人白介素-15基因系列启动子克隆及其荧光素酶报告基因载体的构建

Construction of human IL-15 gene promoter luciferase reporter gene vectors

目的克隆人白介素-15(IL-15)基因系列启动子,构建IL-15基因启动子荧光素酶报告基因载体。方法通过PCR方法从HaCaT细胞基因组DNA中获得IL-15基因编码序列5′上游七段逐步缺失的IL-15启动子序列;双酶切后,分别重组到含萤火虫荧光素酶的报告基因pGL3-Basic载体上,构建IL-15基因系列启动子报告基因载体;用脂质体转染法将含最长启动子序列的报告基因载体转染至HaCaT细胞,并设置空白对照组和LPS组分别处理;24 h后采用双荧光素酶报告基因系统检测其启动子活性。结果经PCR方法扩增出七段逐步缺失的IL-15基因启动子片段,PCR及双酶切鉴定各重组体构建正确;瞬时转染HaCaT细胞后经报告基因检测得知所克隆最长序列具有启动子活性。结论成功构建了IL-15系列启动子报告基因载体,并在HaCaT细胞中初步活性分析得知所克隆IL-15基因调控系列内包含启动子核心区域,为进一步研究IL-15表达调控机制奠定了实验基础。

Purpose To clone series of the human IL-15 gene promoter and to construct seven luciferase reporter gene vectors containing IL-15 promoter regions.Methods Seven DNA fragments of the 5′ flanking region of human IL-15 gene were isolated from genomic DNA of HaCaT cells by polymerase chain reaction（PCR）.After being digested with restriction enzymes,IL-15 gene family promoters were inserted into the luciferase reporter vectors PGL3-Basic.Then the recombinant construct which contains the largest IL-15 promoter region was transiently transfected into HaCaT cells.6 hours later,cells were respectively treated with DMEM,LPS.Then the activity of luciferase was detected.Results Seven-fragments of IL-15 gene promoter were amplified by PCR,and the identification of PCR and double digestion of recombined plasmid was completely correct.The experiment of transient transfection showed that the largest IL-15 promoter region has the significant activity.Conclusion The human IL-15 gene promoter luciferase reporter gene vectors have been constructed successfully.The analysis of activity in HaCaT cells indicated that the cloned IL-15 gene family promoters contain the key cis-regulatory elements.It will be essential to further study the regulation of IL-15 expression.