抗鳗弧菌独特型单克隆抗体dsFv的构建及其噬菌体表面呈现

抗鳗弧菌独特型单克隆抗体 1E10是能够模拟鳗弧菌的保护性表位 ,可以作为疫苗使用的一种单克隆抗体 .利用基因工程抗体技术从抗鳗弧菌独特型单克隆抗体杂交瘤细胞株 1E10中克隆出抗体的重链及轻链可变区基因 (VH 和VL) .通过定点突变技术将VH4 4和VL10 5突变为半胱氨酸并且连接到噬菌体表达载体pCANTAB5E中 ,突变后的VH 和VL 基因位于cpⅢ先导序列和cpⅢ基因之间 ,在LacZ启动子调控之下 ,以融合蛋白的形式被导入细胞间隙 ,依靠链间二硫键组装成二硫键稳定型Fv抗体 (dsFv) .加入辅助噬菌体M13K0 7后 ,dsFv以融合蛋白的形式表达在噬菌体表面 .ELISA测定显示 :dsFv噬菌体能够与抗原结合并且这种结合呈噬菌体浓度依赖 .结果表明 :成功构建出了抗鳗弧菌独特型单克隆抗体dsFv基因并使其在噬菌体表面获得了正确呈现 .该dsFv噬菌体有望成为新一代基因工程疫苗用于预防鱼类鳗弧菌感染

Construction of Disulfide-stabilized Fv Gene of Anti-idiotype Monoclonal Antibody to Vibrio anguillarum and Its Displaying on Phage

Anti-idiotype monoclonal antibody 1E10 can be used as vaccine for fish disease because it can mimic protective epitope of Vibrio anguillarum. V H gene and V L gene of anti-idiotype monoclonal antibody 1E10 were cloned by RT-PCR. The sequence analysis indicated that the fragment of V H gene consisted of 368 bp,which encoded 122 amino acid residues and that fragment of V L gene consisted of 339 bp,which encoded 113 amino acid resdues. V H44 and V L 105 amino acid residues were mutated into the cysteines by site-directed muation method and then were inserted into vector pCANTAB5E. Recombinant pCANTAB5E was introduced into E.coli TG1.When transfected by helper phage M13KO7,the host E.coli cells secreted dsFv which displayed on the surface of filamentous phage. ELISA test showed that this phage-displayed dsFv antibody could bind antigen in a concentration dependent manner.The phage-displayed dsFv may be a new gene engineering vaccine against Vibrio anguillarum in fishery.