鸡源禽流感H9N2亚型河南分离株NS1基因克隆、序列分析及原核表达

Cloning,sequence analysis and prokaryotic expression of NS1 gene of H9N2 influenza virus isolated from Henan

根据H9N2亚型禽流感病毒(AIV)的NS1基因序列,设计1对特异性引物,采用RT-PCR方法扩增AIV的NS1基因序列,将其克隆到原核表达载体pET-32α(+)中,构建重组表达质粒pET-32α(+)-NS1,转化大肠杆菌BL21,IPTG诱导得到NS1融合蛋白;其相对分子质量约28 000,分析显示NS1基因的原核表达载体成功构建,表达蛋白以包涵体形式存在,包涵体经过变性和复性后均可获得单一、高表达量的目的蛋白,为进一步开展关于NS1蛋白的功能,为建立鉴别禽流感疫苗免疫和野毒感染ELISA诊断试剂盒奠定基础。

According to the sequence of nonstructural protein NSl gene of H9N2 avian influenza virus in GenBank,apair of specific primers was designed.NS1 gene was amplified by RT-PCR.The NS1 gene was cloned directionaly into the pET-32α（＋）.The recombinant prokaryotic expression plasmid was consructed and transformed into E.coli BL21.The recombinant protein NS1 were obtained with the induction of IPTG and the molecular weight of the fusion protein was 28 000.Results showed that the prokaryotic expression vector of NS1 gene were successfully constructed,The fusion protein was purified by extracting the inclusion bodies.The results laid the foundation for the further studies on the NS1 protein of H9N2 avian influenza virus and making ELISA diagnosis kit for differentiation infected from vaccinated poultry on the basis of antibody to NS1 protein.