Humanization Of Monoclonal Antibody Against Chikungunya Virus And Study On The Mammalian Cell High Productive Expression

The aim of the study is to express human-mouse chimeric antibody against chikungunya(Chik) virus used for a passive drug for the Chik disease.Both Vl and Vh genes were cloned by RT-PCR. Full length H and L chain genes of chimeric antibody were constructed by fusing Vl and Vh with Cr1 and Ck, respectively. The chimeric antibody expression plasmids were constructed by inserting full length H and L genes into the MCSs of the expression vectors.The plasmids constructed were transfected into the CHO/dhfr' cells, chimeric antibody against Chik virus were delected positive in the supernatant of all the transfected cell cultures. A few high yielding cell clones were obtained by enhancing stepwisely MTX concentration. A stable and high productive cell line with yield of 2mgl-1 was obtained at MTX concentration of 2x10-7 mol/L. The chimeric antibody was purified by affinity chromatography and analyzed by non-reduced and reduced SDS-PAGE respectively. The molecular weights of the bands were consistant with the expected that is 150kD on the non-reduced gel and 50kD and 25kD on the reduced gel. Western blot showed that both the intact antibody molecular and H chain can specifically react with anti-human IgG Fc. Indirect immuno fluorescence assay (IFA) showed that the chimeric antibody has similar capacity of binding Chik virus antigen as mouse McAb. Cell culture neutralizing test (CCNT) with the purified chimeric antibody at six different concentrations from 6.25 to 100 mgl-1 was also undertaken to test whether the antibody is biologically active. The parent mAb was used as a positive control at 4 different concentrations from 7.5 to 60mgl-1. The result showed that the EC50(5O% effect concentration) of purified chimeric antibody was 25mgl-1 while the EC50 of the parent mouse antibody was 15mgl-1.To improve antibody expression in CHO cells, we attempt to express chimeric antibody with Flp-InTM system. A cell line with productivity of 1 ug/ml was attained. But the regret is that the system can 't be amplified. Based on the above, a CHO/dhfr- cell clone with FRT sequence intergrating on the transcription-active site was constructed. A cell clone with productivity of 5mgl-1 was obtained copared to the Flp-In?system with 1mgl-1 and CHO/dhfr- system with 2mgl-1.Flp-InTM system was also used to clarify whether heavy chain constant region genomic gene can improve the expression of chimeric antibody or not. The result shows that the antibody expression from the plasmid carrying cDNA sequence is higher than that from Cr1 genome containing plasmid.