HCV核心蛋白在酵母双杂交系统中的表达及自激活作用检测

Role of HCV core protein in yeast two-hybrid system in reporter gene expression and activation

目的检测用含丙型肝炎病毒(HCV)核心蛋白的cDNA片段构建的酵母双杂交诱饵载体的表达产物对酵母细胞有无毒性作用及对报告基因有无激活作用.方法PCR扩增含HCV核心蛋白不同大小的cDNA片段,分别克隆入pUC19质粒,经测序正确后,再分别亚克隆入酵母双杂交诱饵载体pGBKT7中.将重组质粒导入酵母菌AH109,检测其表达产物在酵母细胞中对报告基因有无激活作用.结果成功获得含HCV核心蛋白的cDNA片段,HCV核心蛋白对酵母菌AH109无毒性,且对报告基因无激活作用.结论利用酵母双杂交Gal4系统3研究与HCV核心蛋白相互作用的蛋白,证实了HCV核心蛋白无自激活功能.

AIM： To construct the bait vector of HCV core protein in yeast two-hybrid system and then to study its effect on the growth of yeast cells and the activation of reporter genes. METHODS： cDNA fragments encoding HCV core region were amplified by PCR and subsequently cloned into pUC19. After verified by sequencing, they were subcloned into the bait vector pGBKT7 of yeast two-hybrid system. These recombinant plasmids were transferred into AH109 and they activated the reporter genes. RESULTS： The fragments of HCV core protein were successfully obtained, which were not toxic to AH109 but could not activate the reporter genes. CONCLUSION： Yeast two-hybrid GAIA system 3 can be utilized to fish HCV core region-interacting protein. HCV core orotein does not have self-activating function.