识别α-synuclein N端结构域的单克隆抗体鉴定及免疫应用  

Identification the monoclonal antibodies recognizing theα-synuclein N-terminal domain and their application in immunotherapy

作　　者：贾焕珍 焦洁 高歌[2] 杨慧[2] Jia Huanzhen;Jiao Jie;Gao Ge;Yang Hui(Central lab,Yanjing Medical College,Capital Medical University,Beijing 101321,China;Department of Neurobiology,School of Basic Medical Sciences,Capital Medical University,Center of Parkinson's Disease Beijing Institute for Brain Disorders,Beijing Key Laboratory of Neural Regeneration and Repair,Key Laboratory for Neurodegenerative Diseases of Ministry of Education,Beijing 100069,China)

机构地区：[1]首都医科大学燕京医学院中心实验室,北京101321 [2]首都医科大学基础医学院神经生物学系北京脑重大疾病研究院帕金森病研究所北京市神经再生修复重点实验室神经变性病教育部重点实验室,北京100069

出　　处：《首都医科大学学报》2023年第6期1022-1028,共7页Journal of Capital Medical University

基　　金：国家自然科学基金项目(81870994)。

摘　　要：目的分析5种识别α-突触核蛋白(α-synuclein,α-syn)N端结构域的单克隆抗体1C16、2B8、2P21、3O18和1J6的特异性,为帕金森病(Parkinson s disease,PD)的早期诊断和免疫治疗提供抗体支持。方法体外蛋白重组技术获得人源α-syn蛋白(human-α-syn,h-α-syn)、鼠源α-syn蛋白(mouse-α-syn,m-α-syn)、β-syn蛋白、N端人源α-syn蛋白(α-syn/N)和去N端人源α-syn蛋白(α-syn/ΔN)。斑点印迹法鉴定5种单克隆抗体的特异性识别结构域,使用蛋白质印迹技术检测其对变性后的纯蛋白和鼠脑组织的识别情况。结果抗体1C16可以识别非变性的h-α-syn纯蛋白和小鼠脑组织中的变性的h-α-syn蛋白,不识别m-α-syn和β-syn。抗体1J6仅识别非变性的h-α-syn及α-syn/N纯蛋白,不识别变性后的全长α-syn纯蛋白和小鼠脑组织中的变性h-α-syn蛋白。结论筛选出的2种单克隆抗体具有特异性,可为本实验下一步生物标志物的酶联免疫吸附法测定检测和针对α-syn的免疫治疗提供基础。Objective To analyze the specificity of five monoclonal antibodies 1C16,2B8,2P21,3O18 and 1J6 that recognizeα-synuclein(α-syn)N-terminal domains,to provide antibody support for early diagnosis and immunotherapy of Parkinson's disease(PD).Methods We generated humanα-syn(h-α-syn),mouseα-syn(m-α-syn),β-syn,N-terminus humanα-syn(α-syn/N)and delete N-terminus humanα-syn(α-syn/ΔN)protein using recombinant protein purification technology in vitro.The specific recognition domains of the five monoclonal antibodies were detected by dot blotting.Using Western blotting to detect the denaturedα-syn from purified protein and mice brain.Results Antibody 1C16 recognized h-α-syn proteins in native form and h-α-syn protein in the mice brain in denatured form rather than m-α-syn andβ-syn.Antibody 1J6 could recognize h-α-syn andα-syn/N protein in native form rather than full-lengthα-syn protein and h-α-syn protein in the mouse brain in denatured form.Conclusion The two selected antibodies are specific to humanα-syn,which provide the basis for the detection of biomarkers by enzyme linked immunosorbent assay and can be used for immunotherapy in PD.

关 键 词：帕金森病 Α-突触核蛋白 α-突触核蛋白N端 蛋白纯化 单克隆抗体 

分 类 号：R394[医药卫生—医学遗传学]