论文部分内容阅读
目的:构建大鼠Caspase 8基因启动子(全长和截短)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 regulatory factor-1,IRF-1)对Caspase 8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Caspase 8基因启动子序列(-1136~+101 nt),将Caspase 8基因启动子插入到荧光素酶报告基因载体pGL3-basic中.将Caspase 8基因启动子全长荧光素酶报告质粒(pGL3-Caspase 8-FL)和大鼠野生型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Caspase 8基因的启动作用.另用生物信息学软件预测Caspase 8基因启动子上IRF-1潜在的结合位点,并构建Caspase 8基因启动子截短的荧光素酶报告质粒(即pGL3-Caspase8-1~4).将上述Caspase 8基因启动子全长和各截短的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-Caspase8-FL和pcDNA3.1-IRF-1共转染HEK293细胞发现,Caspase 8基因启动子活性显著增加.而将pGL3-Caspase 8-FL、pGL3-Caspase 8-1~4和pcDNA3.1-IRF-1共转染HEK293细胞后证实,pGL3-Caspase 8-4的启动活性显著低于pGL3-Caspase 8-2和pGL3-Caspase 8-3.提示IRF-1可能结合在Caspase 8基因启动子的-336~-136 nt区域.结论:本实验成功构建了大鼠Caspase 8基因启动子全长及截短荧光素酶报告质粒,并初步筛查出IRF-1在Caspase 8基因启动子上的结合区域.“,”Objective:To construct luciferase reporter plasmids of full-length and truncated promotors of rat Caspase 8 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 Caspase 8 promoter(-1136~+101 nt) was amplified by PCR and cloned into the luciferase reporter plasmid(pGL3-basic).The recombinant plasmid(pGL3-Caspase 8-FL) and rat interferon regulatory factor-1(IRF-1) expression plasmid(pcDNA3.1-IRF-1) were co-transfected into HEK-293 cells and then the luciferase activity was detected to determine the role of IRF-1 in Caspase 8 gene transcription.Meanwhile,the potential IRF-1 binding sites within Caspase 8 promoter were predicted by bioinformatics software.Based on the predicted results,different luciferase reporter plasmids of truncated Caspase 8 gene promotor that named pGL3-Caspase 8-1~4 were constructed.The promoter luciferase reporter plasmids of pGL3-Caspase 8-FL or pGL3-Caspase 8-1~4 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-Caspase 8-FL and pcDNA3.1-IRF-1 were also co-transfected into HEK293 cells,and then the luciferase activity was detected.The results showed that the transcriptional activity of Caspase 8 gene was increased markedly in response to IRF-1 overexpression.In addition,the plasmids of pGL3-Caspase 8-FL or pGL3-Caspase 8-1~4 and pcDNA3.1-IRF-1 were co-transfected into HEK293 cells,and then the luciferase activity in different groups was determined.The result displayed that the activity of pGL3-Caspase 8-4 was much lower than that in pGL3-Caspase 8-2 and pGL3-Caspase 8-3,indicating that the region of rat Caspase 8 promoter(-336~-136 nt) might contain IRF-1 binding element.Conclusion:The rat full-length and truncated rat Caspase 8 promotor luciferase reporter plasmids were constructed successfully,and the IRF-1 binding region was identified.