摘要
利用重组质粒pCZS转化大肠杆菌,制备出人重组铜-锌超氧化物歧化酶(rhCu-Zn SOD),进一步建立了3株鼠抗rhCu-Zn SOD的单克隆抗体(McAb)杂交瘤细胞株.经鉴定,它们分泌的McAb分别属于IgG_1和IgG_2a亚类,能特异性地识别人Cu-Zn SOD,与pCZS空载菌蛋白无交叉反应.将此McAb制备了免疫亲和层析柱,从大肠杆菌的粗提液中纯化出rhCu-Zn SOD,十二烷基磺酸钠—聚丙烯酰胶凝胺电泳(SDS-PAGE)显示单一条带,纯度>98%,回收率50%,酶比活达10 971.3 U/mg蛋白,rhCu-Zn SOD McAb的制备为Cu-Zn SOD产品的纯化、分析及作用机理研究等提供了有力的工具.
In the study recombinant human Cu-Zn SOD was used as antigen for preparating McAbs. Threehybridoma cell lines, B3,C1,D12 were obtained. The results showed that the McAbs belonged to IgG1and IgG2s subclasses. They can only react with human Cu-Zn SOD. none of them can cross-react withother E. coil proteins. Crude rhCu-Zn SOD from E. cob was purified by immunoaffinity chromatography using McAbC1. The purified enzyme has a single hand in SDS-PAGE, The purity is over 98% andthe recovery is 50%. Its specific activity was 10 971. 2U/mg protein. The McAbs may be of useful toolsfor study of biologic properties and functions of Cu-Zn SOD as well as for purification of rhCu-Zn SOD products.
关键词
单克隆抗体
超氧物歧化酶
铜
锌
<Keyword>Cu-Zn superoxide dismutase Monoclonal antibody Immunoaffinity Chromatography
