慢病毒介导HBV X基因稳定表达HepG2细胞系的建立  

Establishment of a HepG2 cell line stably transduced with a lentivirus expressing the HBV X gene

作　　者：余桂芳[1] 严跃红[1] 王瑞鑫[1] 李显波[1] 曾文铤[2] 朱科伦[2] 

机构地区：[1]广州医学院第五附属医院内一科,广东省广州市510700 [2]广州医学院第一附属医院肝病研究室,广东省广州市510120

出　　处：《世界华人消化杂志》2012年第8期638-643,共6页World Chinese Journal of Digestology

基　　金：广东省科技计划基金资助项目;No.2010B060900092~~

摘　　要：目的:构建乙型肝炎病毒X基因(HBV X)重组慢病毒表达载体,建立稳定表达HBVX蛋白(HBx)的HepG2细胞系.方法:应用PCR法从质粒pIERES2-EGFP-HBV中扩增X基因片段,克隆至慢病毒载体pZac2.1,应用PCR、酶切和测序鉴定正确后,经病毒包装,感染HepG2细胞,经嘌呤霉素筛选稳定表达细胞株,RT-PCR、免疫组织化学、Western blot鉴定HBx的表达.结果:酶切鉴定和基因序列测定证实长度为489bp的HBx基因成功克隆至慢病毒表达载体pZac2.1-HBx;重组慢病毒经包装、纯化后获得滴度为1×108TU/mL,通过感染HepG2细胞株和嘌呤霉素筛选,8-10d形成生长形态良好的单克隆细胞株HepG2-HBx;RT-PCR鉴定显示细胞株HepG2-HBx在3d,14d,30d和2mo后均可见稳定表达的HBx mRNA;利用免疫组织化学和蛋白免疫印迹法鉴定,细胞株HepG2-HBx可稳定表达HBx蛋白.结论:成功构建了HBV X重组慢病毒载体,获得稳定表达HBx的HepG2细胞系,为进一步研究HBx的生物学功能及致病机制提供细胞模型.AIM： To establish a HepG2 cell line stably transduced with a lentivirus expressing the HBV X （HBx） gene for studying the biological function of HBx and its role in hepatocarcinogenesis. METHODS： The HBV X gene was amplified from plasmid pIERES2-EGFP-HBV by PCR. The purified HBx gene fragment was inserted into a lentivirus vector （pZac2.1）, and the insertion was identified by PCR, restriction endonuclease analysis and DNA sequencing. HepG2 cells were then transfected with the packaged recombinant lentivirus, and resistant cell clones were selected with puromycin. The expression of HBx was examined using RT-PCR, immunohistochemistry, and Western blot. RESULTS： Restriction enzyme digestion and DNA sequencing showed that the full-length HBx （489 bp） gene had been successfully subcloned into the lentiviral vector to result in the recombinant vector pZac2.1-HBx. The titre of purified recombinant lentivirus was 1×10 8 TU/mL. Monoclonal cell line HepG2-HBx was produced 8-10 d after transfection with the recombinant lentivirus and selected with puromycin. HBx mRNA could be detected on days 3, 14, 30 and at 2 mo after cell colony formation. Meanwhile, stable expression of HBx protein was verified by immunohistochemistry and Western blot. CONCLUSION： A HepG2 cell line stably transduced with a lentivirus expressing the HBx gene has been successfully generated.

关 键 词：乙型肝炎病毒 X基因 HEPG2 慢病毒 稳定细胞株 

分 类 号：R512.62[医药卫生—内科学]