Abstract： Objective To construct an eukaryotic expression vector pEGFP-N1-CKB and establish a stably transfected NCI-H520 cell line.Methods Human CKB gene was amplified by PCR with human CKB cDNA library as the template and the fragment was combined with plasmid pEGFP-N1.The recombinant expression vector,pEGFP-N1-CKB,was transfected to NCI-H520 using Lipofectamin.The stably transfected cell line was established after G418 selection and the expression level of CKB gene before and after transfection was detected by Western blot.Results After identification by restriction enzyme digestion and sequencing,the eukaryotic expression vector,pEGFP-N1-CKB,was successfully constructed.The expression level of CKB in NCI-H520 transfected by pEGFP-N1-CKB was significantly higher than that in control.CKB gene had a stable transfection in NCI-H520 cells.Conclusions An eukaryotic plasmid encoding CKB (pEGFP-N1-CKB) has been constructed and a cell line expressed CKB stably has been successfully prepared.