乙型肝炎病毒剪接特异性蛋白对病毒自身调控序列的影响  

Effects of the splicing-specific protein on the regulatory elements of hepatitis B virus per se

作　　者：陈婉南[1] 陈金烟[1] 王林[1] 林万松[1] 林建银[1] 林旭[1] 

机构地区：[1]福建医科大学分子医学研究中心福建省高校感染与肿瘤重点实验室,福建福州350004

出　　处：《中华微生物学和免疫学杂志》2008年第4期310-313,共4页Chinese Journal of Microbiology and Immunology

基　　金：基金项目：全国优秀博士学位论文作者专项资金资助项目（200359）;福建省重大科技基金（200217005）;福建省高等学校科技创新团队培育计划基金（FMU-RT001）

摘　　要：目的研究双剪接型2，2kb乙型肝炎病毒（HBV）剪接变异体特异性蛋白TPds对病毒自身调控序列的影响。方法PCR扩增6种HBV启动子／增强子序列，以Kpn I及Xho I位点克隆于pGL3-basic，分别构建萤火虫荧光素酶重组报告载体pGL3-BCP（含HBV基本核心启动子）、pGL3-CP1601（含增强子Ⅱ的核心启动子）、pGL3-XP（X基因最小启动子）、pGL3-XP1071（含增强子I的X基因启动子）、pGL3-SP1（表面抗原大蛋白基因启动子）、pGL3-SP2（表面抗原中蛋白基因启动子）。以FuGENE6将TPds表达载体pcDNA3.1／HisC-TPds或空白载体pcDNA3.1／HisC分别与6种HBV启动子／增强子报告载体共转染Huh7细胞，转染后48h裂解细胞并检测胞内萤火虫荧光素酶活性，实验重复5次，数据以SPSS11.5软件分析。结果在一定的质量比值范围内，与pcDNA3.1／HisC空白载体对照相比，pcDNA3.1／HisC-TPds分别与pGL3，CP1601、pGL3-XP1071、pGL3-SP1、pGL3-SP2共转染后，Huh7细胞内萤火虫荧光素酶活性增高，而pcDNA3.1／HisC-TPds分别与pGL3-BCP或pGL3-XP共转染后，胞内荧光素酶活性无变化。结论TPds蛋白可反式激活HBV启动子SP1、SP2和增强子I、Ⅱ，对基本核心启动子和X基因最小启动子无影响。Objective To investigate the effects of the novel protein TPds encoded by the 2.2 kb doubly spliced variant of hepatitis B virus （HBV） genome on the regulatory elements of HBV. Methods HBV promoters and enhancers were amplified by PCR and respectively cloned into the plasmid pGL3-basic to construct the recombinant firefly luciferase reporter vectors including pGL3-BCP （harboring basal core pro-moter）, pGL3-CP1601 （ core promoter with enhancer H ）, pGL3-XP （ minimal X gene promoter）, pGL3-XP1071 （X gene promoter with enhancer I ）, pGL3-SP1 （promoter of large surface antigen） and pGL3-SP2 （promoter of middle surface antlgen）. The recombinant reporter plasmids were respectively co-transfected with either the pcDNA3.1/HisC-TPds （TPds expression plasmid） or pcDNA3.1/HisC（ empty control） into Huh7 hepatocytes by FuGENE6 transfection reagent. Cells were lysed 48 h post transfection, the intracellu-lar luciferase activities were monitored and calculated by SPSS11.5 software. Results As compared with the empty control of pcDNA3. 1/HisC, the intracellular luciferase activities were elevated when the Huh7 hepatocytes were co-transfected by pcDNA3.1/HisC-TPds with one of the recombinant reporter plasmids as pGL3-CP1601, pGL3-XP1071, pGL3-SP1, or pGL3-SP2, while no changes with pGL3-BCP or pGL3-XP. Conclusion TPds could transactivate the HBV regulatory elements as promoter SP1, SP2, and enhancer I , Ⅱ , while no influences on basal core promoter and minimal X gene promoter.

关 键 词：乙型肝炎病毒 RNA剪接 反式激活 启动子 增强子 

分 类 号：R3[医药卫生—基础医学]