辐射诱导的野生型及突变型PTEN基因重组质粒的构建及鉴定  

Construction and identification of recombinant vectors with radiation-inducible wild-type PTEN and mutant PTEN

作　　者：张勇[1] 王丽[2] 王枫[1] 冯旭东[1] 张燕华[1] 简薇[1] 李荣清[1] 

机构地区：[1]昆明医科大学第一附属医院肿瘤放疗科,昆明650032 [2]成都军区昆明总医院病理科,昆明650032

出　　处：《军事医学》2013年第3期195-200,共6页Military Medical Sciences

基　　金：云南省社会发展科技计划(应用基础研究自筹经费)资助项目(2009ZC148M)

摘　　要：目的构建辐射诱导人野生型及突变型PTEN基因重组质粒并进行鉴定。方法以HeLa细胞总DNA为模板PCR扩增Egr-1基因启动子辐射敏感区,将PCR产物分别定向插入携带野生型PTEN和突变型PTEN蛋白编码基因的表达载体pEGFP-PTEN和pEGFP-PTEN-G129E启动子区,构建具有辐射诱导能力的重组表达质粒pEgr-PTEN和pEgr-PTEN-G129E。同时,将Egr-1基因启动子辐射敏感区亚克隆入pGL3-Promoter载体中,通过荧光素酶报告实验,验证不同照射剂量、照射后不同时间点下人肝癌SMMC-7721细胞中Egr-1启动子区对X线照射的辐射诱导能力。Western印迹检测X线照射对PTEN蛋白表达的影响。结果凝胶电泳分析证实,PCR扩增出Egr-1基因启动子辐射敏感区,构建出具有辐射诱导能力的重组表达质粒pEgr-PTEN和pEgr-PTEN-G129E,经不同酶切实验均证实含有与Egr-1辐射诱导区和PTEN基因相同片段大小的条带。荧光素酶报告实验证实,在不同时间点和不同照射剂量照射下,含Egr-1启动子区报告质粒细胞组的荧光素酶活性均明显高于转染空载体细胞,Western印迹检测表明,转染了pEgr-PTEN和pEgr-PTEN-G129E的细胞组在未接受照射时PTEN蛋白的表达量极低,但在接受8 Gy的X线照射后,PTEN蛋白的表达量明显提高,高于转染了pEGFP-PTEN和pEGFP-PTEN-G129E的细胞。结论成功构建了具有辐射诱导能力的含人野生型及突变型PTEN基因的重组质粒,为今后肿瘤的治疗奠定了基础。Objective To construct and identify the recomhinant vectors with radiation-inducible wild-type PTEN anti mutant PTEN. Methods The Egr-1 promoter was amplified by PCR from HeLa DNA and then inserted into the promoter of pEGFP-PTEN and pEGFP-PTEN-G129E （expressing the fulll-lengtb coding sequences of wild-type PTEN and mutant PTEN, respectively） to produce radiation-inducible pEgr-PTEN and pEgr-PTEN-G129E, respectively. Then the response of Egr-1 promoter to radiation treatment by luciferase report assay was evaluated. The expression of PTEN in different groups of SMMC-7721 cells was detected by Western blotting 24 hours after irradiation. Results The Egr-1 promoter was amplified and restriction analysis proved that the recombinant plasmids pEgr-PTEN and ,pEgr-PTEN-G129E were construc-ted. There was a significant increase in luciferase activity in the pGL3-Egr cells compared with the negative control when exposed to irradiation. PTEN was expressed more highly than in the non-irradiated cells in the pEgr-PTEN and pEgr-PTEN-G129E groups and that of the pEGFP-PTEN and pEGFP-PTEN-G129E groups afrer 8 Gy irradiation.Concluslon The radiation-inducible wild-type PTEN and mutant PTEN expression vectors have been successfully constructed.potentially conducive to the study of cancer therapy.

关 键 词：PTEN 辐射诱导 EGR-1 

分 类 号：R730.55[医药卫生—肿瘤]