高致病性猪繁殖与呼吸综合征病毒N基因真核表达载体的构建及表达

Construction of Eukaryotic Expression Vector and Expression of N Gene of HP-PRRSV

为进一步研究高致病性猪繁殖与呼吸综合征病毒(PRRSV)N蛋白的结构、功能及免疫学特性,提取PRRSV云南分离株A06RNA,设计特异性引物扩增全长N基因,得到长度为372bp的目的片段,克隆至杆状病毒Bac-To-Bac表达载体pFastBac HTB中,转化大肠埃希菌DH10Bac进行同源重组,成功构建了含有N基因的真核表达载体,转染Sf9细胞,回收得到重组杆状病毒。SDS-PAGE和Western blot检测结果表明,表达产物17ku,与预期大小一致,能与PRRSV阳性血清产生特异性反应。

In order to study the N protein structure,function and immunological characteristics of porcine reproductive and respiratory syndrome virus,the A06 RNA was extracted from PRRSV of Yunnan strain. The specific primers were designed to amplify the full-length N gene,372 bp fragment was cloned into ex-pression vector pFastBac HTB of baculovirus Bac-To-Bac,and further homologous recombination in E.coli DH10Bac.The eukaryotic expression vector containing N gene was successfully constructed.Transfection of Sf9 cells,the recovery of recombinant baculovirus were made.SDS-PAGE and Western blot assay indi-cated that recombinant N protein was expressed successfully with molecular weight of 17 ku which was consistent with the expected size and could specifically react with anti PRRSV positive sera.