to obtain extracellular culture supernatant and cells respectively; crushing after cell resuspension, and centrifuging to obtain crushed supernatant in the cells; the hemagglutination titer of the virus-like particles in the extracellular culture supernatant is determined to be 11log2, and the hemagglutination titer of the intracellular disruption supernatant is determined to be 13log 2;
(2) HA, NA, M1 recombinant baculoviruses of P3 generation were combined according to MOI ═ 3: 3: inoculating High five cells (Invitrogen company) in suspension culture, inoculating for 96h, harvesting the cells, and centrifuging to obtain extracellular culture supernatant and cells respectively; crushing after cell resuspension, and centrifuging to obtain crushed supernatant in the cells; the hemagglutination titer of the virus-like particles in the extracellular culture supernatant is determined to be 9log2, and the hemagglutination titer of the intracellular disruption supernatant is 9log 2;
(3) HA, NA, M1 recombinant baculoviruses of P3 generation were combined according to MOI 2: 1: inoculating High five cells (Invitrogen company) in suspension culture, inoculating for 96h, harvesting the cells, and centrifuging to obtain extracellular culture supernatant and cells respectively; crushing after cell resuspension, and centrifuging to obtain crushed supernatant in the cells; the hemagglutination titer of the virus-like particles in the extracellular culture supernatant is determined to be 13log2, and the hemagglutination titer of the intracellular disruption supernatant is 13log 2;
(4) HA, NA, M1 recombinant baculoviruses of P3 generation were combined according to MOI 2: 1: 2 identification of the virus-like particle sample in the extracellular culture supernatant (prepared in step (3)) collected by co-infection of High five cells by SDS-PAGE and Western blot analysis, the primary antibody was His-tagged monoclonal antibody (His-tag of His protein (4C2) monoclonal antibody, Bioword TECHNOLOGY Co., Ltd.), and the secondary antibody was fluorescently-labeled murine secondary antibody (II-A-B) ((III-B800CW Goat anti-Mouse IgG (H + L) Secondary Antibody, LI-COR Biosciences).
The results of SDS-PAGE and Western blot are shown in FIG. 2, and the HA protein is about 70kDa, the NA protein is about 53kDa, and the M1 protein is about 28 kDa.
(5) Virus-like particle purification using sucrose density gradient centrifugation
Preparing sucrose solutions with different concentrations: preparing 20%, 30%, 45% and 60% (m/v) sucrose solution, and filtering by a 0.22 μm filter; respectively adding 20%, 30%, 45% and 60% of sucrose solution into a centrifugal tube from top to bottom, adding the avian influenza virus-like particle sample (the extracellular culture supernatant collected in the step (3)) at the top, centrifuging at 100000 Xg at 4 ℃ for 1 h; after the centrifugation is finished, collecting 20-30% of white transparent bands among the sucrose layers; 10000 Xg, centrifuging at 4 ℃ for 1.5h to remove sucrose; resuspend avian influenza virus-like particles using PBS buffer, and store at 4 ℃. Samples were subjected to subsequent experiments and protein concentrations were determined using the BCA protein quantification kit, approximately 1.96mg/ml protein concentration.
Example 4 Transmission Electron microscopy of the morphological Structure of H7N9-VLP
The sample of avian influenza virus-like particles purified in example 3 (H7N9-VLP) was dropped onto a carbon-coated copper mesh for adsorption and incubated at room temperature for 2 min. Gently absorbing the excessive liquid on the copper mesh by using absorbent paper, drying, negatively dyeing the sample by using 1 wt.% of phosphotungstic acid, and incubating for 10min at room temperature; and then, slowly absorbing the redundant phosphotungstic acid on the copper mesh by using absorbent paper, airing at room temperature, observing round particles with the diameter of about 100nm and a capsule membrane without any genetic substances inside under a transmission electron microscope (figure 3), wherein fiber protrusions are visible on the capsule membrane, the morphological characteristics of the particles are highly similar to those of the natural avian influenza virus, and the result shows that the recombinant baculovirus is successfully assembled into the avian influenza virus-like particles (H7N9-VLP) through coinfection.
Example 5 evaluation of the efficacy of vaccines prepared with H7N9-VLP and white oil adjuvant
(1) Preparation of vaccines
The H7N 9-VLPs harvested in example 3 were combined at an immunizing dose with dadall EOLANE 150 adjuvant according to 1: 2(v/v) to prepare the avian influenza virus-like particle vaccine; wherein each 0.3ml of vaccine contains about 30 μ g H7N9-VLP antigen;
(2) assessment of the immunopotency of vaccines
30 SPF (SPF) chickens (purchased from Xinghua agricultural egg Co., Ltd. in Guangdong province, with production license number SCXK (Guangdong) 2013-. Group 1, 0.3ml of inactivated vaccine of avian influenza whole virus (the source of the vaccine is purchased in the market, and the product is produced by the agricultural large biological medicine company in south China, Guangzhou); group 2 was injected subcutaneously in the neck with 0.3 ml/mouse of avian influenza virus-like particle vaccine (0.3ml vaccine contained about 30 μ g H7N 9-VLP); group 3 was injected with PBS as a blank control. Blood was collected and serum was isolated from all test chickens at 3 weeks after immunization, and the immune serum was subjected to Hemagglutination Inhibition (HI) antibody detection, and A/Chiken/Guangdong/16876/2016 (H7N9) strain (i.e., GD16 strain, provided by poultry disease research laboratory of veterinary medical institute of southern agricultural university, of south China, which was already reported in reference "Shigao, enemy reddish, Qi-protecting Webao, etc.. development of inactivated vaccine against H7N9 subtype recombinant avian influenza virus rGD76 strain [ J7N 9]Animal medical progress, published in 2019,040(008):44-48. ") as four-unit antigen after inactivation. 3 weeks after immunization, challenge with A/Chicken/Guangdong/16876/2016(H7N9) strain, nasal drip, 0.2 ml/body (containing 2X 10)6.0ELD50). Observing the morbidity or mortality of the test chicken every day after the challenge, andrecording time, continuing for 14 days, collecting test chicken larynx and cloaca swab for virus separation on the 5 th day after virus challenge, and counting the protection condition of the vaccine. The results are shown in Table 2.
Table 2 vaccine immunopotency results
The results show that the mean HI antibody titers in the vaccine groups were above 6log2 at 3 weeks post-immunization; after challenge, the non-immunized group died within 2 days after challenge, the vaccine group showed no clinical symptoms within 14 days after challenge, and only 1 chicken throat detoxification was detected in the H7N9 subtype virus-like particle vaccine group on day 5 after challenge.
Example 6H 7N9-VLP and MontanideTMVaccine efficacy assessment of ISA71VG adjuvant preparation
(1) Preparation of vaccines
The H7N9-VLP harvested in example 3 was combined with Montanide at an immunization doseTMISA71VG adjuvant was as follows 3: 7(v/v) to prepare the avian influenza virus-like particle vaccine; wherein each 0.3ml of vaccine contains about 30 μ g H7N9-VLP antigen.
(2) Assessment of the immunopotency of vaccines
20 SPF chickens at 3 weeks of age were randomly assigned to 2 groups, 10 per group. 10 chickens were given an intramuscular injection of avian influenza virus-like particle vaccine, 0.3 ml/chicken (0.3ml vaccine contains about 30 μ g H7N 9-VLP); another 10 chickens were injected with PBS solution as a blank control, 0.3 ml/chicken.
(ii) detection of antibody levels
All test chickens were bled and serum was isolated on days 14 and 19 post immunization. To assess the cross-reactivity of the vaccine sera, the immune sera were separately subjected to a cross-reactivity test with a wild-type H7N9 avian influenza strain, and the hemagglutination-inhibiting antibody (HI) and neutralizing antibody levels were determined. The strains used included: a/Chicken/Guangdong/E157/2017(H7N9) (i.e., E157 strain, disclosed in application No. 201910117092.4, application name "avian influenza vaccine based on multiba baculovirus expression system and preparation and use"), and a/Chicken/Qingyuan/E664/2017(H7N9) (provided by poultry diseases research laboratory of university of agriculture, south china). The HI and neutralizing antibody results are shown in fig. 4.
The results are shown in fig. 4, and the vaccine sera have good cross-reactivity against different wild-type wild strains of avian influenza H7N 9. For the A/Chicken/Guangdong/E157/2017(H7N9) strain, the average HI titer reaches 6.875log2 and the average neutralizing antibody titer reaches 1 at 19 days after immunization: 1706.67, respectively; for the a/Chicken/Qingyuan/E664/2017(H7N9) strain, the mean HI titer reached 8log2, the mean neutralizing antibody titer reached 1: 3413.33.
② toxic substance counteracting protection experiment
3 weeks after immunization, 10 days with A/Chicken/Guangdong/E157/2017(H7N9) strain6.0EID50The medicine is used for counteracting toxic materials, and is inoculated by nasal drip, and the dosage is 0.2ml per unit. And observing the morbidity or mortality of the test chicken every day after the challenge, recording in time for 14 days, collecting swabs of the larynx and the cloaca of the test chicken for virus separation on 3, 5, 7 and 9 days after the challenge, and counting the protection condition of the vaccine. The results are shown in Table 3.
TABLE 3 vaccine challenge protection results
Note: dpc: days post change.
The results show that all the non-immunized groups died within 3 days after challenge, and the vaccine group SPF chickens did not show 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 obviously inhibit detoxification against the attack of the wild type H7N9 subtype avian influenza virus.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> southern China university of agriculture
<120> gene for coding recombinant avian influenza virus HA protein, virus-like particle, vaccine, preparation and application
<130> 1
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 1707
<212> DNA
<213> Artificial
<220>
<223> codon-optimized nucleotide sequence of HA gene
<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> codon-optimized nucleotide sequence of M1 gene
<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