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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。
Staphylococcal protein A (SpA) and Streptococcal protein G (SpG) are the bacterial immunoglobulin (Ig) -binding proteins (IBPs) produced by bacteria that specifically bind to host antibodies ) Of the representative molecules. Both SpA and SpG contain an antibody binding domain consisting of a plurality of highly homologous binding domains, each having a complete IgG binding function. In order to study whether these single-domain random combinations can generate combined molecules with novel binding properties, 7 single binding domains of B2, B3 of A, B, C, D, E and SpG of SpA were randomly combined into phage display After four rounds of screening of this library using human IgG1, 2, 3, and 4 as bait molecules, a single domain-aligned molecular DC that does not exist in the native SpA molecule was obtained. During the screening process, the number of negative control phages decreased and the proportion of phage displaying more than two domains increased. In particular, the selective enrichment of the DC combination and the rigorous selection of its random-linked peptide both showed the effectiveness of the screening and DC The importance of the combination. Phage ELISA further confirmed that D-C binds to human IgG tetra-subclasses more strongly than native SpA molecules. This study, for the first time, obtained a novel combination molecule DC that has the advantages of combining with the human IgG subclass, which not only provides new candidate molecules for IgG purification, preparation, detection and other applications, but also is a bacterial IBP structure Further research on the function provides new means.