耐辐射奇球菌pprM基因在真核细胞中的表达

目的扩增耐辐射奇球菌辐射抗性基因pprM，构建pEGFP-C1-pprM重组载体，转入293T细胞并表达PprM蛋白，为研究原核细胞辐射抗性基因pprM是否提高真核细胞的辐射抗性及其可能存在的作用机制奠定实验基础。方法以无任何突变的pGEX-6p-1-pprM重组质粒为模版，设计引物PCR扩增pprM基因，利用琼脂糖凝胶DNA回收试剂盒纯化回收目的片段，EcoR Ⅰ、BamH Ⅰ双酶切回收的目的片段及质粒pEGFP-C1，将双酶切产物进行连接，连接产物转化至大肠杆菌JM109感受态细胞后涂布于含卡那霉素（Kan）抗性的Luria-Bertani（LB）固体培养基上进行筛选，所筛选的阳性克隆进一步用菌落PCR，EcoR Ⅰ、BamH Ⅰ双酶切及测序鉴定。通过Lipofectamine2000转染试剂将pEGFP-C1-pprM重组质粒转入293T细胞，倒置荧光显微镜观察绿色荧光融合蛋白的表达。裂解细胞抽提蛋白，Western blot进一步验证PprM蛋白的表达。结果菌落PCR结果及双酶切结果显示，在400 bp左右处有一条明显的目的条带，测序结果显示碱基序列与原基因序列一致，载体构建成功。荧光拍照结果显示，pEGFP-C1-pprM重组质粒成功转染293T细胞并表达绿色荧光融合蛋白；Western blot结果显示在40×103处有融合蛋白表达。结论笔者成功将所构pEGFP-C1-pprM重组质粒转入293T细胞，并表达相应蛋白，为后续实验研究原核基因pprM及其产物对真核细胞辐射抗性的影响奠定了良好的实验基础。

Expression of pprM gene from Deinococcus radiodurans in eukaryotic cells

Objective To enhance the radiation resistance of eukaryotic cells and to identify the mechanisms that underlie radiation resistance,the radioresistant pprM gene was amplified from Deinococcus radiodurans and used to construct a recombinant pEGFP-CI-pprM plasmid for the expression of PprM protein in 293T cells.Methods The pprM gene was amplified from pGEX-6p-l-pprM via PCR and purified from agarose gel with a DNA recovery kit.The purified PCR product was digested with EcoR I and BamH I and ligated into the pEGFP-C1 plasmid.Recombinant plasmids were transfected into competent JM109 cells,which were then cultured on LB solid medium that contained kanamycin.Positive clones were identified and characterized via bacterial colony PCR,restriction enzyme digestion,and sequencing analysis.Lipofectamine 2000 reagent was used to transfect pEGFP-C1-pprM plasmids into 293T cells.Green fluorescent fusion protein was observed via fluorescence microscopy and identified by Western blot.Results Bacterial colony PCR and double digestion showed that the target band is approximately 400 bp in length.Sequencing results showed that the base sequence was identical to the original gene sequence,thus indicating the successful construction of the recombinant plasmid.Fluorescence photography results showed that pEGFP-C 1-pprM plasmids were successfully transfected into 293T cells.Western blot results showed that fusion protein is approximately 40×103 in weight.Conelusions The pEGFP-C1-pprM plasmid was transfected into 293T cells,which then successfully expressed PprM protein.This study provides the foundation for future research on the pprM gene and the effects of its products on the radiation resistance of 293T cells.