Construction of the eukaryotic expression vector pIRES2-EGFP-KAI1 and its expression in human biliary carcinoma cells line

Objective To construct the eukaryotic expression vector pIRES2-EGFP-KAI1,and to express KAI1 in human biliary carcinoma cell line.Methods KAI1 gene was cloned from human biliary tissue by RT-PCR and inserted into T-vector.After DNA sequencing verification,it was then sub-cloned into eukaryotic expression vector pIRES2-EGFP.The plasmid was transfected into the QBC939 cells using lipofectamine.The expressed EGFP was observed under fluorescent microscope and the KAI1 protein expression was detected by immunostaining using anti-KAI1 antibody.Results The eukaryotic expression vector pIRES2-EGFP-KAI1 was constructed and transfected successfully into QBC939 cells.The green fluorescence of EGFP was observed in the plasma and nuclei of transfected cells,and KAI1 protein was only found in the plasma.Conclusion The recombinant expression vector pIRES2-EGFP-KAI1 is constructed,and the EGFP and KAI1 gene can be co-expressed in the QBC939 cells.This study leads a foundation for the further research of the function of KAI1 in cell differentiation,growth and tumorigenesis.