N端规则泛素E3连接酶UBR2真核表达载体的构建及验证  

Construction of Eukaryotic Expression Vector of N-end Rule Ubiquit-in Ligase UBR2 and Its Identification

作　　者：刘承波[1] 赵晗 黄慧娟 刘靖 朱彩芬 冯云[1] 

机构地区：[1]云南省第一人民医院(昆明理工大学附属医院),昆明市650032

出　　处：《医学分子生物学杂志》2017年第6期323-326,共4页Journal of Medical Molecular Biology

基　　金：资助项目：云南省卫生科技计划项目（No.2014NS193,2014NS1942014NS195,2014NS196）,云南省科技惠民计划项目（No.2014RA019）,云南省创新团队项目（No.2017HC009）

摘　　要：目的 构建N-端规则泛素E3连接酶UBR2过表达载体,并检测它在HepG2细胞中的过表达效率.方法 通过提取HepG2细胞总RNA, RT-PCR扩增UBR2基因片段,插入到pEGFP-N2载体中,构建真核表达载体pEGFP-N2-UBR2,并转染HepG2细胞,转染后利用荧光显微镜,Western印迹检测UBR2的表达情况.结果 经双酶切和测序鉴定真核表达质粒pEGFP-N2-UBR2构建正确;转染后24 h,利用荧光显微镜观察,在转染pEGFP-N2-UBR2的HepG2细胞中观察到绿色荧光蛋白,在蛋白水平可以检测到UBR2蛋白过表达.结论 成功构建pEGFP-N2-UBR2过表达载体,并在HepG2细胞中验证其过表达效率.为今后研究其在细胞生长和增殖过程中的作用奠定基础.Objective To construct the eukaryotic expression vector of N-end rule ubiquitin ligase UBR2 and detect its expression efficiency in HepG2 cells.Method Total RNA was extracted from HepG2 cells, and then UBR2 gene was amplified by RT-PCR and inserted into the eukaryotic expression vector pEGFP-N2.The recombinant plasmid pEGFP-N2-UBR2 was transfected into HepG2 cells.The expression of UBR2 was observed by fluorescent microscopy and Western blot-ting.Results The eukaryotic vector pEGFP-N2-UBR2 was successfully constructed, which was confirmed by double enzyme digestion and sequencing.Fluorescent microscopy showed green fluores-cent protein in HepG2 cells transfected with pEGFP-N2-UBR2.UBR2 was found to overexpress at the protein level in cells.Conclusion The expression vector pEGFP-N2-UBR2 was successfully constructed and its efficiency to overexpress UBR2 was verified in HepG2 cells, which lays the foundation of the studies on its role in cell growth and proliferation.

关 键 词：N-端规则泛素E3连接酶 UBR2 过表达 

分 类 号：R345[医药卫生—基础医学]