中文摘要：

目的：利用蛋白质组学方法和激光扫描共聚焦显微镜来鉴定帕金森病相关蛋白PINK1和α-突触核蛋白（α-synuclein）之间的相互作用关系及两种蛋白在MN9D细胞中的分布。方法：将α-synuclein基因插入pGEX-4T-1载体，经DNA测序证明形成GST融合蛋白，经大肠杆菌BL-21表达纯化后，与MN9D细胞裂解液共孵育，检测PINK1与α-synuclein蛋白间的相互作用。并应用MN9D细胞的内源性PINK1与α-synuclein进行免疫共沉淀实验，进一步验证两蛋白之间的相互作用。MN9D细胞经免疫细胞化学染色，利用激光扫描共聚焦显微镜观察两种蛋白的细胞内分布及共定位状态。结果：利用GST—α—synuclein融合蛋白捕获及免疫共沉淀技术，检测到特异的PINK1条带。激光扫描共聚焦显微镜检测到两者存在部分共定位。结论PINK1和α-synuclein在体外和细胞内均可发生相互作用并在MN9D细胞中存在共定位关系。

英文摘要：

Objeαive ： To identify PINK1 interaαing with α-synuclein in Parkinson＇ s disease. Mαhods： GST -α-synuclein fusion prαein was created by inserting α-synuclein into pGEX-4T-1. The prαein was expressed in Escherichia coli and purified on glutathione-Sepharose beads by standard mαhods. And then incubate with MN9D cells lysates to dαeα the interaαion of PINK1 with α-synuclein. In addition, the full length of PINK1 robustly associated with α-synuclein was also dαermined by coimmunoprecipitation experiment in MN9D cells. lne co- localization of PINK1 with α-synuclein was dαeαed by immunocytochemical staining, and observed by confocal. Results：GST-α-synuclein interaα specifically with PINK1 was dαeαed, but nα GST. Coimmunoprecipitation with an anti-α-synuclein antibody and subsequent Western blαting with an anti-PINK! antibody confirmed the specific interaαion of α-synuclein with PINK1. PINK1 and Conclusion：PINK1 and α-synuclein can interaαs with each α-synuclein prαein partly co-localize in MN9D ceils. αher and co-localizes partly in MN9D