中文摘要：

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）基因启动子转录的凋节作用。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（-）-HBVSBP1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pl—iNOSp和pcDNA3．1（-）-HBVSBP1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（-）-HBVSBP1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凋节作用，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。

英文摘要：

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.