稳定表达HBx的人肝细胞系7702的建立  

Construction and selection of normal human liver 7702 cells stably expressing HBx in vitro

作　　者：庞丽君 石英 郭向华 乔录新 时红林 王珊珊 刘凯 Pang Lijun;Shi Ying;Guo Xianghua(Beijing Institute of Hepatology,Youan Hospital,Capital Medical University,Beijing 100069)

机构地区：[1]首都医科大学附属北京佑安医院北京市肝病研究所,北京市100069

出　　处：《实用肝脏病杂志》2020年第3期320-323,共4页Journal of Practical Hepatology

基　　金：国家自然科学基金资助项目(编号:81773168);北京市医院管理局青年人才培养“青苗”计划项目(编号:QML20171701);北京市肝病研究所所内基金资助项目(编号:2018-1-1/2018-4-7/Y-2019-1TD)。

摘　　要：目的构建HBx表达载体,筛选可稳定表达HBx的人肝细胞(HL)-7702细胞系。方法采用RT-PCR法扩增HBx基因片段,并将其连接至pIRES载体,经酶切和测序鉴定pIRES-HBx重组质粒序列。然后,分别采用Real-time PCR和Western blot技术检测验证重组质粒的正确性。最后,应用G418抗生素筛选稳定表达HBx的HL-7702细胞系。结果经PCR扩增得到正确的HBx片段,成功构建pIRES-HBx质粒,将重组质粒转染HEK 293细胞和HL-7702细胞均实现了HBx过量表达;经免疫荧光法鉴定,我们获得了稳定大量表达HBx的HL-7702细胞系。结论成功构建的pIRES-HBx表达载体能在HL-7702细胞稳定表达HBx,为后续研究提供了基础实验工具。Objective The aim of this study was to construct and select an HL-7702 cell line stably expressing HBx in vitro.Methods The HBx gene fragment was amplified by RT-PCR and ligated into pIRES vector.The sequence of pIRES-HBx recombinant plasmid was identified by restriction enzyme digestion and sequencing.The recombinant plasmid was further verified by real-time PCR and Western blot,and the HL-7702 cell stably expressing HBx was selected by G418.Results The correct HBx fragment was amplified by PCR,and the pIRES-HBx plasmid was successfully constructed.The recombinant plasmid overexpressed HBx protein in both HEK 293 cells and HL-7702 cells.The HL-7702 cell stably expressing HBx was further verified by immunofluorescence.Conclusions We Successfully construct a pIRES-HBx expression vector and a stably expressing HBx HL-7702 cells,which might provide us a basic experimental tools for further research.

关 键 词：HBVX蛋白 人正常肝-7702细胞 转染 体外 

分 类 号：R735.7[医药卫生—肿瘤]