西尼罗病毒NS1蛋白的原核表达及其免疫原性研究  

Expression of Protein NS1 of West Nile Virus and Identification of Its Immunogenicity

作　　者：王彦芳 杨鹏飞[2] 张丽萍[2] 平芮巾[2] 刑福彬[2] 咸庆杰[2] 胡孔新[2] 

机构地区：[1]德州市中心血站,山东德州253000 [2]中国检验检疫科学研究院

出　　处：《检验检疫学刊》2011年第3期34-37,15,共5页Journal of Inspection and Quarantine

基　　金：国家公益性行业科研专项(200810162);国家质检总局科技项目(2008IK210);传染病国家科技重大专项(2011ZX10004-001)

摘　　要：[目的]采用大肠杆菌表达系统表达西尼罗病毒NS1蛋白,并对表达产物进行免疫原性分析。[方法]将WNV NS1基因克隆入pET30a载体,获得重组表达质粒pET30a-WNV-NS1,并转化入BL21感受态细胞,将表达产物进行SDS-PAGE和W estern-blot分析,经N i+亲和层析纯化、阴离子交换层析后获得纯化的WNV NS1蛋白;将纯化产物免疫新西兰兔,获得的免疫血清作ELISA检测。将重组蛋白包板,检测FOCUS公司商品化试剂盒中的阳性血清。[结果](1)重组WNV NS1蛋白获得表达并纯化成功。(2)W estern blot结果显示表达的蛋白是带有H is标签的目的蛋白。(3)兔免疫血清的间接ELISA结果显示血清效价达到1:50000以上。(4)检测商品化试剂盒中的阳性血清,其OD450吸光值与阴性血清吸光值之比大于2。[结论]重组NS1蛋白能在大肠杆菌系统中获得表达;纯化产物具有免疫原性,而且可以作为抗原检测西尼罗病毒感染的血清,为进一步研究NS1蛋白的生物学特性及西尼罗病毒的诊断试剂奠定了基础。To express protein NS1 of West Nile virus with pET30a and identify immunogenicity of the products,WNV NS1 gene was cloned into the pET30a vector and then the recombinant plasmid pET30a-WNV-NS1 transformed into E.coli BL21.The expressed products were analyzed with SDS-PAGE and Western blot.Purified protein WNV NS1 was obtained by Ni-NTA chromatography and Ion-exchange chromatography.Rabbits were immunized with the purified protein and the immue serum was assayed with ELISA.12 eight-well polystyrene microwell were coated with recombinant WNV NS1 protein and used to detect WNV positive serum in West Nile Virus IgG ELISA kit（FOCUS company,USA）.Recombinant NS1 protein was expressed and purified successfully.Western blot results showed that the expressed product was the target protein which has His tag.Indirect ELISA results showed that the serum titer reached above 1：50000.Coated with recombinant WNV NS1 protein,the absorbance value in OD450 of positive surem was more than two times that of negative serum.Recombinant WNV NS1 protein was able to be expressed in E.coli,and its immunogenicity was verified.It could be used as antigen to detect serum infected by WNV.The results laid the foundation for the further research of the biological characteristics of NS1 protein and diagnostic reagent of WNV.

关 键 词：西尼罗病毒 非结构蛋白NS1 原核表达 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学]