Development of screening system for antibody-displayed phages by photo panning method : isolation of anti-amyloid antibody

本研究将展示噬菌体的光亲和标记和筛选两种异质技术相结合，建立了一种利用光反应配体高效克隆展示噬菌体的方法。光亲和标记引入了配体与其特定结合蛋白之间的交联，通过共价键固定它们的亲和关系。我们使用3-ary1-3-1三氟甲基二氮嘧啶作为光团，因为它似乎最接近于满足有用光团所需的标准。在我们的光化学清洗中，光反应配体带有产生二氧化碳的光载体，二氮嘧啶就像鱼钩上的倒钩，配体就像鱼饵一样被放置在上面。因此，即使经过强烈的洗涤，交联噬菌体也能留在配体上，而几乎所有的非特异性结合噬菌体都被去除。在本研究中，我们建立了光反应筛选法来筛选M13丝状噬菌体中显示抗体的噬菌体。我们利用鸟胺磁珠和以二氮嘧啶为光载体的光反应性抗原肽，以及在n端作为鸟胺结合标签的生物素，成功地筛选了抗体显示噬菌体。我们开发的筛选方法能够克隆出比传统筛选方法灵敏度更高的目标噬菌体。我们发现朊蛋白的某个肽片段抑制了大鼠海马原代培养细胞的生长，并且阿尔茨海默β -淀粉样蛋白n端结构自发改变。我们合成了改变的肽n端，并试图分离出与这种改变反应的抗体显示噬菌体。将合成的肽免疫5只小鼠，获得血清。我们用酶联免疫吸附法证实了血清中抗体的产生。

We developed a method for an efficient cloning of display phages using photoreactive ligand, which was based on the combination of two heterogeneous techniques, photoaffinity labeling and panning of display phages. Photoaffinity labeling introduces a cross-link between a ligand and its specific binding proteins for fixing their affinity relationship through a covalent bond. We used 3-ary1-3-1rifluoromethyl-diazirine as a photophore because it appears to come closest to satisfy the criteria required for useful photophore. In our photochemical panning, the photoreactive ligand baring carbene-generating photophore, diazirine functions as a barb on a hook, onto which ligands are placed as fish bait Thus, the cross-linked phages stay on the ligand even after intensive washing, whereas almost all the unspecific binding phages are removed.In this study, we developed the photoreactive panning method for screening of antibody-displayed phage, which was categorized in M13 filamentous phage. We succeeded the panning of antibody-displayed phage by using avidine-magnet beads and photoreactive antigen peptide, which had diazirine as a photophore, and biotin at N-terminal for a binding tag for avidine. Our developed panning methods enabled to clone the target displayed phage with higher sensitivity than the traditional panning did.We found that a certain peptide fragment of prion protein inhibited the growth of rat primary culture cell of hippocannpus, and also that N-terminal structure of Alzheimer's beta-amyloid peptide altered spontaneously. We synthesized the peptide N-temiinal of which altered and tried to isolate the antibody-displayed phage that was reacted with such alternation. The synthesized peptide was immunized to five mice and their serums were obtained. We confirmed the production of antibody in the senims by ELISA with antigen.