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

发布时间：2018-01-07 13:31

  本文关键词：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　出处：《世界华人消化杂志》2015年28期 　论文类型：期刊论文

  更多相关文章： 乙型肝炎病毒X蛋白 Chang liver细胞 慢病毒载体

【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.
[Abstract]:Objective: to construct the lentivirus expression vector of hepatitis B virus X gene, hepatitis B virus and HBX. A human normal liver cell line Chang liver HBx. stably expressing HBx protein was established. Methods: polymerase chain reaction (PCR) was used. Polymerase chain reaction. HBx gene was amplified from plasmid and cloned into lentivirus p EB-3xflag-GP-Puro vector by PCR. The Chang livers of human hepatocytes were infected by lentivirus packaging and confirmed by restriction endonuclease digestion and sequencing. Then the cell lines expressing HBx protein stably were screened by purine mycin. Finally, the expression of HBx protein was detected by immunofluorescence and Western blot. Results: the HBx gene was successfully cloned into lentivirus expression vector by restriction endonuclease digestion and gene sequencing. The titer of recombinant lentivirus was 1 脳 10 ~ 8 TU/m / L, and the recombinant lentivirus was used to infect Chang liver of human hepatocytes. A monoclonal cell line Chang liver-HBx was obtained by purine mycin screening. Immunofluorescence and Western blot techniques were used to detect the stable expression of HBx protein in cell line Chang liver-HBx. The recombinant lentivirus expression vector of HBx was successfully constructed. Chang liver cell line Chang liver-HBx, which stably expressed HBx, was obtained, which provides a cell model for further study on the malignant transformation of normal hepatocytes induced by HBx.
【作者单位】： 海南医学院分子生物学重点实验室
【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~~
【分类号】：R373.21;Q78
【正文快照】： 核心提示:乙型肝炎病毒X(hepatitis B virus x,HBx)是诱导肝细胞恶性转化的关键分子.研究发现HBx能激活生长信号,或抑制P53的转录调节功能,促进肝细胞的恶性转化.本研究成功构建p EB-GFP-HBx载体,并成功转染Chang Liver细胞,为研究HBx诱导肝细胞恶性转化建立了细胞模型.鲁琰,，

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