乙型肝炎表面抗原基因启动子ⅠDNA结合蛋白1对诱导型一氧化氮合酶基因启动子转录活性的双向调节  

The study of bidirectional regulation effect on inducible nitric oxide synthase gene promoter by hepatitis B virus preS1 promoter DNA-binding protein 1

作　　者：郭风劲[1] 成军[1] 纪冬[1] 刘妍[1] 王琳[1] 张黎颖[1] 宋方洲[2] 

机构地区：[1]北京地坛医院传染病研究所,现在100011 [2]重庆医科大学分子生物学教研室

出　　处：《中华传染病杂志》2007年第5期257-262,共6页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金(30371288);北京市自然科学基金(5042024);第35批博士后科学基金(2004035045)

摘　　要：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 regulating effect of hepatitis B virus （HBV） SP Ⅰ binding protein Ⅰ（SBP1） on inducible nitric-oxide synthase（iNOS） gene promoter activity. Methods Polymerase chain reaction（PCR） technique was employed to amplify the coding sequence of iNOS promoter DNA by using HepG2 genomic DNA as template, and 3 deletion mutants were amplified. The products were cloned into pGEM-T vector, respectively. The iNOS gene and 3 deletion mutants were cut from T-iNOS by Kpn Ⅰ and Xho Ⅰ , and then were cloned into pCAT3-Basic. The constructed vectors were named as pl-iNOSp, p2-iNOSp, p3-iNOSp and p4-iNOSp, respectively. Each of the vectors containing different iNOSp DNA fragments was transfected into the HepG2 cell line and cotransfected into HepG2 cells with pcDNA3.1（- ）-SBP1 by FuGENE 6 transfection reagents. The HepG2 cells transfected with pCAT3-Basic were used as negative control. The activity of chloramphenicol acetyltransferase（CAT） in transfected HepG2 cells was detected by an enzyme-linked immunosorbent assay（ELISA） kit after 48 hours, which would reflect the regulating effect of SBP1 on iNOS gene promoter activity. Results The expression vector pcDNA3.1（- ）-SBP1 and report vector pCAT3-iNOS were constructed and confirmed by restriction enzyme digestion and sequencing. The expression of pcDNA3.1（-）-SBP1 in HepG2 cells up-regulated the activity of pl-iNOSp and downregulated the activity of p3-iNOSp. The inhibiting rate was 31.3%. Conclusions It is suggested that SBP1 can regulate iNOS gene promoter bidirectionally by influencing the binding sites of nuclear factor （NF）-IL6, A activator domain binding site and NF-κB in iNOS gene promoter.

关 键 词：肝炎病毒 乙型 启动区(遗传学) 一氧化氮合酶 白细胞介素6 结合部位 

分 类 号：R512.6[医药卫生—内科学]