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B lymphocyte stimulator (BLyS),a member of the tumor necrosis factor superfamily ofligands,is a crucial survival factor for B cells.We successfully constructed seven mutants of the functionalsoluble fragment of human BLyS (named cBLyS,amino acid 134-285),including three deletion mutants andfour site-directed mutants.All the mutant proteins were expressed in Escherichia coli and purified by Ni-NTA affinity chromatography.The biological activities of these mutants were assessed by the ligand-recep-tor binding assay,B cell proliferation assay and immune effect response in vivo.Our results indicated thatfour residues,H~(218),F~(220),T~(228) and L~(229),are indispensable for the biological activity of cBLyS,whereas tworegions,amino acid 134-148 and amino acid 271-285,are related to the biological activity of BLyS.Theprotein of deletion of amino acid 134-148 leads to a complete defection in raising the antigen-specific IgMtiter.The deletion of amino acid 271-285 reduces the effectiveness compared with the native cBLyS.Thisindicates that the region of amino acid 134-148 is indispensable for cBLyS to function normally.
A member of the tumor necrosis factor superfamily of ligands, is a crucial survival factor for B cells. We successfully constructed seven mutants of the functionalsoluble fragment of human BLyS (named cBLyS, amino acid 134-285), including three deletion mutants and frequently site-directed mutants. All the mutant proteins were expressed in Escherichia coli and purified by Ni-NTA affinity chromatography. Biological enzymes of these mutants were assessed by the ligand-recep-tor binding assay, B cell proliferation assay and Immune effect response in vivo. Our results indicated that fatty residues, H 218, F 220, T 228 and L 229, are indispensable for the biological activity of cBLys, whereas tworegions, amino acid 134-148 and amino acid 271-285, are related to the biological activity of BLyS.Theprotein of deletion of amino acid 134-148 leads to a complete defection in raising the antigen-specific IgMtiter. Deletion of amino acid 271-285 reduces the effectiveness com pared with the native cBLyS.Thisindicates that the region of amino acid 134-148 is indispensable for cBLyS to function normally.