The development of inhibitors for the infection using phage library

已知流感HA在感染的第一步就与宿主细胞上具有α2-3-或α2-6-连接的唾液酸半乳糖苷结合。我们试图设计多肽作为一种通用的抑制物，结合到HA的受体结合口袋上。为了获得模拟唾液酸寡糖的多肽，我们使用了噬菌体展示的随机十五肽文库。噬菌体展示方法是一种利用噬菌体池进行选择的技术。从噬菌体文库中筛选出的HA结合肽有望作为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，它们对HA1和HA3亚型流感病毒对MDCK细胞的感染均有抑制作用。虽然十五肽的理论分子多样性为3.3×10~(-1)，但所用噬菌体文库仅有2.5×10~(-8)种多样性。因此，为了获得改良的HA结合多肽，从选定的序列中制备了亚库。采用定向进化的方法制备亚库，构建两种亚库分别获得HA1和HA3结合肽。通过亚库对HA1和HA3的亲和选择，获得了几个有突变的多肽序列。许多突变噬菌体克隆与HA1和HA3亚型的结合亲和力均高于原始A-1噬菌体。与原始的A-1多肽相比，进化的多肽能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.