通过对免疫显性的精氨酸77和谷氨酸80残基进行基于结构的诱变，同时去除重组葡萄球菌激酶中的T细胞和B细胞表位。

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

作者信息

Xu Ruiguang, He Jintian, Jia Kai, Chen Xi, Liu Jianwei, Zhu Ke

机构信息

College of Life Science, Hebei Normal University, Shijiazhuang 050016, China.

出版信息

Wei Sheng Wu Xue Bao. 2011 May;51(5):692-703.

PMID:21800633

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 DH5alpha. 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.

摘要

目的

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。

方法

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。

结果

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。