Arg77和Glu80定点突变同时去除葡激酶中的T和B细胞抗原表位

【目的】对葡激酶的T和B细胞抗原表位重叠的关键氨基酸Arg77和Glu80进行定点突变以降低葡激酶的免疫原性。【方法】基于Arg77和Glu80的溶剂可及表面积设计葡激酶的突变体;突变体在大肠杆菌DH5α中进行表达。经过三步层析法纯化后,分析突变体的纤溶活性和免疫原性。【结果】免疫学实验提示,葡激酶导致Th2免疫反应;Glu80突变为丙氨酸和丝氨酸减少了溶剂可及表面积,同时去除了部分T和B细胞抗原表位;Arg77突变为天冬酰胺、谷氨酰胺和赖氨酸仅去除了部分T细胞抗原表位;6个组合突变体中,Sak(R77Q/E80A)和Sak(R77Q/E80S)有效去除了部分B和T细胞抗原表位,降低了葡激酶的免疫原性;Sak(R77Q/E80A)and Sak(R77Q/E80S)的纤溶活性和催化效率与r-Sak相当。

Simultaneous removal of T and B cell epitope in recombinant staphylokinase by structure-based mutagenesis of immuno-dominant Arg77 and Glu80 residues

Objective]To reduce immunogenicity of recombined staphylokinase(r-yak),site-directed mutagenesis of Arg77 and Glu80 residue was performed to simultaneously remove T and B cell epitope in r-Sak molecule.Methods The solvent accessible surface areas of residues 77 and 80 in r-Sak were used to analyze rational design of Sak mutation.The Sak mutants were expressed in E coli DHSa.After purified by a 3-step chromatography,their fibrinolytic activities and immunological properties were analyzed.Results]Immunogenicity tests suggested that Sak induced a Th2-type immune response.Substitution of Glu80 with alanine or serine successfully reduced its solvent accessible surface area while simultaneously removing part of the T and B cell epitope.Changing Arg77 to glutamine,asparagine,or lysine removed only part of the T cell epitope.Of six dually substituted variants,Sak(R77Q/E80A)and Sak(R77Q/E80S)variants effectively eliminated part of the B and T cell epitopes,which markedly reduced their immunogenicity.