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Eukaryotic expression method of fish viral hemorrhagic septicemia virus G gene

A technology of viral hemorrhage and expression method, which is applied in the field of eukaryotic expression of fish viral hemorrhagic sepsis virus G gene, can solve the problems of hindering the acquisition of VHSVG gene-encoded protein antibody and is not ideal, and achieves improved protein yield and high efficiency. expressive effect

Inactive Publication Date: 2015-12-09
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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Problems solved by technology

However, due to the different codon preferences of different species, the expression of the unmodified wild-type G gene in vitro is often not ideal, which hinders the acquisition of antibodies to the protein encoded by the VHSVG gene

Method used

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  • Eukaryotic expression method of fish viral hemorrhagic septicemia virus G gene
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  • Eukaryotic expression method of fish viral hemorrhagic septicemia virus G gene

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Experimental program
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Embodiment Construction

[0030] 1 Materials and methods

[0031] 1.1 Strains, cell lines and plasmids

[0032] The VHSV-H strain was isolated and preserved by our laboratory. E.coli DH5α strain, pFastBacdual transposable plasmid and DH10Bac TM CompetentCells competent cells were purchased from Nanjing GenScript Biological Co., Ltd. Sf9 insect cells were provided by Qingdao Weilan Biotech, and myeloma cells SP2 / 0 were preserved by our laboratory.

[0033] 1.2 Reagents

[0034] Fetal bovine serum, CellfectinⅡReagent, insect cell culture medium sf900-ⅡSFM, Grace medium and Lipofectaminetmreagent were purchased from Gibco Company, Razoal RNA extraction kit was purchased from Promega Company; M-MLV reverse transcriptase, ExTaqDNA amplification enzyme, restriction endonuclease Dicer was purchased from TaKaRa Company; T4 ligase and RNA extraction kit were purchased from Promage Company; gel recovery kit and plasmid extraction kit were purchased from Tiangen Company; 6×His protein purification resin Ni-NT...

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Abstract

The invention discloses a eukaryotic expression method of a fish viral hemorrhagic septicemia virus G gene. The method includes the following steps of (1) gene synthesis of a main antigenic domain optimized by codons, (2) construction of a reconstructed baculovirus transfer vector, (3) construction of shuttle plasmids and (4) analysis and identification of reconstructed Bacmid transfection and expression products. By means of the method, efficient expression of the G protein antigenic domain in a Bac-to-Bac baculovirus system; the activity of the expression protein is primarily identified and analyzed through Western blotting, it is proved that the expression protein has immunogenicity, and a foundation is provided for further epitope vaccine development and VHSV molecular diagnosis technology establishment.

Description

Technical field: [0001] The invention relates to a eukaryotic expression method of fish viral hemorrhagic sepsis virus G gene, which belongs to the field of biological genetic engineering. Background technique: [0002] Viral hemorrhagic septicemia virus (viralhemorrhagic septicemiavirus, VHSV) is a severe infectious disease that causes outbreaks of salmon and trout and a variety of marine fishes. The viral genome consists of N (nucleoprotein)-P (phosphoprotein)-M (matrix protein)-G (glycoprotein)-NV (nonstructural protein)-L (polymerase protein) from 3' to 5' gene. [0003] Surface glycoprotein gene G is its important antigen, and it is also the preferred antibody preparation material for various immunological detection tests. However, due to the different codon preferences of different species, the expression of the unmodified wild-type G gene in vitro is often not ideal, which hinders the acquisition of antibodies to the protein encoded by the VHSVG gene. Contents of ...

Claims

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Application Information

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IPC IPC(8): C12N15/47C12N15/866C07K14/145
Inventor 孙涛尹伟力房保海岳志芹梁成珠
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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