Avian influenza virus hemagglutinin antigen, preparation method and application thereof and avian influenza vaccine

A technology of avian influenza virus and hemagglutinin, applied in the direction of virus antigen components, veterinary vaccines, biochemical equipment and methods, etc., can solve the problems of unreachable poison price, cultivation cost, complicated production process and long cycle, etc., and achieve Improve the expression of antigen, improve the broad-spectrum of antigen, and prevent the effect of bird flu

Inactive Publication Date: 2019-01-04
TECON BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, inactivated vaccines for avian influenza are produced using chicken embryos or MDCK whole suspension cells. The vaccine production process using chicken embryos is complicated, and the chicken embryos used may be contaminated by exogenous viruses. It is necessary to inoculate chicken embryos with venom, collect allantoic fluid, and inactivate , the collection of allantoin liquid chicken embryo waste requires high-pressure treatment, which produces more production waste and brings pressure on environmental protection
The production of avian influenza vaccine by MDCK full-suspension cells requires the process of acclimating the seed virus to adapt to the cells, which takes a long period and sometimes fails to reach the expected poison price. The cultivation cost and production process are relatively complicated

Method used

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  • Avian influenza virus hemagglutinin antigen, preparation method and application thereof and avian influenza vaccine

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preparation example Construction

[0039] The present invention also provides a preparation method of the avian influenza virus hemagglutinin antigen. It includes: expressing the gene of the avian influenza virus hemagglutinin antigen in a mammalian expression system. The preparation method can obtain the hemagglutinin protein with broad spectrum and good antigen immunogenicity, and the method is simple to operate and suitable for large-scale production. The host cells used in the present invention can be, for example but not limited to, HEK 293-F cells, HEK 293-E cells, HEK 293-T cells, CHO cells or COS cells, HEK 293-F is preferably used.

[0040] HEK293 is a stable cell line obtained after transfection of human embryonic kidney cells by adenovirus Ad5. HEK293-F is a derivative cell line of HEK293, which has easy transfection, high expression, natural glycosylation modification, allows correct protein folding and related translation Post-modification and other advantages.

[0041]In some preferred embodimen...

Embodiment 1

[0049] Example 1 A sequence expressing hemagglutinin protein

[0050] The HA gene sequence of the H5 subtype of avian influenza that is currently prevalent and has been prevalent in the past five years was downloaded from Genebank for comparison and analysis, and the dominant epitope was selected as the component of the vaccine antigen. According to the partial tropism of HEK 293-F cell codons, the The sequence of the HA gene of the avian influenza virus is optimized and modified to obtain a nucleotide sequence as shown in SEQ ID NO.1, so as to improve the level of HA protein expressed by the target protein. Design restriction sites and artificially synthesize the full length of HA gene.

Embodiment 2

[0051] Example 2 Construction of a recombinant vector expressing hemagglutinin protein

[0052] The amino acid sequence encoding HA protein synthesized above and the fragment with a His tag at the C-terminal were cloned into the eukaryotic transfer vector pcDNA3.1 through the insertion of Sal I and Xho I sites. Use T4 DNA ligase to ligate overnight at 16°C to obtain ligation products, transform Escherichia coli competent DH5α, spread on LB plates containing ampicillin, culture overnight at 37°C, and pick positive colonies on LB plates containing ampicillin Cultivate in culture medium and extract plasmid.

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Abstract

The invention provides an avian influenza virus hemagglutinin antigen, a preparation method and application thereof and an avian influenza vaccine and relates to the technical field of biology. The avian influenza virus hemagglutinin antigen is hemagglutinin protein expressed by a sequence shown as SEQ ID NO. 1 and is also a protein expressed by a mammalian expression system; the avian influenza virus hemagglutinin antigen has wide spectrum and good immunogenicity; after fowls are immunized with the immunogenicity, an antibody of high titer can be produced, and various subtype avian influenzaviruses can be prevented; the preparation method of the avian influenza virus hemagglutinin antigen is suitable for acquiring hemagglutinin protein having wide spectrum and good antigen immunogenicity; the preparation method is simple to perform and is suitable for large-scale production; a vaccine containing the avian influenza virus hemagglutinin antigen has low production cost and good immunization efficiency; after SPF (specific pathogen free) chicken which is 3-4 weeks old is immunized with the vaccine, it can be detected 14 days later that HI antibody GMT (geometric mean titer) measuredby H5 subtype antigen measurement is not less than 1:64 and is higher than that from the sue of conventional vaccines.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to an avian influenza virus hemagglutinin antigen, a preparation method, an application thereof and an avian influenza vaccine. Background technique [0002] Avian influenza virus (AIV) is a single-strand negative-strand virus, spherical or filamentous, with a spherical diameter of 80-120nm. The genome is divided into 8 segments, encoding 10 kinds of proteins, among which HA, NA and M are located on the surface of the virus envelope and are the main protective antigens of AIV. Located under the viral bilayer lipid envelope is the matrix protein M, which is the most abundant protein in the virus particle and constitutes the framework of the viral envelope. On the lipid bilayer membrane, there are two kinds of glycoprotein spikes encoded by viral genes, namely hemagglutinin (HA) and neuraminidase (Neuraminidase, NA), which are used to divide influenza virus subtypes. And its ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/11C12N5/10C12N15/85A61K39/145A61P31/16
CPCA61K39/12A61K2039/552A61P31/16C07K14/005C12N15/85C12N2760/16122C12N2760/16134
Inventor 贺笋李俊辉潘晓梅程兰玲张伟王遵宝
Owner TECON BIOLOGY CO LTD
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