【摘要】 目的　获得稳定表达丙型肝炎病毒 (HCV)结构蛋白的哺乳动物细胞系 ,以便对HCV结构蛋白的性质及其基因免疫效果作进一步的研究。方法　用RT PCR方法扩增HCV结构基因 ,并将其克隆入真核表达载体pcDNA3中 ,用磷酸钙转染法将其转入Sp2 / 0细胞 ,对目的蛋白获得稳定表达的细胞克隆进行免疫组化及细胞全蛋白的Westernblot分析。结果　克隆的基因片段全长约 1 9kb ,包括C及E2基因的全部和E1基因的部分。免疫组化显示表达蛋白位于胞浆中。Westernblot显示C蛋白为一条相对分子质量 (Mr)约 2 0× 10 3 的窄带 ,而E2蛋白为一条Mr 约 70× 10 3 的宽带 ,E1蛋白为一条Mr 约 17× 10 3 的较宽的条带。结论　HCV结构蛋白在哺乳动物细胞中得到稳定表达。更多还原

【Abstract】 Objective Expression of structural protein of hepatitis C virus in mammalian cells stably. Methods The HCV structural gene of typeⅡ was amplified by RT PCR and cloned in to eukaryotic expression vector pcDNA3. Sp2/0 cells were transfected with the constructed vector by calcium phosphate transfection method. The expressed protein was identified by means of immunohistochemistry and Western blot. Results The amplified gene, which is about 1.9kb, includes entire C, E2 gene and part of E1 gene (including the basepare from the first to the 81th of E1 gene and its hydrophilic end). The cells transfected with the constructed vector expressed the HCV structural protein successfully. The expressed protein was found as brown granules in the cytoplasm. In Western blot, three bands could be seen: a single narrow band at the place about 20kD, which is considered as the core protein; the E2 protein was seen as a broad band of approximately 70kD, and an obscure band located at about 17kD, which was considered as the partly expressed E1 protein. Conclusion The HCV structural protein gene cloned in pcDNA3 eukaryotic expression vector can be stably expressed in mammalian cells, which localized in the cytoplasm of the transfected cells. The expression of the HCV structural protein in mammalian cells rollouted the ground for studying the feature of the protein and its immunogenicity.更多还原