The development of inhibitors for the infection using phage library

已知流感HA在感染的第一步结合宿主细胞上具有α2-3-或α2-6-键的唾液酸半乳糖苷。为了获得模拟唾液酸寡糖的多肽，我们利用噬菌体展示随机十五肽库，设计了一种能与HA受体结合口袋结合的通用抑制肽。噬菌体展示法是一种利用噬菌体库的筛选技术。预期从噬菌体文库中选择的HA结合肽将用作甲型流感病毒的抑制剂。本研究从噬菌体展示肽库中筛选出HA结合肽和糖脂结合肽，并对HA 1和HA 3亚型流感病毒感染MDCK细胞进行了抑制实验。虽然十五肽的理论分子多样性计算为3.3 × 10 ^8<19>，但所采用的噬菌体文库只有2.5 × 10 ^8种多样性。因此，为了获得改进的HA结合肽，制备了由所选序列产生的亚文库。采用定向进化方法制备亚文库，构建两种亚文库，获得HA 1和HA 3结合肽。通过使用子文库对HA 1和HA 3进行亲和选择，获得了几个具有突变的肽序列。许多突变体噬菌体克隆显示出比原始A-1噬菌体对HA 1和HA 3亚型更高的结合亲和力。与原始A-1肽相比，进化肽更有效地抑制流感病毒对MDCK细胞的感染。

Influenza HA is known to bind sialylgalactoside having α2-3- or α2-6- linkages on host cells at the first step of infection. We attempted to design peptides as a universal inhibitor that bind to the receptor-binding pocket of HA.In order to obtain peptides which are a mimic of sialyl oligosaccharide, a phage-displayed random pentadecapeptide library have been employed. phage-displayed method is a selection technology using a pool of phage. It was expected that HA-binding peptides selected from the phage library would serve as inhibitors of influenza A virus. In this study, the HA-binding peptides and glycolipid-binding peptides were selected from the phage-displayed peptide libraries, and they inhibited the infection of both HA1 and HA3 subtypes of influenza virus to MDCK cells. Although the theoretical molecular diversity of pentadecapeptide is calculated to be 3.3x10^<19>, the phage library employed has only 2.5x10^8 kinds of diversity. Therefore, in order to obtain improved the HA-binding peptides, sublibrary generated from a selected sequence was prepared. The directed evolution approach was employed to prepare the sublibraries.The two kinds of sublibraries were constructed to obtain HA1- and HA3-binding peptides. Through the affinity selections for HA1 and HA3 using the sublibraries, several peptide sequences having mutations were obtained. Many of the mutant phage clones showed higher binding affinity for both HA1 and HA3 subtypes than the original A-1 phage. The evoluted peptides inhibited the infection of influenza virus to MDCK cells more efficiently than the original A-1 peptide.