【摘要】 目的筛选与脆性X智力低下蛋白(FMRP)相互作用的蛋白,为FMRP生理功能的阐释提供新线索。方法以5月龄人胚海马mRNA为模板制备cDNA,重组于DNA激活结构域载体pGAD10,构建酵母双杂交文库,从中筛选与FMRP相互作用的蛋白。应用酵母双杂交体系对FMRP的相互作用区域进行定位。结果(1)所构建的5月龄人胚海马cDNA酵母双杂交文库含1×106克隆,插入片段平均长度约1.5kb,有效克隆90%;(2)在该文库中筛选到一个与FMRP相互作用的阳性克隆。序列分析表明:该克隆为人的核苷酸二磷酸激酶亚基B(NDK/Nm23-H2)cDNA;(3)NDK/Nm23-H2与缺少外显子12、14-17的FMRP异构体,以及含FMRP外显子1-12的克隆相互作用,而与FMRP外显子1-6、外显子2-7不能直接结合。结论人Nm23-H2在酵母细胞内能与FMRP结合,其相互作用区域定位于FMRP的前1-11个外显子。Nm23-H2具反式调控因子特性,FMRP与Nm23-H2的相互作用提示FMRP参与基因表达调控。这一发现有助于进一步研究FMRP在细胞核内的生物学功能。更多还原

【Abstract】 Objective To investigate the physiological role of fragile X mental retardation protein(FMRP)and screen the proteins interacting with FMRP in human fetal hippocampus cDNA library.Methods Human fetal hippocampus cDNA library was constructed in yeast two -hybrid DAD vector pGAD10.Quality of the library was measured by picking up random colonies as templates for PCR testing.Proteins interacting with FMRP were screened by yeast two -hybrid system.Furthermore,the interaction site of FMRP was mapped in yeast.Results The average length of inserts of the two -hybrid library was1.5kb,and the ratio of recombinant colonies was about 90%.Human NDK/Nm23-H2was found interacting with FMRP.NDK/Nm23-H2interacted with FMRP exon1-12,as well as FMRP isoforms without exon12,and exons14-17.NDK/Nm23-H2couldn’ t interact with FMRP exon1-6and exon2-7fragments.Conclusions Human NDK/Nm23-H2can bind FMRP directly.The interaction site of FMRP is located at its exon1-11.This interaction in vitro might alter the intracellular distribution of NDK/Nm23-H2,and even regulate s the transcription and expression of FMRP.更多还原