乙型肝炎病毒核心启动子下游克隆入Teto序列后其在不同细胞系中转录活性的变化  

Variety of HBV Core Promoter Transcriptional Activity after the Teto Sequence was Cloned into it's Downstream

作　　者：易军[1] 雷俊川[2] 宫卫东[3] 赵亚[2] 姜河[2] 刘忠湘[2] 王岭[1] 

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

出　　处：《生物技术通讯》2010年第6期771-774,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30771890)

摘　　要：目的:探讨将Teto序列克隆入乙型肝炎病毒核心启动子(Cp)下游后对该启动子在不同细胞系中转录活性的影响。方法:利用PCR从质粒pTL-8中扩增7个Teto序列,并将其克隆入T载体pMD19-T/Simple中,测序正确后将7个Teto序列亚克隆入pGL3-Basic/Cp萤光素酶报告基因质粒中Cp启动子的下游,以构建质粒pGL3-Basic/Cp/x7t。将能表达TetR的质粒pTL-8的萤光素酶编码基因切除后,与pGL3-Basic/Cp/x7t共转染肝癌细胞系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,通过双萤光素酶检测系统检测萤光素酶在这些不同细胞系中的活性,以分析将Teto序列克隆入乙型肝炎病毒核心启动子下游后对Cp在不同细胞系中转录活性的影响。结果:构建了质粒pGL3-Basic/Cp/x7t,将其转染HeLa、COS-7、HepG2、MDA-MB-231和HT-29细胞后其相对萤光素酶活性分别为56.14、171.52、211.03、6.11和34.24。结论:将Teto序列克隆入Cp下游并不能明显提高Cp的转录活性,而且破坏了Cp的组织特异性转录活性。Objective： To analyze the transcriptional activity variety of hepatitis B virus core promoter（Cp） after the Teto sequence was cloned into the Cp downstream.Methods： The Teto sequence was amplified from plasmid pTL-8 using PCR and cloned into T vector pMD19-T / Simple.After sequencing,the teto sequence was subcloned into downstream of Cp in pGL3-Basic / Cp.In order to observe the effect of Teto sequence on Cp activity and tis-sue specificity,the plasmid pTL-8 which luciferase encoding sequence was excised and pGL3-Basic / Cp / x7t were cotransfected into different tissue-derived cell lines including HeLa,COS-7,HepG2,MDA-MB-231 and HT-29.The relative luciferase activities of Cp were determined by dual luciferase report assay system.Results： The plas-mid pGL3-Basic / Cp / x7t was successfully constructed.The relative luciferase activities of Cp in HeLa,COS-7,HepG2,MDA-MB-231 and HT-29 cell lines were 56.14,171.52,211.03,6.11 and 34.24 respectively.Conclusion： Clone of Teto sequence into downstream of Cp can＇t increase significantly the activities of Cp and destroy the tissue specificity of Cp.

关 键 词：四环素调控系统 核心启动子 乙型肝炎病毒 肝细胞特异性 萤光素酶 

分 类 号：Q78[生物学—分子生物学] R392.12[医药卫生—免疫学]