Construction Of Eukaryotic Vector And Expression Of Human/mouse Chimeric Anti-body Against Rh-bFGF

AIM: To construct a human-mouse chimeric antibody against rh-bFGF antigen and express in eukaryotic cells.METHODS: The VLand VH genes were amplified and cloned from the hybridoma 1F11 secreting anti-rh-bFGF mouse monoclonal antibody mAb by RT-PCR, The CL and CH genes were amplified and cloned from the plasmid pMDHC and pMDLC.The conjunctions of VL gene and CL gene, VH gene and CH gene were recombinated by fusion PCR, They were inserted into eukaryotic expression vectors pEF-dhfrla (B7) after sequencing respectively. The recombinant plasmids were transformed into CHO-dhfr" cells for expression. The humanization and antigen specificity of expressed products were identified by indirect ELISA, The relative molecular mass (Mr) of expressed products was determined by SDS-PAGE.RESUTS: The variable regions of light and heavy chains were obtained from the 1F11 hybridoma cells. VL gene and CL gene were spliced into whole light chain (about 800bp) and VH gene and CH gene into whole heavy chain (about 1500bp) of chimeric antibody. The cloned antibody genes were approved to be functional by sequencing. Recombinatant expression plasmid of heavy chain pEF-dhfrla(B7)H and recombinatant expression plasmid of light chain pEF-dhfrla(B7)L were constructed; The chimeric antibody could be detected in the cultural supernatant of transformed CHO cells.The humanization and specificity of chimeric antibody against rh-bFGF were identified by ELISA. Finally five strains engineering cells were obtainted by subcloneing and culturing with MTX.The chimeric antibody could be detected in the cultural supernatant of engineering cells with a yield of 5mg/L.CONCLUSION: The mouse-human chimeric antibody against rh-bFGF has been expressed successfully in eukaryotic cells, which lays the foundation for its further clinical research.