河南大学学报（医学版）
河南大學學報（醫學版）
하남대학학보（의학판）
JOURNAL OF HENAN UNIVERSITY (MEDICAL SCIENCE)
2015年
2期
111-114
,共4页
仲念念%朱伶俐%王旋%房娜
仲唸唸%硃伶俐%王鏇%房娜
중념념%주령리%왕선%방나
TAZ基因%重组质粒%HEK293细胞
TAZ基因%重組質粒%HEK293細胞
TAZ기인%중조질립%HEK293세포
TAZ gene%The recombinant plasmid%HEK293 cells
目的：构建重组质粒TAZ‐pcDNA31．及 TAZ‐pEGFP‐C2，并应用Western Blot检测TAZ蛋白在细胞内的表达情况，初步探索TAZ分子促进细胞增殖和迁移的作用机制。方法通过PCR扩增获得 TAZ基因片段，胶回收后连接T载体，蓝白斑筛选，转化，提质粒，酶切，用T4 DNA Ligase连接，亚克隆进入pEGFP‐C2和pcDNA31．获得新的重组质粒，分别转染 HEK293细胞，智能型荧光细胞监测仪观察TAZ分子在细胞内的分布情况，Western Blot检测其在细胞内的表达情况。结果重组质粒经双酶切鉴定和测序证明构建成功，荧光照片显示 TAZ分子分布在细胞核和细胞浆中，Western Blot检测结果表明转染有重组质粒TAZ‐pcDNA31．的HEK293细胞中有大量T AZ蛋白表达。结论成功构建了两种重组质粒，并初步验证了T AZ蛋白的细胞定位，为进一步研究其促进增殖和迁移的作用机制奠定了基础。
目的：構建重組質粒TAZ‐pcDNA31．及 TAZ‐pEGFP‐C2，併應用Western Blot檢測TAZ蛋白在細胞內的錶達情況，初步探索TAZ分子促進細胞增殖和遷移的作用機製。方法通過PCR擴增穫得 TAZ基因片段，膠迴收後連接T載體，藍白斑篩選，轉化，提質粒，酶切，用T4 DNA Ligase連接，亞剋隆進入pEGFP‐C2和pcDNA31．穫得新的重組質粒，分彆轉染 HEK293細胞，智能型熒光細胞鑑測儀觀察TAZ分子在細胞內的分佈情況，Western Blot檢測其在細胞內的錶達情況。結果重組質粒經雙酶切鑒定和測序證明構建成功，熒光照片顯示 TAZ分子分佈在細胞覈和細胞漿中，Western Blot檢測結果錶明轉染有重組質粒TAZ‐pcDNA31．的HEK293細胞中有大量T AZ蛋白錶達。結論成功構建瞭兩種重組質粒，併初步驗證瞭T AZ蛋白的細胞定位，為進一步研究其促進增殖和遷移的作用機製奠定瞭基礎。
목적：구건중조질립TAZ‐pcDNA31．급 TAZ‐pEGFP‐C2，병응용Western Blot검측TAZ단백재세포내적표체정황，초보탐색TAZ분자촉진세포증식화천이적작용궤제。방법통과PCR확증획득 TAZ기인편단，효회수후련접T재체，람백반사선，전화，제질립，매절，용T4 DNA Ligase련접，아극륭진입pEGFP‐C2화pcDNA31．획득신적중조질립，분별전염 HEK293세포，지능형형광세포감측의관찰TAZ분자재세포내적분포정황，Western Blot검측기재세포내적표체정황。결과중조질립경쌍매절감정화측서증명구건성공，형광조편현시 TAZ분자분포재세포핵화세포장중，Western Blot검측결과표명전염유중조질립TAZ‐pcDNA31．적HEK293세포중유대량T AZ단백표체。결론성공구건료량충중조질립，병초보험증료T AZ단백적세포정위，위진일보연구기촉진증식화천이적작용궤제전정료기출。
Objective Two recombinant plasmids , TAZ‐pcDNA3 .1 and TAZ‐pEGFP‐C2 , were established . The protein expression of TAZ in HEK293 cells was detected by Western Blot and the roles of TAZ in promoting cell proliferation and migration were further explored . Methods AZ gene was amplified by PCR , fragments were recovered followed by connection with glue T carrier , blue‐white screening , transformation and extraction of plasmid DNA . Then the plasmid DNA was digested , connected by T 4 DNA Ligase , and then sub‐cloned into pEGFP‐C2 and pcDNA3 .1 to construct new recombinant plasmids . These plasmids were transfected into HEK293 cells to observe the distribution of TAZ using a fluorescence detector . The protein expression was detected by Western Blot .Results By restriction enzyme identification and sequence analysis , the recombinant plasmids were successfully constructed . Fluorescent photos show that the distribution of TAZ molecule was in the nucleus and cytoplasm . Western Blot test results showed that TAZ molecule could induce over‐expression of specific proteins . Conclusion Two recombinant plasmids were successfully constructed . The effects of TAZ over‐expression were validated , which will lay a foundation for revealing the mechanism of TAZ in promoting cell proliferation and migration .
