干扰素调节因子3第二内含子中新启动子在人胚肾AD-293细胞中的功能特征

The functional characteristics of new promoter in the second intron of interferon regulatory factor-3

目的构建包含人干扰素调节因子3(IRF-3)基因第二内含子中新启动子的质粒,研究其功能特征。方法以人类基因组DNA为模板,PCR定向克隆和酶切亚克隆构建IRF-3基因第二内含子内新转录起始位点上游的新启动子基因重组体,并瞬时转染人胚肾AD-293细胞。荧光素酶检测报告基因启动子的活性,计算相对活性单位(RLU)。结果成功构建含有新启动子区的质粒,与pGL3-Basic组相比,新启动子活性明显增强,并定位于新转录起始位点前-317～-110bp,且该区域内存在环腺苷酸应答元件结合蛋白(CREB)和GATA等转录因子结合位点。结论 IRF-3第二内含子内新转录起始位点上游具启动子活性;在-317～-110bp区域存在必需调控元件,并且CREB和GATA等转录因子可能参与其转录调控。

Objective To construct the plasmid of a new promoter in the second intron of human interferon regulatory factor-3（IRF-3） and investigate its functional characteristics.MethodsThe 800 bp fragment in the second intron of the new transcription start site upstream 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.Transfection of AD-293 cells with the promoter-driven luciferase construction was performed to induce luciferase gene expression and the relative luciferase activity unit（RLU）was calculated.Results The plasmid of IRF-3 gene in the second intron promoter was successfully constructed. Compared with pGL3-Basic group,the activity of new promoter was increased and located in front of new transcription start sites between -317 bp and -110 bp,where included CREB and GATA transcription factor binding sites.Conclusion New transcription start site upstream of IRF-3 in the second intron has promoter activity. The necessary regulatory elements exist at -317 bp to -110 bp region.CREB and GATA transcription factors may be involved in this transcriptional regulation.