1型鸭肝炎病毒E53株VP1基因的原核表达及多克隆抗体的制备.docVIP

  1 型鸭肝炎病毒 E53 株 VP1 基因的原核表达 及多克隆抗体的制备# ＊  5 10  （东北农业大学动物医学学院，哈尔滨 150030） 摘要：利用 RT-PCR 技术扩增 1 型鸭肝炎病毒(DHV-1)E53 株 VP1 基因序列，将其连入 pET-30a 载体中，构建原核表达载体 pET-30a-VP1，并转化至宿主菌 Rosetta (DE53) plysS 中，经 IPTG 诱导后，SDS分析表明 VP1 蛋白在大肠杆菌中获得了表达，western blot 表明表达产 物具有良好的反应原性。将表达产物纯化后免疫新西兰大白兔，制备的多克隆抗体可特异性 识别 DHV-1，间接 ELISA 测定多克隆抗体效价达 1:12800，中和实验测定多克隆抗体中和 抗体效价达 1:32。 本研究表达的蛋白和制备的多克隆抗体为 VP1 蛋白功能的进一步研究和 诊断制剂的开发提供了重要数据和材料。 关键词：1 型鸭肝炎病毒；VP1；原核表达；免疫原性；中和活性 中图分类号：R392 15 Prokaryotic Expression of VP1 Protein of duck hepatitis virus type 1 and preparation of polyclonal antibody Fu Peifen, Liu Yan, Li Xin, Wang Junwei *, Ma Bo * (College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China) 20 25 30 35 Abstract: According to the genome sequence of duck hepatitis virus type 1 E53, VP1 gene was amplified by RT-PCR and cloned into prokaryotic expression vector pET-30a. After sequencing, the recombniant expression vector pET-30a-VP1 was transformed into the hostRosetta (DE53) plysS. The fusion protein His-VP1 was expressed after induced by IPTG. Western blot analysis showed that the fusion protein was expressed correctly. The rabbits were immunized with purified recombinant VP1 protein, western blot analysis showed that the sera from immunized rabbits had specific reaction with DHV-1. In addition, the antibody titer was 1:12800 by i-ELISA and neutralization test showed its neutralization antibody titer was up to 1:32. In this study, the expressed recombinant protein and polyclonal antibodies provided important data and material for the further study of VP1 protein function and the development of diagnostics. Key words: duck hepatitis virus type 1; VP1; prokaryotic expression; immunogenicity; neutralization 0 引言 鸭病毒性肝炎（Duck viral hepatitis，DVH）是由鸭肝炎病毒（DHV）引起雏鸭的一种 急性、高度致死性传染病，主要侵害 3 周龄以内的雏鸭，特别是 1 周龄以下的雏鸭最易感染， 死亡率较高，是危害养鸭业最为严重的传染病之一[1]。DVH 特征为病程短促，临床表现为 角弓反张，病变主要为肝脏肿大并伴有出血斑点。该病最先于 1949 年在日本长岛发现[2]， 随后在英格兰，加拿大，德国，日本的其它地区相继爆发，1963 年在中国大陆也发现了此 病。在 118 fang 了 本病有三种血