表达丙型肝炎病毒核心蛋白的人肝癌稳定转染细胞株的建立与鉴定  

Establishment and identification of human hepatocellular carcinoma line stably expressing hepatitis C virus core protein

作　　者：乔卿华[1,2] 吕欣[1] 雷迎峰[1] 杨敬[1] 姚敏[1] 韩佩君[1] 党品香 赵海卫[1] 翁代慧[1] 尹文[1] 

机构地区：[1]第四军医大学基础医学部微生物学教研室,陕西西安710032 [2]中国人民解放军第152医院检验科,河南平顶山467000

出　　处：《细胞与分子免疫学杂志》2014年第11期1137-1141,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：陕西省科技统筹创新工程计划(2014TCL03-08);中国肝炎防治基金会天晴肝病研究基金(CFHPC20132043)

摘　　要：目的建立能表达丙型肝炎病毒核心蛋白(HCV core)的人肝癌细胞SMMC-7721的稳定转染细胞株。方法构建含目的基因HCV core的重组质粒,转染HEK293T细胞,包装获得含ZsGreen和HCV core基因的慢病毒后,感染SMMC-7721人肝癌细胞,采用实时荧光定量PCR检测HCV core mRNA表达,采用免疫荧光细胞化学染色和Western blot法检测HCV core蛋白表达,筛选稳定表达HCV core的细胞株。结果质粒酶切和序列测定证实重组载体构建正确;慢病毒包装48 h后可见清晰ZsGreen表达,感染SMMC-7721细胞后筛选获得稳定转染的细胞株,实时荧光定量PCR检测到HCV core mRNA,免疫荧光细胞化学染色和Western blot法均检测出HCV core蛋白表达。结论成功构建了表达HCV core蛋白的SMMC-7721人肝癌细胞的稳定转染细胞株。Objective To establish SMMC-7721 human hepatocellular carcinoma cell line stably expressing hepatitis C virus（ HCV） core protein. Methods A lentiviral vector containing HCV core gene was constructed and transfected into HEK293 T cells to package recombinant lentivirus（ rLV-core） containing ZsGreen and HCV core genes. The SMMC-7721 cells were infected with the rLV-core. The expression of HCV core mRNA was examined by real-time fluorescent quantitative PCR and the HCV core protein was detected by immunofluorescence cytochemistry and Western blotting. The stably transfected cell line was screened. Results The lentiviral vector was confirmed by enzyme digestion and sequencing. The green fluorescence was seen under fluorescence microscope 48 hours after virus packaging. The SMMC-7721 cell line stably expressing HCV core protein was obtained after infected with the rLV-core. Real-time PCR showed the expression of HCV core mRNA,and both immunofluorescence cytochemistry and Western blotting verified the expression of HCV core protein.Conclusion The SMMC-7721 human hepatocellular carcinoma cell line stably expressing HCV core protein has been established successfully.

关 键 词：丙型肝炎病毒 核心蛋白 肝癌细胞株 慢病毒 稳定表达 

分 类 号：R373.21[医药卫生—病原生物学] R735.7[医药卫生—基础医学]