庚型和丙型肝炎病毒的全长cDNA克隆在体内外的表达与复制(英文)  

Replication and expression of full-length cDNA clones of hepatitis G virus and hepatitis C virus in vitro and in vivo

作　　者：王宏卫[1] 潘欣[1] 戚中田[1] 

机构地区：[1]第二军医大学基础医学部微生物学教研室,上海200433

出　　处：《第二军医大学学报》2004年第4期389-393,共5页Academic Journal of Second Military Medical University

基　　金：This work is supported by the National Natu-ral Science Foundation of China ( No.3 982 5 116;3 0 170 5 14 ;3 0 170 891)

摘　　要：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 。Objective:To research the replication and expression of HGV and HCV in order to better understand the biological difference of the viruses and the interaction with their hosts.Methods:A full length HGV cDNA clone(HGV qz ) and a full length HCV 1a/1b chimeric cDNA clone were used to construct p3.1HGV and p3.1HCV respectively,and to transfect Chang liver cells for investigating the replication and expression of HGV and HCV in vitro .HGV replication and expression in vivo were explored with HGV transgenic mice.Plus and minus strand viral RNAs,protein expression and processing were analyzed by reverse transcription polymerase chain reaction(RT PCR),immunohistochemical staining and Western blotting.Results:The virus minus strand RNAs were found in Chang liver cells transfected with p3.1HCV and some tissues of HGV transgenic mice,but were not seen in Chang liver cells transfected with p3.1HGV.An immunospecific band about 70 000 to HCV NS3 protein was detected by Western blotting in the lysates of Chang liver cells transfected with p3.1HCV and 2 immunospecific bands about 100 000 and 42 000 corresponding to HGV E2 intermediate and HGV E2 proteins were foundin.However,the immunospecific band to HGV E2 protein in Chang liver cells transfected with p3.1HGV was about 310 000 which was corresponding to HGV precursor polyprotein.Conclusion:Some specific factors in cells are very important for processing the virus polyprotein into functional proteins,and the host factors in polyprotein processing are different between HGV and HCV.The replication and polyprotein processing of HGV exist discrepancy between in vivo and in vitro .

关 键 词：庚型肝炎病毒 全长CDNA克隆 表达 复制 丙型肝炎病毒 

分 类 号：R373.21[医药卫生—病原生物学]