Establishment of L929 cell line transfected with human HVEM gene and study of its co-stimulatory effect on human T cell

To establish cell line transfected with human HVEM gene and to investigate its co-stimulatory effect on the in vitro proliferation and activation of T cells,the total length gene encoding human HVEM was amplified from the already constructed plasmid pMD18-T/HVEM,then digested with restriction endonuclease EcoR I and BamH I and inserted into eukaryotic vector pIRES2-EGFP.The recombinant vector was transfected into mouse fibroblast cell line L929 with LipfectAMINETM 2000 and the cells were further selected with G418.Zeocin-resisting cells were harvested for immun of luorescence and flow cytometry analysis to identify the expression of transfected L929/HVEM cell.Effect of L929/HVEM cells on the proliferation and cytokine secretion in vitro was studied by methods of MTT and ELISA.The stable expression of human HVEM on the transfected cell line was identified by flow cytometry analysis.The cell co-culture experiment in vitro showed that L929/HVEM had a positive effect on the proliferation of T cells stimulated by anti-CD3 mAb,prominently through up-regulating the secretion of IL-2,IFN-γ,IL-10 and TNF-α.A cell line L929/HVEM stably expressing the human HVEM gene has been obtained and it can obviously promote the proliferation of T cells and cytokine production in vitro.