pEGFP-prohibitin真核表达重组质粒的构建及其在人肝癌细胞系Hep G2中的表达

肝脏 ›› 2016, Vol. 21 ›› Issue (1): 34-38.

pEGFP-prohibitin真核表达重组质粒的构建及其在人肝癌细胞系Hep G2中的表达

翟嵩, 孙明珠, 王文俊, 李亚萍, 张欣, 李梅党, 双锁

710004 西安交通大学医学院第二附属医院感染科

收稿日期:2015-07-07 发布日期:2020-06-01
通讯作者: 党双锁,Email：dang212@126.com
基金资助:
国家自然科学基金资助项目（81170393）

Establish ment and expression of recom binant pEGFP-prohibitin plasmid in HepG2 cell line

ZHAI Song, SUN Ming-zhu, WANG Wen-jun, LI Ya-ping, ZHANG Xin, LI Mei, DANG Shuang-suo

Department of infectious diseases,thesecond affiliated hospital of medicalschool of Xi’an Jiaotong University,Xi’an 710004,China

Received:2015-07-07 Published:2020-06-01
Contact: DANGShuang-suo,Email：dang212@126.com

摘要/Abstract

摘要： 目的构建pEGFP-prohibitin真核表达质粒,并鉴定其在pEGFP-prohibitin转染的人肝癌细胞系Hep G2中的表达。方法采用高保真PCR扩增获得prohibitin的全长编码序列,构建真核表达载体pEGFP-prohibitin,并通过基因测序和BLAST比对鉴定重组质粒;提取pEGFP-prohibitin和pEGFP-N1质粒,并通过TurboFect转染试剂转染Hep G2细胞;采用实时PCR和Western印迹方法分别在MRNA水平和蛋白质水平检测prohibitin蛋白在转染后Hep G2细胞中的表达。结果成功构建pEGFP-prohibitin真核表达重组质粒,并将其成功转染Hep G2细胞;pEGFP-prohibitin转染组细胞prohibitin的表达量比空载转染组和未转染组细胞明显增加。结论成功构建真核表达载体pEGFP-prohibitin,并证明抗增殖蛋白prohibitin在pEGFP-prohibitin转染人肝癌细胞系Hep G2中高表达。

关键词: prohibitin蛋白, 基因表达, 重组质粒, 转染

Abstract: Objective To construct eukaryotic expression plasmid of enhanced green fluorescent protein plasmids （pEGFP）prohibitin,and to observe its expression in transfected Hep G2 cells. Methods The full coding sequence of prohibitin was am plified by high fidelity poly merase chain reaction（PCR）. Mean w hile,the eukaryotic expression vector of pEGFP-prohibitin was constructed and identified by gene sequencing and basic local align ment search tool（BLAST）. Plasmids of pEGFP-prohibitin and pEGFP-N1 were extracted and transfected into Hep G2 cells by TurboFect oligofectamine reagent,expression of which in transfected Hep G2 cells was assessed by real time PCRat MRNAlevel and western blot at protein level. Results Recombinant pEGFP-prohibitin plasmid was successfully constructed then transfected into Hep G2 cells,prohibitin expression in which was apparently higher than that in Mock-vehicle and non-transfection groups. Conclusion The eukaryotic expression vector of pEGFP-prohibitin was constructed successfully,and prohibitin was confirmed to be up-regulated in Hep G2/pEGFP-prohibitin cells.

Key words: Prohibitin, Gene expression, Recombinant plasmid, Transfection

翟嵩, 孙明珠, 王文俊, 李亚萍, 张欣, 李梅党, 双锁. pEGFP-prohibitin真核表达重组质粒的构建及其在人肝癌细胞系Hep G2中的表达[J]. 肝脏, 2016, 21(1): 34-38.

ZHAI Song, SUN Ming-zhu, WANG Wen-jun, LI Ya-ping, ZHANG Xin, LI Mei, DANG Shuang-suo. Establish ment and expression of recom binant pEGFP-prohibitin plasmid in HepG2 cell line[J]. Chinese Hepatolgy, 2016, 21(1): 34-38.

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