四环素调控系统对乙型肝炎病毒核心启动子活性及组织特异性的影响  

The influences of Tet system on activity and tissue specificity of HBV Cp

作　　者：雷俊川[1] 宫卫东[2] 赵亚[1] 姜河[1] 刘忠湘[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]唐都医院介入科,陕西西安710038 [3]西京医院普通外科,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2009年第12期1103-1105,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30771890)

摘　　要：目的:分析四环素调控系统对乙型肝炎病毒核心启动子(Cp)活性及组织特异性的影响。方法:利用PCR从质粒pTL-8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荧光素酶报告基因质粒中Cp启动子的上游,以构建质粒pGL3-Basic/7t/Cp。将能表达TetR的质粒pTL-8的荧光素酶编码基因切除后,与pGL3-Basic/7t/Cp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,以鉴定四环素调控系统对Cp活性及组织特异性的影响。结果:成功构建了质粒pGL3-Basic/7t/Cp,将其转染入不同组织来源的细胞系后表明将7个teto序列克隆入Cp上游后增强了Cp在各细胞系中的活性。结论:四环素调控系统明显增强了Cp的转录活性。但同时,Cp失去了其组织特异性的特点,即在这些细胞系中均有较强活性。AIM： Analyze the effect of Tetracycline-controlled system on Hepatitis B virus core promoter （Cp） activity and tissue specificity. METHODS： The 7 Teto sequence was amplified from plasmid pTL-8 using polymerase chain reaction （PCR） and cloned into T vector pMD19-T Simple. After sequencing, the 7 teto sequence was subcloned into upstream of Cp in pGL3-Basic/Cp. In order to observe the effect of Tetracycline-controlled system on Cp activity and tissue specificity, the plasmid pTL-8 which luciferase encoding sequence was excised and pGL3-Basic/7t/ Cp were cotransfected into different tissue-derived cell lines including HepG2, Hela, COS-？, MDA-MB-231 and HT-29. The dual luciferase activities were determined by Dual Luciferase Report （DLR） assay system. RESULTS： Have successfully constructed plasmid pGL3-Basic/7t/Cp. The transcriptional activity of Cp was increased significantly after the teto sequence was cloned upstream of Cp. CONCLUSION： The transcriptional activity of Cp is augmented significantly by Tetracycline-controlled system. But the tissue specificity of Cp lost at the same time.

关 键 词：四环素调控系统 CP 肝细胞特异性 荧光素酶 

分 类 号：R392.12[医药卫生—免疫学]