丙型肝炎病毒核心蛋白在昆虫细胞中表达、加工和细胞定位研究

以杆状病毒-昆虫细胞表达系统Bac-to-Bac为模型,就目前存在较多争论的HCV核心蛋白的加工和细胞定位问题进行探讨.根据核心蛋白的亲水性分析结果,设计了三种长度的基因片段(C173、C191和C215),经过PCR扩增、重组转移载体构建、细菌内转座和昆虫细胞转染,获得三种重组病毒(rvBACC173、rvBACC191和rvBACC215)用于表达分析.SDS-PAGE电泳和免疫印迹试验表明,昆虫细胞能够识别核心蛋白第191和192位氨基酸之间的切点并低效率切割;但不能够识别173～191位之间的切点.间接免疫荧光试验表明,截断型核心蛋白C173定位于细胞核内,而C191和C215则停留在细胞浆中.rvBACC173感染的细胞在核内出现单一的"类晶体样结构",电子显微镜分析证实,这种结构是截断型核心蛋白大量转运并沉积在细胞核内形成的蛋白聚合体.试验结果还表明,第173至191之间的疏水性序列负性调节蛋白的表达,并且影响蛋白在细胞内的分布.间接ELISA实验证实,部分纯化的核心蛋白可用作诊断试剂检测人血清中的特异性抗体.

Expression, Processing and Subcellular Localization of Hepatitis C Virus Core Protein in Insect Cells

This study was to investigate the processing and subcellular localization of hepatitis C virus core protein expressed in insect cells.Three constructs that have the same N-terminus' and different C-terminus' were designed and the genes were introduced into the genome of baculovirus via gene transposition.The recombinant baculoviruses were produced by insect cells and were designated as rvBACC 173 ,rvBACC 191 and rvBACC 215 .The SDS-PAGE and Western-blot results showed that the insect cells could produce core protein and cleave it between 191 and 192 residuals,but could not recognize the potential cleavage site located between 173 and 191 residuals.The indirect immunofluorescence assay showed that the products of rvBACC 173 absolutely located in the nuclei of the insect cells,while the other two kinds of products located in the cytoplasm.In addition,we also observed that the Sf9 cells infected with rvBACC 173 had a large"crystal-like" particle in nuclei,and the structure of the particle was revealed by electron microscope.The notable difference between C 173 and C 191 suggests that a stretch of hydrophobic sequence located in C-terminus of C 191 may have negative impact on the protein expression and anchore the protein in cytoplasm.The partially purified core protein of HCV had a good performance used as reagent in indirect ELISA format to detect specific antibodies among blood-donors.