乙型肝炎病毒核心启动子的克隆及其组织特异性的鉴定  

Cloning and tissue specific activity determination of Hepatitis B Virus core promoter

作　　者：雷俊川[1] 宫卫东[2] 赵亚[1] 姜河[1] 易军[3] 

机构地区：[1]第四军医大学基础部病原生物学教研室,陕西西安710032 [2]第四军医大学唐都医院介入科,陕西西安710038 [3]第四军医大学西京医院普通外科,陕西西安710033

出　　处：《中国现代医学杂志》2010年第14期2118-2121,共4页China Journal of Modern Medicine

基　　金：国家自然科学基金资助项目(No:30771890)

摘　　要：目的克隆adr亚型乙型肝炎病毒核心启动子(core promoter,Cp)并鉴定其在肝细胞系的特异性表达。方法利用PCR从质粒pUC19/3HBV扩增adr亚型乙肝病毒核心启动子,并将其克隆入T载体pMD19-T Simple中。测序正确后再亚克隆入pGL3-Basic荧光素酶报告基因载体。将重组载体pGL3-Basic/Cp分别转染肝癌细胞系HepG2、宫颈癌细胞系Hela、绿猴肾细胞系COS-7、乳腺癌细胞系MDA-MB-231和结肠癌细胞系HT-29,通过双荧光素酶检测系统检测荧光素酶在这些不同细胞系中的活性,以确定所克隆的基本核心启动子是否具有肝细胞特异性活性。结果从HBV基因组中成功克隆了Cp,Cp在肝细胞系中具有明显的活性,而在其他组织来源的细胞系中几乎没有活性。结论克隆得到的核心启动子具有明显的肝细胞特异性活性。【Objective】To clone the core promoter（Cp） of hepatitis B virus adr subtype and determine the hepatocyte specific activity of Cp.【Methods】The Cp was amplified from plasmid pUC19/3HBV using PCR and cloned into T vector pMD19-T Simple.After sequencing,the Cp was subcloned into upstream of luciferase encoding sequence in pGL3-Basic plasmid.The recombinant plasmid pGL3-Basic/Cp was transfected into different tissue-derived cell lines including HepG2,Hela,COS-7,MDA-MB-231 and HT-29.The dual luciferase activities were determined by Dual Luciferase Report（DLR） assay system.【Results】The Cp was successfully cloned from HBV genome.Cp had significant activity in live cell lines,but had few in other tissue-derived cell lines.【Conclusions】 The Cp was correctly amplified and has high hepatocyte specific activity.

关 键 词：乙型肝炎病毒(HBV) 核心启动子(Cp) 肝细胞特异性 荧光素酶 

分 类 号：R392.12[医药卫生—免疫学]