人通用转录因子GTFIIF2表达与定位  

Human induced the expression of GTFIIF2 in BL21 and the localization in cells

作　　者：赵健[1] 耿慧武[1] 乔正[1] 李春雨[1] 李克娟[1] 刘晓颖[1] 范礼斌[1] 

机构地区：[1]安徽医科大学生命科学院生物学教研室,合肥230032

出　　处：《安徽医科大学学报》2014年第10期1382-1386,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金青年基金(编号:81201368);安徽省自然科学基金项目(编号:11040606M170;11040606M164);安徽省教育厅自然科学重点科研项目(编号:KJ2010A187)

摘　　要：目的构建不同标签的人通用转录因子IIF多肽2(GTFIIF2)表达载体,观察其细胞内的表达和定位。方法设计GTFIIF2的引物,以全长cDNA序列为模板,利用聚合酶链式反应技术扩增GTFIIF2全长序列,构建pcDNA3.1-FLAG-GTFIIF2和pCDGFP-GTFIIF2质粒,利用Western blot法和免疫荧光法检测其在细胞表达与定位;构建GST-GTFIIF2质粒,转化到BL21菌株,用IPTG诱导剂诱导融合蛋白表达,用SDS-PAGE分析诱导表达情况。结果免疫荧光法检测GTFIIF2蛋白集中分布在COS7细胞核中,在细胞质没有分布;pcDNA3.1-FLAG-GTFIIF2和pCDGFP-GTFIIF2质粒能够在HEK293T细胞中有效表达;GST-GTFIIF2在BL21菌株中很好的诱导表达。结论 GTFIIF2在COS7细胞、HEK293T细胞和BL21感受态细胞中能有效表达,为更好地了解GTFIIF2蛋白在细胞内的功能提供一定的研究基础。Objective To construct recombinant plasmids of GTFIIF2 to detect the expression and the expression and localization of GTIFIIF2 in cell. Methods The primers of GTFIIF2 were designed,GTFIIF2 was amplified by PCR with the template including the full length cDNA fragment of GTFIIF2,to construct pcDNA3. 1-FLAG-GTFIIF2 and pCDGFP-GTFIIF2 vectors,the exprassion and localization of GTFIIF2 were investigated by the method of West-ern blot and immunofluoresent assay. Vector GST-GTFIIF2 was transformed into Eicoli BL21 to observe the expres-sion of fusion protein in the induction of IPTG. Results The protein of GIFIIF2 was expressed efficiently in the nu-cleus of COS7 cells not in the cytoplasm;pcDNA3 . 1-FLAG-GTFIIF2 and pCDGFP-GTFIIF2 plasmid could be effec-tively expressed in HEK293T cells;GST-GTFIIF2 could also be induced in the BL21 strains. Conclusion GTFIIF2 can effectively express in COS7 cells,HEK293T cells and BL21 competent cells. The results provide a better un-derstanding of the basic reasearch of GTFIIF2 functions in cells.

关 键 词：GTFIIF2 基因表达 蛋白定位 BL21 

分 类 号：R341[医药卫生—基础医学] R394.2