Plamid vector, VP7 expresses antigen of blue tongue virus VP7 gene recombined expression, and preparation method

A technology of bluetongue virus and gene recombination, which is applied in the field of preparation of biological preparations, can solve problems such as cumbersome operation, cumbersome antigen extraction, and hidden dangers of biological safety, so as to overcome cumbersome extraction, fast and sensitive regulation, and cumbersome operation Effect

Inactive Publication Date: 2005-01-12
CHECKOUT & QUARANTINE TECH CENT YUNNAN ENTRY &EXIT CHECKOUT & QUARANTINE BUR
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AI Technical Summary

Problems solved by technology

Through the method of tissue culture, the virus is inoculated in the cells and multiplied in large quantities, and the isolated and purified virus is used as the c-ELISA coating antigen. Security risks
It is also reported that VP7 protein has been expressed in prokaryotic and eukaryotic systems using molecular cloning and gene recombination techniques, but the prokaryotic expressed protein has poor antigenicity, and the eukaryotic expressed antigen is cumbersome and costly to extract

Method used

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Experimental program
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Embodiment Construction

[0026] (1) Cloning of bluetongue virus VP7 antigen gene and construction of expression recombinant plasmid

[0027] According to the sequence of VP7 gene of BTV10 type, a pair of primers were designed and synthesized, and the amplification length was 1044bp. VP7-P 1 5'-GAC ACT ATC GCT GAC AGA GCA CT-3', at position 21~43 of S7 gene; VP7-P 2 5'-CAC ATA GGC GGC GCG TGC AAT AG-3', at position 1064~1042 of the S7 gene. PCR products were detected by electrophoresis on 10g / L agarose. According to the instructions of the pMD18-T vector kit (product number: D504CA) produced by Treasure Bioengineering (Dalian) Co., Ltd., the recovered and purified PCR target fragment was inserted into the pMD18-T vector, transformed into E.Coli.JM109 competent cells, and PCR And sequence identification, obtain the positive clone that contains BTV-VP7 gene 1044bp fragment. The gene's accession number in GenBank is: AY386682.

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Abstract

This invention relates to a biological agent testing blue tongue of animals and its preparation method. The agent includes blue tongue virus VP7 gene recombination expression plasmid vectors and a blue tongue virus VPT recombination antigen got from its expression. The preparation method includes: 1, cloning BTV coding group specific antigen VP7 gene fragment to pMD18-T plasmid vector to make up of VP7 gene clone recombination plasmid, 2, sub-cloning plug pBAD / Thio TOPO expression vector, 3, converting TOP10cells, 4, screening the positive clones obtaining BTV VP7 gene segment forward plug with correct read code frame to set up BTV group specific antigen VP7 recombination expression vector 5, cultivating the vector with LB culture media containing 100mug / ml Amp.

Description

technical field [0001] The invention relates to a biological preparation for preparing bluetongue virus diagnostic reagents, especially a biological preparation capable of detecting bluetongue in animals and a preparation method of the biological preparation. Background technique [0002] Bluetongue (Bluetongue, BLU) is caused by bluetongue virus (Bluetongue Virus, BTV) of the genus Orbivirus in the Reoviridae family, and is a serious infectious disease of ruminants transmitted by Culicoides. Since the disease occurred in South Africa in 1905, it has had a major impact on the world's animal husbandry and international trade. The International Office of Epizootics (OIE) identified it as a Class A infectious disease of animals. So far there are 25 serotypes. Its genome contains 10 double-stranded RNA fragments (dsRNA) with different molecular weights and a total of 19218 bp. The 10 fragments of double-stranded RNA of BTV are wrapped in the double-layer protein capsid, encod...

Claims

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Application Information

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IPC IPC(8): A61K39/15C12N15/46C12N15/62C12N15/63G01N33/53
Inventor 花群义徐自忠董俊杨晶焰杨云庆周晓黎贾建军肖荣海龙忠保
Owner CHECKOUT & QUARANTINE TECH CENT YUNNAN ENTRY &EXIT CHECKOUT & QUARANTINE BUR
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