CAJ | 학술논문

目的研究双剪接型2.2 kb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48 h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.
목적 연구쌍전접형2.2 kb을형간염병독(HBV)전접변이체특이성단백TPds대병독자신조공서렬적영향.방법 PCR확증6충HBV계동자/증강자서렬,이Kpn Ⅰ급Xho Ⅰ위점극륭우pGL3-basic,분별구건형화충형광소매중조보고재체pGL3-BCP(함HBV기본핵심계동자)、pGL3-CP1601(함증강자Ⅱ적핵심계동자)、pGL3-XP(X기인최소계동자)、pGL3-XP1071(함증강자Ⅰ적X기인계동자)、pGL3-SP1(표면항원대단백기인계동자)、pGL3-SP2(표면항원중단백기인계동자).이FuGENE6장TPds표체재체pcDNA3.1/HisC-TPds혹공백재체pcDNA3.1/HisC분별여6충HBV계동자/증강자보고재체공전염Huh7세포,전염후48 h렬해세포병검측포내형화충형광소매활성,실험중복5차,수거이SPSS11.5연건분석.결과 재일정적질량비치범위내,여pcDNA3.1/HisC공백재체대조상비,pcDNA3.1/HisC-TPds분별여pGL3-CP1601、pGL3-XP1071、pGL3-SP1、pGL3-SP2공전염후,Huh7세포내형화충형광소매활성증고,이pcDNA3.1/HisC-TPds분별여pGL3-BCP혹pGL3-XP공전염후,포내형광소매활성무변화.결론 TPds단백가반식격활HBV계동자SP1、SP2화증강자Ⅰ、Ⅱ,대기본핵심계동자화X기인최소계동자무영향.
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 promoter),pGL3-CP1601(core promoter with enhancer Ⅱ),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 antigen).The recombinant reporter plasmids were respectively co-transfected with either the pcDNA3.1/HisC-TPds(TPd8 expression plasmid)or pcDNA3.1/HisC(empty control)into Huh7 hepatocytes by FuGENE6 transfection reagent.Cells were lysed 48 h post transfection,the intracellulax 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.Conduslon TPds could transactivate the HBV regulatory elements a8 promoter SP1,SP2.and enhancer Ⅰ,Ⅱ,while no influences on basal core promoter and minimal X gene promoter.