【摘要】 为研究双剪接型2.2kb乙型肝炎病毒(HBV)基因组剪接变异体特异性新蛋白—yTPds的功能,以酵母双杂交系统的诱饵质粒pGBKT7克隆yTPds基因并转化酵母细胞AH109获得相应重组子,滤纸法及液相分析法测酵母转化子内β-半乳糖苷酶(-βgal)的活性,证实yTPds蛋白可反式激活酵母GAL4反应元件(GAL4 responsive ele-ment)。在此基础上,为进一步探讨该蛋白反式激活作用的功能区域,以pGBKT7分段克隆yTPds基因,分别编码yTPds蛋白N端47个氨基酸(yTPds-N47)、N端75个氨基酸(yTPds-N75)、中部28个氨基酸(yTPds-M28)、C端92个氨基酸(yTPds-C92)、C端64个氨基酸(yTPds-C64)以及去除中部28个氨基酸的融合蛋白(yTPds-Fusion)。各重组载体转化酵母细胞AH109获得相应转化子。滤纸法定性检测酵母转化子内-βgal活性,结果显示yTPds-N75、yTPds-M28、yTPds-C92蛋白可反式激活GAL4反应元件,液相分析法显示酵母转化子内-βgal活性强弱为yTPds-M28>yTPds-N75>yTPds-C92>yTPds,提示yTPds蛋白反式激活作用的功能区域是其中部28个氨基酸,该区域对应于HBV preS1蛋白反式激活功能域。此研究证实双剪接型2.2kb乙型肝炎病毒(HBV)基因组剪接变异体特异性新蛋白具有反式激活作用,提示其可能具有重要的致病意义。更多还原

【Abstract】 To investigate the function of TPds,a novel protein encoded by the 2.2kb doubly spliced variant of hepatitis B virus genome,the TPds coding region was cloned into the pGBKT7 bait vector of yeast two-hybrid system and transfected into yeast AH109.The intracellular β-galactosidase activities of the transformants were analyzed by filter assay and liquid culture assay,and the results demonstrated that TPds protein could transactivate GAL4 responsive element.To explore further the transactivating domain of TPds protein,different fragments of TPds gene,which encode the N terminal 47 amino acids,N terminal 75 amino acids,central 28 amino acids,C terminal 92 amino acids,C terminal 64 amino acids and a fusion protein,were subcloned separately into pGBKT7,and transformed into yeast AH109.Qualitative detection by filter assay for the intracellular β-galactosidase activities of the transformants revealed that the proteins of yTPds-N75,yTPds-M28,and yTPds-C92 could transactivate the GAL4 responsive element.Furthermore,quantitative measurement by liquid culture assay demonstrated that the transactivating capacities of each protein increased accordingly as yTPds-M28>yTPds-N75>yTPds-C92>yTPds.Therefore,it was concluded that the transactivating domain of TPds was located within the central region with 28 amino acids,which was overlapped with the transactivating domain of preS1 protein.The results validated the transactivation capacity of the novel protein encoded by 2.2kb doubly spliced variant of hepatitis B virus genome,and implied its potential pathogenicity.更多还原