Abstract: Objective To establish the renca cell strain stably expressing human G250 gene. Methods The complementary sequence of human G250 gene length was amplified by PCR and inserted into the eukaryotic expression vector pIRES-neo using recombinant DNA technology to obtain the recombinant plasmid pIRES-neo-G250. The recombinant plasmid was stably transfected into the mouse renca cells with Lipofectamine 2000. Expression of renca cell strain in mice transfected with the human G250 gene was detected by Western blot and immunofluorescence assay, respectively, after the positive clone was screened by adjusting the G418 concentration. Results Restriction enzyme digestion and sequence analysis revealed that the recombinant plasmid pIRES-neo-G250 was successfully constructed. Western blot showed that the G250 protein was expressed in mouse renca cells stably transfected with pIRES-neo-G250. Flow cytometry and laser confocal microscopy demonstrated that the fluorescence was highly expressed in mouse renca cells stably transfected with pIRES-neo-G250. Conclusion The renca cell strain we established can stably and effectively express the human G250 gene transfected with pIRES-neo-G250 molecules, thus laying a foundation for further study of renal tumor vaccine.