猪细小病毒NS1基因的原核表达

Expression of NS1 of PPV in E.coli

根据GenBank发表的猪细小病毒(porcine parvovirus,PPV)中国株基因序列设计引物,通过PCR方法扩增到了PPV LJL12株NS1全长基因,将其克隆到原核表达载pET30a(+)上,成功构建原核表达载体pET30a-NS1,将其转化到大肠杆菌BL21(DE3)感受态细胞。经IPTG诱导表达后,进行SDS-PAGE和Western blot分析。SDS-PAGE电泳显示NS1在大肠杆菌中得到成功表达,重组蛋白大小约为86ku,与预期大小相符;Western blot分析表明,该蛋白能与PPV阳性血清发生特异性反应,证明该蛋白具有良好生物学活性。NS1在大肠杆菌中成功表达,具有免疫原性,可作为鉴别诊断抗原用于PPV的临床检测。

According to the sequence of porcine parvovirus China strain in GenBank, a pair of primers was designed in order to amplify the NS1 gene of porcine parvovirus LJL12 strain. The gene was cloned into the multiple cloning site of pET30a （＋） vector after double enzyme digested. The recombinant of pET30a-NS1 was transformed into E.coli BL21 （DE3） competent cell and induced to express by IPTG, andthe expressed NS 1 protein was assayed by SDS-PAGE and Western-blot. SDS-PAGE showed that the NS1 protein was successfully expressed in Escherichia coli, with a relative molecular weight of 86 ku accord with anticipate. The results of Western blot showed that this protein was specifically reacted with porcine parvovirus positive serum, indicating the recombinant protein had specificity. NS1 protein was expressed in E. coli and showed good specificity which could be used as antigen of diagnostic assay for detection antibodies of PPV in practice.