大鼠XAF1基因启动子荧光素酶报告质粒的构建及IRF-1结合位点的鉴定

Construction of rat XAF1 promoter and identification of its binding sequence with IRF-1

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 luciferase reporter plasmids of full-length and truncated promotors of rat X-linked inhibitor of apoptosis associated factor 1（XAF1） gene and detect their activity in HEK293 cells in response to interferon regulatory factor-1（IRF-1） overexpression,screening the possible binding sites for IRF-1.Methods： Rat XAF1 promoter（-1497 ～ ＋166 nt） was amplified by PCR and cloned into the luciferase reporter plasmid（pGL3-basic）.The recombinant plasmid（pGL3-XAF1-QC） and rat IRF-1 expression plasmid（pcDNA3.1-IRF-1） were co-transfected into HEK293 cells and then the luciferase activity was detected to comfirm the role of IRF-1 in XAF1 gene transcription.Meanwhile,the possible IRF-1 binding sites within XAF1 promoter were predicted by using bioinformatics software.Based on the predicted results,different luciferase reporter plasmids of truncated XAF1 gene promotor（pGL3-XAF1-1,pGL3-XAF1-2,pGL3-XAF1-3 and pGL3-XAF1-4） were constructed.The promoter luciferase reporter plasmids of pGL3-XAF1-QC or pGL3-XAF1-1,-2,-3,-4 and the plasmid of pcDNA3.1-IRF-1 were co-transfected into HEK293 cells.Then,the luciferase activity was detected to screen the IRF-1 binding sites.Results： It was verified that different kinds of plasmids were all constructed correctly by PCR analysis and nucleotide sequencing.The plasmids of pGL3-XAF1-QC and pcDNA3.1-IRF-1 were co-transfected into HEK293 cells,and then the luciferase activity was detected.The result showed that the transcriptional activity of XAF1 gene was increased markedly in response to IRF-1 overexpression.In addition,when the plasmids of pGL3-XAF1-QC or pGL3-XAF1-1,-2,-3,-4 and pcDNA3.1-IRF-1 were co-transfected into HEK293 cells,the activity of pGL3-XAF1-3 was much lower than that in pGL3-XAF1-1 and pGL3-XAF1-2,indicating that the region of rat XAF promoter（-337 ～-47 nt） might contain IRF-1 binding element.Conclusion： The rat full-length and truncated rat XAF1 promotor luciferase reporter plasmids were constructed successfully,and the IRF-1 binding region was found,which could be beneficial to further studies.