GII型诺如病毒VP2蛋白在昆虫细胞中的表达与定位研究

Study on the expression of Norovirus (GenegroupII) VP2 protein in insect cells and its subcellular localization

摘要:

【目的】利用杆状病毒表达系统表达诺如病毒(GenegroupII) VP2蛋白, 分析其亚细胞定位, 为深入研究VP2蛋白的功能奠定基础。【方法】设计可扩增完整ORF3基因片段的引物P1和P2, 在下游引物中引入6×His标签的编码序列, 从质粒pMD-ORF3中克隆了含有6×His编码序列的ORF3基因, 与pFastBac1载体连接, 构建重组质粒pFB-ORF3, 转化DH10Bac感受态细胞获得重组杆状病毒基因组Bac-ORF3, 脂质体介导转染sf9昆虫细胞获得表达VP2蛋白的重组杆状病毒Ac-VP2, 感染sf9细胞后, 收集病变细胞, 采用抗6×His标签的单克隆抗体作为一抗进行Western blot与间接免疫荧光实验鉴定。【结果】Western blot实验证实Ac-VP2感染的sf9细胞在约29 kD处出现特异性条带; 间接免疫荧光实验证实Ac-VP2感染的sf9细胞出现特异性绿色荧光, 并且VP2主要定位于sf9的细胞核与细胞膜。【结论】诺如病毒VP2蛋白在Ac-VP2感染的sf9细胞中获得成功表达, 并且主要定位于sf9细胞的细胞核与细胞膜。

Abstract:

[Objective] This study is to express Norovirus (GenegroupII) VP2 in insect cells, analysis the subcellular localization of VP2 and lay the foundation of the function study of VP2. [Methods] Norovirus (GenegroupII) ORF3 gene was amplified from plasmid pMD-ORF3 using primers P1/P2 (including 6×His tag coding sequence). The product was cloned into the vector pFastBac1 to construct recombinant plasmid pFB-ORF3. pFB-ORF3 was transformed into competent DH10Bac to get recombinant bacmid Bac-ORF3. Recombinant baculovirus, Ac-VP2, was generated for expressing VP2, by transfecting recombinant Bac-ORF3 with LipofactamineTM 2000 into sf9 insect cells. The expression product was testified by the western blot and indirect immunofluorescence assay with monoclonal antibody against 6×His tag. [Results] The result of western blot showed a 29 kD specific bind in sf9 cells infected by Ac-VP2. Indirect immunofluorescence assay showed the specific green fluorescence in sf9 cells infected by Ac-VP2, and the green fluorescence could localize at nuclear and membrane of sf9 cells. [Conclusion] These results demonstrated that the Norovirus (Gengroup II) VP2 was expressed in sf9 cells successfully and could localize at nuclear and membrane of sf9 cells.