Construction of human G250 eukaryotic expression vector and establishment of stable B16 cell line transfection

Objective To construct the human G250 eukaryotic expression vector and establish the stable B16 cell line expressing human G250 in mice. Methods Human G250 cDNA was sequenced by PCR amplification and inserted into the eukaryotic expression vector pIRES-neo-sig with DNA recombinant techniques.Restriction enzyme position and FLAG tag were added into the vector to obtain the recombinant expression plasmid pIRES-neo-sig-FLAG-G250 which was transfected into B16 cells with Lipofectamine 2000.Positive clones were screened with G418.Expression of human G250 in B16 cells was detected by RT-PCR and immunofluorescence. Results Restriction enzyme and sequence analysis showed that the recombinant plasmid vector pIRES-neo-sig-FLAG-G250 was successfully constructed.The positive expression rate of human G250 in B16 cell line was nearly 100%. Conclusion A human G250 eukaryotic expression vector pIRES-neo-sig-FLAG-G250 can be constructed.Stable transfection of mouse B16 cell line can highly express G250,thus laying a foundation for the application of G250 in immunotherapy for tumors.