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

作　　者：李祯[1] 生秀杰[1] 孙曼[1] 汪志辉[1] 刘启才[2] 

机构地区：[1]广州医科大学附属第三医院妇产科研究所(广东省普通高校生殖与遗传重点实验室),广东广州510150 [2]广州医科大学实验医学研究中心,广东广州510000

出　　处：《中国现代医学杂志》2013年第26期26-29,共4页China Journal of Modern Medicine

基　　金：广州市科技计划资助项目(No:2010GN-E00221)

摘　　要：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.

关 键 词：肝细胞生长因子受体 卵巢癌 启动子 

分 类 号：R737.31[医药卫生—肿瘤]