PEDV流行株重组截短N蛋白间接ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2016.06.007

›› 2016, Vol. 43 ›› Issue (6): 1446-1452.doi: 10.16431/j.cnki.1671-7236.2016.06.007

PEDV流行株重组截短N蛋白间接ELISA检测方法的建立

张博^1,2,3, 李守军^1,2,3, 杨保收^1,2,3

1. 天津农学院动物科学与动物医学学院, 天津 300384;
2. 农业部生物兽药创制重点实验室, 天津 300308;
3. 天津瑞普生物技术股份有限公司研究院, 天津 300308

收稿日期:2015-12-10 出版日期:2016-06-20 发布日期:2016-07-11
通讯作者: 杨保收 E-mail:bsyang@ringpu.com
作者简介:张博(1988-),男,天津人,硕士生,研究方向:兽医生物技术与疫苗,E-mail:2419156562@qq.com
基金资助:
瑞普生物研究院猪流行性腹泻疫苗专项基金项目

Establishment of Indirect ELISA Method for Detecting Recombinant Truncated N Protein of Porcine Epidemic Diarrhea Virus

ZHANG Bo^1,2,3, LI Shou-jun^1,2,3, YANG Bao-shou^1,2,3

1. College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384, China;
2. Key Laboratory of Biogenic Veterinary Drugs Innovation, Ministry of Agriculture, Tianjin 300308, China;
3. Ringpu Biological Research Institute, Tianjin 300308, China

Received:2015-12-10 Online:2016-06-20 Published:2016-07-11

摘要/Abstract

摘要： 为了建立猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)抗体检测的间接ELISA方法,本研究以纯化的原核表达的PEDV截短N蛋白作为包被抗原,建立了PEDV抗体检测的间接ELISA方法,将该方法命名为rnPED-ELISA。该抗原不与其他常见的7种猪病的阳性血清发生交叉反应,批内和批间重复性试验的变异系数均小于13%;rnPED-ELISA相对于血清中和试验(SN)试验的敏感性为93.33%,特异性为90.00%;rnPED-ELISA与TSZ全病毒抗体检测试剂盒的符合率达91.67%。采用rnPED-ELISA方法检测200份临床样品,PEDV抗体阳性检出率为69.5%。本试验建立的rnPED-ELISA方法具有良好的敏感性和特异性,可为免疫猪群抗体监测和猪流行性腹泻流行病学调查提供一种快速、简便的血清学诊断方法。

关键词: 猪流行性腹泻病毒; 重组截短N蛋白; 间接ELISA; 诊断

Abstract: In order to establish an indirect ELISA to detect antibody of porcine epidemic diarrhea virus (PEDV).The experiment using the recombinant and purified truncated N protein as antigen expressed in E.coli BL21(DE3),the indirect ELISA was named rnPED-ELISA.The recombinant truncated N protein antigen showed no cross-reaction with the positive sera of other 7 kinds of swine diseases,CV%of intro-batch duplicativity test and inter-batch duplicativity test were less 13%;Sensitivity and specificity of rnPED-ELISA relative to SN were 93.33% and 90.00%,respectively;rnPED-ELISA compared with TSZ PEDV antibody diagnosis Kit,91.67% concordance was obtained.200 serum samples were detected by this method,the total masculine ratio was 69.5%.Therefore,this rnPED-ELISA based on recombinant truncated N protein antigen had good sensitivity and specificity,could afforded a simple and rapidmeans for assessment of vaccination in the field and investigation of PED epidemiology.

Key words: porcine epidemic diarrhea virus; recombinant truncated N protein; indirect ELISA; diagnosis

中图分类号:

S852.65⁺9.6

张博, 李守军, 杨保收. PEDV流行株重组截短N蛋白间接ELISA检测方法的建立[J]. , 2016, 43(6): 1446-1452.

ZHANG Bo, LI Shou-jun, YANG Bao-shou. Establishment of Indirect ELISA Method for Detecting Recombinant Truncated N Protein of Porcine Epidemic Diarrhea Virus[J]. , 2016, 43(6): 1446-1452.

参考文献

[1] Pensaert M B,Debouck P.A new coronavirus-like particle associated with diarrhea in swine[J]. Arch Virol, l978,58(3):243-247.
[2] Kocherhans R,Bridgen A,Ackermann M,et al.Completion of the porcine epidemic diarrhoea coronavirus(PEDV) genome sequence[J].Virus Genes,2001,23(2):137-144.
[3] Park S J,Kim H K,Song D S,et al.Complete genome sequences of a Korean virulent porcine epidemic diarrhea virus and its attenuated counterpart[J].J Virol,2012,86(10):5964.
[4] Pijpers A,van Nieuwstadt A P,Terpstra C,et al.Porcine epidemic diarrhoea virus as a cause of persistent diarrhoea in a herd of breeding and finishing pigs[J].Vet Rec,1993,132(6):129-131.
[5] Carvajal A,Diego R,Lanza I,et al.Seroprevalence of porcine epidemic diarrhea virus infection among difference types of breeding swine farms in Spain[J].Preventive Veterinary Medicine,1995,23(1):33-40.
[6] Kim S Y,Song D S,Park B K.Differential detection of transmissible gastroenteritis virus andporcine epidemic diarrhea virus by duplex RT-PCR[J].J Vet Diagn Invest,2001,13(6):516-520.
[7] Knuchel M,Ackermann M,Muler H K.An ELISA for detection of antibodies against porcine epidemicdiarrhea virus(PEDV) based on the specific solubility of theviral surface glycoprotein[J].Vet Micobiol,1992,32(3):117-134.
[8] Hofmann M,Wyler R.Enzyme-linked immunosorbentassay for the detection of porcine epidemic diarrhea coronavirusantibodies in swine sera[J].Vet Microbiol,1990,21(2):263-273.
[9] NY/T 54—2002.猪流行性腹泻诊断技术/血清中和试验[S].北京:中国标准出版社,2004.
[10] Song D,Park B.Porcine epidemic diarrhoea virus:A comprehensive review of molecular epidemiology,diagnosis,and vaccines[J]. Virus Genes,2012,44(2):167-175.
[11] 朱迪国,宋建德,袁丽萍,等.2013~2014年全球猪流行性腹泻重大疫情分析[J].中国动物检疫,2014,31(10):42-46.
[12] 张博,李守军,杨保收.猪流行性腹泻病毒部分N基因的厚核表达及表达产物的反应原性分析[J].中国畜牧兽医,2016,43(3):637-643.
[13] 刘云波,赵洪翠,王志成,等.猪病毒性腹泻分子流行病学调查[J].中国畜牧兽医,2013,40(2):204-207.
[14] Beam A,Goede D,Fox A.A porcine epidemic diarrhea virus outbreak in one geographic region of the United States:Descriptive epidemiology and investigation of the possibility of airborne virus spread[J].PLoS One,2015,10(12):158-159.
[15] Steinrigl A,Revilla Fernández S,Stoiber F.First detection,clinical presentation and phylogenetic characterization of prcine epidemic diarrhea virus in Austria[J].BMC Vet Res,2015,11(1):310.
[16] 李一经,刘博,姜艳平,等.双抗体夹心ELISA检测排泄物PEDV[J].东北农业大学学报,2015,46(2):1-10.
[17] Oh J S,Song D S,Yang J S,et al.Comparison of an enzyme-linked immunosorbent assay with serurm neutralization test serodiagnosis of porcine epidemic diarrhea virus infection[J].Journal of Veterinary Science,2005,6(4):349-352.
[18] Hou X L,Yu L Y,Liu J Z,et al.Development and evaluationof enzyme-linked immunosorbent assay based on recombinant nucleocapsid protein for detection ofporcine epidemic diarrhea(PEDV) antibodies[J].Veterinary Microbiology,2005,105(1):9-17.
[19] 石达,陈建飞,时红艳,等.猪流行性腹泻病毒N蛋白与Vero E6 核仁蛋白Fibrillarin的亚细胞定位分析[J].中国预防兽医学报,2011,33(11):833-836.
[20] 张志榜,陈建飞,时洪艳,等.猪流行性腹泻病毒CH/S株M蛋白全长的原核表达及其反应原性分析[J].中国兽医科学,2011,41(1):31-35.

PEDV流行株重组截短N蛋白间接ELISA检测方法的建立

Establishment of Indirect ELISA Method for Detecting Recombinant Truncated N Protein of Porcine Epidemic Diarrhea Virus

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