抗人4-1BB功能性单克隆抗体的研制及生物学特性研究

研制鼠抗人4-1BB功能性单克隆抗体及其在T细胞活化、增殖及信号转导中的作用。以小鼠肾肿瘤细胞转染人4- 1BB基因的细胞株4-1BB/293T为免疫原.采用B淋巴细胞杂交瘤技术,获取分泌特异性4-1BB单抗的杂交瘤细胞株。以体内诱生法产生腹水,Protein G亲和层析法进行纯化,快速定性试纸法鉴定单抗的亚类,MTT法检测单抗对T细胞的刺激作用,流式法检测单抗对T细胞凋亡的影响。结果显示:成功获得1株持续分泌特异性抗人4-1BB单抗的细胞株(命名为5D5).属于IgG2b。该单抗能特异识别人4-1BB分子,介导有效的共刺激信号,体外促进T细胞的增殖,抑制活化诱导的T细胞凋亡。总之,成功获得1株功能性4-1BB单抗,具有重要的研究和潜在应用价值。

Preparation of anti-human 4-1BB monoclonal antibody and analysis of its biological functions

To prepare the monoclonal antibody of human 4-1BB and to study its biological function,the hybridomas were ob- tained by fusing splenocytes of BALB/c mice which was immunized by the transfected cell line 4-1BB/293T with myeloma cells (SP2/0).The monoclonal antibodies were produced by in vivo procedures in mouse peritoneal cavity and purified by protein G affinity chromatography.Fast-strip method was used to analyse the subclass of the mAb.The proliferation of peripheral blood T cells(PBTC)induced by 4-1BB mAb was determined by MTT.Flow cytometry was used to determine the apoptosis of T cells influenced by 5D5.In this way,one hybridoma cell line 5D5 was obtained,which showed the property of secreting anti- human 4-1BB monoclonal antibody continuously and steadily.It belonged to mouse IgG2b.This monoclonal antibody could spe- cifically recognize human 4-1BB molecule and effectively mediate costimulatory signal.It could also promote the proliferation of T cells and inhibit activation induced cell death of PBTC.In a word,one hybridoma cell line 5D5 which can secret functional antibody was developed successfully,It may be of significant value in basic studies and clinical application.