一种蛋白A的Z结构域衍生物的构建及对IgG亲和力的研究

蛋白A是金葡菌中一种重要的致病因子,有较强结合多种哺乳动物的IgG的能力,因此降低机体产生特异性SPA抗体的水平,使金葡菌具免疫逃逸功能.研究一种与IgG亲和力较弱的蛋白A衍生物,对金葡菌疫苗的开发具重要意义.研究表明,蛋白A的Z结构域中位于第13、14位的Phe,Tyr是两个与IgG结合的关键位点.本文首次通过PCR突变技术,将这两个关键氨基酸均改造为Gly,构建突变体ZFY.Discovery Studio3.5受体-配体相互作用力模块分析显示,突变体ZFY与IgG结合力降低到-61.68kcal/mol,仅为原相互作用力的10.69%;构建得到ZFY蛋白,经ITC测定,ZFY与兔IgG的亲和常数降低到1.2×104 M-1,仅为原亲和常数的0.39%,证明了ZFY是一种与IgG亲和力较弱的蛋白A衍生物,为实现新型蛋白A疫苗治疗金葡菌感染奠定基础.

A Z domain of protein A derivative with Reduced Binding Ability to IgG

Protein A is an important virulence factor of S.aureus which binds to the Fc region of mammal IgG makes S.aureus escape from immune and greatly reduces the immunogenicity of protein A.Therefore it is important to develop aprotein A derivative which induces antibody against protein A for vaccine against S.aureus.Research showed that the key sites(F13and Y14of Z domain)of protein A played a major role in protein A binding to IgG molecule.We first made the mutation of the key sites with glycine substitution.Discovery Studio showed that ZFY reduced binding to Fc to-61.68kcal/mol,which was10.69%of that with Z.After the ZFY reconstructed was obtained,ITC analysis experiment showed that the ZFY had the affinity constant of only 1.2×104 M-1 with rabbit IgG,accounting for 0.39% of that of Z,which indicated that the ZFY derivative eliminated the binding to immunoglobulin and provided a novel research concept to develop novel protein A vaccine.