论文部分内容阅读
目的探讨丙型肝炎病毒(HCV)核心(core)蛋白对诱导型一氧化氮合酶(iNOS)基因启动子转录的激活作用。方法利用生物信息学技术确定iNOS基因的启动子区域(iNOSp),聚合酶链反应(PCR)扩增iNOSp的DNA,克隆至真核报告载体pCAT3-Basic中,构建pCAT3-iNOSp报告载体;以该质粒转染正常人肝细胞LO_2,用酶联免疫吸附法(ELISA)检测氯霉素乙酰转移酶(CAT)的表达活性;并与HCV核心蛋白的真核表达载体pcDNA3．1(-)-core共转染LO_2细胞,用ELISA法检测CAT的表达活性。结果成功获得iNOS基因启动子的正确克隆,pCAT3-iNOSp和pcDNA3．1(-)- core瞬时共转染LO_2细胞时,报告基因表达载体中的SV40病毒的即刻早期启动子的转录活性明显提高,CAT表达活性是pCAT3-Basic空载体的11倍,是pCAT3-iNOSp的6倍,重复试验得到了相似的结果。结论成功克隆的iNOS启动子有转录活性;HCV核心蛋白具有对iNOS基因启动子的反式激活作用。HCV核心蛋白在细胞内的表达对于iNOS基因反式激活在HCV感染相关的慢性肝脏疾病的发病过程中具有重要意义。
Objective To investigate the activation of inducible nitric oxide synthase (iNOS) gene promoter by hepatitis C virus (HCV) core protein. Methods The iNOS gene promoter region (iNOSp) was identified by bioinformatics technology. The iNOSp DNA was amplified by polymerase chain reaction (PCR) and cloned into eukaryotic reporter vector pCAT3-Basic to construct pCAT3-iNOSp reporter vector. The plasmids were transfected into normal human hepatocytes LO_2, and the activity of chloramphenicol acetyltransferase (CAT) was detected by enzyme - linked immunosorbent assay (ELISA). The recombinant plasmids were linked with pcDNA3.1 (-) - core The LO 2 cells were co-transfected and the activity of CAT was detected by ELISA. Results The correct cloning of iNOS gene promoter was successfully obtained. The transcript activity of immediate early promoter of SV40 in the reporter gene expression vector was significantly increased when pCAT3-iNOSp and pcDNA3.1 (-) - core were transiently co-transfected into LO_2 cells. CAT expression activity was 11-fold that of pCAT3-Basic empty vector and 6-fold that of pCAT3-iNOSp, and similar results were obtained by repeated experiments. Conclusion The iNOS promoter cloned successfully has transcriptional activity. The HCV core protein has the transactivation effect on the iNOS gene promoter. The intracellular expression of HCV core protein is of great importance for the iNOS gene transactivation in the pathogenesis of chronic liver diseases associated with HCV infection.