Construction, Expression And Primary Biological Function Of Mouse-Human Chimeric Antibody Against CD80

This research was based on the human CD80 antagonist mouse monoclonal antibody (4E5)established by us.We constructed and expressed one mouse-human chimeric antibody against CD80 in CHO cells,and characterized its primary biological function.PartⅠ:construction and expression of mouse-human chimeric antibody against CD80After V_H and V_L gene were amplified by RT-PCR using degenerate primer from 4E5 murine hybridoma cell line,signal peptide sequences of V_H and V_L gene were amplified by SMART-PCR using special primer.Fc,CH1 and Cκgene of human IgG1 were also amplified from pIRES/hu5C11 plasmid.Variable genes containing signal peptide sequences and constant regions of human IgG1 were respectively fused to construct chimeric heavy and light chain by TP-PCR.Chimeric genes were inserted into pIRES to construct co-expressing recombinant plasmid.Recombinant plasmid was firstly transfected into 293T cells using LipofectAMIN kit.Once FCM had detected transient expression of chimeric antibody,recombinant plasmid was transfected into CHO cells with same method,and cells was pressurized by G418 to obtain CHO cell line constantly expressing ch4E5 antibody.Restriction endonuclease digestion and PCR showed that recombinant genes have been cloned into vector pIRES.Chimeric antibody could transiently express in 293T cells.RT-PCR and FCM pointed that chimeric antibody against CD80 specially is stably effectively expressed in CHO cell line successfully. PartⅡ:primary biological function of mouse-human chimeric antibody against CD80CHO-ch4E5 cells supernatant without FCS was purified by protein G affinity chromatography,quantity of purified antibody was detected using Lowry assay,and chimeric antibody was analyzed by SDS-PAGE and FCM.Competition inhibition between ch4E5 and 4E5 was detected by FCM.mCD80 on cancer cells(Daudi,SH2-1 and U937, et al)were identified by ch4E5 antibody,ch4E5 antibody(10μg/ml)how to act on proliferation and apoptosis of Daudi cells(conjunction ratio＞90%)was detected by MTT and FCM.When PBLs were cultured with ch4E5 antibody,change of proliferation and cytokine concentration was detected by MTT and ELISA.Results showed that concentration of chimeric antibody in cells supernatant is 4～5.8mg/L.ch4E5 and 4E5 could compete to bind CD80 antigen with each other,ch4E5 antibody identifies mCD80 on Daudi cells(conjunction ratio 95.5%).When 10μg/ml,ch4E5 effectively inhibits growth(P=0.000067＜0.05)and induces apoptosis of Daudi cells.Proliferation of PBLs in MLR is depressed(P=0.000012＜0.05).Concentration of IL-2(P=0.000156＜0.05)and INF-γ(P=0.000001＜0.05)secreted from PBLs are decreased,and that of IL-10 (P=0.000035＜0.05)is augmented.In conclusion,our study has constructed co-expressing recombinant plasmid pIERS/ch4E5,expressed mouse-human chimeric antibody against human CD80 in CHO cells successfully and set up artwork of cultivation and purification,ch4E5 antibody could depress proliferation of Daudi cells and inhibit mixed lymphocyte reaction(MLR).ch4E5 antibody has potency on tumor immunotherapy and graft rejection.