蓝舌病病毒血清8型NS3蛋白在昆虫杆状病毒系统中的表达与鉴定

Expression of NS3 of Bluetongue Virus in the Baculovirus Expression System

为表达蓝舌病病毒NS3蛋白,将蓝舌病病毒血清8型NS 3序列克隆至pFastBac-HTA载体上,经转座、转染后获得重组杆状病毒rBac-NS3;用间接免疫荧光(IFA)检测NS3蛋白的表达情况,并采用亲和层析进行纯化;以Western Blot和间接ELISA方法鉴定NS3蛋白的反应原性。IFA结果显示,重组杆状病毒rBac-NS3感染的Sf9细胞可以大量表达NS3蛋白。Western Blot结果显示,纯化后的NS3蛋白与抗His单抗和绵羊蓝舌病阳性血清能够发生特异性反应;以纯化的NS3蛋白为包被抗原建立的间接ELISA方法可以区分蓝舌病阳性血清和阴性血清,进一步验证本试验利用杆状病毒表达系统成功表达并纯化出蓝舌病病毒NS3蛋白,表明纯化后的NS3蛋白具有良好的反应原性,可作为开发鉴别诊断试剂盒的备选蛋白及NS3蛋白的结构和生物学功能研究。

In order to express bluetongue virus(BTV)NS3,the ORF of BTV NS 3 was cloned into the pFastBac-HTA vector.By translocation and transfection,the recombinant baculovirus rBac-NS3 was constructed.Recombinant NS3 protein was purified by Ni-NTA Fast Start Kit.The antigenicity of NS3 protein was identified by Western Blot and indirect-ELISA.It was found that the specific fluorescence appeared in the infected cells in IFA test.And the results of Western Blot showed that the purified NS3 protein specifically reacted with anti-His McAb and BTV positive serum.The indirect ELISA,which NS3 was employed as the diagnosis antigen,could differentiate BTV positive serum and negative serum.All these results showed that the purified NS3 protein had good reactogenicity.It laid a foundation for development of the diagnosis kit and the research of structure and biological functions of BTV NS3.