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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 a plasmid containing a new promoter in the second intron of human interferon regulatory factor 3 (IRF-3) gene and study its functional characteristics. Methods The human recombinant genomic DNA was cloned into the upstream of the new transcription initiation site of the IRF-3 gene and subcloned by restriction enzyme digestion. The recombinant plasmid was transiently transfected into human embryo kidney -293 cells. Luciferase reporter assay gene promoter activity, calculate the relative activity unit (RLU). Results The plasmid containing the new promoter region was successfully constructed. Compared with the pGL3-Basic group, the new promoter activity was significantly enhanced and located in the range of -317 ~ -110 bp before the site of new transcription initiation, and the presence of cyclic adenosine Acid response element binding protein (CREB) and GATA and other transcription factor binding sites. CONCLUSION: The upstream of the new transcription start site in IRF-3 has promoter activity. The necessary regulatory elements exist in the -317 ~ -110 bp region, and transcription factors such as CREB and GATA may be involved in transcriptional regulation.