Construction of the eukaryotic expression vector of mouse FasL gene and its expression in HEK293 cells

Objective To construct the eukaryotic expression vector containing Fas ligand(FasL) gene and detect the protein express in its stable transfected HEK293 cell line,which lay the foundation for the establishment of stable dendritic cell model expressing FasL,so as to further research the new method for prevention of graft versus host disease(GVHD) with dendritic cell and T cells.Methods RNA were extracted from mouse spleen and copied into cDNA.The coding sequence of FasL was amplified from the cDNA and cloned into pcDNA3.1(+) vector.The recombinant expression vector pcDNA3.1(+)-FasL was transfected into HEK293 cells by lipofectamine,and the stable cell strains were screened with G418.Then,the protein expression of FasL in the stable cell line was detected by Western blot.Results Restriction endonuclease digestion and sequencing showed that the recombinant eukaryote expression vector containing FasL was constructed successfully.The result of Western blot confirmed the exact expression of FasL in the transfected HEK293 cells,the resistant cell strain which express FasL stably could be constructed after G418 screening.Conclusion The new recombinant expression vector pcDNA3.1(+)-FasL and the HEK 293 cells which could express FasL stably were constructed successfully,which lay the solid foundation for follow-up study of FasL-DC cell.