鸡传染性支气管炎病毒S1蛋白截短表达及间接ELISA抗体检测方法的建立与应用  

Truncated expression of S1 protein and establishment and application of indirect ELISA antibody detection methods for infectious bronchitis virus

作　　者：苗立中[1,2] 祖立闯[2] 王艳[2] 马秀丽[3] 亓丽红[3] 宋敏训[3] 沈志强[3] 韩文瑜[1] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]山东省滨州畜牧兽医研究院,山东滨州256600 [3]山东省农业科学院家禽研究所,山东济南250023

出　　处：《中国兽医学报》2017年第3期426-432,共7页Chinese Journal of Veterinary Science

基　　金：山东省现代农业产业技术体系家禽创新团队计划资助项目(SDAIT-11-16);山东省农业科学院院地科技合作引导计划资助项目(2014YDHZ33);滨州市科技发展计划资助项目(2015ZC0109)

摘　　要：参照IBV S1基因序列,RT-PCR扩增长约867bp的S1基因主要抗原区域,将目的片段定向克隆到pET30α原核表达载体,转化Rossetta表达菌,经IPTG诱导后获得了以包涵体形式表达的重组S1蛋白。重组蛋白纯化后,经免疫印迹检测证明具有良好的免疫活性。以该蛋白作为包被抗原,经间接ELISA反应条件的优化,建立了检测鸡传染性支气管炎病毒(IBV)抗体的间接ELISA检测方法。该方法与其他6种常见禽病病毒(H5亚型禽流感病毒(H5-AIV)、H9亚型禽流感病毒(H9-AIV)、新城疫病毒(NDV)、减蛋综合征病毒(EDSV)、鸡传染性喉气管炎病毒(ILTV)、鸡法氏囊病毒(IBDV))阳性血清不发生交叉反应;批内和批间重复性试验的变异系数分别小于5%和10%;相对于HI试验的符合率、敏感性、特异性分别为91.58%、91.24%和92.31%;相对于IDEXX ELISA的符合率、敏感性、特异性分别为94.55%、95.42%和92.96%;应用该方法检测山东及周边地区2 685份免疫鸡和168份未免疫鸡血清样品,免疫合格率和阳性感染率分别为85.25%和17.26%。本试验截短表达的S1蛋白具有良好的免疫活性,建立的间接ELISA抗体检测方法具有良好的敏感性、特异性、重复性和临床适用性,将为IBV的免疫抗体监测、临床野毒感染快速诊断和流行病学调查提供了一种新的血清学诊断技术,具有良好的推广应用前景。For the establishment of a rapid avian infectious bronchitis virus antibody detection methods,this study based on IBV $1 gene sequence,RT-PCR amplificated about 867 bp of S1 gene major antigenic regions, target fragment was directionally cloning into pET30a prokaryotic expres- sion vector, transformed into Rossetta expression bacteria, the recombinant 81 protein was ob- tained by IPTG induction in inclusion body forms. The purified recombinant protein had good im- munological activity. Using this protein as antigen, established an indirect ELISA method for chicken infectious bronchitis virus antibody detection by optimization of the reaction conditions. The method showed no cross-reaction to other six common poultry diseases （H5-AIV,H9-AIV,NDV,EDSV, ILTV, IBDV）positive sera, The variation coefficients of intra-and inter- batch repeatability tests were less than 5 % and 10% ,respectively;The coincidence rate, sensitivity and specificity of HI were 91.58% ,91.24% and 92.31% ,respectively;The coincidence rate,sensi- tivity and specificity of ELISA IDEXX were 94.55 %, 95.42 % and 92.96%, respectively; using the method to detect the serum samples of 2 685 immunized chickens and 168 chickens without immu- nization collected from Shandong and its surrounding areas, the immune qualified rate and the pos- itive rate were 85.25% and 17.26% respectively. In this study,the truncated expression S1 protein has a good immune activity and the established indirect ELISA method for antibodies detection has good sensitivity, specificity, reproducibility and clinical applicability, it provides a new diagnostic techniques of serology for IBV immune antibody monitoring, clinical wild virus infection rapid di- agnosis and epidemiological investigation.

关 键 词：鸡传染性支气管炎病毒 S1蛋白 截短表达 活性鉴定 间接ELISA 检测 

分 类 号：S852.65[农业科学—基础兽医学]