摘要
本研究利用重组质粒ｐＣＺＳ转化大肠杆菌，制备出入重组Ｃｕ－Ｚｎ超氧化物歧化酶（ｒｈＣｕＺｎ－ＳＯＤ），进一步建立了３株鼠抗ｒｈＣｕＺｎＳＯＤ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ＩｇＧ１和ＩｇＧ２ａ亚类，能特异性地识别人ＣｕＺｎＳＯＤ，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ｒｈＣｕＺｎＳＯＤ，ＳＤＳ－ｐＡＧＥ显示单一条带，纯度＞９８％，回收率５０％，酶化活达１０９７１．３Ｕ／ｍｇ蛋白。ｒｈＣｕＺｎＳＯＤ－ＭｃＡｂ的制备为ＣｕＺｎＪｉＤ产品的纯化和抗辐射等应用研究提供了有效的工具。
n the study,recombinant human CuZnSOD was used as an antigen for the preparation of McAbs. Three hybridoma cell lines B3,C1,D(12) were obtained.The resultsshowed that McAbs belonged to IgG1 and IgG2a subclasses;and they can only react withhuman CuZnSOD,while none of them can cross-react with other E. coli proteins。Crde rhCuZnSOD from E;oli was purified by immunoaffinity chromatography usingMcAbCi.The purified enzyme has a single band in SDSPAGE with a purity of over98%and the recovery rate is 50%。Its specific activity is 10 971.3 U/mg protein。TheMcAbs might be a useful tool for the purification and study on the application ofrhCuZnSOD.
关键词
铜
锌
超氧化物歧化酶
单克隆抗体
抗原
制备
Cuzn-superoxide dismutase Monoclonal antibody Chromatography
immunoaffinity
