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Fusion protein of equine influenza virus H3N8 subtype and preparation method, application and vaccine thereof

A fusion protein, H3N8 technology, applied in antiviral immunoglobulin, veterinary vaccines, biochemical equipment and methods, etc., to achieve good antigenicity, good immunogenicity, and a wide range of applications

Active Publication Date: 2019-01-11
天康生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no subunit vaccine against equine influenza in China

Method used

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  • Fusion protein of equine influenza virus H3N8 subtype and preparation method, application and vaccine thereof
  • Fusion protein of equine influenza virus H3N8 subtype and preparation method, application and vaccine thereof
  • Fusion protein of equine influenza virus H3N8 subtype and preparation method, application and vaccine thereof

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Experimental program
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preparation example Construction

[0047] The present invention also provides a method for preparing the above-mentioned fusion protein, which can be performed by expressing the gene of the above-mentioned fusion protein in the host, for example, but not limited to, expression in E. coli expression system, yeast expression system, insect expression system, plant expression system, or mammalian expression system. Since the protein expressed by mammalian cells is translated and processed, its structure and biological characteristics are closer to natural proteins, so the present invention preferably uses a mammalian expression system to express the fusion The gene for the protein, more preferably the gene for expressing the fusion protein using a CHO cell expression system. CHO cells are Chinese hamster ovary cells. The CHO cell expression system has the following advantages:

[0048] (1) It has accurate post-translational folding and modification functions, and the expressed protein is closest to the natural pro...

Embodiment 1

[0068] Embodiment 1: HA-NA gene design and synthesis

[0069] HA-NA gene design and synthesis: After optimization, the HA gene (GenBank accession number KX815377.1) is designed to be 810bp in length and has the sequence shown in SEQ ID NO.1; the NA gene (GenBank accession number MG132049.1) is optimized The design length is 900bp, and it has the sequence shown in SEQ ID NO.2. The linker sequence is used to connect the HA gene and the NA gene, and the linker sequence has the sequence shown in SEQ ID NO.3. The full length of the HA-NA gene is 1740bp, and has the amino acid sequence shown in SEQ ID NO.4 and the nucleotide sequence shown in SEQ ID NO.5. HA-NA gene synthesis was completed by Shanghai Sangon Biological Company.

Embodiment 2

[0070] Embodiment 2: PEE6.4-HA-NA recombinant plasmid construction

[0071] 2.1 Add restriction sites: Add restriction sites respectively upstream and downstream of the HA-NA gene sequence by PCR amplification: HindⅢ, SmaI, PCR amplification upstream primers are shown in SEQ ID NO.6, PCR amplification downstream Primers are shown in SEQ ID NO.7. PEE6.4-HA-NA plasmid map as figure 1 shown.

[0072] 2.2 Double digestion reaction of HA-NA gene and vector

[0073] 2.2.1 Construct a 50 μL reaction system, mix the components according to the table below, and then bathe in water at 37°C for 2 hours.

[0074]

[0075]

[0076] 2.2.2 Recovery of target DNA fragments: DNA gel recovery kit (purchased from Beijing Laibao Technology Co., Ltd.) was used to recover the enzyme-cut target fragments. The steps are as follows:

[0077] (1) After agarose gel electrophoresis, carefully cut the target DNA band from the agarose gel with a blade, put it into a 1.5mL EP tube, and weigh it. ...

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Abstract

The invention provides a fusion protein of equine influenza virus H3N8 subtype and a preparation method, an application and a vaccine thereof, which belong to the technical field of biology. The fusion protein realizes series connection of an equine influenza virus H3N8 subtype HA gene and a NA gene sequence, and the expressed fusion protein has the advantages of good antigenicity and high expression amount. The fusion protein can be applied to the preparation of vaccines, antibodies and kits for detecting equine influenza viruses. The vaccine containing the fusion protein has good immunogenicity, and can generate an antibody titer having higher titer after animal immunization, so that the animal obtains a better protective effect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein of equine influenza virus H3N8 subtype and its preparation method, application and vaccine. Background technique [0002] Equine Influenza (Equine Influenza, EI) is an acute infectious disease caused by Equine Influenza Virus (EIV), which has the characteristics of acute and highly contagious. The H3N8 subtype is the main epidemic strain and one of the equine respiratory infectious diseases. [0003] Equine influenza viruses belong to the family Orthomyxoviridae, the genus Influenzavirus A. It is a segmented single-stranded negative-sense RNA enveloped virus with a genome length of about 13.6kb. There are many forms of virus particles, most of which are spherical, with a diameter of about 80-120nm, and filaments can be several microns long. The virus particle structure is mainly divided into three layers. The outermost capsule is composed of a double-layer lipid. ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C07K16/10G01N33/569A61K39/145A61P31/16
CPCA61K39/12A61K2039/552A61P31/16C07K14/005C07K16/1018C12N15/85C12N2760/16122C12N2760/16134G01N33/56983
Inventor 贺笋任立松李延涛陈泽良任郭子君
Owner 天康生物制药有限公司
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