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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-2l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。
OBJECTIVE: To identify the interaction between PINK1 and α-synuclein and the distribution of two proteins in MN9D cells by proteomic methods and laser scanning confocal microscopy. Methods: The α-synuclein gene was inserted into pGEX-4T-1 vector and proved to be a GST fusion protein by DNA sequencing. The fusion protein was expressed in E. coli BL-2l and incubated with MN9D cell lysate to detect the protein between PINK1 and α-synuclein Interaction. The co-immunoprecipitation assay of MN9D cells with endogenous PINK1 and α-synuclein was used to further verify the interaction between the two proteins. MN9D cells were immunocytochemically stained and the intracellular distribution and co-localization of the two proteins were observed by laser scanning confocal microscopy. Results: The specific PINK1 band was detected by GST-α-synuclein fusion protein capture and co-immunoprecipitation. Laser scanning confocal microscopy showed some co-localization between the two. Conclusion Both PINK1 and α-synuclein can interact with each other in vitro and in vivo and co-localized in MN9D cells.