猪IFN-α1重组蛋白单克隆抗体杂交瘤细胞株的建立及其生物学特性

Development and biological characteristics of hybridoma cell lines secreting monoclonal antibody against recombinant porcine IFN-α1

应用杂交瘤细胞技术,将用猪IFN-α1重组蛋白免疫的小鼠脾细胞经与骨髓瘤细胞融合、筛选和多次克隆化培养,成功获得了3株能稳定分泌抗猪IFN-α1的单克隆杂交瘤细胞株,分别将其命名为4E9、5B2和5H11。这3株杂交瘤细胞分泌的单克隆抗体经ELISA检测,体外细胞培养上清的效价均可达到1∶104,体内腹水的效价均可达1∶105;SDS-PAGE分析显示,纯化的这3株单克隆抗体的重链分子质量均在60ku以上,轻链均在30ku以上,符合抗体IgG重链、轻链分子的大小;抗体亚类鉴定表明4E9、5B2为IgG2a类,5H11株为IgG2b类,且其抗原结合位点基本一致。细胞染色体分析表明,3株杂交瘤细胞株染色体平均数为88～96条,远高于骨髓瘤细胞或脾细胞的染色体数,从遗传角度证实获得了杂交瘤细胞株;在杂交瘤细胞的稳定性试验中,这3株细胞经多次冻存复苏和传代培养,不同代次细胞上清中的抗体效价均在1∶104～1∶105之间,说明该3株杂交瘤细胞分泌单克隆抗体的性能稳定。

Three hybridoma cell lines secreting stabily anti-rpIFN-α1monoclonal antibody（McAb） were obtained successfully with a series of methods of hybridoma technology,cloning of culture and ELISA screening,and named 4E9,5B2and 5H11strains,respectively.The biological characteristics of the three hybridoma cell lines were analyzed.Titers of 4E9,5B2and 5H11hybridoma lines secreting McAb all reached 1∶105 in ascites,and 1∶104 in the cell supernatant in ELISA detection.Heavy chain molecular weight of the three McAbs was more than 60ku,light chain was more than 30ku by SDS-PAGE analysis. 4E9and 5B2strains were IgG2aisotype,5H11strain was IgG2bisotype by subtype identification,and their antigen-binding site of McAb were all similar.The numbers of chromosome in the three hybridoma cell lines were 88to 96pairs,more than the number of chromosomes in myeloma cell or splenocyte,demonstra- ting that hybridoma cell lines were obtained from the genetic view.By stability testing of the hybridoma, the titer of the three cells secreting antibody was up to 1∶104 to 1∶105 on the different passage cells through passage culturing,indicating that the three hybridoma cells had good stability in secreting McAb.