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目的构建pEGFP-N1-Bex2真核表达质粒,并探索其在U251神经胶质瘤细胞中的表达。方法以正常人脑组织总RNA为模板,采用RT-PCR法获得Bex2的cDNA,并与pEGFP-N1载体连接,构建pEGFP-N1-Bex2真核表达质粒。对pEGFP-N1-Bex2重组质粒的菌液行PCR鉴定、酶切鉴定和测序鉴定。在U251细胞中用脂质体转染法转染pEGFP-N1-Bex2真核表达载体过表达Bex2,分别于转染后12、24、48、72h收集细胞,用Western blot检测Bex2基因的表达情况。结果真核表达质粒pEGFP-N1-Bex2经PCR及双酶切鉴定,均可见387bp的目的基因条带,并单倍正向克隆至质粒中。转染质粒pEGFP-N1-Bex2后荧光显微镜下观察U251细胞内有绿色荧光蛋白表达,Western blot检测见47KD和26KD分别有清晰的外源性Bex2-GFP融合蛋白和GFP蛋白的表达。pEGFP-N1-Bex2在转染U251细胞后12h即有表达,48h达到高峰。结论成功构建pEGFP-N1-Bex2真核表达质粒,瞬时转染后在U251细胞中顺利表达。
Objective To construct pEGFP-N1-Bex2 eukaryotic expression plasmid and explore its expression in U251 glioma cells. Methods The cDNA of Bex2 was obtained by RT-PCR using the total RNA of normal human brain as template. The cDNA of Bex2 was ligated with pEGFP-N1 vector to construct eukaryotic expression vector pEGFP-N1-Bex2. The pEGFP-N1-Bex2 recombinant plasmid was identified by PCR, enzyme digestion and sequencing. Bex2 was overexpressed in U251 cells transfected with pEGFP-N1-Bex2 eukaryotic expression vector by lipofection method. The cells were harvested at 12,24,48,72 h after transfection, and the expression of Bex2 gene was detected by Western blot . Results The eukaryotic expression plasmid pEGFP-N1-Bex2 was identified by PCR and double enzyme digestion. The 387bp gene band was observed and single positive cloned into the plasmid. The expression of green fluorescent protein in U251 cells was observed under a fluorescence microscope after transfection with plasmid pEGFP-N1-Bex2. Western blot showed that the expressed exogenous Bex2-GFP fusion protein and GFP protein were expressed at 47KD and 26KD, respectively. pEGFP-N1-Bex2 was expressed 12h after transfection into U251 cells and peaked at 48h. Conclusion The eukaryotic expression plasmid pEGFP-N1-Bex2 was constructed successfully and transiently transfected into U251 cells.