CN114767845A - Recombinant influenza subunit vaccine - Google Patents

Recombinant influenza subunit vaccine Download PDF

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CN114767845A
CN114767845A CN202210702152.0A CN202210702152A CN114767845A CN 114767845 A CN114767845 A CN 114767845A CN 202210702152 A CN202210702152 A CN 202210702152A CN 114767845 A CN114767845 A CN 114767845A
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CN114767845B (en
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张艳
刘金龙
尹雪
张佳妮
郭梦瑶
张郁
段立津
高辉
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Tianjin Zhongyi Anjian Biotechnology Co ltd
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Abstract

The invention discloses a recombinant influenza subunit vaccine. Belongs to the field of biotechnology. Each dose contains four types of A H1N1, A H3N2, B type Yamagata series and B type Victoria series; the antigen in the vaccine is HA gene and NA gene encoded protein of A type H1N1, A type H3N2, B type Yamagata series and B type Victoria series strains. The invention integrates the Hemagglutinin (HA) gene and the Neuraminidase (NA) gene which are key components of A H1N1, A H3N2, B Yamagata and B Victoria strains on the insect baculovirus genome in a homologous recombination way, reduces/avoids the generation of false positive virus particles, shortens the production period and improves the expression quantity of exogenous genes. The recombinant influenza vaccine prepared by the invention has strong safety and good immune effect, and provides a new idea for the development of influenza vaccines.

Description

Recombinant influenza subunit vaccine
Technical Field
The invention relates to the technical field of biology, in particular to a recombinant influenza subunit vaccine.
Background
Influenza is a respiratory infectious disease caused by influenza virus, and has the characteristics of high morbidity, strong infectivity and high transmission speed. Influenza virus types are classified into type a, type b, type c, and type d. Influenza viruses currently transmitted and prevalent in humans are mainly of the a and b types, with the a H1N1, the a H3N2, the b Yamagata line, and the b Victoria line being the most common. Vaccination with influenza remains one of the most effective means of preventing the development of influenza and controlling the spread of influenza.
Hemagglutinin (HA) of influenza virus is the major antigen responsible for the protective antibody response of the host, and is responsible for the attachment of the virus to oligosaccharide receptors with terminal sialic acids on cells during the early stages of infection. Neuraminidase (NA) has sialic acid hydrolyzing activity. After HA on the surface of the virus is combined with a host cell membrane through a sialic acid receptor, sialic acid needs to be hydrolyzed by NA protein, the connection between the virus and the host cell is cut off, the virus is smoothly released from the host cell, and then the next cell is infected. Hemagglutinin (HA) and Neuraminidase (NA) are two of the most important viral surface glycoproteins for eliciting host immune responses, are the main targets for adaptive immune responses against influenza viruses in humans, and are key components of influenza virus vaccines.
The influenza vaccine produced by the chick embryo has the problems of potential pathogenic factor pollution, harmless treatment and the like, and particularly during the period of high-pathogenicity influenza epidemic, the lack of vaccine preparation raw materials can be caused by the supply problem of the chick embryo, and the great hidden danger exists in the vaccine supply. The potential defects are also the bottleneck for limiting the expansion of the productivity, the improvement of the quality and the improvement of the emergency production capacity of the influenza vaccine. Compared with the influenza vaccine produced by chicken embryos, the influenza vaccine cultured by the cells has the advantages that: the dependence of chick embryo supply is eliminated; the process is easy to automate and scale; the vaccine virus strain has low passage mutation probability in cells, and the antigenicity of the vaccine is closer to that of a self-heating epidemic strain; the production or research and development process adopts a relatively closed bioreactor system, so that the risk of allergy after inoculation can be reduced. The recombinant influenza vaccine produced by adopting the insect baculovirus expression system can carry out correct folding and transcription processing on foreign protein, and has better antigenicity and immunogenicity.
In conclusion, how to provide a recombinant influenza subunit vaccine is a problem that needs to be solved urgently by the technical personnel in the field.
Disclosure of Invention
In view of this, the present invention provides a recombinant influenza subunit vaccine.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aim of the invention is to provide a recombinant influenza subunit vaccine.
The invention integrates the Hemagglutinin (HA) gene and the Neuraminidase (NA) gene which are key components of A H1N1, A H3N2, B Yamagata and B Victoria strains on the insect baculovirus genome in a homologous recombination way, reduces/avoids the generation of false positive virus particles, shortens the production period and improves the expression quantity of exogenous genes.
A recombinant influenza subunit vaccine, each dose contains four types of H1N 1A, H3N 2A, Yamagata series B and Victoria series B;
the antigens in the vaccine are HA genes and NA gene encoded proteins of A type H1N1, A type H3N2, B type Yamagata series and B type Victoria series strains.
The nucleotide sequence of the optimized HA gene of the A type H1N1 strain is shown as SEQ ID NO. 1.
ATGAAAGCTATCCTGGTTGTTATGCTGTACACATTCACAACCGCTAACGCTGACACACTGTGTATCGGTTACCACGCTAACAACTCCACCGACACAGTTGACACAGTCCTGGAAAAGAACGTGACCGTGACACACTCTGTGAACCTCCTGGAAGATAAGCACAACGGCAAGCTGTGCAAGCTCCGTGGCGTGGCTCCCCTGCACCTGGGCAAGTGTAACATCGCTGGTTGGATTCTCGGTAACCCTGAGTGCGAATCCCTGAGCACTGCTCGTAGCTGGTCATACATCGTGGAAACTTCCAACTCCGACAACGGAACTTGCTACCCCGGAGACTTCATCAACTACGAAGAACTGCGTGAGCAGCTCTCTTCTGTGTCATCATTCGAGCGCTTCGAAATCTTCCCAAAGACCAGCTCTTGGCCTAACCACGACAGCGACAACGGAGTTACCGCTGCCTGTCCCCACGCTGGAGCCAAGAGCTTCTACAAGAACCTCATCTGGCTCGTGAAGAAGGGCAAGTCCTACCCAAAGATCAACCAAACCTACATCAACGACAAGGGAAAGGAGGTTCTCGTCCTGTGGGGAATCCACCACCCCCCAACCATCGCTGACCAACAATCACTCTACCAGAACGCTGATGCCTACGTGTTCGTTGGTACATCCCGCTACTCTAAGAAGTTCAAGCCTGAAATCGCCACCCGCCCTAAGGTTCGCGACCAGGAAGGACGTATGAACTACTACTGGACCCTGGTGGAACCTGGCGACAAGATCACTTTCGAGGCCACAGGCAACCTCGTCGCCCCACGTTACGCCTTCACTATGGAACGTGACGCTGGTTCAGGCATCATCATCTCCGACACTCCAGTTCACGACTGTAACACAACTTGTCAAACACCCGAGGGTGCTATCAACACATCTTTGCCATTCCAAAACGTGCATCCTATCACAATCGGTAAATGCCCAAAGTACGTTAAGTCTACAAAGCTCAGATTGGCCACTGGACTCAGGAACGTGCCTTCCATCCAGAGCCGTGGCCTCTTCGGTGCCATCGCTGGTTTCATCGAAGGAGGTTGGACTGGTATGGTGGACGGATGGTACGGCTACCACCACCAGAACGAGCAGGGCTCTGGTTACGCCGCTGACCTCAAGAGCACTCAAAACGCCATTGACAAGATCACCAACAAGGTCAACTCCGTTATCGAAAAGATGAACACTCAGTTCACTGCCGTTGGCAAGGAGTTCAACCACCTGGAGAAGCGTATCGAGAACCTGAACAAAAAGGTGGACGACGGTTTCCTGGACATCTGGACCTACAACGCCGAGCTCCTGGTTCTGCTGGAGAATGAACGCACCCTGGACTACCACGACTCCAACGTCAAGAACCTGTACGAAAAGGTGCGTAACCAACTGAAGAACAACGCCAAGGAGATCGGCAACGGTTGCTTCGAATTCTACCACAAGTGTGACAACACTTGTATGGAGTCCGTCAAGAACGGTACCTACGACTACCCAAAGTACAGCGAAGAGGCTAAGCTGAACCGTGAAAAGATCGACGGCGTCAAGCTCGACTCCACTCGTATCTACCAGATCCTCGCTATCTACTCCACAGTGGCCTCCTCACTGGTGCTGGTGGTGTCCCTGGGTGCTATCTCTTTCTGGATGTGTTCCAACGGTAGCCTGCAGTGCCGCATCTGTATC；SEQ ID NO.1。
The nucleotide sequence of the original HA gene of the A type H1N1 strain is shown in SEQ ID NO. 17.
GGAAAACAAAAGCAACAAAAATGAAGGCAATACTAGTAGTTATGCTGTATACATTTACAACCGCAAATGCAGACACATTATGTATAGGTTATCATGCGAACAATTCAACAGACACTGTGGACACAGTACTAGAAAAGAATGTAACAGTAACACACTCTGTCAATCTTCTGGAAGACAAGCATAACGGAAAACTATGCAAACTAAGAGGGGTAGCCCCATTGCATTTGGGTAAATGTAACATTGCTGGCTGGATCCTGGGAAATCCAGAGTGTGAATCACTCTCCACAGCAAGATCATGGTCCTACATTGTGGAAACATCTAATTCAGACAATGGAACGTGTTACCCAGGAGATTTCATCAATTATGAGGAGCTAAGAGAGCAATTGAGCTCAGTGTCATCATTTGAAAGGTTTGAAATATTCCCCAAGACAAGTTCATGGCCTAATCATGACTCGGACAATGGTGTAACGGCAGCATGTCCTCACGCTGGAGCAAAAAGCTTCTACAAAAACTTGATATGGCTGGTTAAAAAAGGAAAATCATACCCAAAGATCAACCAAACCTACATTAATGATAAAGGGAAAGAAGTCCTCGTGCTGTGGGGCATTCACCATCCACCTACTATTGCTGACCAACAAAGTCTCTATCAGAATGCAGATGCATATGTTTTTGTGGGGACATCAAGATACAGCAAGAAGTTCAAGCCGGAAATAGCAACAAGACCCAAAGTGAGGGATCAAGAAGGGAGAATGAACTATTACTGGACACTAGTAGAACCGGGAGACAAAATAACATTCGAAGCAACTGGTAATCTAGTGGCACCGAGATATGCATTCACAATGGAAAGAGATGCTGGATCTGGTATTATCATTTCAGATACACCAGTCCACGATTGCAATACAACTTGTCAGACACCCGAGGGTGCTATAAACACCAGCCTCCCATTTCAGAATGTACATCCGATCACAATTGGGAAATGTCCAAAGTATGTAAAAAGCACAAAATTGAGACTGGCCACAGGATTGAGGAATGTCCCGTCTATTCAATCTAGAGGCCTATTCGGGGCCATTGCTGGCTTCATCGAAGGGGGGTGGACAGGGATGGTAGATGGATGGTACGGTTATCACCATCAAAATGAGCAGGGGTCAGGATATGCAGCCGATCTGAAGAGCACACAAAATGCCATTGATAAGATTACTAACAAAGTAAATTCTGTTATTGAAAAGATGAATACACAGTTCACAGCAGTTGGTAAAGAGTTCAACCACCTTGAAAAAAGAATAGAGAATCTAAATAAAAAGGTTGATGATGGTTTCCTGGACATTTGGACTTACAATGCCGAACTGTTGGTTCTACTGGAAAACGAAAGAACTTTGGACTATCACGATTCAAATGTGAAGAACTTGTATGAAAAAGTAAGAAACCAGTTAAAAAACAATGCCAAGGAAATTGGAAACGGCTGCTTTGAATTTTACCACAAATGCGACAACACATGCATGGAAAGTGTCAAGAATGGGACTTATGACTACCCAAAATACTCAGAGGAAGCAAAATTAAACAGAGAAAAAATAGATGGAGTAAAGCTGGACTCAACAAGGATCTACCAGATTTTGGCGATCTATTCAACTGTTGCCAGTTCATTGGTACTGGTAGTCTCCCTGGGGGCAATCAGCTTCTGGATGTGCTCTAATGGGTCTCTACAGTGTAGAATATGTATTTAACATTAGGATTTCAGAATCATGAGAAAAACAC；SEQ ID NO.17。
The nucleotide sequence of the optimized NA gene of the A type H1N1 strain is shown as SEQ ID NO. 2.
ATGAACCCCAACCAAAAGATCATCACCATCGGTAGCATCTGCATGACAATCGGAACTGCTAACCTGATCCTGCAGATCGGTAACATCATCTCAATCTGGGTTTCTCACAGCATCCAGATCGGCAACCAGTCCCAAATCGAGACATGTAACAAGTCCGTCATCACATACGAAAACAACACATGGGTTAACCAGACATTCGTGAACATCTCAAACACTAACAGTGCTGCCCGTCAATCCGTGGCCTCAGTGAAGCTCGCTGGTAATAGTAGCCTCTGTCCTGTGTCTGGCTGGGCGATCTACAGCAAGGACAACTCCGTGCGTATTGGTAGCAAGGGCGACGTTTTCGTTATCCGTGAACCCTTCATCTCCTGTAGTCCACTGGAGTGTAGAACCTTTTTCCTCACTCAGGGCGCTCTGCTGAACGACAAGCACTCTAACGGAACCATCAAGGACCGCTCACCCTACAGGACCTTGATGTCCTGTCCAATCGGTGAGGTTCCATCCCCTTACAATTCACGCTTCGAATCTGTTGCTTGGTCCGCTAGCGCCTGCCACGACGGCACTAACTGGCTCACAATTGGAATCAGTGGCCCCGATAGCGGAGCCGTCGCCGTGCTCAAATACAATGGTATCATCACTGATACTATAAAGTCTTGGAGAAACAAGATCCTCCGCACACAAGAGAGCGAGTGCGCCTGCGTCAATGGCTCTTGTTTTACAATCATGACAGATGGTCCAAGCGATGGCCAGGCTTCATACAAAATCTTCCGCATTGAGAAGGGAAAAATTATTAAGAGCGTCGAAATGAAGGCTCCCAACTACCACTATGAAGAATGCTCTTGTTACCCTGACTCATCGGAGATTACTTGCGTGTGCAGGGACAACTGGCATGGCTCTAATAGGCCTTGGGTCTCCTTTAACCAGAACCTAGAATACCAAATGGGCTACATCTGTTCCGGTGTGTTCGGAGACAACCCACGTCCTAACGATAAGACCGGTTCATGCGGTCCCGTGAGTAGTAACGGAGCCAACGGGGTGAAGGGCTTCTCATTCAAATACGGTAACGGCGTCTGGATCGGTAGGACCAAGTCCATCTCTAGCCGTAAGGGTTTTGAAATGATCTGGGACCCTAACGGTTGGACCGGAACTGACAATAAGTTCAGCAAGAAACAGGACATCGTCGGTATTAATGAATGGTCCGGATACTCAGGCTCCTTCGTTCAACACCCCGAACTGACCGGCTTGAACTGTATTCGCCCATGCTTCTGGGTTGAGTTGATCCGTGGACGCCCTGAGGAGAACACCATCTGGACTAGCGGATCATCTATCAGCTTCTGCGGAGTCGATTCTGACATCGTCGGCTGGTCCTGGCCTGACGGAGCCGAGCTGCCATTCACCATTGATAAG；SEQ ID NO.2。
The nucleotide sequence of the original NA gene of the A H1N1 strain is shown in SEQ ID NO. 18.
AGTTTAAAATGAATCCAAACCAAAAGATAATAACCATTGGTTCTATCTGTATGACAATTGGAACGGCTAACTTAATATTACAAATTGGAAACATAATCTCAATATGGGTTAGCCACTCAATTCAAATTGGAAATCAAAGCCAGATTGAAACATGCAATAAAAGCGTCATTACTTATGAAAACAACACTTGGGTAAATCAGACATTTGTTAACATCAGCAACACTAACTCTGCTGCTAGACAGTCAGTGGCTTCCGTGAAATTAGCGGGCAATTCCTCTCTCTGCCCTGTTAGTGGATGGGCTATATACAGTAAAGACAACAGTGTAAGAATCGGTTCCAAGGGGGATGTGTTTGTCATAAGGGAACCATTCATATCATGCTCTCCCTTGGAATGCAGAACCTTCTTCTTGACTCAAGGGGCTTTGCTAAATGACAAACATTCCAATGGAACCATTAAAGACAGAAGCCCATATCGAACCCTAATGAGCTGTCCTATTGGTGAAGTTCCCTCTCCATACAACTCAAGATTTGAGTCAGTCGCTTGGTCAGCAAGTGCTTGTCATGATGGCACCAATTGGCTAACAATTGGAATTTCTGGCCCAGACAGTGGGGCAGTGGCTGTGTTAAAATACAATGGCATAATAACAGACACTATCAAGAGTTGGAGGAACAAGATATTGAGAACACAAGAGTCTGAATGTGCATGTGTAAATGGTTCTTGCTTTACCATAATGACCGATGGACCAAGTGATGGACAGGCCTCATACAAAATCTTCAGAATAGAAAAGGGAAAGATAATCAAATCAGTCGAAATGAAAGCCCCTAATTATCACTATGAAGAATGCTCCTGTTACCCTGATTCTAGTGAAATCACATGTGTGTGCAGGGATAACTGGCATGGCTCGAATCGACCGTGGGTGTCTTTCAACCAGAATCTGGAATATCAGATGGGATACATATGCAGTGGGGTTTTCGGAGACAATCCACGCCCTAATGATAAGACAGGCAGTTGTGGTCCAGTATCGTCTAATGGAGCAAATGGGGTAAAAGGATTTTCATTCAAATACGGCAATGGTGTTTGGATAGGGAGAACTAAGAGCATTAGTTCAAGAAAAGGTTTTGAGATGATTTGGGATCCGAATGGATGGACTGGGACTGACAATAAATTCTCAAAAAAGCAAGATATCGTAGGAATAAATGAGTGGTCAGGGTATAGCGGGAGTTTTGTTCAGCATCCAGAACTAACAGGGCTGAATTGTATAAGACCTTGCTTCTGGGTTGAACTAATAAGAGGACGACCCGAAGAGAACACAATCTGGACTAGCGGGAGCAGCATATCCTTTTGTGGTGTAGACAGTGACATTGTGGGTTGGTCTTGGCCAGACGGTGCTGAGTTGCCATTTACCATTGACAAGTAATTTGTTCAAAAAACT；SEQ ID NO.18。
The nucleotide sequence of the HA gene after optimization of the A type H3N2 strain is shown as SEQ ID NO. 3.
ATGAAAACCATCATCGCCCTGTCCAACATCCTCTGTCTGGTTTTCGCCCAGAAGATCCCAGGTAACGACAACAGCACTGCTACTCTGTGTCTGGGTCACCACGCTGTCCCTAACGGCACAATCGTTAAGACTATCACTAACGACCGTATCGAGGTCACAAACGCCACTGAACTGGTGCAGAACTCATCCATCGGTGAAATCTGTGGTTCACCCCACCAGATCCTCGATGGTGGAAACTGTACCCTGATCGACGCTCTGCTGGGCGACCCCCAATGTGATGGATTCCAGAACAAGGAATGGGACCTGTTCGTTGAGCGCTCCCGCGCTAACTCAAACTGTTACCCCTACGACGTCCCAGACTACGCTAGCCTCCGTTCTCTGGTTGCCTCCTCTGGAACACTGGAGTTCAAGAACGAATCCTTCAACTGGACCGGTGTGAAGCAGAACGGTACCTCAAGCGCCTGTATCCGCGGTTCCAGCAGCAGCTTCTTCTCCCGTCTGAACTGGCTGACATCACTGAACAACATCTACCCTGCTCAAAACGTGACCATGCCCAACAAGGAGCAATTCGACAAGTTGTACATCTGGGGAGTGCACCACCCCGACACAGACAAGAACCAGATCAGTTTGTTCGCCCAATCAAGTGGCCGCATCACCGTGAGCACTAAGCGCAGCCAACAGGCCGTCATCCCTAATATCGGATCTAGACCTCGTATCCGTGATATTCCTTCTAGGATCTCAATCTACTGGACTATCGTTAAGCCAGGTGACATCCTGCTGATCAACTCCACTGGTAACCTGATCGCCCCACGTGGTTACTTCAAGATCCGTTCTGGCAAGTCCTCCATCATGCGTTCTGACGCTCCAATCGGTAAATGTAAGTCTGAGTGCATCACTCCTAACGGATCTATCCCAAACGACAAGCCCTTCCAGAACGTCAACCGCATCACCTACGGAGCCTGTCCTAGGTACGTTAAGCAGTCAACACTCAAGCTGGCTACAGGTATGCGTAACGTTCCTGAGAAGCAAACCCGCGGTATCTTCGGTGCCATCGCCGGCTTCATCGAAAACGGATGGGAGGGAATGGTGGATGGATGGTACGGATTCCGTCACCAAAACTCCGAGGGACGTGGCCAGGCTGCTGACCTCAAGAGCACCCAAGCCGCCATCGACCAGATTAACGGAAAGCTGAACCGCCTGATCGGCAAGACTAATGAAAAATTCCACCAAATCGAAAAGGAATTCTCTGAGGTGGAAGGTCGTGTTCAGGACCTCGAAAAGTACGTGGAGGATACAAAGATCGACCTGTGGTCATACAACGCTGAACTCCTGGTGGCTCTGGAAAACCAGCACACAATCGACCTCACCGATTCAGAAATGAACAAGCTGTTCGAAAAGACAAAGAAGCAGCTGAGGGAAAACGCCGAGGACATGGGTAACGGCTGTTTCAAGATTTACCACAAGTGTGACAACGCCTGCATCGGTTCTATCCGTAACGAGACCTACGACCACAACGTTTACCGCGACGAAGCCCTCAACAACCGTTTCCAGATCAAGGGTGTGGAGCTGAAGAGCGGTTACAAGGACTGGATCTTGTGGATCTCATTCGCTATGAGCTGTTTCCTGCTCTGTATCGCTCTGCTCGGTTTCATCATGTGGGCCTGCCAGAAGGGTAACATCCGTTGTAACATCTGCATC；SEQ ID NO.3。
The nucleotide sequence of the original HA gene of the A type H3N2 strain is shown as SEQ ID NO. 19.
GGATAATTCTATTAACCATGAAGACTATCATTGCTTTGAGCAACATTCTATGTCTTGTTTTCGCTCAAAAAATACCTGGAAATGACAATAGCACGGCAACGCTGTGCCTTGGGCACCATGCAGTACCAAACGGAACGATAGTGAAAACAATCACAAATGACCGAATTGAAGTTACTAATGCTACTGAGTTGGTTCAGAATTCATCAATAGGTGAAATATGCGGCAGTCCTCATCAGATCCTTGATGGAGGGAACTGCACACTAATAGATGCTCTATTGGGGGACCCTCAGTGTGACGGCTTTCAAAATAAGGAATGGGACCTTTTTGTTGAAAGAAGCAGAGCCAACAGCAACTGTTACCCTTATGATGTGCCGGATTATGCCTCCCTTAGGTCACTAGTTGCCTCATCCGGCACACTGGAGTTTAAAAATGAAAGCTTCAATTGGACTGGAGTCAAACAAAACGGAACAAGTTCTGCGTGCATAAGGGGATCTAGTAGTAGTTTTTTTAGTAGATTAAATTGGTTGACCAGCTTAAACAACATATATCCAGCACAGAACGTGACTATGCCAAACAAGGAACAATTTGACAAATTGTACATTTGGGGGGTTCACCACCCGGATACGGACAAGAACCAAATCTCCCTGTTTGCTCAATCATCAGGAAGAATCACAGTATCTACCAAAAGAAGCCAACAAGCTGTAATCCCAAATATCGGATCTAGACCCAGAATAAGGGATATCCCTAGCAGAATAAGCATCTATTGGACAATAGTAAAACCGGGAGACATACTTTTGATTAACAGCACAGGGAATCTAATTGCTCCTAGGGGTTACTTCAAAATACGAAGTGGGAAAAGCTCAATAATGAGATCAGATGCACCCATTGGCAAATGTAAGTCTGAATGCATCACTCCAAATGGAAGCATTCCCAATGACAAACCGTTCCAAAATGTAAACAGGATCACATACGGGGCCTGTCCCAGATATGTTAAGCAAAGCACCCTGAAATTGGCAACAGGAATGCGAAATGTACCAGAGAAACAAACCAGAGGCATATTTGGCGCAATAGCGGGTTTCATAGAAAATGGATGGGAGGGAATGGTGGATGGTTGGTACGGTTTCAGGCATCAAAATTCTGAGGGAAGAGGACAAGCAGCAGATCTCAAAAGCACTCAAGCAGCAATCGATCAAATCAATGGGAAGCTGAATCGATTGATCGGAAAAACCAACGAGAAATTCCATCAGATTGAAAAAGAATTCTCAGAAGTAGAAGGAAGAGTTCAAGACCTTGAGAAATATGTTGAGGACACTAAAATAGATCTCTGGTCATACAACGCGGAGCTTCTTGTTGCCCTGGAGAACCAACATACGATTGACCTAACTGACTCAGAAATGAACAAACTGTTTGAAAAAACAAAGAAGCAACTGAGGGAAAATGCTGAGGATATGGGAAATGGTTGTTTCAAAATATACCACAAATGTGACAATGCCTGCATAGGATCAATAAGAAATGAAACTTATGACCACAATGTGTACAGGGATGAAGCATTAAACAACCGGTTCCAGATCAAGGGAGTTGAGCTGAAGTCAGGGTACAAAGATTGGATCCTATGGATTTCCTTTGCCATGTCATGTTTTTTGCTTTGTATTGCTTTGTTGGGGTTCATCATGTGGGCCTGCCAAAAGGGCAACATTAGATGCAACATTTGCATTTGAGTGCATTAATTAAAAACAC；SEQ ID NO.19。
The nucleotide sequence of the optimized NA gene of the A type H3N2 strain is shown as SEQ ID NO. 4.
ATGAACCCTAACCAGAAGATCATCACCATCGGTAGCGTGAGCCTGACTATCAGCACTATCTGCTTCTTCATGCAGATCGCTATCCTGATCACTACCGTGACACTGCACTTCAAGCAGTACGAATTCAACTCTCCTCCAAACAACCAAGTGATGCTGTGTGAGCCAACCATCATCGAACGTAACATCACAGAGATCGTGTACCTCACCAACACCACCATCGAGAAGGAAATCTGTCCTAAGCCAGCTGAATACCGTAACTGGTCTAAGCCACAGTGTGGTATCACCGGTTTCGCTCCCTTCTCCAAGGACAACTCCATCCGCCTGAGCGCCGGTGGTGACATCTGGGTGACCCGTGAGCCATACGTGTCCTGTGACCTCGACAAGTGTTACCAATTCGCTCTGGGCCAGGGTACAACACTGAACAACGTTCACAGCAACAACACAGTGCGCGACCGTACTCCTTACAGGACTCTGCTGATGAACGAGCTGGGAGTTCCTTTCCACCTGGGTACAAAGCAGGTCTGTATCGCTTGGTCCTCATCTTCCTGCCACGACGGAAAGGCTTGGCTCCACGTTTGTATCACTGGCGACGATAAGAACGCTACAGCCTCATTCATCTACAACGGTCGCTTGGTGGACTCTGTTGTGTCCTGGTCAAACGACATCCTGAGAACTCAGGAAAGCGAATGTGTCTGCATCAACGGAACCTGTACTGTTGTTATGACTGATGGTAACGCCACCGGAAAGGCCGATACAAAGATCCTGTTCATCGAAGAGGGTAAAATCGTTCACACATCCAAGCTGTCTGGTTCCGCCCAACATGTCGAAGAATGTTCTTGTTACCCTCGCTACCCTGGAGTGCGCTGTGTCTGCCGTGACAACTGGAAGGGCAGCAACCGTCCAATCATCGACATCAACATCAAGGACCACTCAATCGTCAGTCGTTACGTCTGCTCCGGACTGGTGGGCGACACACCCCGTAAGTCCGACTCTTCCTCCTCAAGCCACTGTCTCAACCCTAACAACGAAAAGGGCGACCACGGTGTGAAGGGTTGGGCCTTCGACGACGGAAACGACGTTTGGATGGGTCGTACCATCAACGAAACTTCTCGTCTGGGTTACGAGACCTTCAAGGTGGTGGAAGGTTGGTCTAACCCCAAGTCTAAGCTGCAGATCAACCGCCAGGTTATCGTGGACCGTGGCGACCGCTCAGGCTACAGCGGTATCTTCTCAGTTGAAGGTAAAAGCTGTATCAACCGCTGCTTCTACGTGGAATTGATCCGTGGCCGTAAGGAAGAGACTGAAGTCTTGTGGACTTCAAACTCAATCGTTGTTTTCTGTGGCACTTCAGGCACATACGGTACAGGATCTTGGCCTGACGGTGCTAACCTGAGCCTGATGCACATC；SEQ ID NO.4。
The nucleotide sequence of the original NA gene of the A H3N2 strain is shown in SEQ ID NO. 20.
AGTAAAGATGAATCCAAATCAAAAGATAATAACGATTGGCTCTGTTTCTCTCACAATTTCCACAATATGCTTCTTCATGCAAATTGCCATCCTGATAACTACTGTAACATTGCATTTCAAGCAATATGAATTCAACTCCCCCCCAAATAACCAAGTGATGCTGTGTGAACCAACAATAATAGAAAGAAACATAACAGAGATAGTGTATTTGACCAACACCACCATAGAGAAGGAAATATGCCCCAAACCAGCAGAATACAGAAATTGGTCAAAACCGCAATGTGGCATTACAGGATTTGCACCTTTCTCTAAGGACAATTCGATTAGGCTTTCCGCTGGTGGGGACATCTGGGTGACAAGAGAACCTTATGTGTCATGCGATCTTGACAAGTGTTATCAATTTGCCCTTGGACAGGGAACAACACTAAACAATGTGCATTCAAATAACACAGTACGTGATAGAACCCCTTATCGGACTCTATTGATGAATGAGTTGGGTGTTCCTTTCCATCTGGGGACCAAGCAAGTGTGCATAGCATGGTCCAGCTCAAGTTGTCACGATGGAAAAGCATGGCTGCATGTTTGTATAACGGGGGATGATAAAAATGCAACTGCTAGCTTCATTTACAATGGGAGGCTTGTAGATAGTGTTGTTTCATGGTCCAACGATATTCTCAGAACCCAGGAGTCAGAATGCGTTTGTATCAATGGAACTTGTACAGTAGTAATGACTGATGGAAATGCTACAGGAAAAGCTGATACTAAAATACTATTCATTGAGGAGGGGAAAATCGTTCATACTAGCAAATTGTCAGGAAGTGCTCAGCATGTCGAAGAGTGCTCTTGCTATCCTCGATATCCTGGTGTCAGATGTGTCTGCAGAGACAACTGGAAAGGATCCAACCGGCCCATCATAGATATAAACATAAAGGATCATAGCATTGTTTCCAGGTATGTGTGTTCTGGACTTGTTGGAGACACACCCAGAAAAAGCGACAGCTCCAGCAGTAGCCATTGTTTGAACCCTAACAATGAAAAAGGTGATCATGGAGTGAAAGGCTGGGCCTTTGATGATGGAAATGACGTGTGGATGGGGAGAACAATCAACGAGACGTCACGCTTAGGGTATGAAACCTTCAAAGTCGTTGAAGGCTGGTCCAACCCTAAGTCCAAATTGCAGATAAATAGGCAAGTCATAGTTGACAGAGGCGATAGGTCCGGTTATTCTGGTATTTTCTCTGTTGAAGGCAAAAGCTGCATCAATCGGTGCTTTTATGTGGAGTTGATTAGGGGAAGAAAAGAGGAAACTGAAGTCTTGTGGACTTCAAACAGTATTGTTGTGTTTTGTGGCACCTCAGGTACATATGGAACAGGCTCATGGCCTGATGGGGCGAACCTCAGTCTCATGCATATATAAGCTTTCGCAATTTTAGAAAAAGCT；SEQ ID NO.20。
The nucleotide sequence of the HA gene after the optimization of the B-type Yamagata strain is shown as SEQ ID NO. 5.
ATGAAGGCTATCATCGTGCTGCTGATGGTGGTCACTTCAAACGCTGACCGTATCTGCACAGGTATCACTAGCTCTAACTCCCCTCACGTGGTTAAGACCGCTACTCAGGGTGAAGTTAACGTGACAGGAGTGATTCCACTGACTACTACTCCAACCAAGTCCTACTTCGCTAACTTGAAGGGTACTCGCACCCGTGGAAAGTTGTGTCCTGATTGTCTGAACTGTACCGACCTCGACGTTGCCCTGGGTCGCCCAATGTGCGTGGGCACAACCCCAAGTGCCAAGGCTAGCATCCTCCACGAAGTGCGCCCTGTCACCAGCGGTTGTTTCCCTATCATGCACGACCGCACTAAGATCAGACAACTGCCTAACCTGTTGCGTGGATACGAGAAAATCCGTCTGTCCACACAGAATGTTATCGACGCTGAGAAGGCTCCTGGAGGACCTTACAGGTTGGGAACATCAGGCTCCTGCCCTAACGCCACTTCTAAGATCGGTTTCTTCGCCACCATGGCCTGGGCTGTGCCTAAAGACAACTACAAGAACGCTACTAACCCCCTGACAGTTGAGGTGCCTTACATTTGTACTGAAGGTGAGGATCAGATCACCGTTTGGGGTTTCCACAGCGACAACAAGACCCAGATGAAGTCCCTCTACGGAGACTCTAACCCTCAAAAGTTCACATCATCTGCTAACGGTGTCACAACACACTACGTTAGTCAAATCGGTGACTTCCCAGACCAAACTGAGGATGGTGGACTGCCCCAATCAGGTCGCATTGTCGTTGACTACATGATGCAGAAGCCAGGAAAGACTGGCACAATCGTTTACCAACGTGGTGTGCTCCTCCCACAGAAGGTCTGGTGTGCCAGCGGTAGATCAAAGGTTATTAAAGGTAGCCTCCCCCTGATCGGTGAGGCTGACTGCCTGCATGAAGAATACGGTGGTCTCAACAAGTCTAAACCATACTACACAGGTAAACACGCTAAGGCTATCGGTAACTGCCCTATCTGGGTTAAGACTCCCTTGAAGCTCGCTAACGGAACAAAGTACCGTCCACCTGCTAAGCTGTTGAAGGAACGCGGTTTCTTCGGTGCTATCGCTGGTTTCTTGGAAGGTGGATGGGAAGGAATGATCGCTGGATGGCACGGCTACACTAGCCACGGCGCTCACGGAGTGGCCGTGGCTGCTGACCTGAAGTCTACACAGGAAGCCATCAACAAGATCACCAAGAACCTGAACAGCCTCTCAGAGCTGGAAGTTAAGAACCTCCAGCGCCTCTCAGGTGCCATGGACGAACTGCACAACGAAATCCTGGAGCTCGACGAGAAGGTTGATGACCTGAGGGCCGACACCATCAGTAGTCAAATCGAACTGGCCGTGCTCCTGTCTAACGAAGGAATCATCAACAGCGAAGATGAGCACCTGCTCGCTCTGGAACGTAAGCTGAAGAAGATGTTGGGTCCATCCGCTGTTGACATCGGTAACGGCTGTTTTGAAACAAAGCACAAGTGTAACCAAACTTGTCTCGACCGTATCGCCGCCGGTACATTCAACGCTGGTGAGTTCTCACTGCCTACATTTGACAGCCTGAACATCACTGCTGCTAGCCTGAACGACGACGGATTGGACAACCACACAATCTTGCTGTACTACTCCACCGCTGCTAGCTCACTGGCTGTGACTCTGATGTTGGCCATCTTCATCGTTTACATGGTGTCACGTGACAACGTGAGCTGTAGCATCTGCCTG；SEQ ID NO.5。
The nucleotide sequence of the original HA gene of the B-type Yamagata strain is shown as SEQ ID NO. 21.
ATGAAGGCAATAATTGTACTACTCATGGTAGTAACATCCAACGCAGATCGAATCTGCACTGGGATAACATCTTCAAACTCACCTCATGTGGTCAAAACAGCTACTCAAGGGGAGGTCAATGTGACTGGCGTGATACCACTGACAACAACACCAACAAAATCTTATTTTGCAAATCTCAAAGGAACAAGGACCAGAGGGAAACTATGCCCGGACTGTCTCAACTGTACAGATCTGGATGTGGCCTTGGGCAGGCCAATGTGTGTGGGGACCACACCTTCTGCTAAAGCTTCAATACTCCATGAGGTCAGACCTGTTACATCCGGGTGCTTTCCTATAATGCACGACAGAACAAAAATCAGGCAACTACCCAATCTTCTCAGAGGATATGAAAAGATCAGGTTATCAACCCAAAACGTTATCGATGCAGAAAAAGCACCAGGAGGACCCTACAGACTTGGAACCTCAGGATCTTGCCCTAACGCTACCAGTAAAATCGGATTTTTTGCAACAATGGCTTGGGCTGTCCCAAAGGACAACTACAAAAATGCAACGAACCCACTAACAGTGGAAGTACCATACATTTGTACAGAAGGGGAAGACCAAATTACTGTTTGGGGGTTCCATTCGGATAACAAAACCCAAATGAAGAGCCTCTATGGAGACTCAAATCCTCAAAAGTTCACCTCATCTGCTAATGGAGTAACCACGCATTATGTTTCTCAGATTGGCGACTTCCCAGATCAAACAGAAGACGGAGGACTACCACAAAGCGGCAGAATTGTTGTTGATTACATGATGCAAAAACCTGGGAAAACAGGAACAATTGTCTATCAAAGGGGTGTTTTGTTGCCTCAAAAGGTGTGGTGCGCGAGTGGCAGGAGCAAAGTAATAAAAGGGTCATTGCCTTTAATTGGTGAAGCAGATTGCCTTCATGAAGAATACGGTGGATTAAACAAAAGCAAGCCTTACTACACAGGAAAACATGCAAAAGCCATAGGAAATTGCCCAATATGGGTAAAAACACCTTTGAAGCTTGCCAATGGAACCAAATATAGACCTCCTGCAAAACTATTGAAGGAAAGGGGTTTCTTCGGAGCTATTGCTGGTTTCCTAGAAGGAGGATGGGAAGGAATGATTGCAGGTTGGCACGGATACACATCTCACGGAGCACATGGAGTGGCAGTGGCGGCAGACCTTAAGAGTACACAAGAAGCTATAAATAAGATAACAAAAAATCTCAATTCTTTGAGTGAACTAGAAGTAAAGAACCTTCAAAGACTAAGTGGTGCCATGGATGAACTCCACAACGAAATACTCGAGCTGGATGAAAAAGTGGATGATCTCAGAGCTGACACTATAAGCTCACAAATAGAACTTGCAGTCTTGCTTTCCAACGAAGGAATAATAAACAGTGAAGACGAGCATCTATTGGCACTTGAGAGAAAACTAAAGAAAATGCTGGGTCCCTCTGCTGTAGACATAGGAAACGGATGCTTCGAAACCAAACACAAATGCAACCAGACCTGCTTAGACAGGATAGCTGCTGGCACCTTTAATGCAGGAGAATTTTCTCTCCCCACTTTTGATTCATTGAACATTACTGCTGCATCTTTAAATGATGATGGATTGGATAACCATACTATACTGCTCTATTACTCAACTGCTGCTTCTAGTTTGGCTGTAACATTAATGCTAGCTATTTTTATTGTTTATATGGTCTCCAGAGACAACGTTTCATGCTCCATCTGTCTATAAAGAAGGTTAGGCCTTGTATTTTCCTTTATTGTAGTGCTTGTTTGCTTGTCATCATTACAAAGAAAC；SEQ ID NO.21。
The nucleotide sequence of the optimized NA gene of the B-type Yamagata strain is shown as SEQ ID NO. 6.
ATGCTCCCTAGCACAATCCAGACACTCACTCTGTTCTTGACTAGCGGTGGAGTCCTCCTGTCCCTGTACGTGAGCGCTTCTCTGTCATACCTGCTGTACAGCGACATCCTCCTGAAGTTCTCACGCACCGAAGTGACTGCTCCTATCATGCCCCTCGACTGTGCTAACGCCTCAAACGTGCAAGCTGTTAACCGTTCCGCCACAAAGGGTGTTACTCCTTTGCTGCCCGAACCAGAATGGACTTACCCACGTTTGTCATGTCCCGGTAGCACCTTCCAAAAGGCTCTGCTGATCTCCCCACACCGTTTCGGTGAAACCAAAGGTAATTCAGCTCCACTGATCATCCGTGAACCCTTCATTGCTTGTGGACCTAAGGAATGTAAGCATTTCGCTTTGACACATTACGCCGCTCAGCCTGGAGGTTACTACAACGGTACCCGTGAGGACCGTAACAAGCTGAGACACCTGATCAGCGTGAAACTCGGCAAGATCCCAACCGTTGAAAACAGCATCTTCCACATGGCCGCTTGGTCTGGCTCAGCTTGTCACGACGGTCGTGAGTGGACATACATCGGTGTGGACGGTCCAGACAGCAACGCTCTGCTCAAGATCAAGTACGGTGAAGCCTACACAGATACATACCACTCCTACGCTAAGAACATCTTGAGAACTCAGGAATCTGCTTGTAACTGTATTGGCGGAGACTGCTACCTGATGATCACCGACGGTCCTGCCAGCGGCATCTCCGAATGCCGCTTCCTCAAGATCCGCGAAGGTAGGATCATCAAGGAAATTTTCCCAACAGGTCGTGTGAAGCACACTGAAGAATGTACATGTGGCTTCGCCAGTAACAAGACCATTGAATGTGCCTGTCGTGACAACTCATACACTGCTAAGCGTCCTTTCGTGAAGCTGAACGTGGAAACTGACACTGCTGAGATCCGTTTGATGTGCACAAAGACTTACCTGGACACTCCTAGGCCTAACGATGGTTCTATCACAGGTCCTTGCGAATCAGATGGCGACGAAGGTAGCGGTGGAATCAAAGGAGGATTCGTGCACCAACGTATGGCTTCTAAGATCGGCCGTTGGTACTCTCGTACTATGAGCAAGACTAAGCGTATGGGTATGGGTCTCTACGTCAAGTACGACGGTGACCCTTGGACTGACAGCGAGGCCCTGGCTCTGTCTGGTGTGATGGTGAGCATGGAAGAACCTGGTTGGTACAGCTTCGGTTTCGAGATCAAGGACAAGAAGTGCGACGTGCCATGCATCGGTATCGAGATGGTTCATGACGGTGGCAAGACTACCTGGCACTCTGCTGCTACCGCTATCTACTGCCTGATGGGATCTGGACAACTGCTGTGGGACACAGTGACAGGTGTTAACATGACCCTG；SEQ ID NO.6。
The nucleotide sequence of the original NA gene of the B-type Yamagata strain is shown as SEQ ID NO. 22.
CAGAAGCAGAGCATCTTCTCAAAACTGAGGCAAATAGGCCAAAAATGAACAATGCTACCTTCAACTATACAAACGTTAACCTTATTTCTCACATCAGGGGGAGTGTTATTATCACTATATGTGTCAGCTTCATTGTCATACTTACTATATTCGGATATATTGCTAAAATTTTCACGAACAGAAGTAACTGCACCAATAATGCCATTGGATTGTGCAAACGCATCAAATGTTCAGGCTGTGAACCGTTCTGCAACAAAAGGGGTGACACCTCTTCTCCCAGAACCGGAGTGGACGTACCCTCGTTTATCTTGCCCGGGCTCAACCTTTCAGAAAGCACTCCTAATTAGCCCCCATAGATTCGGAGAAACCAAAGGAAACTCAGCTCCCTTGATAATAAGGGAACCTTTTATTGCTTGTGGACCAAAGGAATGCAAACATTTTGCTCTAACCCATTATGCAGCTCAACCAGGGGGATACTACAATGGAACAAGAGAAGACAGAAACAAGCTGAGGCATCTAATTTCAGTCAAATTGGGCAAAATCCCAACAGTAGAAAACTCTATTTTCCACATGGCAGCTTGGAGCGGGTCCGCATGCCATGATGGTAGAGAATGGACTTACATCGGAGTTGATGGCCCAGACAGTAATGCATTGCTCAAAATAAAATATGGAGAAGCATATACTGACACATACCATTCCTATGCAAAAAACATCCTAAGGACACAAGAAAGTGCCTGCAATTGCATCGGGGGAGATTGTTATCTGATGATAACTGATGGCCCAGCTTCAGGGATTAGTGAATGCAGATTCCTTAAGATTCGAGAGGGCCGAATAATAAAAGAAATATTTCCAACAGGAAGAGTAAAACATACTGAGGAATGCACATGCGGATTTGCCAGCAACAAAACCATAGAATGTGCCTGTAGAGATAACAGTTACACAGCAAAAAGACCCTTTGTCAAATTAAATGTGGAGACTGATACAGCGGAAATAAGATTGATGTGCACAAAGACTTATTTGGACACCCCCAGACCAAATGATGGAAGCATAACAGGGCCTTGCGAATCTGATGGGGACGAAGGGAGTGGAGGCATCAAGGGAGGATTTGTTCACCAAAGAATGGCATCCAAGATTGGAAGGTGGTACTCTCGGACGATGTCTAAAACTAAAAGAATGGGGATGGGACTGTATGTAAAGTATGATGGAGACCCATGGACTGACAGTGAAGCCCTTGCTCTTAGTGGAGTAATGGTTTCAATGGAAGAACCTGGTTGGTATTCCTTTGGCTTCGAAATAAAAGATAAGAAATGTGATGTCCCCTGTATTGGGATAGAAATGGTACATGATGGTGGGAAAACGACTTGGCACTCAGCGGCAACAGCCATTTACTGTTTAATGGGCTCAGGACAACTGCTGTGGGACACTGTCACAGGTGTTAATATGACTCTGTAATGGAGGAATGGTTGAGTCTGTTCTAAACCCTTTGTTCCTATTTTGTTTGAACAATTGTCCTTACTGAACTTGATTGTTTCTGAAAAATGCTCTTGTTACTACT；SEQ ID NO.22。
The nucleotide sequence of the optimized HA gene of the Victoria B strain is shown as SEQ ID NO. 7.
ATGAAGGCCATCATCGTGCTGCTCATGGTGGTGACCTCCAACGCTGACCGCATCTGTACAGGTATCACCTCTTCTAACTCCCCCCACGTTGTTAAGACAGCTACACAGGGTGAAGTGAACGTGACAGGTGTGATCCCCTTGACCACAACACCCACTAAGTCACACTTCGCTAACCTGAAGGGCACTGAAACTAGAGGAAAGCTCTGTCCTAAGTGCCTCAACTGCACTGATCTCGATGTCGCCCTCGGTCGTCCTAAGTGTACAGGTAAAATCCCATCAGCCCGTGTGTCTATCCTCCACGAGGTGCGTCCTGTGACTTCAGGTTGCTTCCCCATCATGCACGATCGTACCAAGATCCGTCAGCTGCCAAACTTGCTCCGTGGCTACGAACACGTGAGACTGAGCACTCATAACGTTATCAACACAGAGGACGCCCCTGGCGGTCCTTACGAAATCGGAACCTCCGGATCATGCCTGAACATCACAAACGGAAAGGGATTCTTCGCTACAATGGCTTGGGCCGTTCCAAAGAACAAGACTGCCACTAACCCCCTGACCATCGAAGTTCCATACATTTGTACTGAAGAAGAGGATCAAATCACTGTGTGGGGTTTCCACAGTGACGATGAAACACAAATGGCTCGCTTGTACGGTGACTCTAAGCCTCAAAAGTTCACTTCCAGCGCTAACGGCGTTACCACCCACTACGTCAGCCAAATTGGCGGTTTCCCAAACCAGACAGAGGATGGAGGTCTGCCACAGTCAGGCCGCATCGTCGTTGACTATATGGTGCAAAAGTCCGGAAAAACAGGAACTATCACATACCAGCGTGGAATCCTGCTCCCTCAAAAGGTTTGGTGTGCTAGTGGAAAGTCCAAAGTTATCAAGGGTTCCCTCCCTCTGATCGGTGAAGCCGACTGCCTCCACGAAAAGTACGGAGGACTCAATAAGTCTAAGCCATACTACACCGGTGAGCACGCCAAGGCCATCGGCAACTGTCCCATTTGGGTTAAAACTCCATTGAAACTCGCTAACGGTACAAAGTACAGGCCACCCGCTAAGCTGTTGAAGGAACGTGGCTTCTTCGGAGCCATCGCTGGTTTCCTGGAGGGAGGTTGGGAGGGTATGATCGCTGGCTGGCACGGCTACACTAGTCACGGAGCTCACGGCGTGGCCGTCGCCGCTGACTTGAAGTCTACCCAAGAAGCCATCAACAAGATCACTAAGAACCTGAACTCCCTGTCTGAGCTGGAAGTCAAGAACCTCCAGCGTCTGAGCGGTGCCATGGACGAATTGCACAACGAAATCCTGGAACTGGACGAAAAAGTGGACGACTTGCGTGCTGACACTATTTCAAGCCAGATCGAACTCGCTGTTTTGCTGTCTAACGAGGGTATCATTAATAGCGAGGACGAACACCTCCTGGCTTTGGAACGCAAGCTCAAGAAGATGTTGGGCCCTTCAGCCGTTGAAATCGGTAACGGTTGTTTCGAAACCAAGCACAAGTGTAACCAGACCTGTCTCGACCGTATCGCTGCCGGTACATTCGACGCCGGCGAATTCTCACTGCCTACTTTCGATTCCTTGAACATCACTGCTGCTTCCCTGAACGACGACGGTCTGGACAACCACACTATCCTCCTGTACTACAGCACCGCTGCTTCATCCCTGGCTGTGACTCTGATGATCGCTATCTTCGTTGTGTACATGGTTAGCCGTGATAACGTGAGCTGTAGCATCTGTCTG；SEQ ID NO.7。
The nucleotide sequence of the original HA gene of the Victoria B strain is shown as SEQ ID NO. 23.
ATGAAGGCAATAATTGTACTACTCATGGTAGTAACATCCAATGCAGATCGAATCTGCACTGGGATAACATCGTCAAACTCACCACATGTCGTCAAAACTGCTACTCAAGGGGAGGTCAATGTGACTGGTGTAATACCACTGACAACAACACCCACCAAATCTCATTTTGCAAATCTCAAAGGAACAGAAACCAGGGGGAAACTATGCCCAAAATGCCTAAACTGCACAGATCTGGATGTAGCCTTGGGCAGACCAAAATGCACAGGGAAAATACCCTCTGCAAGGGTTTCAATACTCCATGAAGTCAGACCTGTTACATCTGGGTGCTTTCCTATAATGCATGATAGAACAAAAATTAGACAGCTGCCTAACCTTCTCCGAGGATACGAACATGTCAGGTTATCAACTCACAACGTTATCAATACAGAAGATGCACCAGGAGGACCCTACGAAATTGGAACCTCAGGGTCTTGCCTCAACATTACCAATGGAAAAGGATTCTTCGCAACAATGGCTTGGGCCGTCCCAAAAAACAAAACAGCAACAAATCCATTAACAATAGAAGTACCATACATTTGTACAGAAGAAGAAGACCAAATTACCGTTTGGGGGTTCCACTCTGACGACGAGACCCAAATGGCAAGGCTCTATGGGGATTCAAAGCCCCAGAAGTTCACCTCATCTGCCAACGGAGTGACCACACACTACGTCTCACAGATTGGTGGCTTTCCAAATCAAACAGAAGACGGAGGACTACCACAAAGTGGCAGAATTGTTGTTGATTACATGGTGCAAAAATCTGGAAAAACAGGAACAATTACCTATCAAAGAGGTATTTTATTGCCTCAAAAGGTGTGGTGCGCAAGTGGCAAGAGCAAGGTAATAAAAGGATCCTTGCCCTTAATTGGAGAAGCAGATTGCCTCCATGAAAAATACGGTGGATTAAACAAAAGCAAGCCTTACTACACAGGGGAACATGCAAAGGCCATAGGAAATTGCCCAATATGGGTGAAAACACCCTTGAAGCTGGCCAATGGAACCAAATATAGACCTCCTGCAAAACTATTAAAGGAAAGAGGTTTCTTCGGAGCCATTGCTGGTTTCTTAGAGGGAGGATGGGAAGGAATGATTGCAGGTTGGCACGGATACACATCCCATGGGGCACATGGAGTAGCGGTGGCAGCTGACCTTAAGAGCACTCAGGAGGCCATAAACAAGATAACAAAAAATCTCAACTCTTTGAGTGAGCTGGAAGTAAAGAATCTTCAAAGACTAAGCGGTGCCATGGATGAACTCCACAACGAAATACTAGAACTAGATGAGAAAGTGGATGATCTCAGAGCTGATACAATAAGCTCACAGATAGAACTCGCAGTCCTGCTTTCCAATGAAGGAATAATAAACAGTGAAGATGAACATCTCTTGGCGCTTGAAAGAAAGCTGAAGAAAATGCTGGGCCCCTCTGCTGTAGAGATAGGAAATGGATGCTTTGAAACCAAACACAAGTGCAACCAGACCTGTCTCGACAGAATAGCTGCTGGTACCTTTGATGCAGGAGAATTTTCTCTCCCCACCTTTGATTCACTGAATATTACTGCTGCATCTTTAAATGACGATGGATTGGACAATCATACTATACTGCTTTACTACTCAACTGCTGCCTCCAGTTTGGCTGTAACACTGATGATAGCTATCTTTGTTGTTTATATGGTCTCCAGAGACAATGTTTCTTGCTCCATTTGTCTATAA；SEQ ID NO.23。
The nucleotide sequence of the optimized NA gene of the Victoria B strain is shown as SEQ ID No. 8.
ATGCTGCCTAGCACCATCCAGACACTGACCCTGTTCCTGACTTCCGGAGGTGTGCTCCTGAGCCTGTACGTGTCTGCTTCACTGTCTTACCTGCTGTACAGCGACATCCTCCTGAAGTTCAGCCAGACTGAGATCACAGCTCCAACAATGCCATTGGACTGTGCTAACGCCTCTAACGTGCAAGCTGTCAACCGTTCCGCTACTAAGGGAGCCACCCTGCTGCTCCCTGAGCCTGAGTGGACCTACCCTAGATTGAGCTGTCCTGGCTCCACCTTCCAGAAGGCCCTCCTGATCTCTCCCCACCGTTTCGGTGAAACTAAAGGTAACTCAGCTCCACTGATCATCCGTGAACCCTTCGTCGCTTGTGGCCCTAACGAATGTAAGCACTTCGCTCTGACTCACTACGCCGCTCAGCCTGGAGGCTACTACAACGGTACACGCGGAGACCGTAACAAGCTGCGTCACCTGATCTCTGTGAAGTTGGGAAAGATTCCAACAGTTGAAAACTCTATCTTCCACATGGCTGCTTGGTCAGGATCAGCCTGTCACGACGGAAAGGAATGGACATACATCGGTGTTGACGGACCTGACAACAACGCTTTGCTGAAGGTTAAGTACGGTGAAGCCTACACCGACACCTACCACTCCTACGCTAACAACATCCTCAGAACACAAGAATCAGCTTGTAACTGTATCGGAGGAAACTGCTACCTGATGATCACAGACGGTTCTGCCTCAGGTGTTTCAGAATGTAGGTTCCTGAAGATCCGTGAAGGTCGTATCATTAAGGAAATCTTCCCCACAGGTAGGGTTAAGCACACAGAAGAGTGTACTTGTGGTTTCGCTTCTAACAAGACAATCGAATGTGCTTGTCGTGATAACCGCTACACTGCTAAGAGACCTTTCGTTAAGCTCAACGTTGAGACTGACACAGCTGAAATCCGTTTGATGTGTACAGACACATACCTGGACACACCCCGTCCTAACGACGGTTCCATCACTGGTCCCTGTGAAAGCGACGGTGACGAAGGTAGCGGTGGTATCAAGGGCGGTTTCGTGCACCAGCGTATGAAGTCAAAGATCGGACGTTGGTACTCACGTACTATGTCAAAGACTGAACGCATGGGAATGGGTCTCTACGTTAAATACGGTGGAGACCCTTGGGCTGATTCAGACGCTCTGGTGTTCTCTGGCGTGATGATCTCCATGAAGGAACCAGGTTGGTACAGCTTCGGCTTCGAAATCAAGGACAAGAAGTGTGACGTCCCATGTATCGGTATCGAGATGGTGCACGACGGTGGAAAGGAGACTTGGCACAGCGCCGCTACAGCCATCTACTGCCTGATGGGTAGCGGTCAGCTGCTGTGGGACACCATCACCGGCGTTGACATGGCTCTG；SEQ ID NO.8。
The nucleotide sequence of the original NA gene of the Victoria strain B is shown as SEQ ID NO. 24.
ATGAACAATGCTACCTTCAACTATACAAACGTTAACCCTATTTCTCACATCAGGGGGAGTATTATTATCACTATATGTGTCAGCTTCATTATCATACTTACTATATTCGGATATATTGCTAAAATTCTCACAAACAGAAATAACTGCACCAACAATGCCATTGGATTGTGCAAACGCATCAAATGTTCAGGCTGTGAACCGTTCTGCAACAAAAGGGGCGACACTTCTTCTCCCAGAACCGGAGTGGACATACCCGCGTTTATCTTGCCCGGGCTCAACCTTTCAGAAAGCACTCCTAATTAGCCCTCATAGATTCGGAGAAACCAAAGGAAACTCAGCTCCCTTGATAATAAGGGAACCTTTTGTTGCTTGTGGACCAAATGAATGCAAACACTTTGCTTTAACCCATTATGCAGCCCAACCAGGGGGATACTACAATGGAACAAGAGGAGACAGAAACAAGCTGAGGCATCTAATTTCAGTCAAATTGGGCAAAATCCCAACAGTAGAGAACTCCATTTTCCATATGGCAGCATGGAGCGGGTCCGCGTGCCATGATGGTAAGGAATGGACATATATCGGAGTTGATGGCCCTGACAATAATGCATTGCTCAAAGTAAAATATGGAGAAGCATATACTGACACATACCATTCCTATGCAAACAACATCCTAAGAACACAAGAAAGTGCCTGCAATTGCATCGGGGGAAATTGTTATCTAATGATAACTGATGGCTCAGCTTCAGGTGTTAGTGAATGCAGATTTCTTAAGATTCGAGAGGGCCGAATAATAAAAGAAATATTTCCAACAGGAAGAGTAAAACACACTGAGGAATGCACATGCGGATTTGCCAGTAATAAAACCATAGAATGTGCCTGTAGAGACAACAGGTACACAGCAAAAAGACCTTTTGTCAAATTAAACGTGGAGACTGATACAGCAGAAATAAGATTGATGTGTACAGATACTTATTTGGACACCCCCAGACCAAATGATGGAAGCATAACAGGCCCTTGTGAATCTGATGGGGACGAGGGGAGTGGAGGCATCAAGGGAGGATTTGTTCATCAAAGAATGAAATCCAAGATTGGAAGGTGGTACTCTCGAACGATGTCTAAAACTGAAAGGATGGGGATGGGACTGTATGTCAAGTATGGTGGAGACCCATGGGCTGACAGTGATGCCCTAGTTTTTAGTGGAGTAATGATTTCAATGAAAGAACCTGGTTGGTATTCCTTTGGCTTCGAAATAAAAGATAAGAAATGCGATGTCCCCTGTATTGGGATAGAGATGGTACATGATGGTGGAAAAGAGACTTGGCACTCAGCAGCAACAGCCATTTACTGTTTAATGGGCTCAGGACAGTTGCTGTGGGACACTATCACAGGTGTTGATATGGCTCTGTAA；SEQ ID NO.24。
Further, the amino acid sequence coded by the HA gene of the A type H1N1 strain is shown as SEQ ID NO. 9.
MKAILVVMLYTFTTANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDNGVTAACPHAGAKSFYKNLIWLVKKGKSYPKINQTYINDKGKEVLVLWGIHHPPTIADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVAPRYAFTMERDAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLDSTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI；SEQ ID NO.9。
The amino acid sequence coded by the NA gene of the A type H1N1 strain is shown as SEQ ID NO. 10.
MNPNQKIITIGSICMTIGTANLILQIGNIISIWVSHSIQIGNQSQIETCNKSVITYENNTWVNQTFVNISNTNSAARQSVASVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGTNWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNKILRTQESECACVNGSCFTIMTDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNKFSKKQDIVGINEWSGYSGSFVQHPELTGLNCIRPCFWVELIRGRPEENTIWTSGSSISFCGVDSDIVGWSWPDGAELPFTIDK；SEQ ID NO.10。
The amino acid sequence of HA gene code of the A type H3N2 strain is shown in SEQ ID NO. 11.
MKTIIALSNILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICGSPHQILDGGNCTLIDALLGDPQCDGFQNKEWDLFVERSRANSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVKQNGTSSACIRGSSSSFFSRLNWLTSLNNIYPAQNVTMPNKEQFDKLYIWGVHHPDTDKNQISLFAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAMSCFLLCIALLGFIMWACQKGNIRCNICI；SEQ ID NO.11。
The amino acid sequence coded by the NA gene of the A type H3N2 strain is shown in SEQ ID NO. 12.
MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTNTTIEKEICPKPAEYRNWSKPQCGITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDLDKCYQFALGQGTTLNNVHSNNTVRDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYNGRLVDSVVSWSNDILRTQESECVCINGTCTVVMTDGNATGKADTKILFIEEGKIVHTSKLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIIDINIKDHSIVSRYVCSGLVGDTPRKSDSSSSSHCLNPNNEKGDHGVKGWAFDDGNDVWMGRTINETSRLGYETFKVVEGWSNPKSKLQINRQVIVDRGDRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSNSIVVFCGTSGTYGTGSWPDGANLSLMHI；SEQ ID NO.12。
The amino acid sequence of HA gene code of B-type Yamagata strain is shown in SEQ ID NO. 13.
MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSYFANLKGTRTRGKLCPDCLNCTDLDVALGRPMCVGTTPSAKASILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEKIRLSTQNVIDAEKAPGGPYRLGTSGSCPNATSKIGFFATMAWAVPKDNYKNATNPLTVEVPYICTEGEDQITVWGFHSDNKTQMKSLYGDSNPQKFTSSANGVTTHYVSQIGDFPDQTEDGGLPQSGRIVVDYMMQKPGKTGTIVYQRGVLLPQKVWCASGRSKVIKGSLPLIGEADCLHEEYGGLNKSKPYYTGKHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVDIGNGCFETKHKCNQTCLDRIAAGTFNAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMLAIFIVYMVSRDNVSCSICL；SEQ ID NO.13。
The amino acid sequence coded by the NA gene of the B-type Yamagata strain is shown as SEQ ID NO. 14.
MLPSTIQTLTLFLTSGGVLLSLYVSASLSYLLYSDILLKFSRTEVTAPIMPLDCANASNVQAVNRSATKGVTPLLPEPEWTYPRLSCPGSTFQKALLISPHRFGETKGNSAPLIIREPFIACGPKECKHFALTHYAAQPGGYYNGTREDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGREWTYIGVDGPDSNALLKIKYGEAYTDTYHSYAKNILRTQESACNCIGGDCYLMITDGPASGISECRFLKIREGRIIKEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTKTYLDTPRPNDGSITGPCESDGDEGSGGIKGGFVHQRMASKIGRWYSRTMSKTKRMGMGLYVKYDGDPWTDSEALALSGVMVSMEEPGWYSFGFEIKDKKCDVPCIGIEMVHDGGKTTWHSAATAIYCLMGSGQLLWDTVTGVNMTL；SEQ ID NO.14。
The amino acid sequence of the HA gene code of the Victoria B strain is shown as SEQ ID NO. 15.
MKAIIVLLMVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHVRLSTHNVINTEDAPGGPYEIGTSGSCLNITNGKGFFATMAWAVPKNKTATNPLTIEVPYICTEEEDQITVWGFHSDDETQMARLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGKSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFVVYMVSRDNVSCSICL；SEQ ID NO.15。
The amino acid sequence coded by the NA gene of the Victoria strain B is shown as SEQ ID No. 16.
MLPSTIQTLTLFLTSGGVLLSLYVSASLSYLLYSDILLKFSQTEITAPTMPLDCANASNVQAVNRSATKGATLLLPEPEWTYPRLSCPGSTFQKALLISPHRFGETKGNSAPLIIREPFVACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANNILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRIIKEIFPTGRVKHTEECTCGFASNKTIECACRDNRYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPCESDGDEGSGGIKGGFVHQRMKSKIGRWYSRTMSKTERMGMGLYVKYGGDPWADSDALVFSGVMISMKEPGWYSFGFEIKDKKCDVPCIGIEMVHDGGKETWHSAATAIYCLMGSGQLLWDTITGVDMAL；SEQ ID NO.16。
Further, the concentration of the univalent antigen of the vaccine is 30-300 mu g/ml.
Further, the vaccine antigen is prepared by culturing a recombinant baculovirus carrying the protein-encoding gene.
Further, the recombinant baculovirus is constructed as follows:
(1) constructing a recombinant plasmid:
respectively cloning HA genes and NA genes of H1N 1A, H3N 2A, Yamagata B and Victoria B strains to a transfer vector pOET5.1, respectively adding BglII and NotI to the upstream and downstream of the HA genes, respectively adding BamHI and HindIII to the upstream and downstream of the NA genes to obtain recombinant plasmids pOET5.1-HA-NA-A1, pOE5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2;
(2) construction of recombinant baculovirus:
the recombinant plasmids pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2 are mixed with genome DNA of the FlashBAC ULTRA baculovirus and then are transfected into an insect cell ZY.104 (namely the insect cell Expisf 9) together, and the P0 generation recombinant baculovirus is obtained.
The recombinant baculovirus is constructed by transfecting an insect cell ZY.104 with a Sinofection transfection reagent, a flash BAC ULTRA baculovirus genome DNA and recombinant plasmids (pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2) together, so as to obtain the P0 generation recombinant baculovirus. The method comprises the following specific steps:
(1) the density is 5.5 multiplied by 106~1.5×107cells/ml, insect cells ZY.104 with cell viability above 95% were inoculated in 50ml of Expisf CD medium.
(2) Diluting insect cells ZY.104 to 2.5-5.0 × 10 with culture medium6cells/ml, placed in 250ml cell culture flasks.
(3) And (3) recovering the insect cells ZY.104 in an incubator at 27-28 ℃ for 20-30 min at 110-135 rpm/min.
(4) Preparation of transfection complex: mixing a Sinofection transfection reagent, a flash BAC ULTRA baculovirus genome DNA and a mixture of the 4 recombinant plasmids (pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2) according to the volume ratio of 1-5: 1-5: 1-5, performing vortex mixing.
(5) The transfection complex of step (4) was slowly dripped into the cell culture solution of step (3).
(6) And incubating for 72-96 h at the speed of 110-130 rpm/min in an incubator at 27 ℃ to obtain the P0 generation recombinant baculovirus.
Further, the recombinant plasmids pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2 are mixed according to a volume ratio of 0.1-1: 0.1-1: 0.1-1: 0.1-1 mixture, wherein the total concentration of the recombinant plasmid DNA is 1-50 mug.
The second purpose of the invention is to provide a preparation method of the recombinant influenza subunit vaccine.
A preparation method of a recombinant influenza subunit vaccine comprises the following steps: constructing recombinant baculovirus, proliferating, culturing and harvesting the recombinant baculovirus, obtaining recombinant influenza protein stock solution, and mixing with an adjuvant.
Further, the propagation culture and harvesting of the recombinant baculovirus comprise the following steps:
(1) cell recovery culture: thawing insect cells: transferring insect cells ZY.104 (insect cells Expisf 9) and insect cells ZY.201 (High Five cells) into a sterile centrifuge tube containing 5-15 ml of culture medium, centrifuging at a low speed of 200-500 g for 5-10 min, discarding supernatant, retaining precipitate, then using 10-20 ml of culture medium to resuspend the insect cells, wherein the density of the insect cells after resuspension is more than 1 multiplied by 106cells/ml, respectively placing the resuspended insect cells (insect cells ZY.104 and insect cells ZY.201) into a sterile cell culture flask for suspension culture at 27-28 ℃, wherein the rotation speed of a shaking table is 100-130 rpm/min. When the density of the insect cells grows to 2-4 multiplied by 106cells/ml, and the inoculation density of insect cells is 0.8-1 × 106cells/ml。
The cell recovery culture medium formula comprises: insect cells ZY.104 were revived in Expisf CD medium and insect cells ZY.201 were revived in IB905 medium.
(2) Preparing a seed solution: inoculating the P0 generation recombinant baculovirus into 100ml insect cell ZY.104 culture solution according to MOI 0.01-5, controlling the cell survival rate at 0.5 multiplied by 10 during inoculation5~10×106cells/ml, the cell viability is more than 95%, the cells are cultured for 24-96 h at the temperature of 27-28 ℃, and the cells are detoxified when the cell viability is 40-80%, namely the primary virus seed solution.
(3) Preparing fermentation liquor: the formula of the fermentation liquid culture medium is that 5-90% of Expisf CD culture medium and 5-90% of IB905 culture medium are mixed according to the volume ratio. Inoculating the primary virus seed solution into 300ml of insect cell ZY.104 culture solution according to MOI 0.01-10, and controlling the cell viability rate at 0.5 multiplied by 10 during inoculation5~10×106cells/ml, the cell viability is more than 95%, the cells are cultured for 24-96 h at the temperature of 27-28 ℃, and the cells are detoxified when the cell viability is 40-80%, thus obtaining the secondary virus fermentation liquor.
(4) Fermenting in a cell tank: high-density fermentation is carried out by adopting a 5L cell tank, the charging coefficient of the cell tank is 30-60%, the fermentation temperature is 27-28 ℃, the rotating speed is 80-130 rpm/min, and the tank pressure is kept at 0.01-0.08 Mpa. When insect cells ZY.201 grow to 0.5X 105~25×106When cells/ml, the cell viability is more than 95%, inoculating secondary virus fermentation liquor according to MOI 0.01-10, feeding exogenous nutrient substances in a feeding mode 1-2 h after inoculation, continuing culturing for 12-160 h, and ending fermentation when the cell viability is 40-80%, thus obtaining the recombinant influenza antigen protein harvesting liquor.
The formula of the cell tank fermentation medium is as follows: IB905 medium was used.
The ingredients of the exogenous nutrient added in the fermentation process are as follows: the mass concentration of the composite protein hydrolysis solution is 0.05-1%, the mass concentration of the glucose is 0.01-1%, and the flow rate of the feed supplement is controlled as follows: 0.01 to 0.4 g/L/h.
The composite proteolytic solution comprises the following components: pea protein polypeptide with the mass concentration of 0.1-1%, wheat protein polypeptide with the mass concentration of 0.1-1% and rice protein polypeptide with the mass concentration of 0.1-1%.
The culture mode is a high-density multi-stage continuous fermentation technology, and by limiting the culture medium and the culture process, the growth of insect cells can be promoted, the expression quantity of target protein can be improved, the culture period is greatly shortened, and the production cost of the influenza vaccine is reduced.
Further, the recombinant influenza antigen protein harvest solution is subjected to high-speed centrifugation to be clarified, virus inactivation by a low pH incubation method, ultrafiltration concentration, two-step chromatography treatment and sterilization filtration to obtain a recombinant influenza protein stock solution.
Further, the method comprises the following specific steps:
(1) centrifuging the recombinant influenza antigen protein harvest liquid for 10-30 min by 200-2000 g, and filtering the recombinant influenza antigen protein harvest liquid through a 0.22-0.45 mu m membrane package to remove most impurities to obtain a clarified liquid.
(2) Preparing a concentrated solution: and performing primary ultrafiltration concentration on the clarified solution by using a 100KD ultrafiltration membrane, and performing secondary ultrafiltration concentration on the solution under the membrane by using a 30KD ultrafiltration membrane.
(3) Virus inactivation by low pH incubation: and (3) adjusting the pH value of the concentrated solution to 2-5 by using 1mol/l NaOH, incubating at 24 +/-1 ℃ for 1-21 days, and detecting the titer of a sample after inactivation is finished so that the titer of inactivated viruses is more than or equal to 4.00 Log.
(4) Two-step chromatography treatment: and (3) subjecting the inactivated sample to dextran G-25 gel filtration chromatography, and then carrying out secondary treatment on the sample by using a Q Sepharose XL chromatography medium to obtain a recombinant influenza protein stock solution.
And further, mixing the recombinant influenza protein stock solution with an adjuvant to obtain a semi-finished product of the recombinant influenza subunit vaccine, and bottling and packaging the qualified semi-finished product of the recombinant influenza subunit vaccine to obtain the recombinant influenza subunit vaccine.
Further, the semi-finished product is prepared by mixing the diluted protein antigen stock solution (after purification treatment) with an adjuvant according to a volume ratio of 20-100: 0.01-2, and uniformly stirring at 4-25 ℃ to obtain the semi-finished product.
Further, the recombinant influenza subunit vaccine takes PBD-b-PEO and DOTAP as vaccine adjuvants.
Furthermore, the content of the recombinant influenza vaccine semi-finished product protein antigen is 30-60 mu g/ml.
The recombinant influenza subunit vaccine can be administered by intramuscular injection or nasal spray.
According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: 1. can realize the high-efficiency preparation of tetravalent recombinant influenza vaccines (A H1N1, A H3N2, B Yamagata series and B Victoria series), improve the production efficiency and reduce the production cost. 2. The flash BAC ULTRA system of the baculovirus expression system is used for constructing recombinant bacmids and preparing recombinant influenza strains, so that the generation of false positive virus particles can be reduced/avoided, and the preparation period of the recombinant influenza viruses is greatly shortened. 3. Two high-efficiency expression promoters are adopted to respectively express HA genes and NA genes of 4 strains to produce HA proteins and NA proteins, so that the high-efficiency co-expression of multiple genes is realized in insect cells, and the aim of efficiently preparing influenza vaccines is fulfilled. 4. The recombinant influenza subunit vaccine is prepared by adopting the flash BAC ULTRA system of the baculovirus expression system, frequent plaque screening work is not needed in the production process, the production efficiency is improved, and the application prospect in production is very high. 5. The growth rate of the virus can be efficiently controlled and the expression quantity of the target protein can be improved by adopting a high-density multistage continuous fermentation technology and a heterologous host expression mode. 6. Exogenous nutrient substances are added in the high-density fermentation process, so that the yield of the target protein can be improved. 7. The traditional influenza vaccine preparation process mostly adopts formaldehyde to inactivate viruses, and the invention adopts a low pH incubation method to inactivate the viruses, thereby avoiding/reducing the influence of chemical substances such as formaldehyde on the components of the influenza vaccine and improving the safety of the influenza vaccine.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Insect cells Expisf 9, purchased from siemer feishale science and technology ltd, and now deposited in r 86 zhou yi ancao biotechnology, ltd, research and development center, deposit No. zy.104, north cheng district, tianjin city.
High Five cells were purchased from Saimer Feishale science and technology Co., Ltd, and currently deposited in research and development center of Zhouyi' an biotech, Inc. No. 86 Zhongyi Daokao, North Chen, Tianjin, with deposit number ZY.201.
The Expisf CD medium was purchased from Saimer Feishell science and technology, Inc.
IB905 medium was purchased from Biotechnology, Inc., Yishenke, Zhejiang.
SPF grade healthy BALB/c mice, 6 weeks old, were purchased from Experimental animals technologies, Inc. of Wei Tong Hua, Beijing.
Type A H1N1 (A/Washington/23/2020), available from the U.S. CDC.
Form A H3N2 (A/Darwin/11/2021) available from VIDRL, Australia.
Yamagata type B line (B/Singapore/INFKK-16-0569/2016), purchased from VIDRL, Australia.
Victoria type B line (B/Singapore/WUH 4618/2021) was purchased from ViDRL, Australia.
The required medicament is a conventional experimental medicament, and is purchased from a market channel; the unrecited experimental method is a conventional experimental method, and is not described in detail herein.
Example 1
A recombinant influenza subunit vaccine, each dose contains four types of H1N 1A, H3N 2A, Yamagata series B and Victoria series B;
the antigen in the vaccine is HA gene and NA gene coded protein of A type H1N1, A type H3N2, B type Yamagata series and B type Victoria series strains, wherein,
the nucleotide sequence of the optimized HA gene of the A type H1N1 strain is shown as SEQ ID NO. 1;
the nucleotide sequence of the optimized NA gene of the A H1N1 strain is shown as SEQ ID NO. 2;
the nucleotide sequence of the optimized HA gene of the A type H3N2 strain is shown as SEQ ID NO. 3;
the nucleotide sequence of the optimized NA gene of the A type H3N2 strain is shown as SEQ ID NO. 4;
the nucleotide sequence of the optimized HA gene of the B-type Yamagata strain is shown as SEQ ID NO. 5;
the nucleotide sequence of the optimized NA gene of the B-type Yamagata strain is shown as SEQ ID NO. 6;
the nucleotide sequence of the optimized HA gene of the Victoria B strain is shown as SEQ ID NO. 7;
the nucleotide sequence of the optimized NA gene of the Victoria B strain is shown as SEQ ID No. 8.
The sequences of the HA gene and NA gene detection primers of the target genes of the recombinant influenza tetravalent strains (type A H1N1, type A H3N2, type B Yamagata line and type B Victoria line) are shown in Table 1.
TABLE 1 primer summary sheet
Figure 55233DEST_PATH_IMAGE001
The recombinant influenza subunit vaccine contains 0.5ml per dose, contains H1N 1A, H3N 2A, Yamagata B and Victoria B, and has a content of univalent virus stock solution antigen of 120 μ g/ml.
A preparation method of a recombinant influenza subunit vaccine comprises the following steps:
a construction of recombinant baculovirus
(1) Constructing a recombinant plasmid:
the HA gene and NA gene of the A H1N1 strain are respectively cloned on a pOET5.1 vector, BglII/NotI is respectively added at the upstream/downstream of the HA gene, BamHI/HindIII is respectively added at the upstream and downstream of the NA gene, and the formed recombinant plasmid is pOET5.1-HA-NA-A1.
The HA gene and NA gene of the A H3N2 strain are respectively cloned on a pOET5.1 vector, BglII/NotI is respectively added at the upstream and downstream of the HA gene, BamHI/HindIII is respectively added at the upstream and downstream of the NA gene, and a recombinant plasmid pOET5.1-HA-NA-A2 is formed.
The HA gene and NA gene of the B1 strain (Yamagata series B) were cloned into pOET5.1 vector, BglII/NotI was added to the upstream and downstream of HA gene, BamHI/HindIII was added to the upstream and downstream of NA gene, and the recombinant plasmid was pOET5.1-HA-NA-B1.
The HA gene and NA gene of the B2 strain (Victoria B line) were cloned into pOET5.1 vector, BglII/NotI was added to the upstream and downstream of HA gene, BamHI/HindIII was added to the upstream and downstream of NA gene, and the recombinant plasmid was pOET5.1-HA-NA-B2.
(2) Construction of recombinant baculovirus:
the four recombinant plasmids pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2 are mixed according to the volume ratio of 1:0.5:0.3:1, and the total concentration of the four recombinant plasmid DNAs is 50 mug.
(21) The density is 1.5 multiplied by 107cells/ml insect cells ZY.104 with cell viability above 95% were inoculated in 50ml Expisf CD medium.
(22) Insect cells ZY.104 were diluted to 5X 10 with medium6cells/ml, placed in 250ml cell culture flasks.
(23) The insect cells ZY.104 were revived in an incubator at 27 ℃ for 20min at 110 rpm/min.
(24) Preparation of transfection complex: a mixture of Sinofection transfection reagent, flashBAC ULTRA baculovirus genomic DNA and the 4 recombinant plasmids described above (pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2) were briefly vortex mixed in a volume ratio of 5:5: 3.
(25) Slowly dropping the transfection complex of step (24) into the insect cell culture solution of step (23).
(26) After incubation for 96h at the speed of 110rpm/min in an incubator at the temperature of 27 ℃, the recombinant baculovirus of P0 generation is obtained.
Propagation culture and harvest of B recombinant baculovirus
(1) Cell recovery culture: rapidly taking out the frozen insect cells ZY.104 and insect cells ZY.201 from a liquid nitrogen tank, thawing in a water bath pan at 27 ℃, transferring the thawed cells into a sterile centrifuge tube containing 15ml of fresh culture medium, centrifuging at a low speed of 300g for 5min, discarding supernatant, retaining precipitate, then re-suspending the insect cells with 15ml of fresh culture medium, and ensuring that the density of the re-suspended insect cells is more than 1 x 106cells/ml, the resuspended insect cells (cells ZY.104 and cells ZY.201) were placed in a sterile cell culture flask for suspension culture at 27 ℃ with a shaker speed of 110 rpm/min. When the density of insect cells grows to 2X 106cells/ml, and the inoculation density of insect cells is 1X 106cells/ml。
The cell recovery culture medium formula comprises: insect cells ZY.104 were resuscitated using Expisf CD medium and insect cells ZY.201 were resuscitated using IB905 medium.
(2) Preparing a seed solution: the seed liquid culture medium adopts an Expisf CD culture medium. Fresh P0 generation recombinant baculovirus was inoculated into 100ml insect cell ZY.104 culture solution according to MOI =1, the cell viability at inoculation was controlled at 5.0X 106cells/ml, cell survival rate is more than 95%, culturing for 48h at 27 ℃, and harvesting toxicity when the cell survival rate is 60%, namely the primary virus seed solution.
(3) Preparing fermentation liquor: the formula of the fermentation broth culture medium is that the Expisf CD culture medium and the IB905 culture medium are mixed according to the volume ratio of 50% to 50%. Inoculating the primary virus seed solution into 300ml insect cell ZY.104 culture solution at MOI =2.5, and controlling the cell viability rate at 0.5 × 105cells/ml, cell viability rate > 95%, culturing at 27 deg.C for 96h, and harvesting toxicity when cell viability rate is 50%, i.e. secondary virus fermentation liquid.
(4) Fermenting in a cell tank: high-density fermentation is carried out by adopting a 5L cell tank, the material loading coefficient of the cell tank is 40%, the fermentation temperature is 27 ℃, the rotating speed is 80rpm/min, and the tank pressure is kept at 0.02 Mpa. When the insect cells ZY.201 grow to 1X 106When the cell activity is more than 95% in cells/ml, inoculating secondary virus fermentation liquor according to MOI =2.5, feeding exogenous nutrient substances in a feeding mode after 1.5h after inoculation, continuing culturing for 96h, and ending fermentation when the cell activity is 40% to obtain the recombinant influenza antigen protein harvesting solution.
The formula of the cell tank fermentation medium is as follows: adopting IB905 culture medium, the components of exogenous nutrient substances added in the fermentation process are as follows: the mass concentration of the composite proteolytic solution is 0.1 percent, the mass concentration of the glucose is 0.5 percent, and the feeding flow rate is controlled to be 0.1 g/L/h.
The composite proteolytic solution comprises the following components: pea protein polypeptide with the mass concentration of 0.1 percent, wheat protein polypeptide with the mass concentration of 0.5 percent and rice protein polypeptide with the mass concentration of 1 percent.
C obtaining a recombinant influenza protein stock solution
(1) Preparing a clear solution: the recombinant influenza antigen protein harvest solution is centrifuged for 10min at 200g and is filtered through a 0.45 mu m membrane package to remove most impurities, so as to obtain a clarified solution.
(2) Preparing a concentrated solution: the prepared clarified liquid is firstly subjected to ultrafiltration concentration by using a 100KD ultrafiltration membrane, and then the liquid under the membrane is subjected to ultrafiltration concentration for the second time by using a 30KD ultrafiltration membrane.
(3) Virus inactivation by low pH incubation: adjusting the pH value of the prepared concentrated solution to 2 by using 1mol/l NaOH, incubating the concentrated solution at 24 +/-1 ℃ for 7d, and detecting the titer of a sample after inactivation is finished so that the titer of the inactivated virus is more than or equal to 4.00 Log.
(4) Two-step chromatography treatment: and (3) subjecting the inactivated sample to dextran G-25 gel filtration chromatography, and then carrying out secondary treatment on the sample by using a Q Sepharose XL chromatography medium to obtain a recombinant influenza protein stock solution.
D mixing with adjuvant
Mixing the diluted protein antigen stock solution (after purification treatment) with an adjuvant according to the volume ratio of 100: 2, and uniformly stirring at 4 ℃ to obtain a semi-finished product.
PBD-b-PEO and DOTAP are used as vaccine adjuvants, and the content of the antigen of the semi-finished product of the recombinant influenza vaccine is 30 mu g/ml.
And bottling and packaging the qualified semi-finished product of the recombinant influenza subunit vaccine to obtain the recombinant influenza subunit vaccine.
Example 2
The composition of the recombinant influenza subunit vaccine was the same as in example 1.
The recombinant influenza subunit vaccine contains 0.5ml of each dose, contains H1N 1A, H3N 2A, Yamagata series B and Victoria series B, and the content of the antigen in the monovalent harvest liquid of the recombinant influenza vaccine is 300 mu g/ml.
A preparation method of a recombinant influenza subunit vaccine comprises the following steps:
a construction of recombinant baculovirus
(1) Constructing a recombinant plasmid:
the HA gene and NA gene of the A H1N1 strain are respectively cloned on a pOET5.1 vector, BglII/NotI is respectively added at the upstream/downstream of the HA gene, BamHI/HindIII is respectively added at the upstream and downstream of the NA gene, and the formed recombinant plasmid is pOET5.1-HA-NA-A1.
The HA gene and NA gene of the A H3N2 strain are respectively cloned on a pOET5.1 vector, BglII/NotI is respectively added at the upstream/downstream of the HA gene, BamHI/HindIII is respectively added at the upstream and downstream of the NA gene, and the formed recombinant plasmid is pOET5.1-HA-NA-A2.
The HA gene and NA gene of B1 strain (Yamagata series B) were cloned into pOET5.1 vector, BglII/NotI was added to the upstream and downstream of HA gene, BamHI/HindIII was added to the upstream and downstream of NA gene, and recombinant plasmid pOET5.1-HA-NA-B1 was formed.
The HA gene and NA gene of B2 strain (Victoria B line) were cloned into pOET5.1 vector, BglII/NotI was added to the upstream and downstream of HA gene, BamHI/HindIII was added to the upstream and downstream of NA gene, and recombinant plasmid pOET5.1-HA-NA-B2 was formed.
(2) Construction of recombinant baculovirus:
the four recombinant plasmids pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2 are mixed according to the volume ratio of 0.1:1:1:0.5, and the total concentration of the four recombinant plasmid DNAs is 20 mug.
(21) The density is 5.5 multiplied by 106cells/ml, insect cells ZY.104 with cell viability above 95% were inoculated in 50ml of Expisf CD medium.
(22) Insect cells ZY.104 were diluted to 2.5X 10 with medium6cells/ml, placed in 250ml cell culture flasks.
(23) The insect cells ZY.104 were revived in an incubator at 28 ℃ for 20min at 135 rpm/min.
(24) Preparation of transfection complex: a mixture of Sinofection transfection reagent, flashBAC ULTRA baculovirus genomic DNA and the 4 recombinant plasmids described above (pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2) were briefly vortex mixed in a volume ratio of 3:5: 5.
(25) Slowly dropping the transfection complex of step (24) into the insect cell culture solution of step (23).
(26) After incubation for 96h at 130rpm/min in an incubator at 27 ℃, the recombinant baculovirus of P0 generation is obtained.
Propagation culture and harvest of B recombinant baculovirus
(1) Cell recovery culture: rapidly taking out the frozen insect cells ZY.104 and insect cells ZY.201 from a liquid nitrogen tank, thawing in a 28 deg.C water bath, transferring the thawed insect cells to a sterile centrifuge tube containing 10ml of fresh culture medium, centrifuging at low speed of 200g for 10min, discarding supernatant, retaining precipitate, suspending the insect cells with 15ml of fresh culture medium to ensure that the density of the resuspended insect cells is greater than 1 × 106cells/ml, the resuspended insect cells (insect cells ZY.104 and insect cells ZY.201) were placed in a sterile cell culture flask for suspension culture at 28 ℃ with a shaker speed of 100 rpm/min. When the density of insect cells grows to reach 3X 106cells/ml were passaged at a cell seeding density of 0.8X 106cells/ml。
The cell recovery culture medium formula comprises: insect cells ZY.104 were resuscitated using Expisf CD medium and insect cells ZY.201 were resuscitated using IB905 medium.
(2) Preparing a seed solution: the formula of the seed liquid culture medium adopts an Expisf CD culture medium. Fresh P0 generation recombinant baculovirus was inoculated into 100ml insect cell ZY.104 culture solution according to MOI =5, the cell viability at inoculation was controlled at 5.0X 106cells/ml, cell survival rate is more than 95%, culturing for 24h at 28 ℃, and harvesting toxicity when the cell survival rate is 80%, namely the primary virus seed solution.
(3) Preparing fermentation liquor: the formula of the fermentation broth culture medium is that the Expisf CD culture medium and the IB905 culture medium are mixed according to the volume ratio of 90% to 5%. Inoculating the primary virus seed solution into 300ml insect cell ZY.104 culture solution at MOI =10, and controlling the cell viability rate at 1.0 × 106cells/ml, cell viability rate > 95%, culturing for 72h at 28 ℃, and harvesting toxicity when cell viability rate is 80%, namely secondary virus fermentation liquor.
(4) Fermenting in a cell tank: high-density fermentation is carried out by adopting a 5L cell tank, wherein the charging coefficient of the cell tank is 30 percent, the fermentation temperature is 28 ℃, the rotating speed is 130rpm/min, and the tank pressure is kept at 0.01 Mpa. When ZY.201 cells were grown to 0.5X 105When cells/ml, the cell viability is more than 95%, inoculating secondary virus fermentation liquor according to MOI =10, feeding exogenous nutrient substances in a feeding mode 2h after inoculation, continuing to culture for 12h, and ending fermentation when the cell viability is 80%, thus obtaining the recombinant influenza antigen protein harvest liquor.
The formula of the cell tank fermentation medium is as follows: adopting IB905 culture medium, the components of exogenous nutrient substances added in the fermentation process are as follows: the mass concentration of the composite proteolytic solution is 1 percent, the mass concentration of the glucose is 1 percent, and the feeding flow rate is controlled to be 0.01 g/L/h.
The composite proteolytic solution comprises the following components: pea protein polypeptide with the mass concentration of 1%, wheat protein polypeptide with the mass concentration of 0.1% and rice protein polypeptide with the mass concentration of 0.5%.
C obtaining a recombinant influenza protein stock solution
(1) Preparing a clear solution: the recombinant influenza antigen protein harvest solution is centrifuged for 20min at 2000g and is filtered through a 0.22 mu m membrane package to remove most impurities, so as to obtain a clear solution.
(2) Preparing a concentrated solution: the prepared clarified liquid is firstly subjected to ultrafiltration concentration by using a 100KD ultrafiltration membrane, and then the liquid under the membrane is subjected to ultrafiltration concentration for the second time by using a 30KD ultrafiltration membrane.
(3) Virus inactivation by low pH incubation: adjusting the pH value of the prepared concentrated solution to 3 by using 1mol/l NaOH, incubating the concentrated solution at 24 +/-1 ℃ for 1d, and detecting the titer of a sample after inactivation is finished so that the titer of the inactivated virus is more than or equal to 4.00 Log.
(4) Two-step chromatography treatment: and (3) subjecting the inactivated sample to dextran G-25 gel filtration chromatography, and then carrying out secondary treatment on the sample by using a Q Sepharose XL chromatography medium to obtain a recombinant influenza protein stock solution.
D, mixing with an adjuvant.
And mixing the diluted protein antigen stock solution (after purification treatment) with an adjuvant according to the volume ratio of 20: 2, and uniformly stirring at 25 ℃ to obtain a semi-finished product.
PBD-b-PEO and DOTAP are used as vaccine adjuvants, and the content of the antigen of the semi-finished product of the recombinant influenza vaccine is 45 mu g/ml.
And bottling and packaging the qualified semi-finished product of the recombinant influenza subunit vaccine to obtain the recombinant influenza subunit vaccine.
Example 3
The composition of the recombinant influenza subunit vaccine was the same as in example 1.
The recombinant influenza subunit vaccine contains 0.5ml of each dose, contains H1N 1A, H3N 2A, Yamagata series B and Victoria series B, and the content of the monovalent virus stock solution protein antigen of the recombinant influenza vaccine is 30 mu g/ml.
A preparation method of a recombinant influenza subunit vaccine comprises the following steps:
a construction of recombinant baculovirus
(1) Constructing a recombinant plasmid:
the HA gene and NA gene of the A H1N1 strain are respectively cloned on a pOET5.1 vector, BglII/NotI is respectively added at the upstream/downstream of the HA gene, BamHI/HindIII is respectively added at the upstream and downstream of the NA gene, and the formed recombinant plasmid is pOET5.1-HA-NA-A1.
The HA gene and NA gene of the A H3N2 strain are respectively cloned on a pOET5.1 vector, BglII/NotI is respectively added at the upstream/downstream of the HA gene, BamHI/HindIII is respectively added at the upstream and downstream of the NA gene, and the formed recombinant plasmid is pOET5.1-HA-NA-A2.
The HA gene and NA gene of B1 strain (Yamagata series B) were cloned into pOET5.1 vector, BglII/NotI was added to the upstream and downstream of HA gene, BamHI/HindIII was added to the upstream and downstream of NA gene, and recombinant plasmid pOET5.1-HA-NA-B1 was formed.
The HA gene and NA gene of B2 strain (Victoria B line) were cloned into pOET5.1 vector, BglII/NotI was added to the upstream and downstream of HA gene, BamHI/HindIII was added to the upstream and downstream of NA gene, and recombinant plasmid pOET5.1-HA-NA-B2 was formed.
(2) Construction of recombinant baculovirus:
the four recombinant plasmids pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2 are mixed according to the volume ratio of 1:0.1:0.1:0.1, and the total concentration of the four recombinant plasmid DNAs is 1 mu g.
(21) The density is 1 x 107cells/ml insect cells ZY.104 with cell viability above 95% were inoculated in 50ml Expisf CD medium.
(22) Insect cells ZY.104 were diluted to 2.5X 10 with culture medium6cells/ml, placed in 250ml cell culture flasks.
(23) The insect cells ZY.104 were revived in an incubator at 27 ℃ for 30min at 135 rpm/min.
(24) Preparation of transfection complex: a mixture of Sinofection transfection reagent, flashBAC ULTRA baculovirus genomic DNA and the 4 recombinant plasmids described above (pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2) were briefly vortex mixed in a volume ratio of 5:1: 3.
(25) Slowly dropping the transfection complex of step (24) into the insect cell culture solution of step (23).
(26) After incubation for 72h at 110rpm/min in an incubator at 27 ℃, the recombinant baculovirus of P0 generation was obtained.
Propagation culture and harvest of B recombinant baculovirus
(1) Cell recovery culture: quickly taking out the frozen insect cells ZY.104 and insect cells ZY.201 from a liquid nitrogen tank, respectively thawing in a water bath kettle at 27 ℃, transferring the thawed insect cells to a sterile centrifuge tube containing 5ml of fresh culture medium, centrifuging at a low speed of 500g for 5min, discarding supernatant, retaining precipitate, suspending the insect cells by using 15ml of fresh culture medium to ensure that the density of the resuspended insect cells is more than 1 x 106cells/ml, respectively placing the resuspended insect cells (insect cells ZY.104 and insect cells ZY.201) into a sterile cell culture flask for suspension culture at 27 ℃, and rotating the shaker at 130 rpm/min. When the density of insect cells grows to 4 multiplied by 106Passage is carried out at cells/ml, and the cell seeding density is 1X 106cells/ml。
The cell recovery culture medium formula comprises: insect cells ZY.104 were resuscitated using Expisf CD medium and insect cells ZY.201 were resuscitated using IB905 medium.
(2) Preparing a seed solution: the formula of the seed liquid culture medium adopts an Expisf CD culture medium. Fresh P0 generation recombinant baculovirus was inoculated into 100ml insect cell ZY.104 culture solution according to MOI =0.01, and the cell viability at the time of inoculation was controlled at 5.0X 106cells/ml, the cell viability is more than 95 percent, and the virus is collected when the cells are cultured for 96 hours at 27 ℃ and the cell viability is 40 percent, thus obtaining the primary virus seed solution.
(3) Preparing fermentation liquor: the formula of the fermentation broth culture medium is that the Expisf CD culture medium and the IB905 culture medium are mixed according to the volume ratio of 5 percent to 90 percent. Inoculating the primary virus seed solution into 300ml insect cell ZY.104 culture solution at MOI =0.01, and controlling the cell viability rate at 10 × 106cells/ml, the cell viability is more than 95 percent, and the secondary virus fermentation broth is obtained after the cells are cultured for 24 hours at 27 ℃ and the cell viability is 40 percent.
(4) Fermenting in a cell tank: high-density fermentation is carried out by adopting a 5L cell tank, the material loading coefficient of the cell tank is 60%, the fermentation temperature is 27 ℃, the rotating speed is 130rpm/min, and the tank pressure is kept at 0.08 MPa. When insect cells ZY.201 grow to 25X 106When cells/ml, the cell survival rate is more than 95 percent, inoculating secondary virus fermentation liquor according to MOI 0.01, feeding exogenous nutrient substances in a material supplementing mode 1 hour after inoculation, and continuously culturing for 160 hours, wherein the cell survival rate isEnding fermentation at 60% to obtain recombinant influenza antigen protein harvest liquid.
The formula of the cell tank fermentation medium is as follows: adopting IB905 culture medium, the ingredients of exogenous nutrient substances added in the fermentation process are: the mass concentration of the composite proteolytic solution is 0.05 percent, the mass concentration of the glucose is 0.01 percent, and the feeding flow rate is controlled to be 0.4 g/L/h.
The composite proteolytic solution comprises the following components: pea protein polypeptide with mass concentration of 0.5%, wheat protein polypeptide with mass concentration of 1% and rice protein polypeptide with mass concentration of 0.1%.
C obtaining a recombinant influenza protein stock solution
(1) Preparing a clear liquid: the recombinant influenza antigen protein harvest solution is centrifuged for 30min at 1000g and is filtered through a 0.22 mu m membrane package to remove most impurities, so as to obtain a clarified solution.
(2) Preparing a concentrated solution: the prepared clear solution is firstly subjected to primary ultrafiltration concentration by using a 100KD ultrafiltration membrane, and then the liquid under the membrane is subjected to secondary ultrafiltration concentration by using a 30KD ultrafiltration membrane.
(3) Virus inactivation by low pH incubation: adjusting the pH of the prepared concentrated solution to 5 with 1mol/l NaOH, incubating at 24 +/-1 ℃ for 21d, and detecting the titer of a sample after inactivation is finished so that the titer of the inactivated viruses is more than or equal to 4.00 Log.
(4) Two-step chromatography treatment: and (3) subjecting the inactivated sample to dextran G-25 gel filtration chromatography, and then carrying out secondary treatment on the sample by using a Q Sepharose XL chromatography medium to obtain a recombinant influenza protein stock solution.
D, mixing with an adjuvant.
Mixing the diluted protein antigen stock solution (after purification treatment) with an adjuvant according to the volume ratio of 50: 0.01, and uniformly stirring at 4 ℃ to obtain a semi-finished product.
PBD-b-PEO and DOTAP are used as vaccine adjuvants, and the content of the antigen of the semi-finished product of the recombinant influenza vaccine is 60 mu g/ml.
And bottling and packaging the qualified semi-finished product of the recombinant influenza subunit vaccine to obtain the recombinant influenza subunit vaccine.
Example 4
Evaluation of Effect
The invention selects SPF healthy BALB/c mice, 6 weeks old. The animals were kept in SPF-grade animal houses of Zhongyi Anke biotechnology, Inc. The experiments were grouped into 4 groups including: negative control group: no treatment is carried out; positive control group: injecting a sterilized PBS buffer solution; immunization group a: injecting the recombinant influenza subunit vaccine of the invention; immunization group B: commercially available influenza vaccines. Each group of 60 mice (males and females) was replicated 4 times.
Antibody potency Studies
The immunization mode comprises the following steps: the neck of the mouse is immunized subcutaneously, the immunization dose is 0.5 ml/branch, and the inoculation is carried out for 2 times. The second immunization (14 days) was performed at 14-day intervals after the primary immunization (0 days). Blood was collected from the eye vein of mice 21 days after immunization, and the titer of influenza antibodies (IgA antibody and IgG antibody) in serum was measured.
Study of immune Effect
Virus challenge strain: type A H1N1 (A/Washington/23/2020), type A H3N2 (A/Darwin/11/2021), type B Yamagata line (B/Singapore/INFKK-16-0569/2016) and type B Victoria line (B/Singapore/WUH 4618/2021). Each influenza virus is given a 20 LD50The dose was challenged to mice after immunization (21 days), and the survival rate and weight change of the mice were monitored 14 days after challenge (if the weight loss is more than or equal to 25%, this is an unsatisfactory item). The whole effect evaluation process is monitored to judge whether anaphylactic reaction or abnormal toxicity exists.
TABLE 2 test results
Figure 34690DEST_PATH_IMAGE002
Note: "-" indicates negative.
As can be seen from Table 2, in the same growth cycle, the antibody level in the serum of the mice in the immune group A and the survival rate of the mice after challenge are both higher than those of the commercial vaccines in the immune group B; in the negative control group (not challenged), the survival rate of mice was high; in the positive control group (challenge), the mice lost much weight (weight loss > 25%), and all died 14 days after challenge; immunizing mice in group A and group B to body weight which is normal; the mice have no anaphylactic reaction or abnormal toxicity after the immunization injection. The tests show that the recombinant influenza subunit vaccine provided by the invention has strong safety and good immune protection effect. The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
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tgtatcggtt accacgctaa caactccacc gacacagttg acacagtcct ggaaaagaac 120
gtgaccgtga cacactctgt gaacctcctg gaagataagc acaacggcaa gctgtgcaag 180
ctccgtggcg tggctcccct gcacctgggc aagtgtaaca tcgctggttg gattctcggt 240
aaccctgagt gcgaatccct gagcactgct cgtagctggt catacatcgt ggaaacttcc 300
aactccgaca acggaacttg ctaccccgga gacttcatca actacgaaga actgcgtgag 360
cagctctctt ctgtgtcatc attcgagcgc ttcgaaatct tcccaaagac cagctcttgg 420
cctaaccacg acagcgacaa cggagttacc gctgcctgtc cccacgctgg agccaagagc 480
ttctacaaga acctcatctg gctcgtgaag aagggcaagt cctacccaaa gatcaaccaa 540
acctacatca acgacaaggg aaaggaggtt ctcgtcctgt ggggaatcca ccacccccca 600
accatcgctg accaacaatc actctaccag aacgctgatg cctacgtgtt cgttggtaca 660
tcccgctact ctaagaagtt caagcctgaa atcgccaccc gccctaaggt tcgcgaccag 720
gaaggacgta tgaactacta ctggaccctg gtggaacctg gcgacaagat cactttcgag 780
gccacaggca acctcgtcgc cccacgttac gccttcacta tggaacgtga cgctggttca 840
ggcatcatca tctccgacac tccagttcac gactgtaaca caacttgtca aacacccgag 900
ggtgctatca acacatcttt gccattccaa aacgtgcatc ctatcacaat cggtaaatgc 960
ccaaagtacg ttaagtctac aaagctcaga ttggccactg gactcaggaa cgtgccttcc 1020
atccagagcc gtggcctctt cggtgccatc gctggtttca tcgaaggagg ttggactggt 1080
atggtggacg gatggtacgg ctaccaccac cagaacgagc agggctctgg ttacgccgct 1140
gacctcaaga gcactcaaaa cgccattgac aagatcacca acaaggtcaa ctccgttatc 1200
gaaaagatga acactcagtt cactgccgtt ggcaaggagt tcaaccacct ggagaagcgt 1260
atcgagaacc tgaacaaaaa ggtggacgac ggtttcctgg acatctggac ctacaacgcc 1320
gagctcctgg ttctgctgga gaatgaacgc accctggact accacgactc caacgtcaag 1380
aacctgtacg aaaaggtgcg taaccaactg aagaacaacg ccaaggagat cggcaacggt 1440
tgcttcgaat tctaccacaa gtgtgacaac acttgtatgg agtccgtcaa gaacggtacc 1500
tacgactacc caaagtacag cgaagaggct aagctgaacc gtgaaaagat cgacggcgtc 1560
aagctcgact ccactcgtat ctaccagatc ctcgctatct actccacagt ggcctcctca 1620
ctggtgctgg tggtgtccct gggtgctatc tctttctgga tgtgttccaa cggtagcctg 1680
cagtgccgca tctgtatc 1698
<210> 2
<211> 1407
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgaacccca accaaaagat catcaccatc ggtagcatct gcatgacaat cggaactgct 60
aacctgatcc tgcagatcgg taacatcatc tcaatctggg tttctcacag catccagatc 120
ggcaaccagt cccaaatcga gacatgtaac aagtccgtca tcacatacga aaacaacaca 180
tgggttaacc agacattcgt gaacatctca aacactaaca gtgctgcccg tcaatccgtg 240
gcctcagtga agctcgctgg taatagtagc ctctgtcctg tgtctggctg ggcgatctac 300
agcaaggaca actccgtgcg tattggtagc aagggcgacg ttttcgttat ccgtgaaccc 360
ttcatctcct gtagtccact ggagtgtaga acctttttcc tcactcaggg cgctctgctg 420
aacgacaagc actctaacgg aaccatcaag gaccgctcac cctacaggac cttgatgtcc 480
tgtccaatcg gtgaggttcc atccccttac aattcacgct tcgaatctgt tgcttggtcc 540
gctagcgcct gccacgacgg cactaactgg ctcacaattg gaatcagtgg ccccgatagc 600
ggagccgtcg ccgtgctcaa atacaatggt atcatcactg atactataaa gtcttggaga 660
aacaagatcc tccgcacaca agagagcgag tgcgcctgcg tcaatggctc ttgttttaca 720
atcatgacag atggtccaag cgatggccag gcttcataca aaatcttccg cattgagaag 780
ggaaaaatta ttaagagcgt cgaaatgaag gctcccaact accactatga agaatgctct 840
tgttaccctg actcatcgga gattacttgc gtgtgcaggg acaactggca tggctctaat 900
aggccttggg tctcctttaa ccagaaccta gaataccaaa tgggctacat ctgttccggt 960
gtgttcggag acaacccacg tcctaacgat aagaccggtt catgcggtcc cgtgagtagt 1020
aacggagcca acggggtgaa gggcttctca ttcaaatacg gtaacggcgt ctggatcggt 1080
aggaccaagt ccatctctag ccgtaagggt tttgaaatga tctgggaccc taacggttgg 1140
accggaactg acaataagtt cagcaagaaa caggacatcg tcggtattaa tgaatggtcc 1200
ggatactcag gctccttcgt tcaacacccc gaactgaccg gcttgaactg tattcgccca 1260
tgcttctggg ttgagttgat ccgtggacgc cctgaggaga acaccatctg gactagcgga 1320
tcatctatca gcttctgcgg agtcgattct gacatcgtcg gctggtcctg gcctgacgga 1380
gccgagctgc cattcaccat tgataag 1407
<210> 3
<211> 1698
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgaaaacca tcatcgccct gtccaacatc ctctgtctgg ttttcgccca gaagatccca 60
ggtaacgaca acagcactgc tactctgtgt ctgggtcacc acgctgtccc taacggcaca 120
atcgttaaga ctatcactaa cgaccgtatc gaggtcacaa acgccactga actggtgcag 180
aactcatcca tcggtgaaat ctgtggttca ccccaccaga tcctcgatgg tggaaactgt 240
accctgatcg acgctctgct gggcgacccc caatgtgatg gattccagaa caaggaatgg 300
gacctgttcg ttgagcgctc ccgcgctaac tcaaactgtt acccctacga cgtcccagac 360
tacgctagcc tccgttctct ggttgcctcc tctggaacac tggagttcaa gaacgaatcc 420
ttcaactgga ccggtgtgaa gcagaacggt acctcaagcg cctgtatccg cggttccagc 480
agcagcttct tctcccgtct gaactggctg acatcactga acaacatcta ccctgctcaa 540
aacgtgacca tgcccaacaa ggagcaattc gacaagttgt acatctgggg agtgcaccac 600
cccgacacag acaagaacca gatcagtttg ttcgcccaat caagtggccg catcaccgtg 660
agcactaagc gcagccaaca ggccgtcatc cctaatatcg gatctagacc tcgtatccgt 720
gatattcctt ctaggatctc aatctactgg actatcgtta agccaggtga catcctgctg 780
atcaactcca ctggtaacct gatcgcccca cgtggttact tcaagatccg ttctggcaag 840
tcctccatca tgcgttctga cgctccaatc ggtaaatgta agtctgagtg catcactcct 900
aacggatcta tcccaaacga caagcccttc cagaacgtca accgcatcac ctacggagcc 960
tgtcctaggt acgttaagca gtcaacactc aagctggcta caggtatgcg taacgttcct 1020
gagaagcaaa cccgcggtat cttcggtgcc atcgccggct tcatcgaaaa cggatgggag 1080
ggaatggtgg atggatggta cggattccgt caccaaaact ccgagggacg tggccaggct 1140
gctgacctca agagcaccca agccgccatc gaccagatta acggaaagct gaaccgcctg 1200
atcggcaaga ctaatgaaaa attccaccaa atcgaaaagg aattctctga ggtggaaggt 1260
cgtgttcagg acctcgaaaa gtacgtggag gatacaaaga tcgacctgtg gtcatacaac 1320
gctgaactcc tggtggctct ggaaaaccag cacacaatcg acctcaccga ttcagaaatg 1380
aacaagctgt tcgaaaagac aaagaagcag ctgagggaaa acgccgagga catgggtaac 1440
ggctgtttca agatttacca caagtgtgac aacgcctgca tcggttctat ccgtaacgag 1500
acctacgacc acaacgttta ccgcgacgaa gccctcaaca accgtttcca gatcaagggt 1560
gtggagctga agagcggtta caaggactgg atcttgtgga tctcattcgc tatgagctgt 1620
ttcctgctct gtatcgctct gctcggtttc atcatgtggg cctgccagaa gggtaacatc 1680
cgttgtaaca tctgcatc 1698
<210> 4
<211> 1407
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgaacccta accagaagat catcaccatc ggtagcgtga gcctgactat cagcactatc 60
tgcttcttca tgcagatcgc tatcctgatc actaccgtga cactgcactt caagcagtac 120
gaattcaact ctcctccaaa caaccaagtg atgctgtgtg agccaaccat catcgaacgt 180
aacatcacag agatcgtgta cctcaccaac accaccatcg agaaggaaat ctgtcctaag 240
ccagctgaat accgtaactg gtctaagcca cagtgtggta tcaccggttt cgctcccttc 300
tccaaggaca actccatccg cctgagcgcc ggtggtgaca tctgggtgac ccgtgagcca 360
tacgtgtcct gtgacctcga caagtgttac caattcgctc tgggccaggg tacaacactg 420
aacaacgttc acagcaacaa cacagtgcgc gaccgtactc cttacaggac tctgctgatg 480
aacgagctgg gagttccttt ccacctgggt acaaagcagg tctgtatcgc ttggtcctca 540
tcttcctgcc acgacggaaa ggcttggctc cacgtttgta tcactggcga cgataagaac 600
gctacagcct cattcatcta caacggtcgc ttggtggact ctgttgtgtc ctggtcaaac 660
gacatcctga gaactcagga aagcgaatgt gtctgcatca acggaacctg tactgttgtt 720
atgactgatg gtaacgccac cggaaaggcc gatacaaaga tcctgttcat cgaagagggt 780
aaaatcgttc acacatccaa gctgtctggt tccgcccaac atgtcgaaga atgttcttgt 840
taccctcgct accctggagt gcgctgtgtc tgccgtgaca actggaaggg cagcaaccgt 900
ccaatcatcg acatcaacat caaggaccac tcaatcgtca gtcgttacgt ctgctccgga 960
ctggtgggcg acacaccccg taagtccgac tcttcctcct caagccactg tctcaaccct 1020
aacaacgaaa agggcgacca cggtgtgaag ggttgggcct tcgacgacgg aaacgacgtt 1080
tggatgggtc gtaccatcaa cgaaacttct cgtctgggtt acgagacctt caaggtggtg 1140
gaaggttggt ctaaccccaa gtctaagctg cagatcaacc gccaggttat cgtggaccgt 1200
ggcgaccgct caggctacag cggtatcttc tcagttgaag gtaaaagctg tatcaaccgc 1260
tgcttctacg tggaattgat ccgtggccgt aaggaagaga ctgaagtctt gtggacttca 1320
aactcaatcg ttgttttctg tggcacttca ggcacatacg gtacaggatc ttggcctgac 1380
ggtgctaacc tgagcctgat gcacatc 1407
<210> 5
<211> 1752
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgaaggcta tcatcgtgct gctgatggtg gtcacttcaa acgctgaccg tatctgcaca 60
ggtatcacta gctctaactc ccctcacgtg gttaagaccg ctactcaggg tgaagttaac 120
gtgacaggag tgattccact gactactact ccaaccaagt cctacttcgc taacttgaag 180
ggtactcgca cccgtggaaa gttgtgtcct gattgtctga actgtaccga cctcgacgtt 240
gccctgggtc gcccaatgtg cgtgggcaca accccaagtg ccaaggctag catcctccac 300
gaagtgcgcc ctgtcaccag cggttgtttc cctatcatgc acgaccgcac taagatcaga 360
caactgccta acctgttgcg tggatacgag aaaatccgtc tgtccacaca gaatgttatc 420
gacgctgaga aggctcctgg aggaccttac aggttgggaa catcaggctc ctgccctaac 480
gccacttcta agatcggttt cttcgccacc atggcctggg ctgtgcctaa agacaactac 540
aagaacgcta ctaaccccct gacagttgag gtgccttaca tttgtactga aggtgaggat 600
cagatcaccg tttggggttt ccacagcgac aacaagaccc agatgaagtc cctctacgga 660
gactctaacc ctcaaaagtt cacatcatct gctaacggtg tcacaacaca ctacgttagt 720
caaatcggtg acttcccaga ccaaactgag gatggtggac tgccccaatc aggtcgcatt 780
gtcgttgact acatgatgca gaagccagga aagactggca caatcgttta ccaacgtggt 840
gtgctcctcc cacagaaggt ctggtgtgcc agcggtagat caaaggttat taaaggtagc 900
ctccccctga tcggtgaggc tgactgcctg catgaagaat acggtggtct caacaagtct 960
aaaccatact acacaggtaa acacgctaag gctatcggta actgccctat ctgggttaag 1020
actcccttga agctcgctaa cggaacaaag taccgtccac ctgctaagct gttgaaggaa 1080
cgcggtttct tcggtgctat cgctggtttc ttggaaggtg gatgggaagg aatgatcgct 1140
ggatggcacg gctacactag ccacggcgct cacggagtgg ccgtggctgc tgacctgaag 1200
tctacacagg aagccatcaa caagatcacc aagaacctga acagcctctc agagctggaa 1260
gttaagaacc tccagcgcct ctcaggtgcc atggacgaac tgcacaacga aatcctggag 1320
ctcgacgaga aggttgatga cctgagggcc gacaccatca gtagtcaaat cgaactggcc 1380
gtgctcctgt ctaacgaagg aatcatcaac agcgaagatg agcacctgct cgctctggaa 1440
cgtaagctga agaagatgtt gggtccatcc gctgttgaca tcggtaacgg ctgttttgaa 1500
acaaagcaca agtgtaacca aacttgtctc gaccgtatcg ccgccggtac attcaacgct 1560
ggtgagttct cactgcctac atttgacagc ctgaacatca ctgctgctag cctgaacgac 1620
gacggattgg acaaccacac aatcttgctg tactactcca ccgctgctag ctcactggct 1680
gtgactctga tgttggccat cttcatcgtt tacatggtgt cacgtgacaa cgtgagctgt 1740
agcatctgcc tg 1752
<210> 6
<211> 1398
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atgctcccta gcacaatcca gacactcact ctgttcttga ctagcggtgg agtcctcctg 60
tccctgtacg tgagcgcttc tctgtcatac ctgctgtaca gcgacatcct cctgaagttc 120
tcacgcaccg aagtgactgc tcctatcatg cccctcgact gtgctaacgc ctcaaacgtg 180
caagctgtta accgttccgc cacaaagggt gttactcctt tgctgcccga accagaatgg 240
acttacccac gtttgtcatg tcccggtagc accttccaaa aggctctgct gatctcccca 300
caccgtttcg gtgaaaccaa aggtaattca gctccactga tcatccgtga acccttcatt 360
gcttgtggac ctaaggaatg taagcatttc gctttgacac attacgccgc tcagcctgga 420
ggttactaca acggtacccg tgaggaccgt aacaagctga gacacctgat cagcgtgaaa 480
ctcggcaaga tcccaaccgt tgaaaacagc atcttccaca tggccgcttg gtctggctca 540
gcttgtcacg acggtcgtga gtggacatac atcggtgtgg acggtccaga cagcaacgct 600
ctgctcaaga tcaagtacgg tgaagcctac acagatacat accactccta cgctaagaac 660
atcttgagaa ctcaggaatc tgcttgtaac tgtattggcg gagactgcta cctgatgatc 720
accgacggtc ctgccagcgg catctccgaa tgccgcttcc tcaagatccg cgaaggtagg 780
atcatcaagg aaattttccc aacaggtcgt gtgaagcaca ctgaagaatg tacatgtggc 840
ttcgccagta acaagaccat tgaatgtgcc tgtcgtgaca actcatacac tgctaagcgt 900
cctttcgtga agctgaacgt ggaaactgac actgctgaga tccgtttgat gtgcacaaag 960
acttacctgg acactcctag gcctaacgat ggttctatca caggtccttg cgaatcagat 1020
ggcgacgaag gtagcggtgg aatcaaagga ggattcgtgc accaacgtat ggcttctaag 1080
atcggccgtt ggtactctcg tactatgagc aagactaagc gtatgggtat gggtctctac 1140
gtcaagtacg acggtgaccc ttggactgac agcgaggccc tggctctgtc tggtgtgatg 1200
gtgagcatgg aagaacctgg ttggtacagc ttcggtttcg agatcaagga caagaagtgc 1260
gacgtgccat gcatcggtat cgagatggtt catgacggtg gcaagactac ctggcactct 1320
gctgctaccg ctatctactg cctgatggga tctggacaac tgctgtggga cacagtgaca 1380
ggtgttaaca tgaccctg 1398
<210> 7
<211> 1746
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgaaggcca tcatcgtgct gctcatggtg gtgacctcca acgctgaccg catctgtaca 60
ggtatcacct cttctaactc cccccacgtt gttaagacag ctacacaggg tgaagtgaac 120
gtgacaggtg tgatcccctt gaccacaaca cccactaagt cacacttcgc taacctgaag 180
ggcactgaaa ctagaggaaa gctctgtcct aagtgcctca actgcactga tctcgatgtc 240
gccctcggtc gtcctaagtg tacaggtaaa atcccatcag cccgtgtgtc tatcctccac 300
gaggtgcgtc ctgtgacttc aggttgcttc cccatcatgc acgatcgtac caagatccgt 360
cagctgccaa acttgctccg tggctacgaa cacgtgagac tgagcactca taacgttatc 420
aacacagagg acgcccctgg cggtccttac gaaatcggaa cctccggatc atgcctgaac 480
atcacaaacg gaaagggatt cttcgctaca atggcttggg ccgttccaaa gaacaagact 540
gccactaacc ccctgaccat cgaagttcca tacatttgta ctgaagaaga ggatcaaatc 600
actgtgtggg gtttccacag tgacgatgaa acacaaatgg ctcgcttgta cggtgactct 660
aagcctcaaa agttcacttc cagcgctaac ggcgttacca cccactacgt cagccaaatt 720
ggcggtttcc caaaccagac agaggatgga ggtctgccac agtcaggccg catcgtcgtt 780
gactatatgg tgcaaaagtc cggaaaaaca ggaactatca cataccagcg tggaatcctg 840
ctccctcaaa aggtttggtg tgctagtgga aagtccaaag ttatcaaggg ttccctccct 900
ctgatcggtg aagccgactg cctccacgaa aagtacggag gactcaataa gtctaagcca 960
tactacaccg gtgagcacgc caaggccatc ggcaactgtc ccatttgggt taaaactcca 1020
ttgaaactcg ctaacggtac aaagtacagg ccacccgcta agctgttgaa ggaacgtggc 1080
ttcttcggag ccatcgctgg tttcctggag ggaggttggg agggtatgat cgctggctgg 1140
cacggctaca ctagtcacgg agctcacggc gtggccgtcg ccgctgactt gaagtctacc 1200
caagaagcca tcaacaagat cactaagaac ctgaactccc tgtctgagct ggaagtcaag 1260
aacctccagc gtctgagcgg tgccatggac gaattgcaca acgaaatcct ggaactggac 1320
gaaaaagtgg acgacttgcg tgctgacact atttcaagcc agatcgaact cgctgttttg 1380
ctgtctaacg agggtatcat taatagcgag gacgaacacc tcctggcttt ggaacgcaag 1440
ctcaagaaga tgttgggccc ttcagccgtt gaaatcggta acggttgttt cgaaaccaag 1500
cacaagtgta accagacctg tctcgaccgt atcgctgccg gtacattcga cgccggcgaa 1560
ttctcactgc ctactttcga ttccttgaac atcactgctg cttccctgaa cgacgacggt 1620
ctggacaacc acactatcct cctgtactac agcaccgctg cttcatccct ggctgtgact 1680
ctgatgatcg ctatcttcgt tgtgtacatg gttagccgtg ataacgtgag ctgtagcatc 1740
tgtctg 1746
<210> 8
<211> 1398
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atgctgccta gcaccatcca gacactgacc ctgttcctga cttccggagg tgtgctcctg 60
agcctgtacg tgtctgcttc actgtcttac ctgctgtaca gcgacatcct cctgaagttc 120
agccagactg agatcacagc tccaacaatg ccattggact gtgctaacgc ctctaacgtg 180
caagctgtca accgttccgc tactaaggga gccaccctgc tgctccctga gcctgagtgg 240
acctacccta gattgagctg tcctggctcc accttccaga aggccctcct gatctctccc 300
caccgtttcg gtgaaactaa aggtaactca gctccactga tcatccgtga acccttcgtc 360
gcttgtggcc ctaacgaatg taagcacttc gctctgactc actacgccgc tcagcctgga 420
ggctactaca acggtacacg cggagaccgt aacaagctgc gtcacctgat ctctgtgaag 480
ttgggaaaga ttccaacagt tgaaaactct atcttccaca tggctgcttg gtcaggatca 540
gcctgtcacg acggaaagga atggacatac atcggtgttg acggacctga caacaacgct 600
ttgctgaagg ttaagtacgg tgaagcctac accgacacct accactccta cgctaacaac 660
atcctcagaa cacaagaatc agcttgtaac tgtatcggag gaaactgcta cctgatgatc 720
acagacggtt ctgcctcagg tgtttcagaa tgtaggttcc tgaagatccg tgaaggtcgt 780
atcattaagg aaatcttccc cacaggtagg gttaagcaca cagaagagtg tacttgtggt 840
ttcgcttcta acaagacaat cgaatgtgct tgtcgtgata accgctacac tgctaagaga 900
cctttcgtta agctcaacgt tgagactgac acagctgaaa tccgtttgat gtgtacagac 960
acatacctgg acacaccccg tcctaacgac ggttccatca ctggtccctg tgaaagcgac 1020
ggtgacgaag gtagcggtgg tatcaagggc ggtttcgtgc accagcgtat gaagtcaaag 1080
atcggacgtt ggtactcacg tactatgtca aagactgaac gcatgggaat gggtctctac 1140
gttaaatacg gtggagaccc ttgggctgat tcagacgctc tggtgttctc tggcgtgatg 1200
atctccatga aggaaccagg ttggtacagc ttcggcttcg aaatcaagga caagaagtgt 1260
gacgtcccat gtatcggtat cgagatggtg cacgacggtg gaaaggagac ttggcacagc 1320
gccgctacag ccatctactg cctgatgggt agcggtcagc tgctgtggga caccatcacc 1380
ggcgttgaca tggctctg 1398
<210> 9
<211> 566
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Lys Ala Ile Leu Val Val Met Leu Tyr Thr Phe Thr Thr Ala Asn
1 5 10 15
Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr
20 25 30
Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn
35 40 45
Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val
50 55 60
Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly
65 70 75 80
Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Arg Ser Trp Ser Tyr Ile
85 90 95
Val Glu Thr Ser Asn Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe
100 105 110
Ile Asn Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe
115 120 125
Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp
130 135 140
Ser Asp Asn Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser
145 150 155 160
Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Lys Ser Tyr Pro
165 170 175
Lys Ile Asn Gln Thr Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val
180 185 190
Leu Trp Gly Ile His His Pro Pro Thr Ile Ala Asp Gln Gln Ser Leu
195 200 205
Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Thr Ser Arg Tyr Ser
210 215 220
Lys Lys Phe Lys Pro Glu Ile Ala Thr Arg Pro Lys Val Arg Asp Gln
225 230 235 240
Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys
245 250 255
Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Ala Pro Arg Tyr Ala Phe
260 265 270
Thr Met Glu Arg Asp Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro
275 280 285
Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Glu Gly Ala Ile Asn
290 295 300
Thr Ser Leu Pro Phe Gln Asn Val His Pro Ile Thr Ile Gly Lys Cys
305 310 315 320
Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg
325 330 335
Asn Val Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly
340 345 350
Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr
355 360 365
His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser
370 375 380
Thr Gln Asn Ala Ile Asp Lys Ile Thr Asn Lys Val Asn Ser Val Ile
385 390 395 400
Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His
405 410 415
Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe
420 425 430
Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn
435 440 445
Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu
450 455 460
Lys Val Arg Asn Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly
465 470 475 480
Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val
485 490 495
Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu
500 505 510
Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Asp Ser Thr Arg Ile Tyr
515 520 525
Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val
530 535 540
Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu
545 550 555 560
Gln Cys Arg Ile Cys Ile
565
<210> 10
<211> 469
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Thr
1 5 10 15
Ile Gly Thr Ala Asn Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile
20 25 30
Trp Val Ser His Ser Ile Gln Ile Gly Asn Gln Ser Gln Ile Glu Thr
35 40 45
Cys Asn Lys Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln
50 55 60
Thr Phe Val Asn Ile Ser Asn Thr Asn Ser Ala Ala Arg Gln Ser Val
65 70 75 80
Ala Ser Val Lys Leu Ala Gly Asn Ser Ser Leu Cys Pro Val Ser Gly
85 90 95
Trp Ala Ile Tyr Ser Lys Asp Asn Ser Val Arg Ile Gly Ser Lys Gly
100 105 110
Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser Pro Leu Glu
115 120 125
Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His
130 135 140
Ser Asn Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser
145 150 155 160
Cys Pro Ile Gly Glu Val Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser
165 170 175
Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Thr Asn Trp Leu Thr
180 185 190
Ile Gly Ile Ser Gly Pro Asp Ser Gly Ala Val Ala Val Leu Lys Tyr
195 200 205
Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Lys Ile Leu
210 215 220
Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr
225 230 235 240
Ile Met Thr Asp Gly Pro Ser Asp Gly Gln Ala Ser Tyr Lys Ile Phe
245 250 255
Arg Ile Glu Lys Gly Lys Ile Ile Lys Ser Val Glu Met Lys Ala Pro
260 265 270
Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Ser Glu Ile
275 280 285
Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val
290 295 300
Ser Phe Asn Gln Asn Leu Glu Tyr Gln Met Gly Tyr Ile Cys Ser Gly
305 310 315 320
Val Phe Gly Asp Asn Pro Arg Pro Asn Asp Lys Thr Gly Ser Cys Gly
325 330 335
Pro Val Ser Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Lys
340 345 350
Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg
355 360 365
Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp
370 375 380
Asn Lys Phe Ser Lys Lys Gln Asp Ile Val Gly Ile Asn Glu Trp Ser
385 390 395 400
Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asn
405 410 415
Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Glu
420 425 430
Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val
435 440 445
Asp Ser Asp Ile Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro
450 455 460
Phe Thr Ile Asp Lys
465
<210> 11
<211> 566
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Met Lys Thr Ile Ile Ala Leu Ser Asn Ile Leu Cys Leu Val Phe Ala
1 5 10 15
Gln Lys Ile Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly
20 25 30
His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asn Asp
35 40 45
Arg Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Asn Ser Ser Ile
50 55 60
Gly Glu Ile Cys Gly Ser Pro His Gln Ile Leu Asp Gly Gly Asn Cys
65 70 75 80
Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro Gln Cys Asp Gly Phe Gln
85 90 95
Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser Arg Ala Asn Ser Asn
100 105 110
Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val
115 120 125
Ala Ser Ser Gly Thr Leu Glu Phe Lys Asn Glu Ser Phe Asn Trp Thr
130 135 140
Gly Val Lys Gln Asn Gly Thr Ser Ser Ala Cys Ile Arg Gly Ser Ser
145 150 155 160
Ser Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Ser Leu Asn Asn Ile
165 170 175
Tyr Pro Ala Gln Asn Val Thr Met Pro Asn Lys Glu Gln Phe Asp Lys
180 185 190
Leu Tyr Ile Trp Gly Val His His Pro Asp Thr Asp Lys Asn Gln Ile
195 200 205
Ser Leu Phe Ala Gln Ser Ser Gly Arg Ile Thr Val Ser Thr Lys Arg
210 215 220
Ser Gln Gln Ala Val Ile Pro Asn Ile Gly Ser Arg Pro Arg Ile Arg
225 230 235 240
Asp Ile Pro Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly
245 250 255
Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly
260 265 270
Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala
275 280 285
Pro Ile Gly Lys Cys Lys Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile
290 295 300
Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala
305 310 315 320
Cys Pro Arg Tyr Val Lys Gln Ser Thr Leu Lys Leu Ala Thr Gly Met
325 330 335
Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala
340 345 350
Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly
355 360 365
Phe Arg His Gln Asn Ser Glu Gly Arg Gly Gln Ala Ala Asp Leu Lys
370 375 380
Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Leu
385 390 395 400
Ile Gly Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser
405 410 415
Glu Val Glu Gly Arg Val Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr
420 425 430
Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu
435 440 445
Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe
450 455 460
Glu Lys Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn
465 470 475 480
Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser
485 490 495
Ile Arg Asn Glu Thr Tyr Asp His Asn Val Tyr Arg Asp Glu Ala Leu
500 505 510
Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys
515 520 525
Asp Trp Ile Leu Trp Ile Ser Phe Ala Met Ser Cys Phe Leu Leu Cys
530 535 540
Ile Ala Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile
545 550 555 560
Arg Cys Asn Ile Cys Ile
565
<210> 12
<211> 469
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr
1 5 10 15
Ile Ser Thr Ile Cys Phe Phe Met Gln Ile Ala Ile Leu Ile Thr Thr
20 25 30
Val Thr Leu His Phe Lys Gln Tyr Glu Phe Asn Ser Pro Pro Asn Asn
35 40 45
Gln Val Met Leu Cys Glu Pro Thr Ile Ile Glu Arg Asn Ile Thr Glu
50 55 60
Ile Val Tyr Leu Thr Asn Thr Thr Ile Glu Lys Glu Ile Cys Pro Lys
65 70 75 80
Pro Ala Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Gly Ile Thr Gly
85 90 95
Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly
100 105 110
Asp Ile Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Leu Asp Lys
115 120 125
Cys Tyr Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asn Asn Val His
130 135 140
Ser Asn Asn Thr Val Arg Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met
145 150 155 160
Asn Glu Leu Gly Val Pro Phe His Leu Gly Thr Lys Gln Val Cys Ile
165 170 175
Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val
180 185 190
Cys Ile Thr Gly Asp Asp Lys Asn Ala Thr Ala Ser Phe Ile Tyr Asn
195 200 205
Gly Arg Leu Val Asp Ser Val Val Ser Trp Ser Asn Asp Ile Leu Arg
210 215 220
Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val
225 230 235 240
Met Thr Asp Gly Asn Ala Thr Gly Lys Ala Asp Thr Lys Ile Leu Phe
245 250 255
Ile Glu Glu Gly Lys Ile Val His Thr Ser Lys Leu Ser Gly Ser Ala
260 265 270
Gln His Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg
275 280 285
Cys Val Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Ile Asp
290 295 300
Ile Asn Ile Lys Asp His Ser Ile Val Ser Arg Tyr Val Cys Ser Gly
305 310 315 320
Leu Val Gly Asp Thr Pro Arg Lys Ser Asp Ser Ser Ser Ser Ser His
325 330 335
Cys Leu Asn Pro Asn Asn Glu Lys Gly Asp His Gly Val Lys Gly Trp
340 345 350
Ala Phe Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Asn Glu
355 360 365
Thr Ser Arg Leu Gly Tyr Glu Thr Phe Lys Val Val Glu Gly Trp Ser
370 375 380
Asn Pro Lys Ser Lys Leu Gln Ile Asn Arg Gln Val Ile Val Asp Arg
385 390 395 400
Gly Asp Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser
405 410 415
Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Glu
420 425 430
Glu Thr Glu Val Leu Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly
435 440 445
Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asn Leu
450 455 460
Ser Leu Met His Ile
465
<210> 13
<211> 584
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp
1 5 10 15
Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys
20 25 30
Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr
35 40 45
Thr Thr Pro Thr Lys Ser Tyr Phe Ala Asn Leu Lys Gly Thr Arg Thr
50 55 60
Arg Gly Lys Leu Cys Pro Asp Cys Leu Asn Cys Thr Asp Leu Asp Val
65 70 75 80
Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys Ala
85 90 95
Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile
100 105 110
Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly
115 120 125
Tyr Glu Lys Ile Arg Leu Ser Thr Gln Asn Val Ile Asp Ala Glu Lys
130 135 140
Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn
145 150 155 160
Ala Thr Ser Lys Ile Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro
165 170 175
Lys Asp Asn Tyr Lys Asn Ala Thr Asn Pro Leu Thr Val Glu Val Pro
180 185 190
Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His
195 200 205
Ser Asp Asn Lys Thr Gln Met Lys Ser Leu Tyr Gly Asp Ser Asn Pro
210 215 220
Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser
225 230 235 240
Gln Ile Gly Asp Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro Gln
245 250 255
Ser Gly Arg Ile Val Val Asp Tyr Met Met Gln Lys Pro Gly Lys Thr
260 265 270
Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val Trp
275 280 285
Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile
290 295 300
Gly Glu Ala Asp Cys Leu His Glu Glu Tyr Gly Gly Leu Asn Lys Ser
305 310 315 320
Lys Pro Tyr Tyr Thr Gly Lys His Ala Lys Ala Ile Gly Asn Cys Pro
325 330 335
Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg
340 345 350
Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala
355 360 365
Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly
370 375 380
Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys
385 390 395 400
Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu
405 410 415
Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asp
420 425 430
Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu
435 440 445
Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser
450 455 460
Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu
465 470 475 480
Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile Gly Asn
485 490 495
Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg
500 505 510
Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe
515 520 525
Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp
530 535 540
Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala
545 550 555 560
Val Thr Leu Met Leu Ala Ile Phe Ile Val Tyr Met Val Ser Arg Asp
565 570 575
Asn Val Ser Cys Ser Ile Cys Leu
580
<210> 14
<211> 466
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Met Leu Pro Ser Thr Ile Gln Thr Leu Thr Leu Phe Leu Thr Ser Gly
1 5 10 15
Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Ser Leu Ser Tyr Leu Leu
20 25 30
Tyr Ser Asp Ile Leu Leu Lys Phe Ser Arg Thr Glu Val Thr Ala Pro
35 40 45
Ile Met Pro Leu Asp Cys Ala Asn Ala Ser Asn Val Gln Ala Val Asn
50 55 60
Arg Ser Ala Thr Lys Gly Val Thr Pro Leu Leu Pro Glu Pro Glu Trp
65 70 75 80
Thr Tyr Pro Arg Leu Ser Cys Pro Gly Ser Thr Phe Gln Lys Ala Leu
85 90 95
Leu Ile Ser Pro His Arg Phe Gly Glu Thr Lys Gly Asn Ser Ala Pro
100 105 110
Leu Ile Ile Arg Glu Pro Phe Ile Ala Cys Gly Pro Lys Glu Cys Lys
115 120 125
His Phe Ala Leu Thr His Tyr Ala Ala Gln Pro Gly Gly Tyr Tyr Asn
130 135 140
Gly Thr Arg Glu Asp Arg Asn Lys Leu Arg His Leu Ile Ser Val Lys
145 150 155 160
Leu Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala
165 170 175
Trp Ser Gly Ser Ala Cys His Asp Gly Arg Glu Trp Thr Tyr Ile Gly
180 185 190
Val Asp Gly Pro Asp Ser Asn Ala Leu Leu Lys Ile Lys Tyr Gly Glu
195 200 205
Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala Lys Asn Ile Leu Arg Thr
210 215 220
Gln Glu Ser Ala Cys Asn Cys Ile Gly Gly Asp Cys Tyr Leu Met Ile
225 230 235 240
Thr Asp Gly Pro Ala Ser Gly Ile Ser Glu Cys Arg Phe Leu Lys Ile
245 250 255
Arg Glu Gly Arg Ile Ile Lys Glu Ile Phe Pro Thr Gly Arg Val Lys
260 265 270
His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu
275 280 285
Cys Ala Cys Arg Asp Asn Ser Tyr Thr Ala Lys Arg Pro Phe Val Lys
290 295 300
Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Lys
305 310 315 320
Thr Tyr Leu Asp Thr Pro Arg Pro Asn Asp Gly Ser Ile Thr Gly Pro
325 330 335
Cys Glu Ser Asp Gly Asp Glu Gly Ser Gly Gly Ile Lys Gly Gly Phe
340 345 350
Val His Gln Arg Met Ala Ser Lys Ile Gly Arg Trp Tyr Ser Arg Thr
355 360 365
Met Ser Lys Thr Lys Arg Met Gly Met Gly Leu Tyr Val Lys Tyr Asp
370 375 380
Gly Asp Pro Trp Thr Asp Ser Glu Ala Leu Ala Leu Ser Gly Val Met
385 390 395 400
Val Ser Met Glu Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys
405 410 415
Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp
420 425 430
Gly Gly Lys Thr Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu
435 440 445
Met Gly Ser Gly Gln Leu Leu Trp Asp Thr Val Thr Gly Val Asn Met
450 455 460
Thr Leu
465
<210> 15
<211> 582
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp
1 5 10 15
Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys
20 25 30
Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr
35 40 45
Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Glu Thr
50 55 60
Arg Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp Val
65 70 75 80
Ala Leu Gly Arg Pro Lys Cys Thr Gly Lys Ile Pro Ser Ala Arg Val
85 90 95
Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile
100 105 110
Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly
115 120 125
Tyr Glu His Val Arg Leu Ser Thr His Asn Val Ile Asn Thr Glu Asp
130 135 140
Ala Pro Gly Gly Pro Tyr Glu Ile Gly Thr Ser Gly Ser Cys Leu Asn
145 150 155 160
Ile Thr Asn Gly Lys Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro
165 170 175
Lys Asn Lys Thr Ala Thr Asn Pro Leu Thr Ile Glu Val Pro Tyr Ile
180 185 190
Cys Thr Glu Glu Glu Asp Gln Ile Thr Val Trp Gly Phe His Ser Asp
195 200 205
Asp Glu Thr Gln Met Ala Arg Leu Tyr Gly Asp Ser Lys Pro Gln Lys
210 215 220
Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser Gln Ile
225 230 235 240
Gly Gly Phe Pro Asn Gln Thr Glu Asp Gly Gly Leu Pro Gln Ser Gly
245 250 255
Arg Ile Val Val Asp Tyr Met Val Gln Lys Ser Gly Lys Thr Gly Thr
260 265 270
Ile Thr Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys Val Trp Cys Ala
275 280 285
Ser Gly Lys Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile Gly Glu
290 295 300
Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser Lys Pro
305 310 315 320
Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro Ile Trp
325 330 335
Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro
340 345 350
Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe
355 360 365
Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr
370 375 380
Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr
385 390 395 400
Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu
405 410 415
Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asp Glu Leu
420 425 430
His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg Ala
435 440 445
Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser Asn Glu
450 455 460
Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg Lys
465 470 475 480
Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly Asn Gly Cys
485 490 495
Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg Ile Ala
500 505 510
Ala Gly Thr Phe Asp Ala Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser
515 520 525
Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn His
530 535 540
Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val Thr
545 550 555 560
Leu Met Ile Ala Ile Phe Val Val Tyr Met Val Ser Arg Asp Asn Val
565 570 575
Ser Cys Ser Ile Cys Leu
580
<210> 16
<211> 466
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Met Leu Pro Ser Thr Ile Gln Thr Leu Thr Leu Phe Leu Thr Ser Gly
1 5 10 15
Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Ser Leu Ser Tyr Leu Leu
20 25 30
Tyr Ser Asp Ile Leu Leu Lys Phe Ser Gln Thr Glu Ile Thr Ala Pro
35 40 45
Thr Met Pro Leu Asp Cys Ala Asn Ala Ser Asn Val Gln Ala Val Asn
50 55 60
Arg Ser Ala Thr Lys Gly Ala Thr Leu Leu Leu Pro Glu Pro Glu Trp
65 70 75 80
Thr Tyr Pro Arg Leu Ser Cys Pro Gly Ser Thr Phe Gln Lys Ala Leu
85 90 95
Leu Ile Ser Pro His Arg Phe Gly Glu Thr Lys Gly Asn Ser Ala Pro
100 105 110
Leu Ile Ile Arg Glu Pro Phe Val Ala Cys Gly Pro Asn Glu Cys Lys
115 120 125
His Phe Ala Leu Thr His Tyr Ala Ala Gln Pro Gly Gly Tyr Tyr Asn
130 135 140
Gly Thr Arg Gly Asp Arg Asn Lys Leu Arg His Leu Ile Ser Val Lys
145 150 155 160
Leu Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala
165 170 175
Trp Ser Gly Ser Ala Cys His Asp Gly Lys Glu Trp Thr Tyr Ile Gly
180 185 190
Val Asp Gly Pro Asp Asn Asn Ala Leu Leu Lys Val Lys Tyr Gly Glu
195 200 205
Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala Asn Asn Ile Leu Arg Thr
210 215 220
Gln Glu Ser Ala Cys Asn Cys Ile Gly Gly Asn Cys Tyr Leu Met Ile
225 230 235 240
Thr Asp Gly Ser Ala Ser Gly Val Ser Glu Cys Arg Phe Leu Lys Ile
245 250 255
Arg Glu Gly Arg Ile Ile Lys Glu Ile Phe Pro Thr Gly Arg Val Lys
260 265 270
His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu
275 280 285
Cys Ala Cys Arg Asp Asn Arg Tyr Thr Ala Lys Arg Pro Phe Val Lys
290 295 300
Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Asp
305 310 315 320
Thr Tyr Leu Asp Thr Pro Arg Pro Asn Asp Gly Ser Ile Thr Gly Pro
325 330 335
Cys Glu Ser Asp Gly Asp Glu Gly Ser Gly Gly Ile Lys Gly Gly Phe
340 345 350
Val His Gln Arg Met Lys Ser Lys Ile Gly Arg Trp Tyr Ser Arg Thr
355 360 365
Met Ser Lys Thr Glu Arg Met Gly Met Gly Leu Tyr Val Lys Tyr Gly
370 375 380
Gly Asp Pro Trp Ala Asp Ser Asp Ala Leu Val Phe Ser Gly Val Met
385 390 395 400
Ile Ser Met Lys Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys
405 410 415
Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp
420 425 430
Gly Gly Lys Glu Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu
435 440 445
Met Gly Ser Gly Gln Leu Leu Trp Asp Thr Ile Thr Gly Val Asp Met
450 455 460
Ala Leu
465
<210> 17
<211> 1752
<212> DNA
<213> Influenza Virus (Influenza virus)
<400> 17
ggaaaacaaa agcaacaaaa atgaaggcaa tactagtagt tatgctgtat acatttacaa 60
ccgcaaatgc agacacatta tgtataggtt atcatgcgaa caattcaaca gacactgtgg 120
acacagtact agaaaagaat gtaacagtaa cacactctgt caatcttctg gaagacaagc 180
ataacggaaa actatgcaaa ctaagagggg tagccccatt gcatttgggt aaatgtaaca 240
ttgctggctg gatcctggga aatccagagt gtgaatcact ctccacagca agatcatggt 300
cctacattgt ggaaacatct aattcagaca atggaacgtg ttacccagga gatttcatca 360
attatgagga gctaagagag caattgagct cagtgtcatc atttgaaagg tttgaaatat 420
tccccaagac aagttcatgg cctaatcatg actcggacaa tggtgtaacg gcagcatgtc 480
ctcacgctgg agcaaaaagc ttctacaaaa acttgatatg gctggttaaa aaaggaaaat 540
catacccaaa gatcaaccaa acctacatta atgataaagg gaaagaagtc ctcgtgctgt 600
ggggcattca ccatccacct actattgctg accaacaaag tctctatcag aatgcagatg 660
catatgtttt tgtggggaca tcaagataca gcaagaagtt caagccggaa atagcaacaa 720
gacccaaagt gagggatcaa gaagggagaa tgaactatta ctggacacta gtagaaccgg 780
gagacaaaat aacattcgaa gcaactggta atctagtggc accgagatat gcattcacaa 840
tggaaagaga tgctggatct ggtattatca tttcagatac accagtccac gattgcaata 900
caacttgtca gacacccgag ggtgctataa acaccagcct cccatttcag aatgtacatc 960
cgatcacaat tgggaaatgt ccaaagtatg taaaaagcac aaaattgaga ctggccacag 1020
gattgaggaa tgtcccgtct attcaatcta gaggcctatt cggggccatt gctggcttca 1080
tcgaaggggg gtggacaggg atggtagatg gatggtacgg ttatcaccat caaaatgagc 1140
aggggtcagg atatgcagcc gatctgaaga gcacacaaaa tgccattgat aagattacta 1200
acaaagtaaa ttctgttatt gaaaagatga atacacagtt cacagcagtt ggtaaagagt 1260
tcaaccacct tgaaaaaaga atagagaatc taaataaaaa ggttgatgat ggtttcctgg 1320
acatttggac ttacaatgcc gaactgttgg ttctactgga aaacgaaaga actttggact 1380
atcacgattc aaatgtgaag aacttgtatg aaaaagtaag aaaccagtta aaaaacaatg 1440
ccaaggaaat tggaaacggc tgctttgaat tttaccacaa atgcgacaac acatgcatgg 1500
aaagtgtcaa gaatgggact tatgactacc caaaatactc agaggaagca aaattaaaca 1560
gagaaaaaat agatggagta aagctggact caacaaggat ctaccagatt ttggcgatct 1620
attcaactgt tgccagttca ttggtactgg tagtctccct gggggcaatc agcttctgga 1680
tgtgctctaa tgggtctcta cagtgtagaa tatgtattta acattaggat ttcagaatca 1740
tgagaaaaac ac 1752
<210> 18
<211> 1433
<212> DNA
<213> Influenza Virus (Influenza virus)
<400> 18
agtttaaaat gaatccaaac caaaagataa taaccattgg ttctatctgt atgacaattg 60
gaacggctaa cttaatatta caaattggaa acataatctc aatatgggtt agccactcaa 120
ttcaaattgg aaatcaaagc cagattgaaa catgcaataa aagcgtcatt acttatgaaa 180
acaacacttg ggtaaatcag acatttgtta acatcagcaa cactaactct gctgctagac 240
agtcagtggc ttccgtgaaa ttagcgggca attcctctct ctgccctgtt agtggatggg 300
ctatatacag taaagacaac agtgtaagaa tcggttccaa gggggatgtg tttgtcataa 360
gggaaccatt catatcatgc tctcccttgg aatgcagaac cttcttcttg actcaagggg 420
ctttgctaaa tgacaaacat tccaatggaa ccattaaaga cagaagccca tatcgaaccc 480
taatgagctg tcctattggt gaagttccct ctccatacaa ctcaagattt gagtcagtcg 540
cttggtcagc aagtgcttgt catgatggca ccaattggct aacaattgga atttctggcc 600
cagacagtgg ggcagtggct gtgttaaaat acaatggcat aataacagac actatcaaga 660
gttggaggaa caagatattg agaacacaag agtctgaatg tgcatgtgta aatggttctt 720
gctttaccat aatgaccgat ggaccaagtg atggacaggc ctcatacaaa atcttcagaa 780
tagaaaaggg aaagataatc aaatcagtcg aaatgaaagc ccctaattat cactatgaag 840
aatgctcctg ttaccctgat tctagtgaaa tcacatgtgt gtgcagggat aactggcatg 900
gctcgaatcg accgtgggtg tctttcaacc agaatctgga atatcagatg ggatacatat 960
gcagtggggt tttcggagac aatccacgcc ctaatgataa gacaggcagt tgtggtccag 1020
tatcgtctaa tggagcaaat ggggtaaaag gattttcatt caaatacggc aatggtgttt 1080
ggatagggag aactaagagc attagttcaa gaaaaggttt tgagatgatt tgggatccga 1140
atggatggac tgggactgac aataaattct caaaaaagca agatatcgta ggaataaatg 1200
agtggtcagg gtatagcggg agttttgttc agcatccaga actaacaggg ctgaattgta 1260
taagaccttg cttctgggtt gaactaataa gaggacgacc cgaagagaac acaatctgga 1320
ctagcgggag cagcatatcc ttttgtggtg tagacagtga cattgtgggt tggtcttggc 1380
cagacggtgc tgagttgcca tttaccattg acaagtaatt tgttcaaaaa act 1433
<210> 19
<211> 1737
<212> DNA
<213> Influenza Virus (Influenza virus)
<400> 19
ggataattct attaaccatg aagactatca ttgctttgag caacattcta tgtcttgttt 60
tcgctcaaaa aatacctgga aatgacaata gcacggcaac gctgtgcctt gggcaccatg 120
cagtaccaaa cggaacgata gtgaaaacaa tcacaaatga ccgaattgaa gttactaatg 180
ctactgagtt ggttcagaat tcatcaatag gtgaaatatg cggcagtcct catcagatcc 240
ttgatggagg gaactgcaca ctaatagatg ctctattggg ggaccctcag tgtgacggct 300
ttcaaaataa ggaatgggac ctttttgttg aaagaagcag agccaacagc aactgttacc 360
cttatgatgt gccggattat gcctccctta ggtcactagt tgcctcatcc ggcacactgg 420
agtttaaaaa tgaaagcttc aattggactg gagtcaaaca aaacggaaca agttctgcgt 480
gcataagggg atctagtagt agttttttta gtagattaaa ttggttgacc agcttaaaca 540
acatatatcc agcacagaac gtgactatgc caaacaagga acaatttgac aaattgtaca 600
tttggggggt tcaccacccg gatacggaca agaaccaaat ctccctgttt gctcaatcat 660
caggaagaat cacagtatct accaaaagaa gccaacaagc tgtaatccca aatatcggat 720
ctagacccag aataagggat atccctagca gaataagcat ctattggaca atagtaaaac 780
cgggagacat acttttgatt aacagcacag ggaatctaat tgctcctagg ggttacttca 840
aaatacgaag tgggaaaagc tcaataatga gatcagatgc acccattggc aaatgtaagt 900
ctgaatgcat cactccaaat ggaagcattc ccaatgacaa accgttccaa aatgtaaaca 960
ggatcacata cggggcctgt cccagatatg ttaagcaaag caccctgaaa ttggcaacag 1020
gaatgcgaaa tgtaccagag aaacaaacca gaggcatatt tggcgcaata gcgggtttca 1080
tagaaaatgg atgggaggga atggtggatg gttggtacgg tttcaggcat caaaattctg 1140
agggaagagg acaagcagca gatctcaaaa gcactcaagc agcaatcgat caaatcaatg 1200
ggaagctgaa tcgattgatc ggaaaaacca acgagaaatt ccatcagatt gaaaaagaat 1260
tctcagaagt agaaggaaga gttcaagacc ttgagaaata tgttgaggac actaaaatag 1320
atctctggtc atacaacgcg gagcttcttg ttgccctgga gaaccaacat acgattgacc 1380
taactgactc agaaatgaac aaactgtttg aaaaaacaaa gaagcaactg agggaaaatg 1440
ctgaggatat gggaaatggt tgtttcaaaa tataccacaa atgtgacaat gcctgcatag 1500
gatcaataag aaatgaaact tatgaccaca atgtgtacag ggatgaagca ttaaacaacc 1560
ggttccagat caagggagtt gagctgaagt cagggtacaa agattggatc ctatggattt 1620
cctttgccat gtcatgtttt ttgctttgta ttgctttgtt ggggttcatc atgtgggcct 1680
gccaaaaggg caacattaga tgcaacattt gcatttgagt gcattaatta aaaacac 1737
<210> 20
<211> 1441
<212> DNA
<213> Influenza Virus (Influenza virus)
<400> 20
agtaaagatg aatccaaatc aaaagataat aacgattggc tctgtttctc tcacaatttc 60
cacaatatgc ttcttcatgc aaattgccat cctgataact actgtaacat tgcatttcaa 120
gcaatatgaa ttcaactccc ccccaaataa ccaagtgatg ctgtgtgaac caacaataat 180
agaaagaaac ataacagaga tagtgtattt gaccaacacc accatagaga aggaaatatg 240
ccccaaacca gcagaataca gaaattggtc aaaaccgcaa tgtggcatta caggatttgc 300
acctttctct aaggacaatt cgattaggct ttccgctggt ggggacatct gggtgacaag 360
agaaccttat gtgtcatgcg atcttgacaa gtgttatcaa tttgcccttg gacagggaac 420
aacactaaac aatgtgcatt caaataacac agtacgtgat agaacccctt atcggactct 480
attgatgaat gagttgggtg ttcctttcca tctggggacc aagcaagtgt gcatagcatg 540
gtccagctca agttgtcacg atggaaaagc atggctgcat gtttgtataa cgggggatga 600
taaaaatgca actgctagct tcatttacaa tgggaggctt gtagatagtg ttgtttcatg 660
gtccaacgat attctcagaa cccaggagtc agaatgcgtt tgtatcaatg gaacttgtac 720
agtagtaatg actgatggaa atgctacagg aaaagctgat actaaaatac tattcattga 780
ggaggggaaa atcgttcata ctagcaaatt gtcaggaagt gctcagcatg tcgaagagtg 840
ctcttgctat cctcgatatc ctggtgtcag atgtgtctgc agagacaact ggaaaggatc 900
caaccggccc atcatagata taaacataaa ggatcatagc attgtttcca ggtatgtgtg 960
ttctggactt gttggagaca cacccagaaa aagcgacagc tccagcagta gccattgttt 1020
gaaccctaac aatgaaaaag gtgatcatgg agtgaaaggc tgggcctttg atgatggaaa 1080
tgacgtgtgg atggggagaa caatcaacga gacgtcacgc ttagggtatg aaaccttcaa 1140
agtcgttgaa ggctggtcca accctaagtc caaattgcag ataaataggc aagtcatagt 1200
tgacagaggc gataggtccg gttattctgg tattttctct gttgaaggca aaagctgcat 1260
caatcggtgc ttttatgtgg agttgattag gggaagaaaa gaggaaactg aagtcttgtg 1320
gacttcaaac agtattgttg tgttttgtgg cacctcaggt acatatggaa caggctcatg 1380
gcctgatggg gcgaacctca gtctcatgca tatataagct ttcgcaattt tagaaaaagc 1440
t 1441
<210> 21
<211> 1821
<212> DNA
<213> Influenza Virus (Influenza virus)
<400> 21
atgaaggcaa taattgtact actcatggta gtaacatcca acgcagatcg aatctgcact 60
gggataacat cttcaaactc acctcatgtg gtcaaaacag ctactcaagg ggaggtcaat 120
gtgactggcg tgataccact gacaacaaca ccaacaaaat cttattttgc aaatctcaaa 180
ggaacaagga ccagagggaa actatgcccg gactgtctca actgtacaga tctggatgtg 240
gccttgggca ggccaatgtg tgtggggacc acaccttctg ctaaagcttc aatactccat 300
gaggtcagac ctgttacatc cgggtgcttt cctataatgc acgacagaac aaaaatcagg 360
caactaccca atcttctcag aggatatgaa aagatcaggt tatcaaccca aaacgttatc 420
gatgcagaaa aagcaccagg aggaccctac agacttggaa cctcaggatc ttgccctaac 480
gctaccagta aaatcggatt ttttgcaaca atggcttggg ctgtcccaaa ggacaactac 540
aaaaatgcaa cgaacccact aacagtggaa gtaccataca tttgtacaga aggggaagac 600
caaattactg tttgggggtt ccattcggat aacaaaaccc aaatgaagag cctctatgga 660
gactcaaatc ctcaaaagtt cacctcatct gctaatggag taaccacgca ttatgtttct 720
cagattggcg acttcccaga tcaaacagaa gacggaggac taccacaaag cggcagaatt 780
gttgttgatt acatgatgca aaaacctggg aaaacaggaa caattgtcta tcaaaggggt 840
gttttgttgc ctcaaaaggt gtggtgcgcg agtggcagga gcaaagtaat aaaagggtca 900
ttgcctttaa ttggtgaagc agattgcctt catgaagaat acggtggatt aaacaaaagc 960
aagccttact acacaggaaa acatgcaaaa gccataggaa attgcccaat atgggtaaaa 1020
acacctttga agcttgccaa tggaaccaaa tatagacctc ctgcaaaact attgaaggaa 1080
aggggtttct tcggagctat tgctggtttc ctagaaggag gatgggaagg aatgattgca 1140
ggttggcacg gatacacatc tcacggagca catggagtgg cagtggcggc agaccttaag 1200
agtacacaag aagctataaa taagataaca aaaaatctca attctttgag tgaactagaa 1260
gtaaagaacc ttcaaagact aagtggtgcc atggatgaac tccacaacga aatactcgag 1320
ctggatgaaa aagtggatga tctcagagct gacactataa gctcacaaat agaacttgca 1380
gtcttgcttt ccaacgaagg aataataaac agtgaagacg agcatctatt ggcacttgag 1440
agaaaactaa agaaaatgct gggtccctct gctgtagaca taggaaacgg atgcttcgaa 1500
accaaacaca aatgcaacca gacctgctta gacaggatag ctgctggcac ctttaatgca 1560
ggagaatttt ctctccccac ttttgattca ttgaacatta ctgctgcatc tttaaatgat 1620
gatggattgg ataaccatac tatactgctc tattactcaa ctgctgcttc tagtttggct 1680
gtaacattaa tgctagctat ttttattgtt tatatggtct ccagagacaa cgtttcatgc 1740
tccatctgtc tataaagaag gttaggcctt gtattttcct ttattgtagt gcttgtttgc 1800
ttgtcatcat tacaaagaaa c 1821
<210> 22
<211> 1555
<212> DNA
<213> Influenza Virus (Influenza virus)
<400> 22
cagaagcaga gcatcttctc aaaactgagg caaataggcc aaaaatgaac aatgctacct 60
tcaactatac aaacgttaac cttatttctc acatcagggg gagtgttatt atcactatat 120
gtgtcagctt cattgtcata cttactatat tcggatatat tgctaaaatt ttcacgaaca 180
gaagtaactg caccaataat gccattggat tgtgcaaacg catcaaatgt tcaggctgtg 240
aaccgttctg caacaaaagg ggtgacacct cttctcccag aaccggagtg gacgtaccct 300
cgtttatctt gcccgggctc aacctttcag aaagcactcc taattagccc ccatagattc 360
ggagaaacca aaggaaactc agctcccttg ataataaggg aaccttttat tgcttgtgga 420
ccaaaggaat gcaaacattt tgctctaacc cattatgcag ctcaaccagg gggatactac 480
aatggaacaa gagaagacag aaacaagctg aggcatctaa tttcagtcaa attgggcaaa 540
atcccaacag tagaaaactc tattttccac atggcagctt ggagcgggtc cgcatgccat 600
gatggtagag aatggactta catcggagtt gatggcccag acagtaatgc attgctcaaa 660
ataaaatatg gagaagcata tactgacaca taccattcct atgcaaaaaa catcctaagg 720
acacaagaaa gtgcctgcaa ttgcatcggg ggagattgtt atctgatgat aactgatggc 780
ccagcttcag ggattagtga atgcagattc cttaagattc gagagggccg aataataaaa 840
gaaatatttc caacaggaag agtaaaacat actgaggaat gcacatgcgg atttgccagc 900
aacaaaacca tagaatgtgc ctgtagagat aacagttaca cagcaaaaag accctttgtc 960
aaattaaatg tggagactga tacagcggaa ataagattga tgtgcacaaa gacttatttg 1020
gacaccccca gaccaaatga tggaagcata acagggcctt gcgaatctga tggggacgaa 1080
gggagtggag gcatcaaggg aggatttgtt caccaaagaa tggcatccaa gattggaagg 1140
tggtactctc ggacgatgtc taaaactaaa agaatgggga tgggactgta tgtaaagtat 1200
gatggagacc catggactga cagtgaagcc cttgctctta gtggagtaat ggtttcaatg 1260
gaagaacctg gttggtattc ctttggcttc gaaataaaag ataagaaatg tgatgtcccc 1320
tgtattggga tagaaatggt acatgatggt gggaaaacga cttggcactc agcggcaaca 1380
gccatttact gtttaatggg ctcaggacaa ctgctgtggg acactgtcac aggtgttaat 1440
atgactctgt aatggaggaa tggttgagtc tgttctaaac cctttgttcc tattttgttt 1500
gaacaattgt ccttactgaa cttgattgtt tctgaaaaat gctcttgtta ctact 1555
<210> 23
<211> 1749
<212> DNA
<213> Influenza Virus (Influenza virus)
<400> 23
atgaaggcaa taattgtact actcatggta gtaacatcca atgcagatcg aatctgcact 60
gggataacat cgtcaaactc accacatgtc gtcaaaactg ctactcaagg ggaggtcaat 120
gtgactggtg taataccact gacaacaaca cccaccaaat ctcattttgc aaatctcaaa 180
ggaacagaaa ccagggggaa actatgccca aaatgcctaa actgcacaga tctggatgta 240
gccttgggca gaccaaaatg cacagggaaa ataccctctg caagggtttc aatactccat 300
gaagtcagac ctgttacatc tgggtgcttt cctataatgc atgatagaac aaaaattaga 360
cagctgccta accttctccg aggatacgaa catgtcaggt tatcaactca caacgttatc 420
aatacagaag atgcaccagg aggaccctac gaaattggaa cctcagggtc ttgcctcaac 480
attaccaatg gaaaaggatt cttcgcaaca atggcttggg ccgtcccaaa aaacaaaaca 540
gcaacaaatc cattaacaat agaagtacca tacatttgta cagaagaaga agaccaaatt 600
accgtttggg ggttccactc tgacgacgag acccaaatgg caaggctcta tggggattca 660
aagccccaga agttcacctc atctgccaac ggagtgacca cacactacgt ctcacagatt 720
ggtggctttc caaatcaaac agaagacgga ggactaccac aaagtggcag aattgttgtt 780
gattacatgg tgcaaaaatc tggaaaaaca ggaacaatta cctatcaaag aggtatttta 840
ttgcctcaaa aggtgtggtg cgcaagtggc aagagcaagg taataaaagg atccttgccc 900
ttaattggag aagcagattg cctccatgaa aaatacggtg gattaaacaa aagcaagcct 960
tactacacag gggaacatgc aaaggccata ggaaattgcc caatatgggt gaaaacaccc 1020
ttgaagctgg ccaatggaac caaatataga cctcctgcaa aactattaaa ggaaagaggt 1080
ttcttcggag ccattgctgg tttcttagag ggaggatggg aaggaatgat tgcaggttgg 1140
cacggataca catcccatgg ggcacatgga gtagcggtgg cagctgacct taagagcact 1200
caggaggcca taaacaagat aacaaaaaat ctcaactctt tgagtgagct ggaagtaaag 1260
aatcttcaaa gactaagcgg tgccatggat gaactccaca acgaaatact agaactagat 1320
gagaaagtgg atgatctcag agctgataca ataagctcac agatagaact cgcagtcctg 1380
ctttccaatg aaggaataat aaacagtgaa gatgaacatc tcttggcgct tgaaagaaag 1440
ctgaagaaaa tgctgggccc ctctgctgta gagataggaa atggatgctt tgaaaccaaa 1500
cacaagtgca accagacctg tctcgacaga atagctgctg gtacctttga tgcaggagaa 1560
ttttctctcc ccacctttga ttcactgaat attactgctg catctttaaa tgacgatgga 1620
ttggacaatc atactatact gctttactac tcaactgctg cctccagttt ggctgtaaca 1680
ctgatgatag ctatctttgt tgtttatatg gtctccagag acaatgtttc ttgctccatt 1740
tgtctataa 1749
<210> 24
<211> 1408
<212> DNA
<213> Influenza Virus (Influenza virus)
<400> 24
atgaacaatg ctaccttcaa ctatacaaac gttaacccta tttctcacat cagggggagt 60
attattatca ctatatgtgt cagcttcatt atcatactta ctatattcgg atatattgct 120
aaaattctca caaacagaaa taactgcacc aacaatgcca ttggattgtg caaacgcatc 180
aaatgttcag gctgtgaacc gttctgcaac aaaaggggcg acacttcttc tcccagaacc 240
ggagtggaca tacccgcgtt tatcttgccc gggctcaacc tttcagaaag cactcctaat 300
tagccctcat agattcggag aaaccaaagg aaactcagct cccttgataa taagggaacc 360
ttttgttgct tgtggaccaa atgaatgcaa acactttgct ttaacccatt atgcagccca 420
accaggggga tactacaatg gaacaagagg agacagaaac aagctgaggc atctaatttc 480
agtcaaattg ggcaaaatcc caacagtaga gaactccatt ttccatatgg cagcatggag 540
cgggtccgcg tgccatgatg gtaaggaatg gacatatatc ggagttgatg gccctgacaa 600
taatgcattg ctcaaagtaa aatatggaga agcatatact gacacatacc attcctatgc 660
aaacaacatc ctaagaacac aagaaagtgc ctgcaattgc atcgggggaa attgttatct 720
aatgataact gatggctcag cttcaggtgt tagtgaatgc agatttctta agattcgaga 780
gggccgaata ataaaagaaa tatttccaac aggaagagta aaacacactg aggaatgcac 840
atgcggattt gccagtaata aaaccataga atgtgcctgt agagacaaca ggtacacagc 900
aaaaagacct tttgtcaaat taaacgtgga gactgataca gcagaaataa gattgatgtg 960
tacagatact tatttggaca cccccagacc aaatgatgga agcataacag gcccttgtga 1020
atctgatggg gacgagggga gtggaggcat caagggagga tttgttcatc aaagaatgaa 1080
atccaagatt ggaaggtggt actctcgaac gatgtctaaa actgaaagga tggggatggg 1140
actgtatgtc aagtatggtg gagacccatg ggctgacagt gatgccctag tttttagtgg 1200
agtaatgatt tcaatgaaag aacctggttg gtattccttt ggcttcgaaa taaaagataa 1260
gaaatgcgat gtcccctgta ttgggataga gatggtacat gatggtggaa aagagacttg 1320
gcactcagca gcaacagcca tttactgttt aatgggctca ggacagttgc tgtgggacac 1380
tatcacaggt gttgatatgg ctctgtaa 1408
<210> 25
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
atgaaagcta tcctggttg 19
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gatacagatg cggcactgca 20
<210> 27
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
atgaacccca accaaaag 18
<210> 28
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
cttatcaatg gtgaatggca gctc 24
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
atgaaaacca tcatcgccct 20
<210> 30
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gatgcagatg ttacaacgga tgtt 24
<210> 31
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
atgaacccta accagaagat cat 23
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gatgtgcatc aggctcaggt t 21
<210> 33
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
atgaaggcta tcatcgtgct gc 22
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
caggcagatg ctacagctca 20
<210> 35
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
atgctcccta gcacaatc 18
<210> 36
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
cagggtcatg ttaacacctg tc 22
<210> 37
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
atgaaggcca tcatcgtgct gctc 24
<210> 38
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
cagacagatg ctacagctca cg 22
<210> 39
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
atgctgccta gcaccatcca g 21
<210> 40
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cagagccatg tcaacgc 17

Claims (6)

1. A recombinant influenza subunit vaccine, characterized in that each dose contains four types of H1N 1A, H3N 2A, Yamagata type B and Victoria type B;
the antigen in the vaccine is HA gene and NA gene encoded protein of A type H1N1, A type H3N2, B type Yamagata series and B type Victoria series strains;
the nucleotide sequence of the optimized HA gene of the A type H1N1 strain is shown as SEQ ID NO. 1;
the nucleotide sequence of the optimized NA gene of the A H1N1 strain is shown as SEQ ID NO. 2;
the nucleotide sequence of the optimized HA gene of the A type H3N2 strain is shown as SEQ ID NO. 3;
the nucleotide sequence of the optimized NA gene of the A type H3N2 strain is shown as SEQ ID NO. 4;
the nucleotide sequence of the optimized HA gene of the B-type Yamagata strain is shown as SEQ ID NO. 5;
the nucleotide sequence of the optimized NA gene of the B-type Yamagata strain is shown as SEQ ID NO. 6;
the nucleotide sequence of the optimized HA gene of the Victoria B strain is shown as SEQ ID NO. 7;
the nucleotide sequence of the optimized NA gene of the Victoria B strain is shown as SEQ ID No. 8.
2. The recombinant influenza subunit vaccine of claim 1 wherein the amino acid sequence encoded by the HA gene of the H1N1 strain a is set forth in SEQ ID No. 9;
the amino acid sequence coded by the NA gene of the A type H1N1 strain is shown in SEQ ID NO. 10;
the amino acid sequence coded by the HA gene of the A type H3N2 strain is shown as SEQ ID NO. 11;
the amino acid sequence coded by the NA gene of the A type H3N2 strain is shown in SEQ ID NO. 12;
the amino acid sequence coded by the HA gene of the B-type Yamagata strain is shown as SEQ ID NO. 13;
the amino acid sequence coded by the NA gene of the B-type Yamagata strain is shown as SEQ ID NO. 14;
the amino acid sequence coded by the HA gene of the Victoria B strain is shown as SEQ ID NO. 15;
the amino acid sequence coded by the NA gene of the Victoria B strain is shown in SEQ ID NO. 16.
3. The recombinant influenza subunit vaccine of claim 1, wherein the monovalent antigen concentration of the vaccine is 30 to 300 μ g/ml.
4. A recombinant influenza subunit vaccine according to claim 1 wherein the vaccine antigen is prepared by culturing a recombinant baculovirus carrying the gene encoding the protein.
5. The recombinant influenza subunit vaccine of claim 4, wherein the recombinant baculovirus is constructed as follows:
(1) constructing a recombinant plasmid:
respectively cloning HA genes and NA genes of H1N 1A, H3N 2A, Yamagata B and Victoria B strains to a transfer vector pOET5.1, respectively adding BglII and NotI to the upstream and downstream of the HA genes, respectively adding BamHI and HindIII to the upstream and downstream of the NA genes to obtain recombinant plasmids pOET5.1-HA-NA-A1, pOE5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2;
(2) construction of recombinant baculovirus:
the recombinant plasmids pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2 are mixed with genome DNA of the flash BAC ULTRA baculovirus and then are transfected into insect cells ZY.104 together, and the P0 generation recombinant baculovirus is obtained.
6. The recombinant influenza subunit vaccine of claim 5, wherein the recombinant plasmids pOET5.1-HA-NA-A1, pOET5.1-HA-NA-A2, pOET5.1-HA-NA-B1 and pOET5.1-HA-NA-B2 are present in a volume ratio of 0.1 to 1: 0.1-1: 0.1-1: 0.1-1 mixture, wherein the total concentration of the recombinant plasmid DNA is 1-50 mug.
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CN111420044A (en) * 2020-05-11 2020-07-17 中逸安科生物技术股份有限公司 Tetravalent influenza virus subunit vaccine and preparation method thereof
CN111920944A (en) * 2020-10-12 2020-11-13 天津中逸安健生物科技有限公司 Preparation method of influenza virus subunit vaccine stock solution
CN113583981A (en) * 2021-09-29 2021-11-02 易慧生物技术(上海)有限公司 Chicken embryo culture method-derived influenza virus purification method, tetravalent influenza virus subunit vaccine and application thereof

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CN111420044A (en) * 2020-05-11 2020-07-17 中逸安科生物技术股份有限公司 Tetravalent influenza virus subunit vaccine and preparation method thereof
CN111920944A (en) * 2020-10-12 2020-11-13 天津中逸安健生物科技有限公司 Preparation method of influenza virus subunit vaccine stock solution
CN113583981A (en) * 2021-09-29 2021-11-02 易慧生物技术(上海)有限公司 Chicken embryo culture method-derived influenza virus purification method, tetravalent influenza virus subunit vaccine and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115725660A (en) * 2022-10-09 2023-03-03 天津中逸安健生物科技有限公司 Preparation method of recombinant influenza subunit vaccine

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