干扰素调节因子-3启动子区序列分离、鉴定及在人类胚胎肾-293细胞中启动活性分析

Isolation and Identification of Promoter Sequence of Interferon Regulatory Factor-3 and Detection of Its Promotor Activity in Human Embryonic Kidney-293 Cells

目的构建表达干扰素调节因子-3(IRF-3)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(human embryonic kidney,HEK)-293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IRF-3转录起始位点上游1 000 bp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IRF-3启动子的重组报告质粒。转染HEK-293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IRF-3基因转录起始位点上游1 000 bp的启动区的表达质粒。IRF-3的启动子与正常的pGL-3基本质粒比较,其RLU增加了42.2倍。其上游启动子区序列中含多个转录因子结合序列如GATA-1、Sp1和E2F等。结论IRF-3转录起始位点上游序列在HEK-293细胞中具有较强的启动活性。

Objective To construct a luciferase reporter plasmid containing interferon regulatory factor 3 （ IRF - 3 ） human gene promoter and to evaluate promoter activity in human embryonic kidney（HEK） -293 cells. Methods The 1 000 bp fragment was amplified by PCR with human genomic DNA as a template and was directionally cloned into pGL3 - basic multiple cloning sites to construct the luciferase reporter plasmid pGL3 - pIRF - 3. Transfection of HEK - 293 cells with the promoter - driven lucife-rase construct was performed to induce luciferase gene expression and calculate the relative luciferase activity unit （RLU）. Promoter sequence of 1 000 bp upstream of transcription initiation site of IRF - 3 was analyzed by using Promoter 2.0 Prediction software. Results DNA sequencing and restriction endonuclease analysis verified the successful construction of the plasmid pGL3 - pIRF - 3. This IRF - 3 promoter exhibited a strong promoter activity with an increase of 42. 2 - fold of RLU in HEK - 293 cells when compared with pGL - 3 basic vector. The transfection experiment confirmed that the levels of its activation were significantly higher than that in controls in HEK - 293 cells. Function analysis of IRF - 3 promoter disclosed several GATA - 1 and specific protein 1 （ Sp1 ） sites and E2F in minimal promoter region. Conclusion The plasmid pGL3 - pIRF - 3 promoter is successfully constructed and has a strong basal promoter activity in HEK- 293 cells.