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

我们开发了一种利用光反应配体高效克隆展示噬菌体的方法，该方法基于两种不同的技术相结合，即展示噬菌体的光亲和标记和平移。光亲和标记引入了配体与其特定结合蛋白之间的交联键，以通过共价键固定它们之间的亲和关系。我们使用3-芳基-1-3-1三氟甲基-二氮杂环作为发光体，因为它似乎最接近于满足有用发光体所需的标准。在我们的光化学淘洗中，光反应配体包括卡宾产生的发光团，二氮杂环的作用就像钩子上的倒钩，配体被放在上面作为鱼饵，因此，即使在密集的洗涤后，交联的噬菌体仍然留在配体上，而几乎所有非特异性结合的噬菌体都被去除了。在本研究中，我们发展了光反应淘洗方法来筛选抗体展示噬菌体，归类为M13丝状噬菌体。我们成功地利用亲和素磁珠和以二氮杂环为发光团的光反应性抗原肽，以及N端的生物素作为亲和素的结合标签，成功地获得了抗体展示噬菌体。我们开发的PAN方法能够以比传统PAN方法更高的灵敏度克隆目标展示噬菌体。我们发现Pron蛋白的某一肽片段抑制了原代培养的大鼠海马神经细胞的生长，并且阿尔茨海默氏病β-淀粉样多肽的N-末端结构自发改变。我们合成了N-末端改变的多肽，并试图分离与这种改变发生反应的抗体展示噬菌体。将合成的多肽免疫5只小鼠，获得它们的血清。用抗原酶联免疫吸附试验证实了抗体的产生。

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.