H-2K^d四聚体的制备及其在检测特异性同种CTLs中的应用  

Preparation of GAD/H-2K^d tetramer for specific alloreactive CTLs detection

作　　者：陈雪玲[1,2] 严永祥[2] 唐婕[2] 翁秀芳[2] 吴雄文[2] 

机构地区：[1]石河子大学医学院免疫学教研室,832002 [2]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》2011年第5期424-427,共4页Immunological Journal

基　　金：国家自然科学基金项目(81060227);石河子大学人才启动基金(RCZX200829)

摘　　要：目的制备H-2Kd限制性胰岛GAD抗原肽/H-2Kd四聚体并用于检测相应的特异性同种CTL。方法以原核表达的sH-2Kd-BSP为重链,β2m为轻链,与人工合成的GAD抗原肽在体外利用稀释法进行共折叠复性得到单体。然后在BirA酶作用下对其进行生物素化,通过凝胶过滤层析法纯化生物素化的复合物单体,再与Strep-tavidin-FITC按4∶1比例混合形成四聚体;取Balb/c小鼠(H-2Kd)巨噬细胞加载胰岛GAD抗原肽,作为刺激细胞,取C3H小鼠脾细胞(H-2KK)作为效应细胞,在体外进行混合淋巴细胞培养,以获得针对H-2Kd限制性胰岛GAD抗原肽的特异性同种CTL。结果该四聚体能够与长期混合淋巴细胞培养出的同种特异性T细胞结合。结论成功构建胰岛GAD抗原肽/H-2Kd四聚体,并可用于针对H-2Kd限制性胰岛GAD抗原肽特异性同种CTL的检测。Major histocompatibility complex（MHC） tetramer technology offers a powerful means to study specific T cell populations of interest.Our aim was to prepare GAD/H-2Kd tetramer for specific alloreactive CTLs detection.The prokaryotic sH-2Kd-BSP heavy chain and the β2m light chain were refolded by dilution method in presence of a synthetic antigenic peptide（SYQPLGDKV） to form a complex in vitro.The complex was biotinylated in presence of BirA enzyme and purified by gel-filtration chromatography,and then mixed with Strep-tavidin-FITC at a ratio of 4∶1 to produce a tetramer.For generation of peptide-specific alloreactive CTLs,C3H splenocytes were co-cultured with Balb/c M loaded with the GAD peptide in vitro.The peptide-specific alloreactive CTLs were stained with GAD/H-2Kd tetramer.Thus,the GAD/H-2Kd tetramer has constructed successfully and can to detect H-2Kd restrictive GAD peptide-specific alloreactive CTLs.

关 键 词：GAD抗原肽/H-2Kd四聚体 特异性CTL 同种反应 

分 类 号：R392.33[医药卫生—免疫学]