基于非洲猪瘟病毒MGF505-3R蛋白的间接ELISA检测方法的建立  

Establishment of an indirect ELISA detection method based on African swine fever virus MGF505-3R protein

作　　者：全吉玛 杨福荣 李栋凡 田永祥 杨克礼 李畅 刘威 郭锐 袁芳艳 高婷 刘泽文 姚敏 周丹娜 QUAN Jima;YANG Furong;LI Dongfan;TIAN Yongxiang;YANG Keli;LI Chang;LIU Wei;GUO Rui;YUAN Fangyan;GAO Ting;LIU Zewen;YAO Min;ZHOU Danna(College of Animal Science and Technology,Yangtze University,Jingzhou 434000,China;College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;Institute of Animal Husbandry and Veterinary Medicine,Hubei Academy of Agricultural Sciences,Wuhan 430064,China)

机构地区：[1]长江大学动物科学技术学院,湖北荆州434000 [2]华中农业大学动物医学院,武汉430070 [3]湖北农业科学研究院畜牧兽医研究所,武汉430064

出　　处：《黑龙江畜牧兽医》2024年第17期57-63,114,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：湖北省科技重大专项(2022ABA002);湖北省农业技术产业体系项目([2020]-09533);湖北省农业科技创新中心资助项目(2021-620-000-001-017)。

摘　　要：为了建立检测非洲猪瘟病毒(ASFV)的间接ELISA检测方法,试验基于ASFV MGF505-3R基因序列,构建pET-28a(+)-3R原核表达系统和pFastBac1-3R杆状病毒表达系统,分别通过37℃诱导5 h和28℃培养5 d获得3R重组蛋白,以此为包被抗原建立快速检测ASFV的间接ELISA检测方法,对该检测方法的反应条件、阴阳性临界值、敏感性、特异性及重复性进行检测,并用该检测方法与ASFV抗体ELISA检测试剂盒对70份临床血清样品进行符合率验证试验。结果表明:原核表达系统与杆状病毒表达系统表达的3R重组蛋白抗原特异性均良好。利用杆状病毒表达系统表达的3R重组蛋白为包被抗原建立的间接ELISA检测方法的最佳条件为:包被浓度为2μg/mL,血清稀释度为1∶80,5%BSA 37℃封闭2 h,ASFV阳性血清(一抗)37℃孵育90 min,辣根过氧化物酶(HRP)标记的羊抗猪IgG(二抗)37℃孵育45 min。该间接ELISA检测方法的批间和批内变异系数均小于10%;与猪流行性腹泻病毒(PEDV)、猪圆环病毒2型(PCV-2)、猪繁殖与呼吸综合征病毒(PRRSV)等常见病原阳性血清无交叉反应;该检测方法的临界值为0.38,ASFV阳性血清稀释至640倍检测仍高于临界值。该检测方法与ASFV抗体ELISA检测试剂盒的阳性符合率、阴性符合率、整体符合率分别为92%(37/40)、86%(26/30)、90%(63/70)。说明试验基于3R重组蛋白建立的间接ELISA检测方法具有良好的特异性、敏感性与重复性,可以用于临床血清样品中ASFV抗体的检测。In order to establish an indirect ELISA method for the detection of African swine fever virus(ASFV),in the experiment,based on the ASFV MGF505-3R gene sequence,the pET-28a(+)-3R prokaryotic expression system and pFastBac1-3R baculovirus expression system were constructed,and the 3R recombinant protein was obtained by induction at 37℃for 5 h and 28℃for 5 days,respectively,which were the coated antigen;an indirect ELISA method for the rapid detection of ASFV was established;and the reaction conditions,positive and negative cut-offs,sensitivity,specificity and repeatability of the detection method were detected,and the coincidence rate verification test of 70 clinical serum samples was carried out with the detection method and the ASFV antibody ELISA detection kit.The results showed that the specificity of the 3R recombinant protein antigen expressed by the prokaryotic expression system and the baculovirus expression system was good.The optimal conditions for the indirect ELISA detection method using the 3R recombinant protein expressed in the baculovirus system were as follows:coating concentration of 2μg/mL,serum dilution of 1:80,blocking of 5%BSA at 37℃for 2 h,incubation of ASFV-positive serum(primary antibody)at 37℃for 90 min,and incubation of horseradish peroxidase(HRP)-labeled goat anti-porcine IgG(secondary antibody)at 37℃for 45 min.The indirect ELISA detection method had a coefficient of variation of less than 10%between batches and within assays,and had no cross-reactivity with the positive sera of common pathogens such as Porcine epidemic diarrhea virus(PEDV),Porcine circovirus type 2(PCV-2)and Porcine reproductive and respiratory syndrome virus(PRRSV),and the cut-off value of the detection method was 0.38,and the detection of ASFV positive serum diluted to 640 times was still higher than the cut-off value.The positive coincidence rate,negative coincidence rate,and overall coincidence rate of the detection method with the ASFV antibody ELISA detection kit were 92%(37/40),86%(26/30),and 90%(63/70

关 键 词：非洲猪瘟病毒(ASFV) 原核表达系统 杆状病毒表达系统 3R蛋白 间接ELISA 

分 类 号：S852.659.1[农业科学—基础兽医学]