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目的本研究拟通过免疫学方法寻找帕金森病(PD)患者血清中IgG针对的黑质区域靶蛋白,初步探索血清IgG在帕金森病发病机制中的作用。方法分别收集临床确诊的PD(10人)和正常对照组(10人)血清,用免疫亲和层析法分离出血清IgG,提取正常SD大鼠黑质膜蛋白及胞浆蛋白制备抗原,用免疫沉淀法筛选出与血清IgG相互作用的蛋白,SDS-PAGE电泳进行蛋白分离后获得差异蛋白条带,质谱鉴定差异蛋白并进行生物信息学分析。结果 PD组与正常对照组的血清IgG仅与大鼠黑质结构中的膜蛋白发生结合,而与胞浆蛋白不发生结合,在两组膜蛋白的免疫沉淀中可以找到一个差异性条带,相对分子质量约为60 000。利用离子阱串联质谱(HPLC-IRON-TRAP-MS/MS)及生物信息学分析,获得了2个候选蛋白:α微管蛋白(α-tubuin)和细胞角蛋白(cytokeratin)。结论 PD患者血清IgG在大鼠黑质结构中的靶蛋白为膜蛋白,与正常组比较,PD血清IgG与膜蛋白的结合存在差异,表明在黑质结构中膜蛋白可能与血清IgG结合参与PD的发病与进展过程。
ObjectiveThis study intends to find the target protein in the substantia nigra of Parkinson’s disease (PD) by immunological methods and to explore the role of serum IgG in the pathogenesis of Parkinson’s disease. Methods Sera of clinically diagnosed PD (10 human) and normal controls (10 human) were collected respectively, serum IgG was separated by immunoaffinity chromatography, antigens were extracted from the plasma membrane proteins of the substantia nigra of normal SD rats, The proteins that interact with serum IgG were screened by immunoprecipitation. The bands of different proteins were obtained after SDS-PAGE electrophoresis, and the differential proteins were identified by mass spectrometry and bioinformatics analysis. Results The serum IgG of PD group and normal control group only bound to the membrane protein in the substantia nigra of rats, but not to the cytoplasmic protein. A differential band could be found in the immunoprecipitation of the two groups of membrane proteins. The relative molecular mass is about 60 000. Two candidate proteins, α-tubuin and cytokeratin, were obtained by using ion trap tandem mass spectrometry (HPLC-IRON-TRAP-MS / MS) and bioinformatics analysis. Conclusion The serum IgG of PD patients is a membrane protein in the substantia nigra of rats. Compared with the normal group, there is a difference in the binding between PD serum IgG and membrane proteins, indicating that membrane proteins in the substantia nigra may bind to serum IgG and participate in PD The incidence and progress of the process.