猪生殖与呼吸综合征病毒HN6株ORF5基因的克隆及其在大肠杆菌中的表达

Cloning and expression of ORF5 gene of porcine reproductive and respiratory syndrome virus HN6 strain in Escherichia coli

用RT-PCR方法从河南分离的1株(HN6)猪生殖与呼吸综合征病毒(PRRSV)核酸中扩增缺失N端疏水序列的基因片段dORF5(deleting ORF5),并将其克隆到pMD18-T载体中测序,再亚克隆到原核表达载体pET-32a上。重组质粒转化大肠杆菌BL21,用不同浓度IPTG分别于37℃诱导,经SDS-PAGE分析,所表达的融合蛋白的分子质量约31.4 ku,薄层扫描分析显示,表达量占菌体总蛋白含量的28.8%。Western-blotting结果表明,重组蛋白可被PRRSV阳性血清所识别。证实,该蛋白可用于PRRSV的诊断。

The dORF5 gene segment deleting N-terminal hydrophobic sequence was amplified from total RNA of porcine reproductive and respiratory syndrome virus（PRRSV） HN6 strain by RT-PCR,and the amplicon was cloned into pMD18-T vector and sequenced. Then the gene was subcloned into prokaryotic expression vector pET-32a. The recombinant fusion proteins E-dORF5 were highly expressed in Escherichia coli BL21 in the forms of inclusion bodies induced by different concentration of IPTG at 37 ℃,and made up 28.8% of the total mass of bacterial proteins scanned by Imaging Den-sitometer. The expressed protein was identified to be 31.4 ku by SDS-PAGE analysis. Western-blotting showed that the recombinant protein could react with the porcine polyclonal antibody against PRRSV, indicating that the protein could be used as antigen in diagnostic assay for the detection of antibodies against PRRSV.