人IgG四亚类对噬菌体展示Ig结合蛋白单结构域随机组合文库的体外进化筛选

金黄色葡萄球菌蛋白A(Staphylococcal protein A,SpA)和链球菌蛋白G(Streptococcal protein G,SpG)是细菌产生的特异结合宿主抗体的细菌免疫球蛋白结合蛋白(Immunoglobulin(Ig)-binding proteins,IBPs)的代表分子。SpA和SpG均包含由多个序列高度同源的结合结构域重复组成的抗体结合区,各单结构域都具有完全的结合IgG的功能。为研究这些单结构域随机组合能否产生具有新结合特性的组合分子,将SpA的A、B、C、D、E以及SpG的B2、B3共7个单结合结构域随机组合构建成噬菌体展示文库后,应用人IgG1、2、3、4为诱饵分子对该文库进行4轮筛选,获得了SpA天然分子中不存在的单结构域排列组合分子D-C。在筛选过程中,阴性对照噬菌体的逐渐减少、展示两个结构域以上的噬菌体比例不断增多,尤其是D-C组合的选择性富集和其随机连接肽的严格筛选都显示了筛选的有效性和D-C组合的重要性。噬菌体ELISA进一步证实D-C与人IgG四亚类的结合能力远强于天然SpA分子。该研究应用分子进化技术首次获得了一种与人IgG四亚类具有结合优势的新型组合分子D-C,不仅可为IgG纯化、制备、检测等方面的应用提供新的候选分子,还为细菌IBP结构功能的进一步研究提供新的手段。

In vitro evolutional selection of a combinatorial phage library displaying randomly-rearranged various binding domains of SpA and SpG with four human IgG subclasses

Protein A and protein G are two well-defined immunoglobulin(Ig)-binding proteins(IBPs),which show affinity for specific sites on Ig of mammalian hosts.Protein A and protein G contained several highly homologous IgG-binding domains which had been demonstrated to have function to bind to IgG.Whether combinations of Ig-binding domains of various IBPs could produce useful novel binding properties remains interesting.We constructed a combinatorial phage library which displayed randomly-rearranged A,B,C,D and E domains of protein A,B2 and B3 domains of protein G.Four rounds molecular evolution of this library directed by all four human IgG subclasses respectively generated a common arrangement of D-C respectively which didn’t exist in SpA.The dynamic loss of control phages and increase of the phages displaying two or more binding domains,especially the selective enrichment of D-C and strict selection of its linking peptides demonstrated the efficient molecular evolutions and the significance of the selected D-C arrangement.The phage binding assays confirmed that D-C possessed a binding advantage with four human IgG subclasses compared to SpA.In this work,a novel combination of Ig-binding domains,D-C,was obtained and presented the novel Ig binding properties which provided a novel candidate molecule for the purification,production and detection of IgG antibodies and a new approach for the further study of structures and functions of IBPs.