权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

通过照相淘选法开发抗体展示噬菌体筛选系统：抗淀粉样蛋白抗体的分离

基本信息

批准号：
18590114
负责人：
SADAKANE Yutaka
金额：
$ 2.5万
依托单位：
Kyushu University of Health and Welfare
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590114/
关键词：
photoreaction diazirine antibody-displayed phage amyloid proteome panning screening peptide polymerization

项目摘要

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.

我们开发了一种利用光反应性配体高效克隆展示质粒的方法，该方法基于两种异质性技术的结合，即光亲和标记和展示质粒的淘选。光亲和标记在配体和其特异性结合蛋白之间引入交联，以通过共价键固定它们的亲和关系。我们使用3-芳基-3-1三氟甲基-二氮杂环丙烯作为载体，因为它似乎最接近于满足有用载体所需的标准。在我们的光化学淘选中，光反应性配体暴露于产生卡宾的载体，二氮丙啶起着钩上的倒钩的作用，配体被放置在其上作为鱼饵。因此，即使在强烈的洗涤后，交联的载体仍然停留在配体上，而几乎所有的非特异性结合载体都被去除。其被归类为M13丝状噬菌体。我们成功地用亲和素磁珠和光反应性抗原肽淘选了抗体展示噬菌体，其中光反应性抗原肽以二氮丙啶为载体，在N-末端以生物素作为亲和素的结合标签。本研究建立的淘选方法比传统的淘选方法具有更高的灵敏度，能够克隆到靶向噬菌体，发现朊蛋白的某个肽段抑制了原代培养的大鼠海马细胞的生长，同时发现阿尔茨海默病β-淀粉样肽的N端结构发生了自发的改变。我们合成了N端发生改变的肽，并试图分离与这种改变反应的抗体展示噬菌体。将合成的多肽免疫5只小鼠，获得血清。用抗原ELISA法证实血清中有抗体产生。