AP-1和ETS-1位点缺失的DC-SIGN启动子荧光素酶报告质粒的构建及其活性研究  

Construction of luciferase reporter vectors harboring DC-SIGN promoters without AP-1 or ETS-1 transcription factor binding site and detection of their activity

作　　者：靳昌忠[1] 李杰[1] 姚航平[1] 吴南屏[1] 

机构地区：[1]浙江大学医学院附属第一医院传染病诊治国家重点实验室,杭州310003

出　　处：《中华微生物学和免疫学杂志》2009年第12期1075-1079,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（30872241）;卫生部十一五“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项（2008ZX10001-007）

摘　　要：目的研究AP-1和ETS-1转录因子结合位点（TFBS）缺失对树突状细胞特异性胞间黏附分子-3结合非整合素（DC—SIGN）启动子活性的影响，探讨DC—SIGN表达的机制。方法从人外周血中提取全基因组DNA，设计DC-SIGN启动子序列上下游引物、转录因子结合位点两侧的引物及其相应的连接引物，利用PCR方法，通过不同的引物组合扩增出转录因子结合位点两侧的片段，再用连接引物连接两片段，得到转录因子结合位点缺失的DC—SIGN启动子序列，通过双酶切、连接的方法，将得到的DC—SIGN启动子序列定向克隆入荧光素酶报告质粒，将上述质粒通过脂质体转染Hacat和293细胞，48h后检测荧光素酶的活性。结果体外扩增得到的转录因子结合位点缺失的DC—SIGN启动子序列以及重组的荧光素酶报告质粒经双酶切、基因测序鉴定正确，荧光素酶活性检测结果显示AP-1位点缺失使DC—SIGN启动子活性下降20％（293细胞）和10％（Hacat细胞），带增强子的DC—SIGN启动子活性下降了40％～50％；ETS—1位点缺失的普通型和带增强子型DC—SIGN启动子的活性几乎消失。结论ETS-1转录因子结合位点在DC—SIGN启动子活性表达中起重要作用，AP-1转录因子结合位点作用不大。Objective To detect the role of AP-1 or ETS-1 transcription factor binding sites （TFBS） in activity of DC-specific intercellular adhesion moleeule-3-grabbing nonintegrln （DC-SIGN） promoter. Methods Genomic DNA was extracted from peripheral blood. Complete DC-SIGN promoter and those without AP-1 or ETS-1 TFBS were amplified by PCR using designed primers. The PCR products were digested with MLu Ⅰ and Bgl Ⅱ and then ligated into MLu Ⅰ and Bgl Ⅱ digested pGL-3/Basic and pGL-3/Enhancer vectors. Transfection in Hacat and 293 cells was accomplished with Trans Fast liposome. Activity of luciferase was detected after 48 h. Results The DC-SIGN promoters without AP-1 or ETS-1 TFBS and the recombined pGL-3 vectors were correctly constructed. The activity of DC-SIGN promoters without AP-1 was reduced 20% （293） and 10% （Haeat）, which was 40%-50% with enhancer. The activity of DC-SIGN prorooters without ETS-1 was nearly vanished, no matter with or without enhancer. Conclusion ETS-I TFBS, not AP-1 TFBS, plays an important role in activity of DC-SIGN promoter.

关 键 词：HIV DC-SIGN 启动子 转录因子结合位点 报告质粒 

分 类 号：R686[医药卫生—骨科学]