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

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 pEB3xflag-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 pEB3xflag-GP-Puro vector. The titer of purified recombinant lentivirus was 1 × 10 TU/mL. 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 鲁 琰, 朱明月, 张雪儿, 李 伟, 董 栩, 陈 栘, 林 波, 郭峻莉, 李孟森 在线投稿: http://www.baishideng.com/wcjd/ch/index.aspx 帮助平台: http://www.wjgnet.com/esps/helpdesk.aspx DOI: 10.11569/wcjd.v23.i28.4482 世界华人消化杂志 2015年10月8日; 23(28): 4482-4489 ISSN 1009-3079 (print) ISSN 2219-2859 (online) © 2015年版权归百世登出版集团有限公司所有. 基础研究 BASIC RESEARCH ® ■同行评议者 高国全, 教授, 中 山大学中山医学 院生物化学教研 室; 高英堂, 研究 员 , 天 津 市 第 三 中心医院 鲁琰, 等. 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 2015-10-08|Volume 23|Issue 28| WCJD|www.wjgnet.com 4483 ■研发前沿 研究HBx诱导肝 癌发生的机制是 防治肝癌的热点 和重点问题 . 尽 管研究已经发现 HBV通过HBx的 作用促进肝细胞 恶性转化 , 但是 HBx通过怎样的 机制促进肝细胞 恶性转化 , 目前 并不清楚 , 如何 预警HBV相关肝 癌发生是亟待解 决的问题. 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. © 2015 Baishideng Publishing Group Inc. All rights reserved.