Construction of the transfected cell line expressing human CD83 and its function study

To construct the 293 transfectant expressing human CD83 molecule and explore its effects on monocytes,the gene encoding human CD83 was amplified by RT-PCR from mature dendritic cells,and then the target gene fragment was inserted into pIRES2-EGFP vector.The recombinant vector was transfected into 293 cells with Lipofect 2000 and the stable cell line expressing human CD83 was established by GFP reporter assay and CD83 expression.After purified from PBMC by MACS sorting,monocytes were stimulated with LPS in the presence or absence of 293/CD83 or 293/mock transfectant,and then the status of cell activation as well as TNF-α level in supernatant was analyzed 24 h later.The in vitro experiments showed that 293/CD83 cells promoted activation and TNF-α expression of LPS-stimulated monocytes.All this data suggested that 293/CD83 cells have co-stimulatory effects on LPS-stimulated monocytes.