HCV结构蛋白在昆虫细胞中的表达及病毒样颗粒的形成

Expression of HCV structural proteins and assembly of HCV-like particles in insect cells

目的 在昆虫细胞中同时表达HCV的核心蛋白C和包膜蛋白 (E1、E2 )以形成病毒样颗粒 (VLP)。方法 利用PCR技术得到HCV的核心蛋白和包膜蛋白基因 ,用基因重组技术分别构建表达HCVC、E1和E2蛋白的重组杆状病毒 ,用SDS PAGE电泳分析HCV结构蛋白在昆虫细胞中的表达。电镜观察昆虫细胞内VLP的形成。结果 得到了能表达HCVC蛋白和同时表达E1、E2蛋白的重组杆状病毒reBV/C和reBV/E1、E2。reBV/C和reBV/E1、E2共感染的昆虫细胞同时表达了C、E1和E2蛋白 ,分子量分别为 2 0kD ,35kD和 6 6kD。电镜观察到表达HCV结构蛋白的昆虫细胞质中存在大量的直径为 4 0～ 6 0nm的VLP ,对照细胞无此结构。结论 在昆虫细胞中表达的HCVC。

Objective To study simultaneous expression of all three HCV structural proteins in insect cells and to investigate the assembly of expressed proteins into HCV like particles. Methods HCV structural protein encoding sequences C, E1 and E2 were amplified by PCR. Recombinant baculoviruses were constructed with recombinant DNA techniques and used to co infect insect cells. Expression of HCV structural proteins in the insect cells was analyzed by SDS PAGE. The virus like particles (VLPs) in insect cells were visualized by electron microscopy (EM). Results The recombinant baculoviruses reBV/C and reBV/E1,E2 were constructed. Insect cells co infected with reBV/C and reBV/E1,E2 expressed HCV C, E1 and E2 proteins with the molecular weight of 20kD, 35kD and 66kD respectively. Electron microscopy of insect cells co infected with reBV/C and reBV/E1,E2 demonstrated spherical particles (40 to 60 nm in diameter) similar to the HCV virions from sera or hepatic tissues of HCV infected humans. Conclusion HCV structural proteins simultaneously expressed in the insect cells could assemble into HCV like particles.