弓形虫酵母双杂交cDNA文库构建及与AMA1羧基端相互作用蛋白的筛选

Construction of yeast two-hybrid cDNA library of Toxoplasma gondii and screen of AMA1 c-terminal interacting proteins

为筛选弓形虫AMA1基因羧基端(AMA1c)在弓形虫中的相互作用分子,本研究采用MatchmakerTM Gold酵母双杂交系统,构建了弓形虫RH株酵母双杂交cDNA文库,文库滴度达到4.26×107cfu/mL。以构建的pGBKT7-AMA1c诱饵质粒,使用PEG/LiAc法转化酵母菌株Y2H,利用表型筛选法检测AMA1c的自激活作用并进行酵母双杂交筛选,得到阳性克隆,根据序列比对分析结果,进一步进行共转化验证。结果表明,诱饵蛋白在酵母双杂交系统中具有无毒性和自激活作用,而且通过酵母双杂交筛选共得到13个与AMA1c蛋白相互作用的虫体蛋白。本研究结果为进一步研究AMA1c的功能奠定了基础。

To screen the interaction protein to C-terminal fragment of AMA1（AMA1c） in T.gondii, Yeast two-hybrid cDNA library of T.gondii was construced by using the Clontech’s MatchmakerTM Gold Yeast Two-Hybrid System kit in this study. The titer of the amplified library was 4.26×107 cfu/mL. The bait vector （pGBKT7-AMA1c） was transformed into the yeast strain Y2H by PEG/LiAc method and its self-activation was tested by the phenotype assay. The constructed library was screened then as described in the manufacturer’s instruction. The results showed that pGBKT7-AMA1c had no toxicity to the yeast cells and could not induce self-activation in yeast two-hybrid system. Moreover, a total of 13 AMA1c interacting proteins were screened from the library by this system with AMA1c as bait. This study laid a solid foundation for the further research on the function of AMA1c.