基于B细胞表位肽的水貂阿留申病毒抗体ELISA检测方法的建立及其在流行病学调查中的应用
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摘要
为建立水貂阿留申病(AD)抗体间接ELISA检测方法,本研究通过生物信息学预测筛选多个阿留申病毒(ADV)VP2结构蛋白保守的B细胞表位,设计人工诊断抗原肽SD,经原核表达、纯化后作为ELISA的包被抗原,建立了间接ELISA检测方法。结果显示该方法特异性好、敏感性高,与对流免疫电泳(CIEP)方法的符合率达93.2%。利用该方法对我国主要养貂区域的2190份血清样品进行AD血清学调查,结果显示山东、大连、河南、吉林、黑龙江AD阳性率分别为80.41%、82.14%、69.81%、71.24%、78.41%,表明AD在我国已呈高发态势。本研究建立的ELISA方法显著提高了AD阳性检出率,对貂场AD的净化及养貂产业的健康发展具有重要意义。
In order to establish an assay for rapid detection of the antibodies against Aleution disease virus(ADV), several conserved B cell epitopes of the structural protein VP2 of ADV were screened by bioinformatics prediction, and the indirect ELISA was developed for the antibody detection with the prokaryoticaly expressed and purified artificially designed peptide antigen SD as the coated antigen. The results showed that this method was specific and sensitive, and the coincidence rate was 93.2% with counter immunoelectrophoresis. Moreover, a total of 2,190 serum samples collected from mink farms were tested by the established assay and the positive rates of Aleution disease(AD) were 80.41%, 82.14%, 69.81%, 71.24% and 78.41% in Shandong, Dalian, Henan, Jilin and Heilongjiang provinces, respectively, indicating that AD had a high incidence in these areas.The method had the advantages of high specificity and sensitivity, and obviously improves the positive detection rate, and was of great practical significance for eradication of AD in mink farms and for the healthy development of the mink industry.
In order to establish an assay for rapid detection of the antibodies against Aleution disease virus(ADV), several conserved B cell epitopes of the structural protein VP2 of ADV were screened by bioinformatics prediction, and the indirect ELISA was developed for the antibody detection with the prokaryoticaly expressed and purified artificially designed peptide antigen SD as the coated antigen. The results showed that this method was specific and sensitive, and the coincidence rate was 93.2% with counter immunoelectrophoresis. Moreover, a total of 2,190 serum samples collected from mink farms were tested by the established assay and the positive rates of Aleution disease(AD) were 80.41%, 82.14%, 69.81%, 71.24% and 78.41% in Shandong, Dalian, Henan, Jilin and Heilongjiang provinces, respectively, indicating that AD had a high incidence in these areas.The method had the advantages of high specificity and sensitivity, and obviously improves the positive detection rate, and was of great practical significance for eradication of AD in mink farms and for the healthy development of the mink industry.
引文
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[9]Chen Xiao-wei,Song Cai-ling,Liu Yun,et al.Development of an enzyme-linked immunosorbent assay based on fusion VP2[J].J Clin Microbiol,2016,54(2):439-442.
[10]Ma Fan-shu,Zhang Lei,Wang Yang,et al.Development of a peptide ELISA for the diagnosis of Aleutian mink disease[J].PLo S One,2016,11(11):e165793.
[11]逄凤娇,俞正玉,何孔旺,等.猪丁型冠状病毒重组N蛋白间接ELISA抗体检测方法的建立[J].中国预防兽医学报,2017,39(06):461-465.
[12]张卓.水貂阿留申病毒分离鉴定及其致病性研究和诊断方法的建立[D].哈尔滨:东北农业大学,2015.
[13]Kolaskar A S,Ku Lkarni-Kale U.Prediction of three-dimensional structure and mapping of conformational epitopes of envelope glycoprotein of Japanese encephalitis virus[J].Virology,1999,261(1):31-42.
[2]Knuuttila A,Aronen P,Saarinen A,et al.Development and evaluation of an enzyme-linked immunosorbent assay based on recombinant VP2 capsids for the detection of antibodies to aleutian mink disease virus[J].Clin Vaccine Immunol,2009,16(9):1360-1365.
[3]贾赟,刘钊,宋大贺,等.水貂阿留申病毒Taq Man-MGB荧光定量PCR检测方法的建立与应用[J].中国预防兽医学报,2016,38(11):898-902.
[4]Castelruiz Y,Blixenkrone-Moller M,Aasted B.DNA vaccination with the Aleutian mink disease virus NS1 gene confers partial protection against disease[J].Vaccine,2005,23(10):1225-1231.
[5]Porter D D,Larsen A E,Porter H G.The pathogenesis of Aleautian disease of mink in vivo viral replication and the host antibody response to viral antigen[J].J Exp Med,1969,130(3):575-593.
[6]Wright P F,Wilkie B N.Detection of antibody in Aleutian disease of mink:comparison of enzyme-linked immunosorbent assay and counter immunoelectrophoresis[J].Am J Vet Res,1982,43(5):865-868.
[7]Cho H J,Ingram D G.Antigen and antibody in Aleutian disease in mink.II.The reaction of antibody with the Aleutian disease agent using immunodiffusion and immunoelectroosmophoresis[J].Can J Comp Med,1973,37(3):217-223.
[8]Yi Li,Cheng Yue-ning,Zhang Miao,et al.Identification of a novel Aleutian mink disease virus B-cell epitope using a monoclonal antibody against VP2 protein[J].Virus Res,2016,223:39-42.
[9]Chen Xiao-wei,Song Cai-ling,Liu Yun,et al.Development of an enzyme-linked immunosorbent assay based on fusion VP2[J].J Clin Microbiol,2016,54(2):439-442.
[10]Ma Fan-shu,Zhang Lei,Wang Yang,et al.Development of a peptide ELISA for the diagnosis of Aleutian mink disease[J].PLo S One,2016,11(11):e165793.
[11]逄凤娇,俞正玉,何孔旺,等.猪丁型冠状病毒重组N蛋白间接ELISA抗体检测方法的建立[J].中国预防兽医学报,2017,39(06):461-465.
[12]张卓.水貂阿留申病毒分离鉴定及其致病性研究和诊断方法的建立[D].哈尔滨:东北农业大学,2015.
[13]Kolaskar A S,Ku Lkarni-Kale U.Prediction of three-dimensional structure and mapping of conformational epitopes of envelope glycoprotein of Japanese encephalitis virus[J].Virology,1999,261(1):31-42.