慢病毒-HBx表达载体的构建及其在人正常肝细胞系Chang liver的稳定表达  

Construction of a lentiviral expression vector of HBx and its stable expression in human Chang liver cell line

作　　者：鲁琰[1] 朱明月[1] 张雪儿 李伟[1] 董栩[1] 陈栘 林波[1] 郭峻莉[1] 李孟森[1,3] 

机构地区：[1]海南医学院分子生物学重点实验室 海南省肿瘤发生与干预重点实验室,海南省海口市571199 [2]海南医学院 [3]海南医学院肿瘤研究所,海南省海口市570102

出　　处：《世界华人消化杂志》2015年第28期4482-4489,共8页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;Nos.81560450;81360307;81260306;81160261;31060164;海南省社会发展专项基金资助项目;No.2015SF03;海南省重点科技基金资助项目;No.DZXM20110038;海南医学院培育基金资助项目;No.HY2013-19;海南医学院大学生创新计划基金资助项目;N o.HYCX2014031~~

摘　　要：目的:构建乙型肝炎病毒X基因(hepatitis B virus x,HBx)慢病毒表达载体,建立稳定表达HBx蛋白的人正常肝细胞系Chang liverHBx.方法:应用聚合酶链式反应(polymerase chain reaction,PCR)方法从质粒中扩增HBx基因,并克隆到慢病毒p EB-3xflag-GP-Puro载体上,经PCR、酶切和测序鉴定正确后经慢病毒包装感染人肝细胞Chang liver,再用嘌呤霉素筛选出稳定表达HBx蛋白的细胞株,最后用免疫荧光和Western blot技术检测HBx蛋白的表达.结果:酶切鉴定和基因测序证实HBx基因成功克隆到慢病毒表达载体上,重组慢病毒经包装纯化后获得滴度为1×108 TU/m L,用包装好的重组慢病毒感染人肝细胞Chang liver,经嘌呤霉素筛选获得单克隆细胞株Chang liver-HBx,利用免疫荧光和Western blot技术检测发现细胞株Chang liver-HBx可稳定表达HBx蛋白.结论:成功构建了HBx的重组慢病毒表达载体,获得了稳定表达HBx的Chang liver细胞系Chang liver-HBx,为进一步研究HBx诱导正常肝细胞恶性转化提供细胞模型.AIM：To construct a lentiviral expression vector carrying the hepatitis B virus x（HBx） gene and to test its expression in the human Chang liver cell line.METHODS：The HBx gene was amplified from plasmid pcDNA3.1-HBx by PCR.The purified HBx gene fragment was inserted into the pEB-3xflag-GP-Puro lentiviral vector,and the inserted fragment was identified by PCR,restriction endonuclease digestion and DNA sequencing.The recombinant vector was then transfected into Chang liver cells.Puromycin was applied to screen stable cell clones,and the expression of HBx in Chang liver cells was examined by immunofluorescence and Western blot.RESULTS：Restriction enzyme digestion and DNA sequencing showed that the HBx gene had been successfully subcloned into the pEB-3xflag-GP-Puro vector.The titer of purified recombinant lentivirus was 1×10~8 TU/mL.HBx was produced after transfection with the recombinant lentivirus vector and screening with puromycin.Stable expression of HBx protein was present in Chang liver-HBx cells.CONCLUSION：The Chang liver-HBx cell line that can stably express the HBx gene has been successfully generated;this model cell may be applied to study the role of HBx in the malignant transformation of liver cells.

关 键 词：乙型肝炎病毒X蛋白 CHANG liver细胞 慢病毒载体 

分 类 号：R373.21[医药卫生—病原生物学] Q78[医药卫生—基础医学]