西尼罗病毒E蛋白在杆状病毒/昆虫细胞表达系统中的表达及其鉴定

Expression of E protein of West Nile virus by baculovirus expression vector system/insect cell system and identification of expressed product

目的利用杆状病毒/昆虫细胞表达系统表达西尼罗病毒(West Nile virus,WNV)囊膜E蛋白,并进行鉴定。方法利用分子克隆技术将WNV E基因克隆至杆状病毒供体质粒p Fast Bac1中,构建重组供体质粒p Fast Bac-E,转化大肠埃希菌DH10Bac,构建重组穿梭质粒Bac-E,转染sf9细胞,收获含WNV E基因的重组杆状病毒Ac MNPV-E,采用间接免疫荧光及Western blot法检测E蛋白的表达。结果重组供体质粒p Fast Bac-E经PCR及双酶切鉴定证明构建正确,重组穿梭质粒Bac-E经PCR鉴定证明构建正确,转染约96 h后,sf9细胞体积变大、变圆,细胞颗粒化,进而从培养板上脱落,漂浮,收获的第1代Ac MNPV-E经PCR鉴定为阳性。感染重组杆状病毒Ac MNPV-E的sf9细胞周围出现绿色荧光,表达的目的蛋白可与兔抗WNV E蛋白多克隆抗体及鼠抗His单克隆抗体特异性结合,在相对分子质量约53 000处可见目的蛋白条带。结论利用杆状病毒/昆虫细胞表达系统成功表达了WNV E蛋白,为WNV快速诊断方法及新型疫苗的建立奠定了基础。

Objective To express the envelope（E） protein of West Nile virus（WNV） by using baculovirus expression vector system（BEVS）/ insect cell（IC）system and identify the expressed product. Methods WNV E gene was cloned into baculovirus donor plasmid p Fast Bac1 by molecular cloning technique,and the constructed recombinant plasmid p Fast BacE was transformed to competent E. coli DH10 Bac. The obtained shuttle plasmid Bac-E was transfected to sf9 cells. Recombinant baculovirus Ac MNPV-E containing WNV E gene was harvested and determined for expression of E protein by indirect immunofluorescence assay（IFA） and Western blot. Results Both PCR and restriction analysis proved that recombinant donor plasmid p Fast Bac-E was constructed correctly. The sf9 cells were enlarged,round,granulated then fell off the culture plate and floated about 96 h after transfection. The harvested Ac MNPV-E of passage 1 was identified as positive by PCR. Green fluorescence was observed around sf9 cells infected with recombinant baculovirus Ac MNPV-E.The expressed protein showed specific binding to rabbit anti-WNV E polyclonal antibody and mouse anti-His monoclonal antibody,showing a protein band with relative molecular mass of about 53 000. Conclusion WNV E protein was successfully expressed by BEVS / IC system,which laid a foundation of development of a method for rapid diagnosis of WNV as well as novel vaccines.