Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

Using antibody phage display technique, a human single chain antibody to va scular endothelial growth factor (VEGF) has been cloned. The antibody expre ssion reached 45% of the total bacterial proteins. The purification and ref olding of the antibody were completed in one step by using gel filtration c hromatograph, ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with i ts receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with hi gher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provide d a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.