CD2AP启动子的克隆和活性分析  

Construction of promoter CD2AP and analysis of its promoter activity in human embryonic kidney-293T cells

作　　者：顾曼丽[1] 卢春[2] 

机构地区：[1]南京医科大学第一附属医院河西分院妇保科,210036 [2]南京医科大学微生物学和免疫学系

出　　处：《江苏医药》2010年第17期2063-2065,共3页Jiangsu Medical Journal

摘　　要：目的构建表达CD2AP基因转录起始位点上游启动子的表达质粒,转染人类胚胎肾(HEK)-293T细胞,评价其启动子活性。方法以人全血细胞总DNA为模板,PCR扩增CD2AP转录起始位点上游2082bp的启动子区片段。亚克隆此片段至无启动子活性的pGL-3基本载体荧光素酶报告基因上游的多克隆位点,构建含CD2AP启动子的重组报告质粒。转染HEK-293T细胞,行荧光素酶活性检测,计算相对活性单位(RLU)。生物信息学分析转录因子结合位点。结果酶切,测序鉴定证实成功构建含有CD2AP基因转录起始位点上游2082bp的启动区的表达质粒。CD2AP的启动子与正常的pGL-3基本质粒比较,其RLU增加了74.8倍。其上游启动子区序列中含多个转录因子结合序列如AP1、Sp1、CREB和GATA-1等。结论 CD2AP转录起始位点上游序列在HEK-293T细胞中具有较强的启动活性。Objective To construct a luciferase reporter plasmid containing CD2AP human gene promoter and evaluate its activity in human embryonic kidney（HEK）-293T cells.Methods The 2082 bp fragment was amplified by PCR with human genomic DNA as a template and directionally cloned into pGL3-basic multiple cloning sites to construct the luciferase reporter plasmid pGL3-2k.Transfection of HEK-293T cells with the promoter-driven luciferase construct was performed to induce luciferase gene expression and to calculate the relative luciferase activity unit（RLU）.Promoter sequence of 2082 bp upstream of transcription initiation site of CD2AP was analyzed with TFSEARCH ver.1.3 software.Results DNA sequencing and restriction endonuclease analysis verified the successful construction of the plasmid pGL3-2k.This CD2AP promoter exhibited a strong promoter activity with an increase of 74.8-fold of RLU in HEK-293T cells when compared with pGL-3 basic vector.The transfection experiment confirmed that the levels of its activation were significantly higher than those in the controls in HEK-293T cells.Function analysis of CD2AP promoter disclosed several AP1,Sp1,CREB and GATA-1 sites in minimal promoter region.ConclusionThe plasmid pGL3-2k promoter has been successfully constructed and has a strong basal promoter activity in HEK-293T cells.

关 键 词：CD2相关蛋白 启动子 

分 类 号：R692[医药卫生—泌尿科学]