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目的探讨HBV表面抗原基因启动子ⅠDNA结合蛋白1(SBP1)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,PCR分别扩增iNOSp和3个缺失体的基因序列,分别克隆至报告基因表达载体pCAT3-Basic中,构建pCAT3-iNOSp报告载体;以构建的这4种报告质粒分别转染人肝母细胞瘤细胞系HepG2,用ELISA检测氯霉素乙酰转移酶(CAT)的表达活性;并与构建的真核表达载体pcDNA3.1(-)-HBV SBP1共转染HepG2细胞系,用ELISA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,其中p1-iNOSp和pcDNA3.1(-)-HBV SBP1瞬时共转染HepG2细胞时,iNOS启动子的转录活性上升1.54倍;p3-iNOSp启动子和pcDNA3.1(-)-HBV SBP1瞬时共转染HepG2细胞时,iNOS启动子的转录活性下降31.3%,重复实验得到相似结果。结论克隆的新基因SBP1在细胞内的表达,对iNOS基因反式激活iNOS启动子的转录活性具有明显的双向调节作用,双向调节的部位是iNOS启动子的核因子(NF)-IL6、A-激活域结合位点(AABS)和NF-κB这3个结合位点。
Objective To investigate the regulatory effect of HBV surface antigen gene promoter Ⅰ DNA binding protein 1 (SBP1) on inducible nitric oxide synthase (iNOS) gene promoter transcription. Methods The iNOS gene promoter region (iNOSp) and three deletion mutants were sequenced by bioinformatics techniques. The gene sequences of iNOSp and three deletions were amplified by PCR and cloned into the reporter gene expression vector pCAT3-Basic The constructed pCAT3-iNOSp reporter vector was constructed. The four reporter plasmids were transfected into human hepatoblastoma cell line HepG2 respectively, and the expression activity of chloramphenicol acetyltransferase (CAT) was detected by ELISA. The HepG2 cell line was co-transfected with pcDNA3.1 (-) - HBV SBP1 and the expression of CAT was detected by ELISA. Results The correct clones of iNOS gene promoter and three deletion mutants were successfully obtained. The transcript activity of iNOS promoter increased by 1.54 fold when p1-iNOSp and pcDNA3.1 (-) - HBV SBP1 were transiently co-transfected into HepG2 cells. The p3 iNOSp promoter and pcDNA3.1 (-) - HBV SBP1 transiently co-transfected HepG2 cells, iNOS promoter activity decreased by 31.3%, repeated experiments to obtain similar results. Conclusion The expression of a novel gene SBP1 in the cell has obvious bidirectional regulation on the transactivation of iNOS promoter in iNOS gene. The site of bidirectional regulation is the nuclear factor (NF) -IL6 of iNOS promoter, Activation domain binding sites (AABS) and NF-κB these three binding sites.