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Indirect ELISA (enzyme-linked immunosorbent assay) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) antibody through tandem repeat expression of GP5 dominant antigen epitopes

A technology of tandem expression and dominant antigen, applied in the field of animal epidemiology, can solve the problem of low expression level

Inactive Publication Date: 2013-09-04
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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Problems solved by technology

The immunogenicity of GP5 is similar to M protein and N protein, but its expression level is lower in PRRSV-infected cells

Method used

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  • Indirect ELISA (enzyme-linked immunosorbent assay) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) antibody through tandem repeat expression of GP5 dominant antigen epitopes
  • Indirect ELISA (enzyme-linked immunosorbent assay) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) antibody through tandem repeat expression of GP5 dominant antigen epitopes
  • Indirect ELISA (enzyme-linked immunosorbent assay) method for detecting PRRSV (porcine reproductive and respiratory syndrome virus) antibody through tandem repeat expression of GP5 dominant antigen epitopes

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Embodiment 1

[0034] 1) According to the amino acid characteristics of the identified linear conserved epitope (GP530) and highly variable dominant epitope (GP5190) of the PRRSV antigen, design 2 copies of the repeated sequence for tandem expression, and introduce EcoRI restriction enzyme digestion into the sequence site and XhoI restriction enzyme site to obtain recombinant DNA. The amino acid sequence is: NASNDSSSHLGSGSTRVSAEQWGRPGGGSNANDSSSHLGSGSTRVSAEQWGRP, where GGGS is linker.

[0035] The sequence of the recombinant DNA is shown in SEQ ID No.2.

[0036] 2) Construction of positive recombinant plasmid pGEX-6p-GP5

[0037] The recombinant DNA synthesized by the gene and the prokaryotic expression vector pGEX-6p-1 were double digested with EcoRI and XhoI, and then connected overnight with T4 DNA ligase. After transforming DH5α competent cells, the plasmid was picked and shaken for double enzymes For cut identification, the positive recombinant plasmid was sent to Bao Biological Engine...

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Abstract

The invention relates to an indirect ELISA (enzyme-linked immunosorbent assay) method for detecting a porcine reproductive and respiratory syndrome antibody, which comprises the following steps: by using a pGEX-6p-1 prokaryotic expression vector, performing tandem repeat on two epitopes to improve the antigen activity of an expressed protein, thus constructing a gene engineering bacterium BLpGEX-6p-GP5 capable of realizing secretory expression of the GP5 protein dominant antigen epitopes, wherein one epitope is a linear conservative neutralizing epitope (epitope B) of a screened PRRSV (porcine reproductive and respiratory syndrome virus) GP5 protein, which can be identified by a monoclonal antibody and can also be identified by porcine anti-PRRSV neutralizing serum, and the other epitope is a high-variability immunodominant epitope (A); and purifying and renaturing the expressed recombinant protein, and coating an ELISA plate, thus establishing the indirect ELISA method for detecting a PRRSV antibody to detect the PRRSV antibody level in porcine serum. Results show that the method has the characteristics of favorable repetitiveness and high specificity and can be used for PRRSV serological search.

Description

technical field [0001] The invention relates to an indirect ELISA method for detecting PRRSV antibody, which belongs to the field of animal infectious diseases. Background technique [0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious disease caused by PRRS virus (PRRSV) characterized by reproductive failure in pregnant sows and respiratory symptoms in piglets. The disease was first reported in the United States in 1987, and spread rapidly to all parts of the world, causing huge economic losses to the pig farming industry. The disease was first discovered in Taiwan in 1991. In 1996, Guo Baoqing reported that the disease was prevalent in China. It is still an important disease that endangers the pig industry in my country. [0003] PRRSV belongs to the genus Arterivirus of the family Arteriviridae. It is a single-stranded positive-sense RNA virus with an envelope. There are currently two serotypes: the American type and the European typ...

Claims

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Application Information

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IPC IPC(8): C07K14/08C12N15/40C12N15/70G01N33/68G01N33/543C12R1/93
Inventor 陈如敬周伦江车勇良王隆柏吴学敏魏宏严山刘玉涛
Owner INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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