稳定表达HBx的人肝细胞系7702的建立

doi:10.3969/j.issn.1672-5069.2020.03.005

实用肝脏病杂志 ›› 2020, Vol. 23 ›› Issue (3): 320-323.doi: 10.3969/j.issn.1672-5069.2020.03.005

• 实验性肝炎 • 上一篇

稳定表达HBx的人肝细胞系7702的建立

庞丽君,石英,郭向华,乔录新,时红林,王珊珊,刘凯

100069 北京市首都医科大学附属北京佑安医院北京市肝病研究所

发布日期:2020-05-27
通讯作者: 刘凯,E-mail:liukaihj@163.com
作者简介:庞丽君,女,36岁,医学博士,助理研究员。主要从事肝癌相关分子机制研究。E-mail:pang_lijun2012@163.com
基金资助:
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)

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

Pang Lijun, Shi Ying, Guo Xianghua, et al.

Beijing Institute of Hepatology, Youan Hospital, Capital Medical University, Beijing 100069

Published:2020-05-27

摘要/Abstract

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。

关键词: HBVx蛋白, 人正常肝-7702细胞, 转染, 体外 , ,

Abstract: 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.

Key words: HBx, HL-7702 cells, Transfection, In vitro

庞丽君,石英,郭向华,乔录新,时红林,王珊珊,刘凯. 稳定表达HBx的人肝细胞系7702的建立[J]. 实用肝脏病杂志, 2020, 23(3): 320-323.

Pang Lijun, Shi Ying, Guo Xianghua, et al.. Construction and selection of normal human liver 7702 cells stably expressing HBx in vitro[J]. Journal of Practical Hepatology, 2020, 23(3): 320-323.

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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

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