携带FLAG标签的人BTG2真核表达载体的构建及其在HeLa细胞中的表达 

目的：构建人B细胞易位基因2（BTG2）真核表达载体，并在HeLa细胞中表达携带FLAG标签的BTG2蛋白，为阐明BTG2基因的功能提供实验工具。方法：利用PCR法获得全长BTG2片段，将扩增得到的片段插入真核细胞表达载体pcDNA3.1(+)的多克隆位点；然后按照FLAG序列设计并合成寡核苷酸片段，插入pcDNA3.1(+)-BTG2载体，构建pcDNA3.1(+)-FLAG-BTG2载体；将以上重组质粒转染HeLa细胞，将细胞分为转染pcDNA3.1(+)空载体组、转染pcDNA3.1(+)-BTG2组和转染pcDNA3.1(+)-FLAG-BTG2组，采用抗FLAG抗体的Western blotting法，检测FLAG-BTG2融合蛋白在HeLa细胞中的表达水平。结果：将全长BTG2片段链接于pcDNA3.1(+)质粒，经限制性核酸内切酶BamH Ⅰ酶切分析及DNA测序证实载体序列准确；该载体转染HeLa细胞后用抗FLAG 抗体进行Western blotting法检测，在空载体组和pcDNA3.1(+)-BTG2组HeLa细胞中检测不到FLAG融合蛋白的表达，而在转染pcDNA3.1(+)-FLAG-BTG2组中检测到FLAG-BTG2融合蛋白的表达。结论：成功构建了pcDNA3.1(+)-FLAG-BTG2真核表达载体，并能在HeLa细胞中有效表达携带FLAG标签的BTG2蛋白。

Construction of human BTG2 eukaryotic expression vector with FLAG tag and its expression in HeLa cells

Objective To construct an eukaryotic expression vector of human B-cell translocation gene 2(BTG2),to express the FLAG-tagged BTG2 protein in HeLa cells,and to supply an experimental tool for investigating the function of BTG2 gene.Methods The full-length BTG2 fragment was obtained by PCR and inserted into the multiple cloning site of pcDNA3.1(+)vector.Oligo DNA encoding FLAG tag was designed and inserted into pcDNA3.1(+)-BTG2 to construct another vector pcDNA3.1(+)-FLAG-BTG2.The HeLa cells were divided into pcDNA3.1(+)empty vector group,pcDNA3.1(+)-BTG2 group and pcDNA3.1(+)-FLAG-BTG2 group.The HeLa cells were transfected with recombinant plasmids.Western blotting using anti-FLAG antibody was performed to detect the expression of FLAG-BTG2 protein in HeLa cells.Results The sequence of the vector was verified by both BamH Ⅰ endonuclese digestion and DNA sequencing.The Western blotting analysis confirmed that FLAGfused BTG2 was detected in pcDNA3.1(+)-FLAG-BTG2 group but not in empty vector or pcDNA3.1(+)-BTG2 groups.Conclusion The eukaryotic expression vector pcDNA3.1(+)-FLAG-BTG2 is successfully constructed and FLAG-tagged BTG2 protein is expressed in HeLa cells.