sH-2K^d肽复合物的构建及多克隆抗体的制备  

Construction of soluble H-2K^d-peptide complex and preparation of polyclonal antibody

作　　者：杨新星[1] 郝友华[1] 张正茂[1] 宋娜[1] 杨东亮[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院临床免疫研究室,武汉430030

出　　处：《免疫学杂志》2007年第4期436-439,共4页Immunological Journal

基　　金：国家重点基础研究发展计划项目973计划资助(20014CB510008)

摘　　要：目的构建可溶性的H-2Kd肽复合物,并制备抗MHC-Ⅰ(H-2Kd)分子多克隆抗体。方法原核高效表达的sH-2Kd-BSP和β2 m蛋白,在抗原肽(乙型肝炎病毒的核心蛋白HBc131-139)的存在下,体外复性折叠成可溶性的H-2Kd-β2m-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。应用纯化的该复合物单体免疫C57BL/6小鼠制备多克隆抗体。结果①Western blot检测显示获得了生物素化的sH-2Kd-HBc复合物单体。②Western blot检测显示抗MHC-Ⅰ(H-2Kd)分子多克隆抗体与复合物单体有很好的抗原抗体反应;Balb/C小鼠脾脏冰冻切片的免疫组化结果:在细胞膜表面见棕色着色。③Dot-ELISA检测该多克隆抗体的滴度可以达到1∶3 200。结论成功构建了具有完整构象的生物素化sH-2Kd-HBc复合物单体,制备了抗MHC-Ⅰ(H-2Kd)分子多克隆抗体。Objective To construct soluble H-2K^d-peptide complex and prepare anti-major histocompatibility complex（MHC）-Ⅰ （H- 2K^d ） polyclonal antibody. Methods Proteins sH-2K^d-BSP and β2m were obtained by effective prokaryotic expression. After being purified, the proteins were refolded into sH-2K^d-β2m-peptide complex in the presence of an antigenic peptide （Hepatitis B virus core 131-139） by dilution method. The complex then was biotinylated by BirA enzyme and purified via the gel-fdtration chromatography. The purified complex was injected into C57BL/6 mice to prepare polyclonal antibody. Results ①The biotinylated sH-2K^d-HBc complex was identified by Western blotting. ②Western blotting and immunohistochemistry showed that the antibody could bind to the complex specifically. ③The Dot-ELISA titer of the polyclonal antibody was about 1 ： 3200. Conclusion The soluble H-2K^d-peptide complex is constructed and the anti- MHC-Ⅰ （ H-2K^d ） polyclonal antibody is prepared successfully.

关 键 词：sH-2K^d-β2m-抗原肽复合物 多克隆抗体 

分 类 号：R392[医药卫生—免疫学]