摘要
目的制备HBc-HLA-A ＊ 1101-β2m复合物单体和四聚体,并对其特异性进行了鉴定。方法原核高效表达的HLA-A ＊ 1101-BSP和β2m蛋白,在抗原肽（乙型肝炎病毒的核心蛋白HBc88-96）的存在下,体外复性折叠成可溶性的HLA-A ＊ 1101抗原肽复合物单体,经BirA酶作用并通过凝胶过滤层析法纯化复合物单体;然后将此复合物单体与藻红蛋白标记的链霉亲和素按一定比例耦合构建成四聚体;最后用流式细胞仪检测分析该四聚体结合特异性CTL的能力。结果Western blot和Dot-ELISA检测显示所制备的HLA-A ＊ 1101抗原肽复合物单体具有天然构象,且可被生物素化;流式细胞仪分析显示所制备的四聚体可与HLA-A11^＋供者的抗原特异性CTL结合。结论成功制备了具有完整构象的生物素化HLA-A ＊ 1101抗原肽复合物单体;此四聚体可以特异检测抗原特异性的细胞毒性T细胞。
Objective：To prepare HLA-A ＊ 1101-HBc complex monomer and tetramer, and identify the specificity of the tetramer in detection of HLA-A11 restricted, HBV-specific cytotoxic T lymphocytes （CTL）. Methods ： Proteins HLA-A ＊ 1101 -BSP and β2m were obtained by effective prokaryotic expression. After being purified, they were refolded into HLA-A ＊ 1101-β2m-peptide complex in the presence of an antigenic peptide（ Hepatitis B virus core 88-96） by dilution method. The complex then was biotinylated by BirA enzyme and purified the gel-filtration chromatography. Tetramers were generated by mixing the complex with PE-Streptavidin at a molar ratio of 5： 1. The capactiy of the tetramer to detect specific CTL was analysed by Flow cytometry. Results：The refolding and biotinylation of HLA-A ＊ 1101 -HBc complex was successfully performed by Western blot and Dot-ELISA. Flow cytometry analysis indicated that the tetramer could bind to specific CTL from HLA-A11^＋ donor. Conclusion：We successfully prepared the biotinylated HLA-A ＊ 1101-peptide complex monomer and tretamer. The tetramer could be used to detect antigen specific CTLs.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2007年第4期352-354,356,共4页
Chinese Journal of Immunology
基金
国家自然科学基金资助项目(批准号:30400412)
