睾丸特异的核孔蛋白BS—63与transportin（karopherin beta2）结合区域的确定以及体外结合的证明

目的：确定睾丸特异的核孔蛋白BS－63与transportin(karopherin beta2)的结合区域,体外验证BS－63与transportin的结合作用。方法：将BS－63 C端分成不同长度的片段,采用酵母双杂交技术确定与transportin阳性克隆的结构区域。分别于大肠杆菌中表达上述相互作用片段,并进一步运用pull down实验在体外验证BS－63与transportin的结合作用。结果：BS－63 C端与transportin相结合的区域从起始的1．6kb确定至包括一个Ran binding domain(RanBD)在内0．6kb范围内。Pulldown以及Western blot证实此区域在体外可与transportin相结合。结论：在生精细胞中,位于胞则的睾丸特异的核 孔蛋白BS－63将被转动物向核内的转运过程可能是由transportin协助完成的。

Determination of the binding site of testis-specific nucleoporin BS-63 to transportin (karopherin beta 2) and the proof of their combination in vitro

Objective To locate the binding site of testis-specific nucleoporin BS-63t o transportin(karo ph e-rin beta2)and confirm their combination in vitro.Met hods Constructed different fragments of C term-inal BS-63was employed to loc alize the binding site of the testis-specific nucleoporin BS-63to tran s-porti n by yeast two -hybrid system technique pull-down test was used to identify the interaction betw e-en the purified expressed fragments of BS-630.6K and tra nsportin in vitro.Results BS-63binding site to transportin was shortened from 1.6kb to0.6kb which included a Ran binding domain(Ran BD).SDS-PAGE and Western blot tests confirmed the recombinant purified protein coded by0.6kb fragm en t of BS-63cDNA could interact with transportin in vitro.Conclusions In germ cells,the function of the testis-specific nucleoporin BS-63localized at cytoplasmic side of NPC importing cargoes into nucl-ear may be accomplished by transportin cooperation.