hnRNPK与Nef相互作用并有利于细胞表面CD4的表达  

hnRNPK Interacts with Nef and Facilitates the Cell Surface Expression of CD4

作　　者：魏金梅[1] 范小琴[1] 熊海庭 高学娟[1] 刘小会[1] 刘朗夏[1] 

机构地区：[1]广东省高校功能蛋白质研究重点实验室暨南大学生命与健康工程研究院,广州510632

出　　处：《中国生物工程杂志》2015年第4期17-22,共6页China Biotechnology

摘　　要：目的:前期不同的研究分别证明HIV蛋白Nef下调宿主细胞表面受体CD4的表达,以及Nef与宿主细胞蛋白heterogeneous nuclear ribonucleoprotein K(hnRNPK)存在相互作用。因此提出了两个值得研究探讨的重要问题:(1)hnRNPK是否参与调节细胞表面CD4的表达?(2)Nef是否通过hnRNPK调节细胞表面CD4的表达?方法:利用半体外GST-pulldown技术验证Nef与hnRNPK存在相互作用。通过瞬时转染的方式将HIV-1Nef表达在He La-CD4细胞里,同时利用siRNA干扰技术敲低hnRNPK,最后运用流式细胞技术检测细胞表面CD4的表达水平。结果:(1)GST-pulldown结果验证了Nef与hnRNPK存在相互作用;(2)Nef的表达使细胞表面CD4水平下降约75%;(3)不管是否有Nef,hnRNPK的敲低都使细胞表面CD4表达水平明显下降(50%);同样的,Nef下调CD4的作用也不受hnRNPK敲低的影响。结论:(1)hnRNPK与Nef相互作用;(2)hnRNPK有利于细胞表面CD4的表达,其与Nef的下调作用的关系尚不明确,Nef对CD4的下调作用可能涉及有其他因素参与的复杂调控。Objective： Previous studies have respectively demonstrated that HIV-1 Nef down-regulated the cell surface expression of CD4, and that Nef interacted with the host protein hnRNPK. The purpose is to investigate （1） if hnRNPK regulates the surface expression of CD4, and （2） if hnRNPK is involved in the down-regulation of CD4 by HIV-1 Nef. Methods： in vitro GST-pulldown assay was used to confirm the interaction of HIV-1 Nef with hnRNPK. HIV-1 Nef was expressed in HeLa-CD4 cells by transient transfection, hnRNPK was knocked down by means of siRNA, and the cell surface expression level of CD4 was assessed by using flow cytometry. Results： （1） The GST-pulldown assay has successfully confirmed the Nef-hnRNPK interaction; （2） Nef ectopic expression resulted in about 75 percent reduction of the cell surface CD4 expression; （3） hnRNPK knockdown reduced dramatically the cell surface expression of CD4（50% of reduction） regardless Nef was present or not. Similarly, the effect of Nef on the cell surface expression of CD4 was not affected by hnRNPK knockdown. Conclusion： （1） hnRNPK interacts with Nef; （2） hnRNPK facilitates the cell surface expression of CD4, but the relationship between this effect and the down-regulation of CD4 expresion by Nef remains unclear, probably reflecting the complex regulation involving other factors.

关 键 词：分子生物学 NEF CD4 hnRNPK 流式细胞术 

分 类 号：Q71[生物学—分子生物学]