| CD83, a 45KD type transmembrane glycoprotein, belongs to immunoglobulin Superfamily(IgSF). CD83 is a significant functional molecule which expressed on mature dendritic cells and activated B,T lymphocytes and has been implicated to play an important role during T cell development in the thymus. It is also detected in immature DCs. Soluble CD83(sCD83) is mainly produced by proteolysis of membrane CD83(mCD83) ,and can be detected in the serum of health adults and found its activity of immunosuppression.sCD83 can inhibits DC maturation and DC-mediated T cellprolixferation in vitro and in vivo. sCD83 can remarkably inhibit the development of experimental autoimmuneec ephalomyelitis(EAE) and allogenetic graft rejection,which show great prospect in its therapeutic value on this kind of diseas. It is reported that CD83 can bind monocytes and a subset of activated CD8+ T cells,also some research shows that induction of CD83+CD14+ cells by exposure of monocytes to IFN-αwhich does not have a typical DC phenotype, but have a similar phagocytosis,but GM-CSF drives monocytes to CD14low CD83+ DCSIGN- myeloid cells with differential effects on T-cell subsets.Therefore the role of CD83 and how the signal transmission is still need to further study.1 Construction of Human CD83 Transfectant Cell LineThe gene encoding entire Human CD83 molecule was obtained by RT-PCR using the mRNA of mature DC cells as templates.Digested with EcoRI and BglII,the PCR product was cloned into corresponding region of pIRES2-EGFP vector that have bothe G418 resistant gene and GFP reporter gene.The recombinant plasmid CD83/ pIRES2- EGFP was transfected into 293 cellline with lipofection and then selected by G418.RT-PCR and flow cytometry analysis were used to assay the expression stability and efficiency of the target molecule,A stable cell line expressing the human CD83 was established successfully.2. Preparation of monoclonal antibody against CD83After pretreatment with mitomycin, L929/CD83, a transgenic cell line, which highly expressing CD83 molecule, was used to immunize BALB/c mice. According to the hybridoma technique, the immunized mice spleencytes were fused with mouse myeloma cells (SP2/0). L929/CD83 and 293/CD83 was used as positive screening cell line. Through repeated sub-cloning and screening, one hybridoma (9D8) was eventually obtained. The hybridoma grew well after long-term culturing and storage in liquid nitrogen. Fast-strip analysis showed that subclass of 9D8 was IgG1 and the light chain belonged toκ. After primed with pristine,ascite was induced in BALB/c mice by intraperitoneal injection of well-grown hybridoma and the output is average to 5ml each mouse. Affinity chromatography was used to purify mAb, and the concentration of protein is 1.5~2.0mg/mL. Indirect immunofluorescence assay suggested the engagement of purified CD83 mAb to cells was 0.2-1μg/1×106 cells. The results of western blot showed that the strap was specific. Competition experiment indicated that 9D8 recognized a different antigen epitope from that of commercial mAb HB15e. Pheno- typic analysis suggested that 9D8could well recognize CD83 molecule expressed on L929/CD83 , Daudi, 8226,activated T cell and mature DC.In conclution, the full-length CD83 gene cloning, Construction of recombinant vector and stable expression in cell lines has laid a good foundation for further study,Also,one hybridoma continously and steadily secreting specific anti-CD83 mAbs was eventually obtained.It can recognize membrane and soluble CD83 Specially, therefore all the results laid material basis for the further study of CD83 and its biological function ,meanwile the mAb 9D8 may has potential clinical value. |
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