CAJ | 학술논문

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-GTFI-IF2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。
목적구건불동표첨적인통용전록인자 IIF다태2(GTFIIF2)표체재체，관찰기세포내적표체화정위。방법설계GTFIIF2적인물，이전장cDNA서렬위모판，이용취합매련식반응기술확증 GTFIIF2전장서렬，구건 pcDNA3.1-FLAG-GTFIIF2화 pCDGFP-GTFIIF2질립，이용 Western blot법화면역형광법검측기재세포표체여정위；구건GST-GTFI-IF2질립，전화도BL21균주，용IPTG유도제유도융합단백표체，용SDS-PAGE분석유도표체정황。결과면역형광법검측GTFIIF2단백집중분포재COS7세포핵중，재세포질몰유분포；pcDNA3.1-FLAG-GTFIIF2화pCDGFP-GTFIIF2질립능구재HEK293T세포중유효표체；GST-GTFIIF2재BL21균주중흔호적유도표체。결론 GTFIIF2재 COS7세포、HEK293T세포화BL21감수태세포중능유효표체，위경호지료해GTFIIF2단백재세포내적공능제공일정적연구기출。
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.