Improvement of neutralizing activity of anti-canine parvovirus antibodies through directed evolution.

通过定向进化提高抗犬细小病毒抗体的中和活性。

• 批准号: 15580281
• 负责人: IWATA Akira
• 金额: $ 1.73万
• 依托单位: Nippon Institute for Biological Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580281/
• 关键词: dog; parovovirus; treatment; neutralizing antibody; baculovirus; ScFv; caninized antibodies; 中和; モノクローナル抗体

(1)Characterization of monoclonal antibodies (mAb) to canine parvovirus (CPV)Five mAbs (CPla - 5a) reacting to CPV has characterized through ELISA, agglutination activity of antigen-coated latex microspheres, Western blotting analysis, inhibitory activity to CPV's hemagglutination (HI), neutralizing activity against CPV (VN). The amino acid sequence of complementarity determining region (CDR) of each mAb was analyzed through cloning of cDNA of each V domain. The amino acid sequence of CDR was classified three types, and concomitantly, the biological activity of the mAbs correlated to the sequence.(2)Expression of a canine immunoglobulin G (IgG) with recombinant baculoviruses.Previously, we have cloned cDNA of canine IgG. In this study, we expressed IgG by baculovirus vector system. To express the canine IgG, two systems were prepared. The one was a recombinant baculovirus which has dual promoter and expresses both heavy and light chain at the same time. The another was co-infection wit … More h two recombinant baculoviruses which independently express light or heavy chain. The amount of secreted IgG in both system was the same, around 0.1 μg/mL. The recombinant IgG can be purified with protein A column, together with heavy chains. The IgG was reacted with anti-IgG2 antisera (Bethyl Lab. Inc.), so this molecule is IgG2 subclass.(3)Construction of caninized antibody.As homology of amino acid sequence of frame work region to canine IgG is highest among the mAbs, CP2a was selected as a donor of the CDR sequence. The caninized CP2a antibody was expressed by baculovirus system and characterized its binding property with donor CP2a by Western blotting. The caninized CP2a reacted with CPV proteins equally with CP2a, in the same peptide region (219-292) of VP2 proteins.(4)Expression of single chain V fragment (scFv)ScFv molecules was constructed with CP1a because of its highest HI and VN activity, and expressed in baculovirus system. The expression was confirmed with Western blotting using histidine hexamer tag, but the HI and VN activities were not observed. To adapt the directed evolution method to improve the property of the antibody, more basic data on structure-activity relationship of these antibodies were necessary to establish the platform. Less

（1）抗犬细小病毒（CPV）单克隆抗体（mAb）的鉴定通过ELISA、抗原包被乳胶微球的凝集活性、Western印迹分析、对犬细小病毒（CPV）血凝抑制活性（HI）、对犬细小病毒（CPV）的中和活性（VN））对5株与犬细小病毒（CPV）反应的mAbs（CPla - 5a）进行了表征。通过克隆每个V结构域的cDNA，分析每个mAb的互补决定区（CDR）的氨基酸序列。CDR的氨基酸序列分为三类，其生物学活性与序列相关。（2）利用重组杆状病毒表达犬IgG。本研究利用杆状病毒载体系统表达IgG。为了表达犬IgG，制备了两种系统。一种是具有双启动子的同时表达重链和轻链的重组杆状病毒。另一种是合并感染， ...更多信息 h两种独立表达轻链或重链的重组杆状病毒。两种系统中分泌IgG的量相同，约为0.1 μg/mL。重组IgG可以与重链一起用蛋白A柱纯化。使IgG与抗IgG 2抗血清（Bethyl Lab. Inc.）、所以该分子是IgG 2亚类。（3）犬源化抗体的构建：由于所制备的单克隆抗体与犬IgG的框架区氨基酸序列同源性最高，因此选择CP 2a作为CDR序列的供体。利用杆状病毒系统表达了犬源化CP 2a抗体，并通过Western blotting鉴定了其与供体CP 2a的结合特性。犬源化的CP 2a与CPV蛋白在VP 2蛋白的相同肽段（219-292）上具有相同的反应性。（4）单链V片段（scFv）的表达由于CP 1a具有最高的HI和VN活性，因此用CP 1a构建了单链V片段（scFv）分子，并在杆状病毒系统中表达。用组氨酸六聚体标签的蛋白质印迹法证实表达，但未观察到HI和VN活性。为了适应定向进化方法对抗体进行性能改进，需要更多的抗体构效关系基础数据来建立平台。少

项目成果

イヌ化抗体の作製方法および使用

犬化抗体的制备和使用方法

• 发表时间: 2003