穿孔素基因启动子的克隆及体外区域性高甲基化

目的 克隆穿孔素基因启动子区域(PRF1),并对PRF1进行体外区域性高甲基化,为探讨穿孔素基因调控序列高甲基化能否引起穿孔素表达下调奠定基础.方法 PCR扩增穿孔素基因启动子区域(PRF1)后,先将PRF1克隆到T载体,再定向亚克隆至报告基因栽体;然后对PRF1片段进行体外区域性高甲基化.结果 (1)克隆PRF1到报告基因载体pGL3-Basic后双酶切及测序鉴定结果正确.(2)对PRF1片段进行体外区域性高甲基化后鉴定显示PRF1片段区域性高甲基化完全.结论 成功克隆PRF1至报告基因载体pGL3-Basic并对PRF1片段进行体外区域性高甲基化,为今后研究奠定了基础.

Construction of a reporter gene vector and methylation of perforin promoter in vitro

Objective To construct a luciferase reporter gene vector of perforin promoter and methylate it in vitro. Methods The promoter of the human peffofin was amplified by PCR, cloned into pMD18-T vector and subcloned into pGL3-Basie vector, and then con-finned by restriction mapping and DNA sequencing. The regions of interest were excised with the appropriate restriction endonucleases, then it were methylated with methylase Sss I(M. SssI)and S-adenosymethioine(SAM), and methylation was confirmed by digestion with appropri-ate methylation sensitive enzyme AciI and agrose gel electrophoresis, and then the fragment was relegated back into the promoter-reporter constructor pGL3-Basic. Results The result of DNA sequencing showed that the sequence of cloned promoter was right. The result of diges-tion methylation with appropriate methylation sensitive enzyme showed that perforin promoter was completely methylated. Conclusion The promoter of perforin was successfully cloned and completely methylated in vitro, which provided an important basis for the study of transfec-tion.