Expression, purification and preliminary identification of hDll1~(ext)-Fc fusion protein

Objective To obtain the CHO-S cell lines expressing hDll1ext-Fc(human Delta-like-1 extracellular domain-Fc) fusion protein and the target protein with biological activity.Methods The hDll1ext gene was amplified through PCR with the primers designed according to the known sequences, then inserted into pIRES2-EGFP-Fc vector, transformed into the competent cells. The positive clones were screened for sequencing. The recombinant plasmid pIRES2-EGFP-hDll1ext-Fc was transfected into the CHO-S cells, and then the high expressed cell lines were screened and the interest protein was purified with affinity rProtein A column. The biological activity of hDll1ext-Fc was determined by Dual-Luciferase Reporter Assay and the express of Hes1 which is a downstream factor in Notch pathway. Results We obtained the eukaryotic expression vector pIRES2-EGFP-hDll1ext-Fc, high expressed cell lines and high purified hDll1ext-Fc fusion protein. The experiment of biological activity showed that soluble hDll1ext-Fc activated Hes1 reporter gene and up-regulated the expression of Hes1. Conclusion The recombinant plasmid pIRES2-EGFP-hDll1ext-Fc is constructed and the fusion protein is expressed, which lays the important foundation of further study on its biological function.