Cloning of Human Germinal Center Kinase Gene and Construction of Its Transgenic Cells

Objective To clone human germinal center kinase(GCK) gene and construct recombinant plasmid vector carrying the target gene which can be expressed stably in mammal cell line HEK293.Methods Human GCK gene was amplified by PCR using the pCMV5-GCK as template and then confirmed by DNA-sequence analysis.Digested with the restriction endonucleases BglⅡ and SalI,the human GCK gene was inserted into pIRES2-EGFP plasmid vector.The recombinant pIRES2-EGFP plasmid vector was cotransfected into the HEK293 cell in the context of Lipfect2000.After 72 hours,HEK293 cell line stably expressing human DC-SIGN protein was selected in the presence of G418.Result The full-length of human GCK gene was cloned and the recombinant pIRES2-EGFP plasmid vector was constructed,the HEK293 cell line expressing human GCK was successfully transfected and selected.Results of RT-PCR and Western blot indicated that HEK293 transgenic cells could stably express human GCK protein.Conclusion Cloning of human GCK gene and construction of the recombinant pIRES2-EGFP plasmid vector and HEK293 transgenic cell line stably expressing GCK protein could contribute to further biological function research.