负载HCMV pp65抗原肽的HLA-A*1101-GPI四聚体的制备和鉴定  

Preparation and identification of HLA-(A*1101)-GPI tetramer loading with human cytomegalovirus pp65 antigen peptide

作　　者：李丰耀[1] 查庆兵[1] 徐丽慧 迟晓云[1] 贾仟涛[1] 何贤辉[1] 曾耀英[1] 

机构地区：[1]暨南大学生命科学技术学院组织移植与免疫实验中心,广州510632 [2]生物工程研究所,广州510632

出　　处：《现代免疫学》2006年第6期459-464,共6页Current Immunology

基　　金：国家自然科学基金资助项目(30230350;30371651和30572199)

摘　　要：为体外复性制备负载人巨细胞病毒(HCMV)pp65抗原肽的HLA-A*1101-GPI四聚体,研究优化HLA-A*1101重链与生物素化酶底物肽融合蛋白(HLA-A*1101-BSP)在大肠杆菌中的诱导表达的温度、时间和诱导剂IPTG浓度,并以抗HLA-A*0201抗血清进行免疫印迹鉴定HLA-A*1101-BSP的表达水平。将初步纯化的HLA-A*1101-BSP与β2-微球蛋白(β2m)和HCMV抗原肽pp6516-24(GPISGHVLK,简称GPI)一起利用稀释法进行重折叠复性,获得可溶性HLA-A*1101-GPI单体,经生物素化和纯化后,与Streptavidin-PE结合成四聚体。流式细胞仪分析显示HLA-A*1101-GPI四聚体具有与特异性细胞毒T细胞(CTL)的结合活性,表明成功获得可溶性HLA-A*1101-GPI四聚体,为研究HLA-A*1101限制性CTL的免疫应答打下基础。The HLA-A ＊ 1101-GPI tetramer loading with human cytomegalovirus （HCMV） pp65 antigen peptide was prepared by using method of refolding in vitro, and the expression of heavy chain of HLA-A ＊ 1101 fused with biotinylation enzyme （Bi-rA） substrate （HLA-A ＊ 1101 BSP） in E. coli was optimized in inducing temperature and time as well as the concentration of IPTG for induction. This tetramer was identified by immuno-blotting with anti-HLA-A＊0201 serum. In the present study, HLA-A＊ 1101-BSP and β2m were used as the heavy and light chain respectively, to refold in the presence of an HCMV anti genic peptide pp6516 21 （GPISGHVLK, GPI） with dilution method. The soluble monomer of HLA-A ＊ 1101-GP1 obtained was then combined with Streptavidin-PE to form tetramer after biotinylation and purification. It was found that the tetramer formed could bind specifically with cytotoxic T lymphocytes, as demonstrated by flow cytometry, indicating the successful preparation of HLA-A＊ 1101 GPI tetramerand thus providing the basis of further studies on the HLA-A＊ 1101 restricted immune responses against HCMV infection.

关 键 词：HLA-A＊1101 四聚体 流式细胞术 巨细胞病毒 

分 类 号：R392.11[医药卫生—免疫学]