犬瘟热病毒R/20-8株核衣壳蛋白基因的克隆和表达

应用RT-PCR技术扩增出犬瘟热病毒(CDV)核衣壳(N)蛋白基因的高度保守序列,将其克隆至质粒pMD18-T中,获得了重组质粒pMD18-T-N。将N基因的目的片段克隆到表达载体pGEX-4T-1中谷胱甘肽转移酶(GST)基因的下游,并将该重组质粒转化大肠杆菌BL21株,经IPTG诱导,N基因融合蛋白获得了高效表达。SDS-PAGE电泳和Western-blot分析结果显示,表达产物的分子质量为55 ku,与CDV标准阳性血清呈阳性反应。表明,大肠杆菌表达的CDV N蛋白在免疫原性上与天然N蛋白具有一定的相似性,可作为诊断用抗原。

Cloning and expression of nucleocapsid protein gene of canine distemper virus CDV/R/20-8 strain

A highly conserved sequence of nucleocapsid protein gene of canine distemper virus(CDV) was amplified by RT-PCR,then the product was cloned into pMD18-T vector to construct recombinant plasmid pMD18-T-N and sequenced.The gene was inserted into the downstream of GST of pGEX-4T-1 vector.The plasmid pGEX-4T-1-N was transformed into Escherichia coli BL21 for expression under induction with IPTG.SDS-PAGE analysis showed that the molecular weight of the expressed product was 55ku.Western-blot analysis showed that the recombinant protein had a positive reaction with standard positive serum against CDV.The results indicated that the expressed N protein was similar to the natural nucleocapsid protein on immunogenicity,which could be used as antigen for diagnosis.