Construction and expression of single chain Fv gene against domain II of human VEGF receptor II / 生物工程学报

ABSTRACT

The genes encoding for the light and heavy chain variable regions (V(H) and V(L)) has been cloned by RT-PCR from a murine hybridoma that produced monoclonal antibody (mAb) Ycom1D3, which was against domain III of human vascular endothelial growth factor receptor II (KDRIII) and were then connected to each other by a short peptide linker containing 15 amino acids (Gly4Ser)3 using splice-overlap extensive PCR. The recombinant Ycom1D3-ScFv gene was cloned into the expression vector pAYZ and induced to express in E. coli 16C9. SDS-PAGE and Western blot analysis showed that the recombinant Ycom1D3-ScFv gene was expressed in E. coli 16C9 and the relative molecular weight of the fusion protein is 30kD which was consistent with the theoretically predicted value. ScFv expression was in the form of an inclusion body and the purified fusion protein was obtained after a series of purification steps including cell breakage, inclusion body solubilization, TALON metal affinity chromatography and protein refolding. Flow cytometric analysis showed that the ScFv fragment can react with human umbilical vein endothelial cells (HUVECs) which express KDR on the cell surface. In Conclusion, Recombinant Ycom1D3-ScFv gene has been successfully constructed and expressed in E. coli 16C9, which could be useful in both diagnostic and therapeutic applications.