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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

发表时间:2013-06-15  浏览量:1044  下载量:476
全部作者: 付培芬,刘琰,李鑫,母晓宇,董井泉,王君伟,马波
作者单位: 东北农业大学动物医学学院
摘 要: 利用RT-PCR技术扩增1型鸭肝炎病毒(duck hepatitis virus type 1,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。经异丙基-β-D-硫代吡喃半乳糖苷(isopropyl β-D-1-thiogalactopyranoside,IPTG)诱导后,SDS-PAGE分析表明VP1蛋白在大肠杆菌中成功表达,Western blotting表明该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1:12 800,中和实验测定多克隆抗体中和抗体效价达1:32. 该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: 兽医免疫学;1型鸭肝炎病毒;VP1;原核表达;免疫原性;中和活性
Title: Prokaryotic expression and polyclonal antibody preparation of VP1 gene of duck hepatitis virus type 1 E53
Author: FU Peifen, LIU Yan, LI Xin, MU Xiaoyu, DONG Jingquan, WANG Junwei, MA Bo
Organization: College of Veterinary Medicine, Northeast Agricultural University
Abstract: According to the genome sequence of duck hepatitis virus type 1 (DHV-1) E53, VP1 gene was amplified by RT-PCR and cloned into prokaryotic expression vector pET-30a to construct pET-30a-VP1 vector. After sequencing, the recombniant expression vector pET-30a-VP1 was transformed into the host Rosetta(DE53)plysS. SDS-PAGE analysis indicated that the VP1 fusion protein was expressed in Escherichia coli successfully after induced by isopropyl β-D-1-ihiogalactopyranoside (IPTG). Western blotting analysis showed that the fusion protein was expressed correctly. Then the rabbits were immunized with purified recombinant VP1 protein and Western blotting analysis showed that the sera from immunized rabbits had specific reaction with DHV-1. In addition, the antibody titer was 1:12 800 by indirect ELISA, and neutralization test showed that its neutralization antibody titer was up to 1:32. Therefore, the expressed recombinant protein and polyclonal antibodies from this study provided important data and material for the further study of VP1 protein function and the development of diagnostics.
Key words: veterinary immunology; duck hepatitis virus type 1; VP1; prokaryotic expression; immunogenicity; neutralization
发表期数: 2013年6月第11期
引用格式: 付培芬,刘琰,李鑫,等. 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[J]. 中国科技论文在线精品论文,2013,6(11):1058-1063.
 
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