西尼罗病毒E蛋白的原核表达及其多克隆抗体的制备

利用PCR技术扩增西尼罗病毒(WNV)E蛋白基因并将其插入原核表达载体pET-30a(+),构建重组质粒pET-30a(+)-WNV-E,并转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导目的蛋白表达,对诱导条件进行优化,利用His镍柱亲和层析法纯化目的蛋白,纯化后的蛋白经SDS-PAGE与Westernblot鉴定正确后,免疫BALB/c小鼠制备多克隆抗体并进行间接免疫荧光鉴定。结果显示:WNV E蛋白在大肠杆菌中以包涵体形式被成功表达,蛋白最佳诱导条件为37℃,0.8 mmol/L IPTG诱导5 h,经间接免疫荧光鉴定多克隆抗体能特异性识别WNV E蛋白。本研究成功表达并纯化了WNV E蛋白,证明该蛋白具有良好的免疫原性,为WNV抗原、抗体检测方法的建立和新型疫苗的研发及相关研究奠定了基础。

Prokaryotic expression and polyclonal antibody preparation of West Nile virus E protein

The gene encoding West Nile virus(WNV)E protein was amplified by PCR and inserted into the prokaryotic expression vector pET-30a(+),constructing the recombinant plasmid pET-30a(+)-WNV.The constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3)and induced with IPTG.Meanwhile,the induction condition was optimized.The expressed recombinant E protein was purified by His nickel column affinity chromatography and identified by SDS-PAGE and Western-blot.Then,the purified E protein was used as immunogen for BALB/c mice by intramuscular injection route after mixed with Freund adjuvant.The prepared polyclonal antibody was detected for specificity by indirect immunofluorescence assay(IFA).The results showed that the E protein was expressed in a form of inclusion body in E.coli.The optimal induction of the protein was induced at 37℃and 0.8 mmol/L IPTG for 5 h.In conclusion,the WNV E protein was successfully expressed and purified.This protein has good immunogenicity,which lays the foundation for the establishment of detection methods of WNV antigen and antibody,the development of new vaccines and related research.