Expression, purification and biological activity assay of humanized single-chain Fv dimers against hepatocellular carcinoma in Pichia pastoris

AIM:To express the humanized single-chain Fv dimers against hepatocellular carcinoma(BDM diabody) in Pichia pastoris and assay their biological activity and function.METHODS:The yeast expression plasmid pGAPZαA-BDM was constructed and trans-formed into Escherichia coli(E.coli) strain DH5α.The recombinant vector plasmid was then amplified,sequenced and transformed into Pichia pastoris strain GS115 to express BDM diabody in yeast.The expression product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),Western blot,enzymelinked immunosorbant assay(ELISA) and immunohistochemistry.RESULTS:SDS-PAGE and Western blot analysis showed that the BDM diabody was successfully expressed in Pichia pastoris.The yield of BDM was 30 mg/L,approximately 300 times as high as that in E.coli.The expression product showed significantly stronger binding to hepatocellular carcinoma cells than single-chain variable fragment(scFv) antibody.The purified diabody could specially bind to tumor antigens of hepatocellular carcinoma.CONCLUSION:Biologically active humanized diabody against hepatocellular carcinoma is successfully prepared and can be used for targeted diagnosis and therapy of hepatocellular carcinoma.