真核表达质粒pEGFP-N1-Bex2的构建及其在U251细胞中的表达

Construction of eukaryotic expression vector pEGFP-N1-Bex2and its expression in U251glioma cells

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 the eukaryotic expression vector pEGFP-N1-Bex2 and its expression in U251 glioma cells. Methods The Bex2 cDNA was obtained from human brain tissue by RT-PCR and cloned into pEGFP-N1 vector at the BamH I/EcoR I site. The eukaryotic expression vector pEGFP-NI-Bex2 was identified by PCR, enzyme digestion and sequencing. The pEGFP-N1- Bex2 constructed was transfected into U251 glioma cells by lipofectamine 2000. The expression of Bex2-GFP was tested by Western blot analysis at 12,24,48 and 72 h after transfection. Results The target gene band at a length of about 387 bp was observed in recombinant plasmid pEGFP-N1-Bex2 by PCR and restriction analysis and cloned into vector correctly. The green fluorescence of expressed Bex2 was observed under fluorescence microscope. The expressions of Bex2-GFP and GFP were tested at 47 KD and 26 KD bands using Western blot analysis. The exogenous Bex2 gene expression was detected and reached the peak level at 48 h after transfection. Conclusion The eukaryotic expression vector pEGFP-N1-Bex2 has been constructed successfully, which can be expressed in U251 glioma cells.