生物素化的HLA-A*2402-抗原肽复合物的制备  

Preparation of biotinylated HLA-A*2402-peptide complex

作　　者：卢小玲[1] 吴雄文[1] 翁秀芳[1] 李青[1] 梁智辉[1] 龚非力[1] 

机构地区：[1]华中科技大学同济医学院免疫学系,武汉430030

出　　处：《免疫学杂志》2006年第1期78-81,共4页Immunological Journal

基　　金：国家自然科学基金(30271201);科技部"973"计划(2001CB510008)资助项目

摘　　要：目的制备生物素化的HLA-A*2402-抗原肽复合物。方法将原核高效表达的sHLA-A*2402-BSP融合蛋白及β2m和HLA-A*2402限制性抗原肽EB病毒BRLF1蛋白中的九肽NH2-TYPVLEEMF-COOH进行稀释、复性、折叠,形成HLA-A*2402-抗原肽复合物单体,并在BirA酶作用下进行生物素化,使生物素结合到HLA-A*2402-抗原肽复合物中H链C端的BSP序列上形成生物素化的sHLA-A*2402-抗原肽复合物单体。利用特异性单克隆克体(W6/32和兔抗人2β微球蛋白抗体)及链霉亲合素进行ELISA和Western blot,检测稀释复性和生物素化的折叠产物。结果折叠复合物中,主要含有HLA-A*2402-肽复合物单体及β2m两种成分,其中HLA-A*2402-肽复合物单体可生物素化。结论成功制备出生物素化的HLA-A*2402-抗原肽复合物单体,为进一步构建四聚体及制备人工抗原提呈细胞奠定了基础。Objective To prepare biofinylated HLA-A ＊ 2402-peptide complex. Methods The BirA substrate pepfide （BSP） containing H chain of HLA-A ＊ 2402 and β2 m were expressed highly as insoluble aggregates in E. coli, and then the two subunits refolded to form an HLA-A ＊ 2402-peptide complex by dilution method in the presence of an antigenic peptide [ NH2- TYPVLEEMF-COOH of EB virus BRLF1 ]. The refolded complex was biotinylated by the BirA enzyme. The refolded and biotinylated products were detected by ELISA and Werstern blotting with mAb （W6/32 and rabbit anti-hmnan β2m antibody） and streptavidin. Results The refolded complexes was composed of HLA-A ＊ 2402-peptide complex and β2 m. The HLA-A ＊ 2402-peptide complex could be biotinylated. Conclusion The refolding and biotinylation of HLA-A ＊ 2402-peptide complex are successfully achieved and proved by our practical immunological methods. This study lays a foundation for the preparation of HLA-peptide tetramer and artificial antigen presenting cell.

关 键 词：HLA—A＊2402-抗原肽复合物 生物素化 BirA酶 

分 类 号：R392[医药卫生—免疫学]