Ⅰ型鸭肝炎病毒VP1蛋白优势抗原区的鉴定及抗体检测ELISA方法的建立  

Development of the indirect ELISA for detecting the antibody of duck hepatitis virus type I(DHV-I) coating with truncated VP1 of DHV-I

作　　者：董井泉 张蕾[1] 马波[1] 师东方[1] 王君伟[1] 

机构地区：[1]东北农业大学动物医学院,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2014年第7期542-546,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省"十二五"科技攻关项目(GA09B302);高校博士学科点专项基金(20102325110004);黑龙江省青年基金(QC04C32)

摘　　要：为鉴定Ⅰ型鸭肝炎病毒(DHV-Ⅰ)VP1蛋白优势抗原区及建立DHV-Ⅰ抗体检测ELISA方法,本研究以重组质粒pEasy-VP1为模板,将其分为5段(VP1-a^e)及全长VP1经PCR扩增,并分别克隆于pET-32a(+)进行原核表达。经western blot分析表明,截短表达的蛋白VP1-c(80 aa^150 aa)抗原性良好。将其纯化作为包被抗原建立了DHV-Ⅰ抗体的间接ELISA检测方法。经反应条件优化确定:VP1-c包被浓度为2μg/mL,DHV-Ⅰ抗血清稀释度为1∶20,其临界值为0.208。该方法能够特异性检测DHV抗体,与其他常见的几种鸭病阳性血清无交叉反应;其批内和批间重复性试验变异系数均小于9%。应用建立的ELISA方法对48份鸭血清进行了检测,与中和试验相比较,符合率为86.9%。表明该方法可以初步应用于DHV抗体的检测和血清流行病学调查。To develop a method for detecting antibodies against duck hepatitis virus type Ⅰ（DHV-Ⅰ）, an indirect enzyme-linked immunosorbent assay （ELISA） was established using the purified antigenic dominant region of DHV-Ⅰ VP1 as coating antigen expressed in E.coli. Under the optimal conditions including working concentrations of coating antigen and serum samples, the detection results showed that the assay was specific to detect the antibody against DHV-Ⅰ in duck serum and no cross-reaction with positive antiserum from other related duck disease virus. Furthermore, comparing with the serum neutralization test, the coincidence rate was 86.9%. The established ELISA method provided a simple and rapid serological diagnostic technique.

关 键 词：鸭肝炎病毒 VP1蛋白 抗原优势区 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]