纳米尺度神经细胞膜蛋白单分子免疫细胞化学三维定位方法的建立  

A new immunocytochemistry method to situate the single membrane protein molecule on the cell surface in nanoscale

作　　者：郁毅刚[1] 徐如祥[1] 姜晓丹[1] 柯以铨[1] 

机构地区：[1]第一军医大学珠江医院全军神经医学研究所神经外科,广州510282

出　　处：《中华微生物学和免疫学杂志》2004年第8期618-623,共6页Chinese Journal of Microbiology and Immunology

基　　金：全军十五医学重点科研基金 ( 0 1Z0 5 4) ;国家自然科学基金 ( 3 0 2 70 491)资助项目

摘　　要：目的　建立纳米尺度神经细胞膜表面蛋白单分子三维标记的免疫细胞化学方法。方法　应用原子力显微镜 ,对分布在云母表面的抗NMDAR1亚基IgG 葡萄球菌蛋白A 胶体金颗粒免疫标记复合物分子进行扫描 ,明确其特定的三维结构作为对照标准 ,然后扫描结合了免疫标记复合物分子的神经细胞膜表面 ,对表面形貌作出对比后确定目的抗原的定位和复合物三维结构。并与激光共聚焦免疫荧光方法对比。结果　在空白云母表面 ,免疫复合物分子在原子力显微镜下呈现出均匀分散粒径为 4 9nm的特征性球形结构。在神经元表面结合免疫标记物后可以发现有大量的粒径为 5 3nm的球形或双球形 (短棒状 )结构 ,颗粒均匀 ,截面为双峰或单峰。而在LSCM下荧光呈颗粒点状分布 ,为多个FITC聚集显色的结果。结论　原子力显微镜下免疫胶体金标记技术可以在纳米尺度对目的膜受体蛋白进行定位和表面三维结构测定。NMDA受体蛋白可以结合 1个或 2个胶体金复合物 ,与LSCM相比 ,原子力显微镜下胶体金标记方法可以对单个膜NMDA受体蛋白抗原进行定位、定性、定量标记测定。ObjectiveTo establish a new immunocytochemistry method which could 3D earmark the membrane protein on the cell surface in nanoscale. MethodsThe anti-NMDAR1-IgG-SPA-Gold complexes molecule distributed on the surface of mica was scanned by atomic force microscopy and analyzed characteristic 3D conformation by particle software. The 3D conformation was contrasted with the results of the scanned neuron membrane surface that combined IgG-SPA-gold complexes molecules by atomic force microscopy and the scaned result by LSCM. Then the NMDAR protein is situated under characteristic globe of colloid gold particle. ResultsThe 3D conformation of IgG-SPA-G scanned through mica surface shows the average diameter of globe particles is 49nm. The results combined with IgG-SPA-G shows that the immunity complex are globe or cosh shapes. Its average diameter is 53nm. The long axis section is a typical two apexes structure. ConclusionAtomic force microscopy could determine the situation and 3D conformation of NMDAR protein on neuron membrane surface in nano scale. NMDAR single molecule could combine one or two gold particles. Colloid gold particle scanned by atomic force microscopy is a new method in immunocytochemistry.

关 键 词：纳米尺度 神经细胞膜蛋白 单分子 免疫细胞化学 三维定位方法 胶体金 

分 类 号：R392[医药卫生—免疫学]