中文摘要：

目的筛选及鉴定发动蛋白-1（Dynamin-1）的脯氨酸和精氨酸富集域（PRD）功能域在大鼠神经突触小体中的相互作用蛋白。方法构建Dynamin-1PRD功能域的重组原核表达质粒pGEX-4T-2-PRD，通过大肠杆菌表达系统进行诱导表达；谷胱甘肽琼脂糖凝脂柱纯化得到融合蛋白GST—PRD，继而利用谷胱甘肽巯基转移酶沉淀技术，筛选出GST．PRD融合蛋白与分离提取的大鼠神经突触小体的相互作用蛋白，并通过液相质谱技术结合数据库，对上述获得的相互作用蛋白进行分析、鉴定。结果通过构建pGEX-4T-2-PRD重组质粒及诱导表达，成功纯化出了融合蛋白GST—PRD；同时成功提取了大鼠神经突触小体，经谷胱甘肽巯基转移酶沉淀技术联合液相质谱分析，获得在大鼠神经突触小体中与Dynamin-1的PRD域有相互作用的蛋白共35个，分别属于突触囊泡相关蛋白、细胞骨架蛋白、代谢酶及其他类的蛋白。结论本研究获得的与Dynamin-1的PRD域有相互作用的蛋白共35个。

英文摘要：

Objective Synaptic vesicles complete of synaptic vesicles cycle by exocytosis and endocytosis. The retrieval of synaptic vesicle membrane after exocytosis is essential for the maintenance of synaptic transmission in central nervous system synapses. Dynamin-1 is a 96kD multidomain GTPase enzyme that is crucial for the fission stage of synaptic vesicle recycling and vesicle traffick. Several models range from viewing Dynamin-1 strictly as a mechanochemical enzyme to considering it as a regulatory protein for the recruitment of the downstream binding partners responsible for scission. To address the role of Dynamin-1 and its interaction proteins in synaptic vesicle endocytosis, we screened and identified interaction proteins of Dynamin-1 PRD functional domain in rat brain synaptosomes. Methods pGEX-4T-2-PRD, a prokaryotic expression plasmid of PRD functional domain, was constructed into Dynamin-1. GST-PRD fusion proteins were obtained by Escherichia coli （E. coli） expression system combined glutathione agarose purified column. Rat brain synaptosomal fractions were isolated by ultracentrifugation. Glutathione S-transferase（GST） pull-down assay was employed to screen interaction proteins between rat brain synaptosome and GST-PRD fusion proteins. Subsequently, these proteins were identified using liquid chromatography spectroscopy （LC-MS）. Result We successfully purified the GST-PRD fusion proteins and extracted rat brain synaptosome. Thirty-five interaction proteins of Dynamin-1 PRD functional domain in rat synaptosome were isolated and identified, consisting of synaptic vesicle-associated proteins, cytoskeletal proteins, metabolic enzymes and other proteins. Conclusion Here we reported a comprehensive set of candidate proteins that are closely related to synaptic vesicle recycling. The study has laid the foundation for clarifying the function, regulatory mechanism of Dynamin-I and cracking the pathway/protein network of synaptic vesicle recycling. [