人PIRH2b与ARF4相互作用的初步研究

Preliminary Study on the Interaction between Human PIRH2b and ARF4

目的利用酵母双杂交技术筛选PIRH2b的相互作用蛋白。方法以PIRH2b为诱饵蛋白,利用酵母双杂交技术筛选人胎肝cDNA文库,用GST-pull down验证PIRH2b与ARF4在体外的相互作用,并用绿色荧光蛋白标记PIRH2b,红色荧光蛋白标记ARF4,观察两者在肝癌细胞株Hep3B中的亚细胞定位。结果利用酵母双杂交筛选到一个能与PIRH2b相互作用的蛋白ARF4,GST-pull down验证了两者在体外的相互作用,荧光标记共定位结果显示两个蛋白共定位于Hep3B细胞的核周区域。结论首次发现并证实了PIRH2b与ARF4的相互作用,PIRH2b对ARF4的功能可能有重要影响。

Objective To clone and identify human PIRH2b interacting proteins by using yeast two-hybrid screening technique. Methods A human fetal liver cDNA library was screened by employing Clontech yeast two-hybrid system 3, with PIRH2b as the bait. GST-pull down assay was performed to verify the specific interaction between PIRH2b and ARF4 in vitro. Green fluorescent pro- tein-fused PIRH2b and red fluorescent protein-fused ARF4 were constructed to examine PIRH2b and ARF4 subcellular localizations in Hep3B cells. Results ARF4 was identified as a PIRH2b interacting protein by yeast two-hybrid screening and the interaction was confirmed in vitro by pull-down assay. Moreover, PIRH2b and ARF4 were found to co-localize in cytoplasm surrounding the nucleus in Hep3B cells. Conclusion ARF4 was a PIRH2b interacting protein and PIRH2b may play an important role in ARF4 function.