摘要
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BamHⅠ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。
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＆amp;nbsp;both BamH Ⅰ endonuclese digestion and DNA sequencing. The Western blotting analysis confirmed that FLAG-fused 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.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2014年第6期1149-1154,共6页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金青年基金资助课题(81302240)
甘肃省科技计划项目资助课题(1308RJYA056)
兰州大学中央高校基本科研业务费项目资助课题(lzujbky-2013-134)
