应用酵母双杂交系统筛选小鼠PIAS2相互作用蛋白

Screening PIAS2-interacting proteins in the mouse using the yeast two-hybrid system

目的:应用酵母双杂交技术,从小鼠精原细胞cDNA文库中筛选与PIAS2(protein inhibitor of activatedSTAT2,PIAS2)相互作用的未知蛋白。通过对其相互作用蛋白的筛选和研究,探讨PIAS2在精子发生中的作用机制,为临床男性不育症的治疗和诊断提供理论基础。方法:以pGBKT7-PIAS2为诱饵质粒,从小鼠精原细胞cDNA文库中筛选出与pGBKT7-PIAS2相互作用的阳性克隆,对验证后的阳性克隆进行测序及生物信息学分析,进一步采用直接酵母双杂交进行相互作用的验证。结果:经过筛选、测序、同源性分析及直接酵母双杂交验证,发现了与PIAS2相互作用的8个候选蛋白:Cyfip2、Psmb3、Nme1、nischarin、Nsun5、Ints10、Gnb2l1及Ndufaf3。结论:应用酵母双杂交系统,共筛选得到了8个不同的基因,其编码蛋白与PIAS2相互作用,可能与男性生殖调控相关。对上述蛋白与PIAS2的相互作用研究有助于揭示PIAS2的作用机制。

Objective： To screen and identify PIAS2-interacting proteins from the mouse spermatogonial cDNA library using the yeast two-hybrid system,and to investigate the action mechanism of PIAS2 in spermatogenesis.Methods： With pGBKT7-PIAS2 as a bait plasmid,the positive clones interacting with pGBKT7-PIAS2 were screened from the mouse spermatogonial cDNA library,the inserted fragments were sequenced and underwent bioinformatic analysis,and their interaction was verified using the yeast two-hybrid system.Results： Through screening,sequencing,homology analysis and yeast two-hybrid verification,we obtained 8 different candidate proteins interacting with PIAS2,including Cyfip2,Psmb3,Nme1,nischarin,Ints10,Nsun5,Gnb211 and Ndufaf3.Conclusion： Eight different genes were successfully obtained using the yeast two-hybrid system,and their encoding proteins interacted with PIAS2,which might be related with male fertility regulation.Our findings have offered some new clues to the action mechanism of PIAS2 in spermatogenesis.