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

Inactive Publication Date: 2014-08-06
INST OF ANIMAL HUSBANDRY & VETERINARY FUJIAN ACADEMY OF AGRI SCI
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AI Technical Summary

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

[0033] 1) Based on the identified PRRSV antigen linear conservative table (GP530) and high mutation advantageous tables (GP5190) amino acid characteristics, design two copy numbers with repeated sequences to express, and introduce ECORI restricted enzyme cutting in the sequenceSites and XHOI restricted enzyme cutting points to obtain reorganization DNA.The amino acid sequence is listed as: NASNSSSSHLGSGSAEQWGRPGGGSNSSSSSSSSSSSSSSSGSGSAEQWGRP, where GGGS is LINKER.

[0034] The sequence of the reorganization DNA is shown in SEQ ID No.2.

[0035] 2) Construction of positive recombinant plasmid PGEX-6P-GP5

[0036] PGEX-6P-1 with genetic synthesis reorganization DNA and genuine expression vector PGEX-6P-1 use ECORI and XHOI for dual enzyme cutting and connect to the night with T4 DNA connection enzymes.Cut the identification to determine the sequence of the positive reorganized granules.The sequencing identification as a positive reorganization is PGEX-6P-GP5.

[0037] 3) The express...

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Abstract

The invention relates to an indirect ELISA method for detecting porcine reproductive and respiratory syndrome antibodies, that is, using the pGEX-6p-1 prokaryotic expression vector and utilizing a linear conserved neutralizing epitope (epitope B) of the PRRSV GP5 protein that has been screened ) can be recognized by monoclonal antibody and can also be recognized by pig anti-PRRSV neutralizing serum; the other is a highly variable immunodominant epitope (A). Genetically engineered bacteria BLpGEX-6p-GP5 expressing the dominant antigenic epitope of GP5 protein, and the expressed recombinant protein was purified and refolded and coated with ELISA plate, and an indirect ELISA method for detecting PRRSV antibody was established to detect PRRSV in pig serum The results show that this method has the characteristics of good repeatability and high specificity, and can be used for PRRSV serological investigation.

Description

Technical field [0001] The invention involves an indirect ELISA method for detecting PRRSV antibodies, which belongs to the field of animal infectious diseases. Background technique [0002] Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a high contact infectious infectious disease characterized by Pregnancy sow breeding disorders and piglet symptoms characterized by PRRSV.The disease was first reported in the United States in 1987 and spread quickly to all parts of the world, causing huge economic losses to the pig breeding industry.The disease was first discovered in 1991 in Taiwan in my country. In 1996, Guo Baoqing reported that the disease was popular in China. It is still an important epidemic that endangers the pig industry in my country. [0003] PRRSV is a genus of arteritis virus virus virus. It is a single -stock chain RNA virus with a capsule. There are currently two serum types: American and European types.The genome size is about 15KB, including 8 op...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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