猪繁殖与呼吸综合征病毒ORF5基因原核表达载体的构建与鉴定

doi:10.16431/j.cnki.1671-7236.2015.11.010

Abstract

Abstract: The present study was designed to construct recombinant plasmids,which could express porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene.RNA was extracted from spleen and lung samples of the suspected pigs which were infected with PRRSV.According to PRRSV ORF5 gene,a pair of primers was designed for RT-PCR amplification.The ORF5 target gene was cloned into pMD19-T vector and then the recombinant pMD19-ORF5 was achieved.According to the sequencing results and the characteristics of expression vectors,a pair of primers with NcoⅠand XbaⅠenzyme cleavage sites was designed.Target fragment dORF5 was amplified and then connected to pProEXHTb and pNZ8149 vectors,respectively.And recombinant HTb-dORF5/DE3 and pNZ8149-dORF5/NZ3900 was induced by IPTG and Nisin,respectively,and analyzed by SDS-PAGE and Western blotting.Recombinant HTb-dORF5/DE3 induced by 1.5 mmol/L IPTG was expressed in the highest quantity.There were specific band at about 22 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.Recombinant pNZ8149-dORF5/NZ3900 induced by 20 ng/mL Nisin was expressed in the highest quantity.There were specific band at about 19 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.The IFA result showed specific green fluorescence.This study successfully constructed recombinant plasmids HTb-dORF5 and pNZ8149-dORF5 and expressed,the result laid a solid foundation for further development of PRRS vaccines.

Key words: porcine reproductive and respiratory syndrome virus (PRRSV); ORF5 gene; expression; Escherichia coli; Lactococcus lactis

CLC Number:

S852.65

ZHOU Yu-zhao, MA Zhi-liang, SUN Ting-ting, YANG Jie, ZHANG Xiao-miao, CHAI Jun, ZHANG Na-na, ZHANG Yi-fang. Construction and Identification of PRRSV ORF5 Gene Prokaryotic Expression Vector[J]. , 2015, 42(11): 2880-2887.

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Construction and Identification of PRRSV ORF5 Gene Prokaryotic Expression Vector

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