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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 recombinant plasmids of wild-type and mutant PTEN induced by radiation. Methods The total RNA of HeLa cells was used as a template to amplify the radiation sensitive region of Egr-1 promoter. The PCR products were inserted into the expression vectors pEGFP-PTEN and pEGFP-PTEN-G129E carrying wild-type PTEN and mutant PTEN protein respectively Promoter region to construct recombinant expression plasmids pEgr-PTEN and pEgr-PTEN-G129E with radiation-induced ability. At the same time, the promoter region of Egr-1 promoter was subcloned into pGL3-Promoter vector and luciferase reporter assay was used to verify Egr-1 activation in human hepatocellular carcinoma SMMC-7721 cells at different irradiation doses at different time points Radiation-induced ability of sub-regions to X-ray irradiation. Effect of X - ray irradiation on the expression of PTEN protein by Western blotting. Results The gel electrophoresis analysis confirmed that the recombinant plasmid pEgr-PTEN and pEgr-PTEN-G129E with the ability of inducing radiation were constructed by PCR amplification of the radiation-sensitive region of Egr-1 promoter. Egr-1 radiation-induced region and PTEN gene the same fragment size of the band. Luciferase reporter assay confirmed that, at different time points and different irradiation doses, the Egr-1 promoter region reported plasmid cell group luciferase activity was significantly higher than transfected empty vector cells, Western blot detection showed that the transfer PTEN protein expression was very low in the cells transfected with pEgr-PTEN and pEgr-PTEN-G129E, but the expression of PTEN protein was significantly higher than that of the transfected cells Cells of pEGFP-PTEN and pEGFP-PTEN-G129E were obtained. Conclusion The recombinant plasmids containing wild-type and mutant PTEN gene with radiation-induced ability were successfully constructed, which laid the foundation for the future treatment of tumors.