鼠抗人4-1BB单克隆抗体的研制及其生物学特性的鉴定  

Preparation of anti-human 4-1BB monoclonal antibody and characterization of its biological activities

作　　者：周桓[1,2] 张光波[2] 董秋明[2] 於葛华[2] 徐颖[2] 张学光[2] 

机构地区：[1]苏州大学附属第四医院检验科,江苏无锡214062 [2]苏州大学医学生物技术研究所,江苏苏州215007

出　　处：《细胞与分子免疫学杂志》2011年第9期993-996,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：江苏省卫生厅重点实验室项目(K0622);国家自然科学基金资助项目(30330540)

摘　　要：目的:鼠抗人4-1BB分子功能性单克隆抗体(mAb)的研制及其生物学特性的鉴定。方法:以高表达人4-1BB分子的转基因细胞293T/4-1BB为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,并以293T/4-1BB为阳性抗体筛选细胞,293T/mock为阴性抗体筛选细胞,经免疫荧光标记和流式细胞术(FCM)分析、反复筛选和多次克隆化培养,筛选出特异分泌鼠抗人4-1BB分子mAb的杂交瘤细胞株;采用W estern b lot、间接免疫荧光法、竞争结合抑制试验、3H掺入和细胞凋亡分析等方法对mAb进行生物学特性的鉴定。结果:成功获得3株持续、稳定分泌鼠抗人4-1BB mAb的杂交瘤细胞株,命名为1G5、4B11和9F11。对mAb的生物学功能的研究结果表明,3株mAb均能识别活化T细胞和单核细胞来源的树突状细胞(Mo-DC)表达的4-1BB分子。mAb 4B11能有效地激发T细胞体外增殖、抑制其凋亡,并能促进Mo-DC体外扩增,上调CD80、CD83、CD86和CD1α的表达。结论:获得的3株分泌鼠抗人4-1BB mAb的杂交瘤,所分泌的抗体具有特异性地识别人4-1BB分子;其中4B11 mAb具有激发T细胞增殖及促进Mo-DC体外扩增与成熟的作用,为激发型4-1BB mAb。AIM： To prepare an anti-human 4-1BB functional monoclonal antibody and to characterize its biological activities.METHODS： A stable human 4-1BB molecule transfected cell line 293T/4-1BB was used as an antigen to immunize BALB/c mice.By means of the cell fusion by hybridoma technique and multiple cell subcloning and repeated screening with 293T/4-1BB as the antibody screening positive cell while 293T/mock as the negative cell.The hybridoma cell lines specifically secreting anti-4-1BB monoclonal antibodies were selected.Then their characteristics and its biological activities were investigated by Western blot,fast-strip routine Ig subclass typing method,indirect immunofluorescence,competitive inhibition test,3H-TdR and cell apoptosis analysis.RESULTS： Three hybridoma cell lines 1G5,4B11 and 9F11 with the property of secreting specific anti-4-1BB monoclonal antibody continuously and steadily were successfully obtained.These monoclonal antibodies could bind to human 4-1BB epitopes on activated T cells and monocytoes and DC.Additionally,mAb 4B11 could promote T proliferation and enhance the growth and maturation of Mo-DC.CONCLUSION： Three hybridoma cell lines which secrete anti-4-1BB monoclonal antibodies steadily have been established.These monoclonal antibodies could specifically recognize 4-1BB molecule and mAb 4B11 had a potent function to promote T proliferation cell as well as to enhance the growth and maturation of Mo-DC in vitro.

关 键 词：4-1BB 杂交瘤 共刺激信号 单克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]