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

(1)犬细小病毒（canine parvovirus， 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 blotting分析、对CPV的血凝抑制活性（HI）、对CPV的中和活性（VN）鉴定了5种与CPV反应的单克隆抗体（cpa - 5a）。通过克隆各V结构域cDNA，分析各单抗的互补决定区（CDR）氨基酸序列。CDR的氨基酸序列被划分为三种类型，同时单克隆抗体的生物活性也与该序列相关。(2)重组杆状病毒表达犬免疫球蛋白G （IgG）。此前，我们已经克隆了犬IgG的cDNA。本研究采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。其中一种是具有双启动子，同时表达重链和轻链的重组杆状病毒。另一种是与两种独立表达轻链或重链的重组杆状病毒共感染。两种系统的IgG分泌量相同，均在0.1 μg/mL左右。重组IgG可以用蛋白A柱和重链纯化。IgG与抗igg2抗血清（Bethyl实验室）反应。所以这个分子是IgG2亚类。(3)犬化抗体的构建。由于框架区氨基酸序列与犬IgG的同源性在单克隆抗体中最高，因此选择CP2a作为CDR序列的供体。犬化CP2a抗体通过杆状病毒系统表达，Western blotting检测其与供体CP2a的结合特性。犬化CP2a与CPV蛋白在VP2蛋白的同一肽区（219-292）发生相同的反应。(4)单链V片段（scFv）的表达由于其具有最高的HI和VN活性，CP1a构建了scFv分子，并在杆状病毒系统中表达。用组氨酸六聚体标记Western blotting证实表达，但未观察到HI和VN活性。为了适应定向进化方法来提高抗体的性能，需要更多的抗体结构-活性关系的基础数据来建立平台。少

(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