Cloning, Prokaryotic Expression, Purification And Fusion Protein Active Detection Of Human Soluble CD83 Gene

Objective:The glycoprotein CD83 has a molecular mass of 40–45 kDa and belongs to the Ig superfamily. The function of CD83 remains largely unknown. CD83 expression on mature dendritic cells and activated lymphocytes suggests that CD83 might be involved in activating immune effector cells. Soluble forms of CD83(sCD83) have been detected in the serum of healthy donors, but the mechanism of how these soluble forms of CD83 are generated are fully understood. Studies suggest that the mechanism for the generation of sCD83 is shedding of cell surface-associated CD83. Latest studies suggest that soluble forms of cell surface proteins can also result from alternative splicing. Recombinant soluble CD83 strongly inhibited MLR or DC-mediated allogeneic T cell proliferation, moreover, sCD83 was able to prevent experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. Results suggest that soluble CD83 could have an inhibitory function. To perform functional studies and to elucidate its mode of action it is vital to obtain recombinant expressed and highly purified CD83 molecules.Therefore,the gene of human soluble CD83 was expressed as a 6×His fusion protein (6×His-sCD83) and the protein was purified under native conditions.The fusion protein was purified by Ni-NTA affinity chromatography and detected in vitro activity.Methods:Soluble CD83 gene was obtained from a healthy human PBMCs by RT-PCR, then cloned into expression vector pET-32a, thus forming recombinant plasmid , which was transformed into E.coli BL21 (DE3)and induced by IPTG. SDS-PAGE and Western blot were used to test and identify expressed (His)6 fusion proteins. The expression product in inclusion body was purified by Ni-NTA affinity chromatography. With mixed lymphocyte reaction detect the fusion protein in vitro activity.Results : Restriction analysis and sequencing confirmed that the recombinant plasmid expressing soluble CD83 gene was successfully constructed. Both SDS-PAGE and Western blot analysis showed soluble CD83 gene were expressed, and the molecular weight of the product was about 32 000.The fusion protein production accounted approximately for 45% of total bacterial protein .The recombinant (His)6 fused proteins were purified by Ni-NTA affinity chromatography. Mixed lymphocyte reaction experimental results indicated that the fusion protein has in vitro activity.Conclusion: Soluble CD83 gene from human PBMCs was successfully cloned and expressed in E.coli BL21(DE3).The purified proteins are obtained by Ni-NTA affinity chromatography. The fusion protein has in vitro activity.