Preparation And Identification Of Recombinant Humanized Anti-VEGF Antibody

Vascular endothelial growth factor(VEGF)is one of the most important growth factors for regulation of vascular development and angiogenesis.Signal transduction involves binding to tyrosine kinase receptors and results in endothelial cell proliferation,migration,and new vessel formation which leads to the growth of tumor cell lines.Therefore,an anti-VEGF monoclonal antibody has been approved as an important direction for tumor vascular therapy,which has demonstrated therapeutic utility in blocking VEGF-induced angiogenesis.The new drugs could inhibit the growth of tumor cells by blocking the formation and growth of microvessels.At present,the research on the role and expression of VEGF in tumors is mainly achieved by xenograft tumor or vitro cell.It has been reported in kidney carcinoma,breast carcinoma,melanoma,hepatocellular carcinoma,gastric carcinoma,bladder carcinoma,endometrial carcinoma and embryonal carcinoma.New drugs can inhibit the growth of tumor cells by blocking the formation and growth of microvascular.A variety of marketed anti-VEGF antibody drugs have been proven to have a good blocking effect on angiogenesis of primary bronchogenic carcinoma,breast carcinoma and colon carcinoma.In this study,the human anti-VEGF antibody with better activity was prepared by synthesize the optimized gene and other improvements.a variety of marketed anti-VEGF antibody drugs have been proven to have a good blocking effect on angiogenesis of primary bronchogenic carcinoma,breast carcinoma and colon carcinoma.In this study,the human anti-VEGF antibody with better activity was prepared by synthesize the optimized gene and other improvements.In this study,the sequence of human anti-VEGF antibody was obtained from GenBank and optimized with preferred condon.The sequence was assembled with the signal sequence,heavy chain and light chain of human constant region.DNA sequences of heavy chain and light chain and multiple protein was obtained.Expression vectors pcDNA-VEGF1 H,pcDNA-VEGF1 V,pcDNA-VEGF2 H,pcDNA-VEGF2 V,ptt5-VEGF1 H and ptt5-VEGF1 V were constructed respectively.Two expression vectors,pcDNA-VEGF1 H + pcDNA-VEGF1 V and pcDNA-VEGF2 H +pcDNA-VEGF2 V and HEK-293 cells was made following assisted transfection with polyethylenimine(PEI).C6 glioma cells was treated by the secreted protein in the supernatants.MTT confirmed that the proliferation of C6 was significantly inhibited(P<0.01).pcDNA-VEGF1 H + pcDNA-VEGF1 V,pcDNA-VEGF2 H + pcDNA-VEGF2 V,ptt5-VEGF1 H + ptt5-VEGF1 V and HEK 293-F cells was made following assisted transfection with PEI.SDS-PAGE confirmed that the the secreted protein in the supernatant of cell culture had expressed after 24h-72 h.Protein obtained by transfection with pcDNA-VEGF1 H + pcDNA-VEGF1 V and HEK-293 F was isolated and purified by affinity chromatography method.The results of SDS-PAGE and Western-blot confirmed that the purified protein was consistent with protein sample.The concentration of anti-VEGF antibody was 200 ng / ml by indirect ELISA.MTT tests the activity and biological function of the purified protein,and it shows that the protein can significantly inhibit the proliferation of C6,which is similar to the protein sample(P<0.05~0.01).This study generated the preparation and purification process of antibody at the laboratory level,which provided basic data for medium-scale trial and antibody production by manufactures in the future.