神经生长抑制因子相互作用蛋白的分子克隆、鉴定及其生物学特性

为了探讨神经生长抑制因子(Neuronal growth inhibitory factor，GIF)与Alzheimer’s病(Alzheimer’s disease，AD)的关系，将GIF的cDNA全基因克隆到载体pHyblex中,运用酵母双杂交系统从Alzheimer’s病人脑cDNA文库中筛选出与GIF相互作用蛋白的cDNA克隆。免疫共沉淀和蛋白质印迹实验进一步验证了该蛋白在体内与GIF相互作用的特异性。克隆并鉴定了其中1个与GIF特异性结合的蛋白，与人细胞核dUTP焦磷酸酶(DUT)同源。进一步构建了重组表达质粒pGEX-4T-1／DUT，转化大肠杆菌BL21，经谷胱甘肽-Sepharose 4B亲和层析、凝血酶酶切和Sephacryl S100纯化，得到纯度95％以上的dUTPase蛋白。体外生物学活性检测表明，表达的dUTPase蛋白可以与GIF共同作用嗜铬细胞瘤株(pheochromocytoma)PC12，对细胞的生长产生抑制作用。

Molecular Cloning and Identification and Biological Activity of GIF-interacting Protein(s)

In order to examine the elusive functional mechanism of GIF (Neuronal growth inhibitory factor,GIF) and elucidate the possible relationship between GIF and Alzheimer's disease,we constructed bait1 plasmid (pHyblex-GIF) by cloning GIF cDNA directly in frame with plasmid pHyblex,and used the yeast two-hybrid system to screen Alzheimer's disease human brain cDNA library and found the GIF-interacting proteins.The final results from coimmunoprecipitation and western blotting experiments confirmed that interacting proteins specifically binds to GIF.After sequencing the nucleotide of the putative positive plasmids and searching for homologues,we found that one of these is the part of human nuclear dUTPase protein sequence.Then the dUTPase genes are cloned into pGEX-4T-1,the fusion expression vector of GST,and highly expressed in E.coli BL21.The proteins dUTPase and GIF were purified and obtained by affinity chromatography,thrombin digestion and gel filtration on Sephacryl S100.It demonstrated that the proteins dUTPase and GIF had the growth inhibitory activity on co-cultured neuron in vitro.The inhibitory curve was very similar to the GIF.It's possible that dUTPase is one of the proteins interacting with GIF in Alzheimer's disease human brain extracts.