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Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) method established based on classical swine fever virus NS3

A kind of swine fever virus, indirect technology, applied in the direction of viral peptides, recombinant DNA technology, botanical equipment and methods, etc., can solve the problems of CSFV genetic engineering marker vaccine restrictions, low sensitivity, and pig industry hazards

Inactive Publication Date: 2010-08-11
HUNAN AGRICULTURAL UNIV
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  • Application Information

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Problems solved by technology

Although the CSFV E2 genetically engineered vaccine can provide protection to susceptible pigs, but in actual clinical practice, E2 rns The differential diagnosis ELISA established for the antigen has a defect of low sensitivity (75-80%) in serological detection, which limits the practical clinical application of the CSFV genetically engineered vaccine
The existence of the above problems has seriously hindered the CSFV purification work, making CSFV exist in pigs in my country for a long time and causing great harm to the pig industry

Method used

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  • Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) method established based on classical swine fever virus NS3
  • Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) method established based on classical swine fever virus NS3
  • Indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) method established based on classical swine fever virus NS3

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Experimental program
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Embodiment Construction

[0029] 1. Preparation of coating antigen:

[0030] 1.1 Design and synthesis of specific primers: Use Primer 5.0 primer analysis software to design a pair of primers fP and rP based on the gene sequence of the NS3 coding region in the CSFV full gene sequence included in GenBank (GenBank accession number: AF351433). Sac I and Xho I restriction sites are expected to amplify 5,142-7,191bp (2,049bp in length) gene fragments in the CSFV genome.

[0031] Upstream primer fP: 5′-GAT GAGCTC GGGCCTGCCGTTTGCAAGAAG-3'; downstream primer rP: 5'-TCTC CTCGAG TTATAGACCAACTACCTGTTTTAGTGC-3'. Both the upstream and downstream primers were synthesized by Shanghai Shenggong Biological Engineering Technology Co., Ltd.

[0032] 1.2 Construction of pETNS3 prokaryotic expression vector: NS3 specific upstream and downstream primers were used to amplify NS3 gene fragments from plasmid pPOHCLV (containing full-length HCLV cDNA), and restriction endonucleases SacI and Xho I (purchased from Dalian Bao Biologic...

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Abstract

The invention relates to an indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) method established based on classical swine fever virus (CSFV). The swine fever virus recombination protein NS3 is obtained by a gene clone expression technique and the indirect ELISA method is established by using the recombination protein NS3 as the envelope antigen. The method comprises the steps of preparing the antigen recombination protein NS3, establishing indirect ELIS, establishing a judging standard and carrying out clinical serology detection. The invention can be used for detecting specific antibody of CSFV NS3 in the swinery so as to judge the antibody level of CSFV NS3 in the swinery and the condition of natural infection of CSFV in the swinery, and provides a detection method for identifying ELISA in the marker vaccine in the CSFV E2 gene engineering. At present, the method firstly adopts CSFV NS3 to establish the ELISA for detecting the CSFV antibody in the swinery worldwide. The sensitivity and the specificity are obviously higher than the ELISA established based on Ems.

Description

Technical field: [0001] The invention belongs to the field of veterinary biotechnology, and is based on the classical swine fever virus (Classical Swine Fever Virus CSFV) recombinant protein NS3 as a coating antigen, and establishes an indirect ELISA method for detecting CSFV antibodies. Background technique: [0002] Classical Swine Fever CSF (Classical Swine Fever CSF) is an acute febrile infectious disease caused by CSFV. Pigs of all ages can be infected. The disease is listed as a Class A infectious disease by the International Organization for Animal Health (OIE). List it as a type of infectious disease. Although the disease has been eliminated in North America, Oceania and some European countries, it is still widespread in other pig-raising countries and has caused serious harm to the pig industry. my country's long-term immunization of pigs with Hog Cholera Lapizined Virus (HCLV) vaccine has basically controlled the pandemic of swine fever, but swine fever is widespread i...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569C12N15/40C12N15/70C07K14/18
Inventor 余兴龙蒋大良李润成葛猛
Owner HUNAN AGRICULTURAL UNIV
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