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目的 :研究 HGV和 HCV的复制和表达。 方法 :利用 HGV全长 c DNA克隆 (HGVqz)及 HCV1 a/ 1 b嵌合体 c DNA克隆分别构建表达质粒 p3.1 HGV和 p3.1 HCV并转染张氏肝细胞 ,以 HGVqz克隆建立 HGV转基因小鼠。分别应用 RT- PCR、免疫组化及 Western印迹分析病毒正、负链 RNA,蛋白表达和剪切。结果 :在转染 p3.1 HCV的张氏肝细胞及 HGV转基因小鼠某些组织中可检出相应病毒的负链 RNA,但在转染 p3.1 HGV的张氏肝细胞中未能检出 HGV负链 RNA。Western印迹在转染 p3.1 HCV的张氏肝细胞中检测到针对 HCV NS3蛋白、相对分子质量约 70 0 0 0的特异性条带 ,在 HGV转基因小鼠某些组织中检测到 2条特异性条带 ,相对分子质量约 4 2 0 0 0和 1 0 0 0 0 0 ,分别相当于 HGV E2蛋白及其剪切中间体。然而 ,在转染p3.1 HGV的张氏肝细胞中检测到针对 HGV E2蛋白的特异性条带 ,其相对分子质量约为 31 0 0 0 0 ,相当于 HGV整个前体蛋白。结论 :HGV的表达与复制在体内、外存在差异。细胞内某些特异性因子在病毒前体蛋白剪切中起重要作用 ,HGV和 HCV前体蛋白剪切所需宿主因子可能存在差异 ,故两种病毒体外培养时的嗜性细胞株并不完全一致
Objective: To study the replication and expression of HGV and HCV. Methods: The expression plasmids p3.1 HGV and p3.1 HCV were constructed by HGV full-length c DNA clone (HGVqz) and HCV1 a / 1 b chimeric c DNA clone respectively and transfected into HepG2 cells. The HGV transgene Mouse. RT-PCR, immunohistochemistry and Western blotting were used to analyze positive and negative strand RNA, protein expression and cleavage respectively. Results: Negative stranded RNA of the corresponding virus was detected in some tissues of Zhang hepatocytes and HGV transgenic mice transfected with p3.1 HCV, but failed to detect in Zhang hepatocytes transfected with p3.1 HGV HGV minus strand RNA. Western blot detected specific NS3 protein with a relative molecular weight of about 70 000 in Zhang hepatocytes transfected with p3.1 HCV, and detected 2 specific genes in some tissues of HGV transgenic mice Sexual bands, the relative molecular masses of about 4200 and 100000, respectively, equivalent to the HGV E2 protein and its cleavage intermediates. However, the specific band of HGV E2 protein was detected in Chang3 hepatoma cells transfected with p3.1 HGV. The relative molecular weight of HGV E2 protein was about 31 000, which is equivalent to the entire precursor protein of HGV. Conclusion: The expression and replication of HGV are different in vivo and in vitro. Some specific intracellular factors play an important role in the prod- uct cleavage of the precursor protein. The host factors required for the cleavage of HGV and HCV precursor proteins may be different. Therefore, the in vitro culture of the two tropic cell strains is not complete Consistent