干扰素基因刺激因子启动子的克隆、鉴定及活性分析

Construction,identification and promoter activity of stimulator of interferon genes

目的克隆和鉴定干扰素基因刺激因子(STING)基因上游启动子序列,并评价其在人胚肾HEK-293T细胞中的启动活性。方法以人全血DNA为模板,PCR扩增STING启动子区2154bp序列。将此片段亚克隆至pGL-3基本载体荧光素酶报告基因上游的多克隆位点,构建重组报告质粒pGL-3/STING。转染HEK-293T细胞,检测其荧光素酶活性,并利用生物信息学方法分析转录因子结合位点。结果经酶切、测序鉴定,成功构建了含有STING启动子序列的表达质粒。与pGL-3基本质粒相比,pGL-3/STING启动子的相对荧光素酶活性增加(P<0.01)。STING启动子区域含SRY、HSF、AP1、Sp1/3、环磷酸腺苷反应元件结合蛋白和GATA-1等多个转录因子结合序列。结论 STING转录起始位点上游2154bp区域在HEK-293T细胞中具有较强的启动活性。

Objective To clone and identify the promoter sequences of stimulator of interferon genes（STING）,and evaluate its activity in HEK-293 Tcells.Methods The 2154 bp fragment was amplified by PCR with human genomic DNA as a template and then subcloned into multiple cloning sites of pGL-3basic vector to construct the luciferase reporter plasmid pGL-3/STING.After pGL-3/STING was transfected into HEK-293 Tcells,the relative luciferase activity was detected and binding sites of transcriptional factors were analyzed by bioinformatics methods.Results Enzyme digestion and DNA sequencing verified that the recombinant plasmid containing promoter sequences of STING was successfully constructed.Compared with pGL-3basic vector,the relative luciferase activity was increased after transfection of pGL-3/STING in HEK-293Tcells（P0.01）.The binding sequences of SRY,HSF,AP1,Sp1/3,CREB and GATA-1 were included in the promoter region of STING.Conclusion The upstream region of transcriptional start site of STING has a strong promoter activity in HEK-293 Tcells.