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目的探讨乙型肝炎病毒(HBV)前-S2(pre-S2)蛋白对诱导型一氧化氮合酶(iNOS)基因启动子转录的调节作用。方法利用生物信息学技术确定iNOS基因的启动子区域(iNOSp)和3个缺失突变体的基因序列,聚合酶链反应(PCR)分别扩增iNOSp和3个缺失突变体的基因序列,分别克隆至报告基因表达载体pCAT3-Basic中,构建pCAT3-iNOSp载体;以构建的这4种报告基因表达载体,分别转染人肝母细胞瘤细胞系HepG2,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;并与真核表达载体pcDNA3.1(-)-HBV pre-S2共转染HepG2细胞系,用ELISA法检测CAT的表达活性。结果成功获得iNOS基因启动子和3个缺失突变体的正确克隆,p1-iNOSp、p3-iNOSp启动子和pcDNA3.1 (-)-HBV pre-S2瞬时共转染HepG2细胞时,iNOS启动子的转录活性明显下降,HBV pre-S2蛋白对p1- iNOSp、p3-iNOSp表达活性的抑制率分别是54.7%和79.5%,p2-iNOSp、p4-iNOSp与pcDNA3.1(-)-HBV pre-S2共转染HepG2细胞后,HBV pre-S2蛋白对p2-iNOSp、p4-iNOSp表达活性没有明显的调节作用。重复试验得到了相似的结果。结论HBV pre-S2蛋白在细胞内的表达对iNOS启动子的转录活性具有明显的下调作用。
Objective To investigate the regulatory effect of hepatitis B virus pre-S2 protein on the transcription of inducible nitric oxide synthase (iNOS) gene promoter. Methods The gene sequences of iNOS gene promoter region (iNOSp) and three deletion mutants were determined by bioinformatics techniques. The gene sequences of iNOSp and three deletion mutants were amplified by polymerase chain reaction (PCR) and cloned into Reporter gene expression vector pCAT3-Basic to construct pCAT3-iNOSp vector; The constructed four reporter gene expression vector were transfected into human hepatoblastoma cell line HepG2, enzyme-linked immunosorbent assay (ELISA) detection of chloramphenicol (CAT). The eukaryotic expression vector pcDNA3.1 (-) - HBV pre-S2 was co-transfected into HepG2 cell line, and the activity of CAT was detected by ELISA. Results The correct cloning of the iNOS gene promoter and 3 deletion mutants was successfully obtained. When the p1-iNOSp, p3-iNOSp promoter and pcDNA3.1 (-) - HBV pre-S2 were transiently co-transfected into HepG2 cells, the iNOS promoter The inhibitory rate of HBV pre-S2 protein on the expression of p1-iNOSp and p3-iNOSp was 54.7% and 79.5%, respectively. The inhibitory rates of p2-iNOSp, p4-iNOSp and pcDNA3.1 (- The co-transfection of HepG2 cells, HBV pre-S2 protein p2-iNOSp, p4-iNOSp expression did not significantly regulate the activity. Repeated trials yielded similar results. Conclusion The expression of HBV pre-S2 protein in cells has a significant down-regulation effect on the transcriptional activity of iNOS promoter.