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目的:构建大鼠X染色体连锁的凋亡抑制蛋白相关因子1(X-linked inhibitor of apoptosis associated factor 1,XAF1)基因启动子(全长和截断)荧光素酶报告质粒,并观察在人胚肾细胞HEK293中过表达干扰素调节因子-1(interferon regulatoryfactor-1,IRF-1)对XAF1基因启动活性的影响,同时,筛选其可能的IRF-1结合位点。方法:采用PCR技术,扩增出大鼠XAF1基因启动子序列(-1497～+166 nt),将XAF1基因启动子插入荧光素酶报告基因载体pGL3-basic中获得pGL3-XAF1-QC,与大鼠野生型IRF-1表达质粒(pcDNA3.1-IRF-1)共转染HEK293细胞,检测其荧光素酶活性,确定IRF-1对XAF1基因的启动作用。同时,应用生物信息学软件预测XAF1基因启动子上IRF-1潜在的结合位点,并构建截断的XAF1基因启动子荧光素酶报告质粒(pGL3-XAF1-1、pGL3-XAF1-2、pGL3-XAF1-3和pGL3-XAF1-4)。将上述全长和各截断的XAF1基因启动子荧光素酶报告质粒和IRF-1过表达质粒共转染HEK293细胞,再行荧光素酶活性测定,筛选IRF-1的结合位点。结果:菌液PCR及核酸测序证实,上述荧光素酶报告质粒均构建成功。将pGL3-XAF1-QC和pcDNA3.1-IRF-1共转染HEK293细胞发现,XAF1基因启动子活性显著增加。而将pGL3-XAF1-QC、pGL3-XAF1(1～4号)和pcDNA3.1-IRF-1共转染HEK293细胞后证实,pGL3-XAF1-3的启动活性显著低于pGL3-XAF1-1和pGL3-XAF1-2。提示IRF-1可能结合在大鼠XAF1基因启动子的-337～-47 nt区域。结论:本实验成功构建了大鼠全长及截断的XAF1基因启动子荧光素酶报告质粒,并初步筛查出IRF-1在XAF1基因启动子上的结合区域,为后续研究奠定了基础。
OBJECTIVE: To construct a luciferase reporter plasmid containing X-linked inhibitor of apoptosis associated factor 1 (XAF1) gene linked to X chromosome in human embryonic kidney The effect of interferon regulatory factor-1 (IRF-1) overexpression on the activation of XAF1 gene in HEK293 cells was studied. Meanwhile, the possible IRF-1 binding sites were screened. Methods: The promoter region of XAF1 gene (-1497 ~ + 166 nt) was amplified by PCR. The promoter of XAF1 gene was inserted into luciferase reporter gene vector pGL3-basic to obtain pGL3-XAF1-QC, The wild-type IRF-1 expression plasmid (pcDNA3.1-IRF-1) was co-transfected into HEK293 cells to detect the luciferase activity and determine the promoter of XAF1 by IRF-1. At the same time, bioinformatics software was used to predict the potential binding sites of IRF-1 on the promoter of XAF1 gene and to construct a truncated XAF1 promoter luciferase reporter plasmid (pGL3-XAF1-1, pGL3-XAF1-2, pGL3- XAF1-3 and pGL3-XAF1-4). The full-length and each truncated XAF1 promoter luciferase reporter plasmid and IRF-1 overexpression plasmid co-transfected HEK293 cells, and then determination of luciferase activity screening IRF-1 binding sites. Results: Bacterial PCR and nucleic acid sequencing confirmed that the luciferase reporter plasmids were constructed successfully. Co-transfection of pGL3-XAF1-QC and pcDNA3.1-IRF-1 into HEK293 cells showed that the promoter activity of XAF1 gene was significantly increased. The co-transfection of pGL3-XAF1-QC, pGL3-XAF1 (1-4) and pcDNA3.1-IRF-1 into HEK293 cells confirmed that the promoter activity of pGL3-XAF1-3 was significantly lower than that of pGL3-XAF1-1 pGL3-XAF1-2. It is suggested that IRF-1 may bind to the -337 ~ -47 nt region of rat XAF1 gene promoter. Conclusion: The full-length and truncated XAF1 gene promoter luciferase reporter plasmid was successfully constructed in this experiment and the binding region of IRF-1 promoter on XAF1 gene was screened out initially, which laid the foundation for further study.