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

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

摘要:

摘要【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DH5? 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。

关键词:关键词：葡激酶，T细胞抗原表位，B细胞抗原表位，免疫原性，蛋白质工程

Abstract:

Abstract [Objective] To reduce immunogenicity of recombined staphylokinase (r-Sak), 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 DH5α. 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.

Key words:Keywords: staphylokinase, T cell epitope, B cell epitope, immunogenicity, protein engineering