人eIF4A3真核表达载体构建及稳定表达细胞株筛选  

Construction of human eIF4A3 eukaryotic expression plasmid and screening of stable cell line

作　　者：李向云[1,2] 徐祥[2] 李秉煦[1] 黄宏[2] 徐云升[1] 

机构地区：[1]温州医学院附属第一医院皮肤科,浙江温州325000 [2]第三军医大学大坪医院野战外科研究所一室 创伤、烧伤与复合伤国家重点实验室,重庆400042

出　　处：《第三军医大学学报》2011年第18期1897-1899,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金(30972800)~~

摘　　要：目的构建人eIF4A3重组质粒载体,筛选高表达人eIF4A3的稳定细胞株。方法 RT-PCR克隆eIF4A3基因,将获得的基因片段分别连接到含有HA和c-myc标签的质粒载体上,转染HeLa细胞,经G418筛选获稳定表达细胞株,在体外翻译体系中进行体外翻译,通过免疫印迹鉴定eIF4A3的表达。结果成功构建人eIF4A3的真核表达载体,可在HeLa细胞中稳定表达,且能在体外翻译体系中翻译eIF4A3蛋白质。功能研究初步证明eIF4A3可以抑制荧光素酶报告基因的表达。结论获得了可稳定高表达人eIF4A3的HeLa细胞株及可用于体外翻译的真核表达载体。Objective To construct the human eIF4A3 recombinant vector and screen the stable cell line over-expressing human eIF4A3.Methods Human eIF4A3 gene was cloned by RT-PCR.Gene fragments were linked to HA and c-myc labeled plasmid vectors to transfect HeLa cells.A stable cell line was generated by screening G418.In vitro translation was conducted in an in vitro translation system.Expression of eIF4A3 was identified by Western blotting.Results The human eIF4A3 eukaryotic expression vector was successfully constructed,which was stably expressed in HeLa cells and could translate the eIF4A3 protein in an in vitro translation system.Preliminary function study showed that eIF4A3 could inhibit the expression of luciferase reporter gene.Conclusion A HeLa cell line over-expressing human eIF4A3 we generated can be used as an eukaryotic expression vector in an in vitro translation system.

关 键 词：真核起始因子4A3 稳定细胞株 体外翻译 

分 类 号：R341[医药卫生—基础医学] R394.33