Jα33基因工程蛋白与多克隆抗体的制备及初步应用  

Preparation of the anti-Ja33 polyclonal antibody and its preliminary application

作　　者：肖睿璟[1,2] 吴孟君[3] 王梅 彭妹 郑颖城 郑小玲 何玉玲[1,2] 谭锦泉[1,2] 

机构地区：[1]武汉大学医学结构生物学研究中心 [2]武汉大学基础医学院免疫系,湖北武汉430071 [3]宜昌市中心人民医院肾病内科,湖北宜昌443003

出　　处：《现代免疫学》2012年第5期365-370,共6页Current Immunology

基　　金：国家自然基金项目(30730054;30971279;30901363);中央高校基本科研业务费专项资金(301274622)

摘　　要：为了获得同时识别人类和小鼠MAIT细胞的特异性抗体,我们制备了MAIT细胞TCRα链Jα33融合蛋白及其抗体,为MAIT细胞在炎症性肠道疾病(IBD)中作用的研究提供有利的工具。用Jα33的全长序列为引物经PCR扩增Jα33串联体,扩增的串联体片段连接进入T载体,阳性克隆转入pGEX-4T-1表达载体中原核表达GST-Jα33融合蛋白,纯化后的融合蛋白免疫新西兰大白兔制备anti-Jα33多克隆抗体。并分别用Western blot和RT-PCR检测IBD模型小鼠MAIT细胞Jα33的表达。成功获得分子量约为38 kD的融合蛋白,免疫大白兔后得到抗Jα33的多克隆抗体,Western blot结果显示Jα33多克隆抗体能检测到人外周血单个核淋巴细胞表面Jα33的表达,而HeLa中则无Jα33的表达;MAIT细胞Jα33在IBD小鼠肠组织中表达量明显低于正常对照。成功制备了能同时识别人类和小鼠MAIT细胞Jα33的多克隆抗体,为研究MAIT细胞的定位和功能开启了方便之门。To obtain specific antibodies which can identify human and mouse MAIT cells, we produced a polyclone antibody a gainst TCR Jα33 from mouse MAIT cells. This antibody facilitating the study of MAlT cells in IBD. Jα33 concatemer was amplified by PCR using the Jα33 full sequence as primers. And amplified concatemers were ligated into the T vector. Then posi tive clones were transferred to pGEX4T1 expression vector to produce the GSTJα33 fusion protein. The purified fusion pro tein was used to immunize New Zealand white rabbits and produced the antiα33 polyclonal antibody. Respectively, we detec ted the specificity of the antiJa33 antibody with Western blot and immunofluorescence, we successfully obtained the fusion pro tein of the molecular weight about 38kD. After immunized the white rabbit we got an antiJα33 polyelonal antibody. Western blot results showed that this Jα33 polyclonal antibody could detect the expression of Jα33 on the surface of human peripheral blood mononuclear cells. However, HeLa cells have no expression of Jα33. The expression of MAIT cell＇s a chain in the intes tine of IBD model was lower than that in the normal controls. We successfully produced a polyelonal antibody that can recognize human and mouse MAIT cells, which will open the door for the research of the localization and function of MAIT cells.

关 键 词：MAIT细胞 抗体制备 

分 类 号：R392-33[医药卫生—免疫学]