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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 an expression plasmid expressing upstream promoter of transcriptional initiation site of interferon regulatory factor-3 (IRF-3) gene and then transfect it into human embryonic kidney (HEK) -293 cells to evaluate its promoter activity. Methods The total DNA of human whole blood cells was used as a template to amplify a 1 000 bp promoter region upstream of the IRF-3 transcriptional start site. The fragment was subcloned into the multiple cloning site upstream of the pGL-3 basic vector luciferase reporter gene without promoter activity to construct a recombinant reporter plasmid containing the IRF-3 promoter. HEK-293 cells were transfected with luciferase activity assay to calculate relative activity units (RLU). Bioinformatics analysis of transcription factor binding sites. Results Restriction analysis and sequencing confirmed that the expression plasmid containing the promoter region of 1 000 bp upstream of the transcription start site of IRF-3 gene was successfully constructed. Compared with the normal pGL-3 basic plasmid, the promoter of IRF-3 increased the RLU by 42.2-fold. Its upstream promoter region contains multiple transcription factor binding sequences such as GATA-1, Sp1 and E2F. Conclusion The upstream sequence of IRF-3 transcription initiation site has strong priming activity in HEK-293 cells.