Xu Ruiguang, He Jintian, Jia Kai, Chen Xi, Liu Jianwei, Zhu Ke
College of Life Science, Hebei Normal University, Shijiazhuang 050016, China.
Wei Sheng Wu Xue Bao. 2011 May;51(5):692-703.
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.
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 DH5alpha. After purified by a 3-step chromatography, their fibrinolytic activities and immunological properties were analyzed.
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.
为降低重组葡激酶（r-Sak）的免疫原性，对第77位精氨酸和第80位谷氨酸残基进行定点诱变，以同时去除r-Sak分子中的T细胞和B细胞表位。
利用r-Sak中第77位和第80位残基的溶剂可及表面积来分析Sak突变的合理设计。Sak突变体在大肠杆菌DH5α中表达。经三步层析纯化后，分析其纤溶活性和免疫学特性。
免疫原性测试表明，Sak诱导Th2型免疫反应。用丙氨酸或丝氨酸取代第80位谷氨酸成功降低了其溶剂可及表面积，同时去除了部分T细胞和B细胞表位。将第77位精氨酸变为谷氨酰胺、天冬酰胺或赖氨酸仅去除了部分T细胞表位。在六个双重取代变体中，Sak（R77Q/E80A）和Sak（R77Q/E80S）变体有效消除了部分B细胞和T细胞表位，显著降低了它们的免疫原性。
