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Prokaryotic expression protein of VP73 gene from African swine fever virus and preparation method thereof

An African swine fever virus, prokaryotic expression technology, applied in the field of the preparation of genetic engineering products, can solve the problems of impossible eradication of wild hosts, high mortality, catastrophic consequences, etc.

Inactive Publication Date: 2011-06-22
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
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Problems solved by technology

Due to the high infectivity and high mortality rate of the disease, it has caused huge economic losses to the pig industry, and the wild host cannot be eradicated, so the prevention and control of the disease is facing great challenges
[0003] Although ASF has never occurred in my country, with the increasing frequency of international exchanges and import and export trade, the risk of ASF spreading across borders is increasing. If sufficient attention is not paid, once the disease occurs, it will lead to disastrous consequences.
Due to the complexity of the ASF protective immune response, there is no ideal vaccine for active or passive immunization, and strict diagnostic and quarantine measures are mainly used for prevention and control. Therefore, it is very necessary to establish a rapid and non-infectious diagnostic method for our country

Method used

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  • Prokaryotic expression protein of VP73 gene from African swine fever virus and preparation method thereof
  • Prokaryotic expression protein of VP73 gene from African swine fever virus and preparation method thereof

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Embodiment Construction

[0078] The specific embodiment of the prokaryotic expression protein of a kind of African swine fever virus VP73 gene provided by the invention and preparation method thereof is as follows:

[0079] 1. Artificially synthesized VP73 gene sequence;

[0080] 2. Directional cloning of VP73 gene into pET32a prokaryotic expression vector;

[0081] 3. Induced expression of VP73 gene in Escherichia coli;

[0082] The recombinant expression vector pET32a-VP73 was transformed into Escherichia coli E.coliBL21(DE3) for expression.

[0083] 4. Affinity chromatography purification of VP73 gene expression protein, mainly including:

[0084] (1) The overnight culture of the E.coli BL21 / pET32aVP73 transformant was transferred to 200 ml of freshly prepared TB liquid medium containing Amp (100 μg / ml) at 1% inoculum size, and cultured with shaking at 37° C. at 250 rpm.

[0085] (2) Wait for its OD 600 When it reaches 0.8-1.0, add IPTG to a final concentration of 0.2mmol / L, transfer to 30°C, s...

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Abstract

The invention relates to a method for preparing a genetic engineering product, in particular to a prokaryotic expression protein of VP73 gene from African swine fever virus (ASFV) and a preparation method thereof. The preparation method comprises: artificially synthesizing the whole-length sequence of VP73 gene according to the sequence of the VP73 gene from ASFV in GenBank, constructing a recombinant expression vector pET32a-VP73, sequencing, verifying, transforming prokaryotic expression recipient bacteria E.coli BL21(DE3), and inducing expression by isopropyl-1-thio-beta-d-galactopyranoside (IPTG), wherein the molecular weight of the recombinant fusion protein is about 65KD. Protein purified by nickel column affinity chromatography can undergo a specific immune imprinting reaction with ASFV positive serum and avoid cross reaction with viruses such as swine fever virus, hog cholera virus, porcine circovirus, porcine reproductive and respiratory syndrome virus, swine influenza virus and pseudorabiesvirus. Experiments show the expressed protein has high detection sensitivity, and high specificity. When the antigen is used for detection, risk of spreading poison is avoided. And the antigen can be used as a detection antigen for use in an enzyme-linked immuno sorbent assay (ELISA) method for identifying an ASFV antibody.

Description

(1) Technical field [0001] The invention relates to a preparation method of a genetic engineering product, in particular to a prokaryotic expression protein antigen of a VP73 gene sequence for identifying an African swine fever virus antibody and a preparation method thereof. (2) Background technology [0002] African swine fever (ASF) is an acute, febrile, highly contagious infectious disease of pigs caused by African swine fever virus (ASFV). The main clinical symptoms include high fever, short course of disease, high mortality, extensive bleeding of internal organs, and functional changes of the respiratory system and nervous system. Due to the high infectivity and high mortality rate of the disease, which has caused huge economic losses to the pig industry, and the wild host cannot be eradicated, the prevention and control of the disease is facing great challenges. [0003] Although ASF has never occurred in my country, with the increasing frequency of international exc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/34C12N15/70C07K1/22C12R1/19
Inventor 滕达王建华李秋霞杨雅麟田子罡
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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