Expression, Purification And Characterization Of Human Ds-diobady Against BFGF In Pichia Pastoris

Object: In order to improve the expression of human anti-bFGF ds-Diabody in Pichiapastoris and study theirs biological activity, the ds-Diabody gene was constructed intoexpression vector pPICZαA.Methods: The recombinant plasmid pPICZαA-ds-Diabody was linearized with Bgl II andelectroporated into Pichia pasporis strain GS115．The transformants were induced by methanol,and the ds-Diabody was expressed. The expression products were purified by affinitychromatography of Ni-Seproase6FF and ion exchange chromatography of DEAE Sepharose FF.The binding activity to bFGF was assayed by indirect ELISA. The inhibitive effects ofds-Diabody on tumor cell proliferation were detected by CCK-8. Transwell assay andwound-healing assay detected its influence on the migration and invaion of tumor cells. Matrigelassay detected its influence on tube rates of vascular endothelial cells in vitro. Western blotanalyzed its role on the mechanism of tumor cells.Results: The results of SDS-PAGE and Western-blot showed that anti-bFGF ds-Diabodywas high level expressed successfully and the yeild was about158mg/L. The target protein waspurified from the expression products and the purity was more than95%. The results of ELISAshowed that the purified ds-Diabody could bind to bFGF with improved binding activity andshowed high stability and good biological activity under37℃．The results of CCK8showed thatthe purified ds-Diabody could inhibit the proliferation of Skov3cells, MCF-7cells, A375cellsand A549cells in a dose-dependent manner in vitro, and the inhibitive rate was24.3%,56.5%,43.4%and35.4%, respectively. The results of wound-healing assay showed that the ds-Diabodycould inhibit the migration of MCF-7cells, which was demonstrated in the FGF2-triggeredsignal pathways. Matrigel assay showed that the ds-Diabody could inhibit tube formation ofHUVEC cell. Transwell analysis showed that the ds-Diabody could effectively decrease themigration of HUVEC cells and the invasion of tumor cells. The phosphorylation of Akt andMAPK were inhibited by ds-Diabody in a dose dependent manner.Conclusion: The results demonstrated that the ds-Diabody against FGF-2can be high levelexpressed in Pichia pastoris with good binding activity and high stability. The anti-bFGFds-Diabody could effectively inhibit proliferation and invasion of a variety of tumor cells，andinhibit tube formation of vascular endothelial cells in vitro, which may have therapeutic potentialin cancers.