鸭坦布苏病毒NS3蛋白原核表达及其间接ELISA检测方法的建立与应用  

Prokaryotic Expression of NS3 Protein of Duck tembusu virus and Establishment and Application of Its Indirect ELISA Detection Method

作　　者：安而立 嵇辛勤[1,2] 阮涌 陈泽辉[1,2,3,4] 曹伟 王晗晗 罗晓宇[1,2,3,4] 龙丹丹 陈佳琪 杨纯培 吴宗豪[5] 王丽娟[5] 姚碧琼 AN Er-Li;JI Xin-Qin;RUAN Yong;CHEN Ze-Hui;CAO Wei;WANG Han-Han;LUO Xiao-Yu;LONG Dan-Dan;CHEN Jia-Qi;YANG Chun-Pei;WUZong-Hao;WANG Li-Juan;YAOBi-Qiong(Faculty of Animal Sciences,GuiZhou University,Guiyang 550025,China;Guizhou Institute of Animal Disease,GuiZhou University,Guiyang 550025,China;Key Laboratory of Plateau Mountain Animal Genetics,Breeding and Reproduction,Ministry of Education,Guizhou University,Guiyang 550025,China;Key Laboratory of Animal Genetics,Breeding and Reproduction,Guiyang 550025,China;County Animal Disease Prevention and Control Center,Sansui 556500,China)

机构地区：[1]贵州大学动物科学学院,贵阳550025 [2]贵州省动物疫病研究所,贵阳550025 [3]贵州大学高原山地动物遗传育种与繁殖教育部重点实验室,贵阳550025 [4]贵州省动物遗传育种与繁殖重点实验室,贵阳550025 [5]三穗县动物疫病预防控制中心,三穗556500

出　　处：《农业生物技术学报》2024年第10期2437-2446,共10页Journal of Agricultural Biotechnology

基　　金：贵州省科技支撑计划项目(黔科合支撑[2022]一般137)。

摘　　要：为了建立一种能够快速检测鸭坦布苏病毒病(Duck tembusu virus,DTMUV)抗体的间接ELISA检测方法,本研究构建了pET-28a-NS3的原核重组表达载体以诱导表达NS3蛋白,以NS3蛋白作为包被抗原,对试验条件进行优化,建立了一种检测DTMUV抗体的间接ELISA方法,使用所建立ELISA方法对30份攻毒试验血清样品及150份采集的血清样品进行检测。结果显示,所建立间接ELISA方法批内与批间变异系数(CV)分别为1.49%~4.31%和1.22%~6.07%,符合国标;该方法可特异性地检测出DTMUV抗体,且与禽流感病毒(Avian influenza virus,AIV)H5亚型、AIV H7亚型、新城疫病毒(Newcastle disease virus,NDV)、鸭圆环病毒(Duck circovirus,DuCV)和鸭甲肝病毒1型(Duck hepatitis A virus,DHAV-1)阳性血清均无交叉反应;所建立方法对攻毒试验样品阳性、阴性检出数与商品化试剂盒检测符合率为100%,对养殖场采集样品阳性、阴性检出数与商品化试剂盒检测符合率为97.33%。综上所述,本研究所建立的DTMUV NS3蛋白抗体间接ELISA检测方法具有良好的特异性与重复性,为DTMUV流行病学调查提供了快速、有效的检测方法。To develop an indirect ELISA method for rapid detection of Duck tembusu virus(DTMUV)antibodies,in this study,the prokaryotic recombinant expression vector pET-28a-NS3 was constructed,and NS3 protein obtained through induced expression was used as the coated antigen.After optimizing a series of conditions,an indirect ELISA method for detecting DTMUV antibodies was established.Antibody analysis was performed on 30 serum samples for challenge test and 150 collected serum samples by the established ELISA method.The results showed that the intra-assay coefficient of variation and the coefficient of variation(CV)of the indirect ELISA method was 1.49%~4.31% and 1.22%~6.07%,respectively,which met the national standard.DTMUV antibody could be specifically detected by this method.And the indirect ELISA method had no cross-reaction with Avian influenza virus(AIV)H5 subtype,AIV H7 subtype,Newcastle disease virus(NDV),Duck Circovirus(DuCV)and Duck hepatitis A virus(DHAV-1)positive sera.The coincidence rate of positive and negative detection numbers of challenge test samples and commercial kit was 100%,and the coincidence rate of positive and negative detection numbers of farm samples and commercial kit was 97.33%.In summary,the DTMUV NS3 protein antibody indirect ELISA method established in this study has good specificity and repeatability,and provides a rapid and effective detection method for the investigation of DTMUV epidemiological situation.

关 键 词：鸭坦布苏病毒(DTMUV) NS3蛋白 ELISA 

分 类 号：S855.3[农业科学—临床兽医学]