grp78真核表达载体构建及其稳定转染HeLa细胞系建立  

Establishment of Eukaryotic Expression Vector of Human grp78 and Its Stable Expression in HeLa Cell Line

作　　者：王恬[1] 陶涛[1] 杨润峰[1] 吴章颖[1] 李伟[1] 廖书杰[1] 王世宣[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院妇产科妇科肿瘤实验室,武汉市430030

出　　处：《医学分子生物学杂志》2011年第4期309-312,共4页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.81001152,81071663,30901586）

摘　　要：目的构建人grp78基因真核表达载体，并建立稳定高表达grp78的人宫颈癌HeLa细胞系。方法用RT—PCR方法从人宫颈癌HeLa细胞中扩增grp78基因编码区，将PCR产物克隆到pcDNA3．1（＋）真核表达载体，构建重组质粒pcDNA3．1（＋）／grp78并测序鉴定。用构建成功的pcDNA3．1（＋）／grp78真核表达载体转染入人宫颈癌HeLa细胞，经G418筛选获得grp78稳定高表达的HeLa细胞系，并用RT—PCR及Western印迹方法鉴定。结果成功构建pcDNA3．1（＋）／grp78真核表达载体，筛选获得稳定高表达人grp78的HeLa细胞系。结论grp78真核表达载体的成功构建和稳定高表达grpTS的HeLa细胞系的建立为进一步研究grp78的功能奠定了基础。Objective To construct eukaryotic expression vector of human grp78 and establish its stable transfected HeLa cell line. Methods The CDS of grp78 was amplified from HeLa cell line by RT-PCR and cloned into pcDNA3.1 （ ＋ ） . The recombinant plasmid pcDNA3.1 （ ＋ ） /grp78 was sequenced. HeLa cells were transfeeted with pcDNA3.1 （ ＋ ） /grp78 and selected with G418. The expression of grp78 was identified by RT-PCR and Western Blot. Results The eukaryot- ic expression plasmid of pcDNA3.1 （ ＋ ） /grp78 was successfully constructed and grp78 stably ex- pressing HeLa cell line was established. Conclusion The recombinant eukaryotic expression vector pcDNA3.1 （ ＋ ） /grp78 and the established Hela cell line stably expressing grp78 may be used for further study of grp78 function in vitro.

关 键 词：人grp78基因 载体构建 转染 基因表达 

分 类 号：R349.64[医药卫生—基础医学]