Tec真核表达载体构建及其对细胞信号的影响  

Tec eukaryotic expression vector construction and its effect on cell signal transduction pathway

作　　者：李菲菲[1] 刘雷[2] 郑红[1] 周颖 余科科 程洁 王本忠[2] 汪思应[1] 

机构地区：[1]安徽医科大学基础医学院病理生理教研室,合肥230032 [2]安徽医科大学第一附属医院普外科,合肥230022

出　　处：《安徽医科大学学报》2005年第3期206-210,共5页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金(编号:00044202;01043709);安徽省人才开发基金(编号:2002Z035)

摘　　要：目的构建人Tec酪氨酸蛋白激酶真核表达载体;研究Tec参与肝细胞再生的可能信号途径。方法经PCR扩增、酶切、连接等基因重组技术将人Tec插入pCDNA3.1(hismycTec)真核表达载体中,并经酶切、PCR、测序鉴定;用脂质体法将构建的真核表达载体瞬时转染Hela细胞,用Western方法检测Tec蛋白是否表达。用荧光素酶报告基因系统,检测Tec是否参与HGF刺激信号途径的活化。结果构建的Tec真核表达载体使Hela细胞高表达Tec蛋白;并发现Tec在HGF介导下,对Elk信号分子活化的报告质粒中荧光素酶的表达有明显增强作用。结论构建了Tec真核表达载体,并能使HGF介导的MAPK途径活化,提示Tec可能参与HGF介导肝细胞增殖的信号调控。Objective To construct eukaryotic expression vector of Tec tyrosine protein kinase, and explore Tec involvement in liver rege neration signal transduction. Methods Human TeccDNA was subclo ned into eukaryotic (expression) vector pCDNA 3.1 flagged with ( his-myc.) The vector was identified by restriction (endonuclease,) ( PCR,) sequencing methods. The vector was then transfected transiently into Hela cell by Lipofectamine. The expression of Tecprotein was detected by western blot in the transfected cells line. Results Abundant Tec protein expressed in Hela cell transfeced with pcDNA 3.1(his-myc-Tec) vector. The luci ferase activation in WB-F344 cell transfected with Elk signal molecular was promoted by Tec with HGF simulation. Conclusion We successfully co nstruct human Tec eukaryotic expression vector. The vector may activate MAPK sig nal pathway via HGF. Tecmay be involved in cell proliferation signal pathway me diated by HGF.

关 键 词：真核表达载体构建 TEC 细胞信号 Hela细胞 酪氨酸蛋白激酶 Western pcDNA3 基因重组技术 MAPK途径 信号途径 荧光素酶 肝细胞再生 PCR扩增 肝细胞增殖 HGF 瞬时转染 脂质体法 基因系统 增强作用 信号分子 信号调控 c蛋白 

分 类 号：R780.2[医药卫生—口腔医学] R329.453[医药卫生—临床医学]