人肝细胞生长因子受体启动子荧光素酶表达载体的构建及活性测定

Construction of luciferase expression vector containing C-MET and characterization of the promoter activity

目的构建含人肝细胞生长因子受体(C-MET)启动子-223^+60区域的荧光素酶报告基因质粒,并测定其在卵巢癌细胞株中的活性。方法应用PCR法扩增获得含有人C-MET基因启动子-223^+60区域的DNA片段,将其连接至荧光素酶质粒pGL3-Basic中,得到pGL3-C-MET-Promoter重组质粒;经限制性内切酶酶切及测序得以鉴定;将该质粒转染4株卵巢癌细胞,检测细胞中荧光素酶的活性。结果双酶切及测序结果证实pGL3-C-MET-Promoter重组质粒插入片段序列正确,克隆的C-MET基因片段具有启动子活性,在卵巢癌细胞株OVCAR3细胞中的活性最高。结论成功构建了pGL3-C-MET-Promoter报告基因重组质粒,为进一步研究C-MET对卵巢癌生物学行为的影响奠定了基础。

[Objectives] To make a luciferase reporter gene construct with promoter region -223～ ＋60 from human C-MET gene, and to determine its activity in the ovarian cancer cell lines. [Methods] The - 223 ～＋60 region of C-MET promoter was amplified by polymerase chain reaction （PCR） and recombinated into pGL3-Basic vector. The recombinated plasmid pGL3-C-MET-Promoter was confirmed by restriction analysis and sequencing. Transcient transfected pGL3-C-MET-Promoter into four ovarian cancer cell lines and detected its activity. [Results] The results showed that the sequence of cloned promoter was right, the promoter construct has higher activity in OVCAR3 cells compared to others. [Conclusion] The luciferase reporter gene vector containing C-MET promoter has been successfully constructed and it establishes the foundation to future research on the influence to the biology behavious of ovarian cancer.