利用基因重组抗原研制人CD20单克隆抗体及其功能的研究

Preparation and function assay of monoclonal antibodies against human CD20 by recombinant CD20 antigen

目的 利用基因重组抗原免疫小鼠 ,制备人CD2 0单克隆抗体 ,研究其特异性和功能。方法 用表达重组人CD2 0基因的细胞NIH 3T3免疫BALB c小鼠 ,取其脾细胞与Sp2 0细胞融合 ,以间接免疫荧光法筛选杂交瘤上清。免疫沉淀法及流式细胞术鉴定其识别抗原的相对分子质量 (Mr)和特异性 ;以MTT法、流式细胞术及形态学方法检测诱导细胞凋亡和抑制细胞增殖的功能。结果 利用杂交瘤技术获得了 1株抗人CD2 0的单克隆抗体 1 2 8,它具有CD2 0抗体特异的细胞反应谱 ,其识别的抗原Mr 为 33× 10 3 。MTT实验证实 1 2 8能显著抑制Daudi和Raji细胞生长。结论 利用基因重组抗原制备了 1株能够稳定分泌抗人CD2 0的单克隆抗体的杂交瘤细胞株 1 2 8,此单抗具有抑制CD2 0阳性细胞增殖和诱导其凋亡的功能。

Objective To obtain monoclonal antibodies (McAb) against human CD20. Mothods BALB/c mice were immunized with CD20-expressing NIH-3T3 cells. The spleen cells of immunized mice were used to prepare the McAb by hybridoma techniques. CD20-McAb was selected by indirect immunofluorescence experiments on Daudi cells with cultural supernatant of hybridoma cells. Immunoprecipitation and flow cytometry (FCM) analysis were used to identify the surface molecules on Daudi cells recognized by the prepared antibody. Apoptosis assay and cell proliferation inhibition were examined by MTT, FCM and microscopy. Results A monoclonal antibody against CD20(1-28) was produced. The antibody recognized 33kD molecules on the surface of Daudi cells. FCM analysis indicated that 1-28 accorded with CD20-McAb in specific reaction with cell lines. Functional assay revealed that 1-28 could markedly inhibited the growth of Daudi cells and Raji cells, induced apoptosis of Daudi cells. Conclusion A hybridoma cell line 1-28 secreting CD20-McAb stably was obtained by recombinant CD20 antigen, and the CD20-McAb inhibited proliferation and induced apoptosis of CD20 positive cells.