Construction, Expression And Characterization Of A Human-Mouse Chimeric Antibody Against Human CD25

Basing on the anti-human CD25 hybridoma cell established by our laboratory, we constructed a human-mouse chimeric antibody expression plasmid against human CD25 and then expressed it in CHO cell. The aim was to establish a CHO cell line which stably expressed chimeric antibody against human CD25 and characterize its biological activity and lay foundation for the further development of safe and effective therapeutic antibody, and we also hoped to achieve progress in the study of genetically engineered antibody.Firstly, total RNA was extracted from the murine anti-human CD25 hybridoma cell line, the VH and VL gene of the hybridoma cells were amplified by RT-PCR with the degenerate primers binding to signal peptide sequences of the heavy and light variable chain and primers binding to constant region sequences. Human antibody heavy and light constant region were cloned by PCR using plasmid PAG4622 as a template. The mouse variable region and human constant region were spliced by PCR method, the heavy and light human-mouse chimeric genes were ligated with plasmids pOptiVEC and pcDNA3.3 respectively and cotransfected 293T cells throμgh lipofectomine method.The antibody expression lever was determined by sandwich ELISA; the antigen binding activity of the chimeric antibody was characterized by FCM. The results showed that the chimeric antibody could bind to the antigen and had human antibody constant region. The eukaryotic expression plasmids of chimeric antibody were constructed succesffuly.Secondly, plasmids pOptiVEC-H and pcDNA3.3-L were cotransfected CHO-dhfr- cells for stable expression.Quantitative ELISA result showed that the antibody expression lever was 103ng/ml when MTX concertration was 300nM;220μg antibody was harvested in total; the antibody purity was more than 97% analysed by SDS-PAGE; Western blotting result showed that the chimeric antibody had human heavy and light chain constant region; Dot Blotting and Western blotting results showed that the chimeric antibody could bind to CD25 molecule specifically; the competitive analysis showed that there was a competitive relationship between the chimeric and parental antibody and they may recognize the same epitope; CCK result showed that T lymphocytes proliferation could be inhibited by the chimeric antibody; biocore results showed that the affinity of chimeric antibody was about 1.9×1010l/mol; the cell line 1C1 could stably secret antibody for 3 months continuously.In summary, we constructed and expressed a human-mouse chimeric antibody against human CD25 in CHO cells successfully, which laid the foundation for the development of genetically engineered antibody against human CD25.