猪繁殖与呼吸综合征病毒GP5基因的亚克隆及原核表达

本研究从河北省保定地区6份疑似感染猪繁殖与呼吸综合征病毒(PRRSV)病猪的肺脏中,通过RT-PCR获得PRRSV GP5蛋白完整的基因片段,克隆至pMD18-T载体。经测序鉴定正确后,再设计一对引物从重组载体pMD18-T-GP5中扩增出缺失了N端30个氨基酸残基的dGP5的编码序列dORF5,克隆至原核表达载体pGEX-6p-1,构建原核重组质粒(pGEX-6p-dGP5),转化至E.coli BL21感受态细胞。经IPTG诱导后,SDS-PAGE电泳可检测到分子质量为40ku的目的蛋白。经Western blot分析表明,该蛋白与PRRS阳性血清具有良好的免疫反应性,为进一步研究PRRS新型基因工程疫苗和诊断试剂奠定了基础。

Prokaryotic Expression and Subcloning of ORF5 Gene of PRRSV

The porcine reproductive and respiratory syndrome virus isolated from the lung materials of six swine.The specific ORF5 gene amplified by RT-PCR was cloned into pMD18-T vector.The dORF5 gene segment deleting N-terminal hydrophobic sequence was cloned into prokaryotic expression vector pGEX-6p-1 and the recombinant plasmid named pGEX-6p-dGP5 was transformed into E.coli BL21.SDS-PAGE indicated that the recombinant fusion protein was about 40 ku,after the recombinant plasmid was induced by IPTG.The protein dGP5 showed a strong immunological reaction to the PRRSV-positive sera in Western blot analysis,which may be helpful for the design of new vaccines,as well as the detection of antibodies against PRRSV.