三种白癜风抗原肽HLA-A*0201四聚体的构建和临床初步应用  

Construction of three HLA-A*0201-peptide tetramers and their preliminary application in detection of vitiligo-specific cytotoxic T lymphocytes

作　　者：吴俊琪[1] 石佳 董剑峰 蒋亦秀 庄顺红[1] 杨军[4] 

机构地区：[1]浙江大学金华医院检验科,浙江金华321000 [2]北京旷博生物技术有限公司,北京100176 [3]金华市第五医院皮肤科,浙江金华321000 [4]杭州师范大学医学院公共卫生系,浙江杭州310016

出　　处：《浙江大学学报（医学版）》2014年第5期553-558,共6页Journal of Zhejiang University(Medical Sciences)

基　　金：浙江省金华市科技局社会发展类重点科研项目(2010-3-056)

摘　　要：目的：体外构建三种白癜风抗原肽HLA-A ＊ 0201复合物四聚体,并用其检测白癜风患者外周血中抗原特异性细胞毒性CD8＋T细胞（CTL）.方法：取原核表达HLA0201＊-BSP和β2M蛋白,分别同MelanA 26-35、gp100 209-217、tyrosinase 1-9三种白癜风抗原肽进行体外复性折叠,形成可溶性抗原肽HLA-A＊0201复合物,经BirA酶作用并通过凝胶过滤层析法纯化得复合物单体,再与藻红蛋白标记的链霉亲合素按4：1比例耦合成四聚体,并采用斑点印迹法对上述构建的四聚体进行鉴定.运用流式细胞术检测分析该四聚体结合白癜风患者外周血中特异性CTL的能力.结果：斑点印迹检测显示三种具有天然构象的生物素化的白癜风抗原肽HLA-A＊0201四聚体复合物构建成功;流式细胞术检测结果显示所制备的四聚体可与白癜风患者血液中抗原特异性CTL结合.结论：成功制备了三种具有完整构象的生物素化白癜风抗原肽HLA-A＊ 0201四聚体,此四聚体均可检测白癜风抗原特异性CTL.Objective： To construct vitiligo-specific HLA-A ＊ 0201-peptide tetramers and to apply the constructed tetramers in detection of vitiligo-specific cytotoxie T lymphocytes （CTL）. Methods： Proteins HLA-A0201 ＊-BSP and β2M were obtained by effective prokaryotic expression. The purified proteins were refolded with vitiligo antigen peptides MelanA 26-35, gp100 209-217, and tyrosinase 1-9, respectively to form HLA- A ＊ 0201-peptide complex, The complex was biotinylated by BirA enzyme and purified by gel-filtration chromatography. The tetramers were generated by mixing the complex with phycoerythrin （PE）-streptavidin at a ratio of 4：1 and identified by Dot-blot assay. The capacity of tetramer to detect vitiligo-specific CTL was analyzed by flow cytometry. Results： The biotinylation of vitiligo-specific HLA-A＊ 0201-peptide tetramers were successfully performed by Dot-blot. Flow cytometry analysis indicated that the tetramer effectively bound to specific CTL from peripheral blood of patients with vitiligo. Conclusion： Three kinds of biotinylated vitiligo-specific HLA-A＊ 0201-peptide tetramers have been constructed successfully. The tetramer can detect antigen specific CTL from patients with vitiligo.

关 键 词：白癜风 HLA-A抗原 β2微球蛋白 抗原抗体复合物 T淋巴细胞 细胞毒性 聚合物 肽类 

分 类 号：R758.41[医药卫生—皮肤病学与性病学]