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

(1)抗犬细小病毒(CPV)五株单抗(CPLA-5a)的鉴定：通过酶联免疫吸附试验、包被胶乳微球的凝集活性、Western blotting分析、抗CPV血凝抑制活性(HI)、抗CPV中和活性(VN)等鉴定。通过克隆各V区基因，分析各单抗的互补决定区(CDR)的氨基酸序列。CDR的氨基酸序列分为三种类型，单抗的生物学活性与该序列相关。(2)犬免疫球蛋白G基因在重组杆状病毒中的表达。在本研究中，我们利用杆状病毒载体系统表达了免疫球蛋白。为表达犬免疫球蛋白，制备了两种表达系统。其中一株是同时表达重链和轻链的具有双启动子的重组杆状病毒。另一种是与…合并感染两种独立表达轻链或重链的重组杆状病毒。两种系统的分泌量基本相同，均为0.1μg/mL左右。重组免疫球蛋白可用蛋白A柱结合重链进行纯化。将免疫球蛋白与抗-IgG2抗血清反应。(3)犬化抗体的构建由于与犬免疫球蛋白的同源性最高，因此选择CP2a作为CDR序列的供体。在杆状病毒系统中表达了犬化的CP2a抗体，并用Western blotting鉴定了其与供体CP2a的结合特性。(4)利用具有最高HI和VN活性的CP1a构建了单链V片段(ScFv)单链抗体分子，并在杆状病毒系统中进行了表达。用组氨酸六聚体标签进行Western blotting证实表达，但未观察到HI和VN活性。为了采用定向进化的方法来提高抗体的性质，需要更多关于这些抗体构效关系的基础数据来建立平台。较少

(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