基于猪非典型瘟病毒E2蛋白的间接ELISA检测方法的建立
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摘要
猪非典型瘟病(APPV)是新近发现能引起仔猪先天震颤的病原体,血清学检测方法亟待构建。本研究制备了APPV原核表达的E2重组蛋白,以其作为包被抗原,建立了间接ELISA检测方法。结果表明,最佳抗原包被浓度为10 g/mL,最佳血清稀释度为1∶40,阴阳性临界值D_(450)=0.243,灵敏度达到1∶64;与猪瘟、猪伪狂犬病、猪繁殖与呼吸综合征、猪流感及圆环病毒病的阳性血清均不发生交叉反应,特异性好;批内变异系数最大值为3.797%,批间变异系数最大值为5.425%,重复性好。利用建立的间接ELISA方法对50份猪临床血清样本进行检测,阳性率为12%。本研究基于E2蛋白建立的间接ELISA方法为猪非典型瘟病毒感染的检测提供了可靠的工具,也为开展APPV相关的研究工作提供了基础材料。
Atypical porcine pestivirus(APPV) is a newly discovered pathogen which cause the congenital tremor of piglets,and the serological detection method needs to be established urgently.E2 protein is the major antigen of neutralizing antibodies against APPV and one of the most significant target of serological detection.In this study,the recombinant E2 protein of prokaryotic expression was coated as the antigen to establish an indirect ELISA method.In result,the optimal antigen coating concentration was 10 g/mL,and the optimal serum dilution was 1/40.The positive and negative critical value was D450=0.243 and the sensitivity of the indirect ELISA was 1/64.There were no cross reactions with the positive sera with classical swine fever virus,porcine pseudorabies virus,porcine reproductive and respiratory syndrome virus,swine influenza virus and porcinecircovirus virus,respectively,showing that it had a good specificity of detection.The coefficient of maximum variation was 3.797% and 5.425% in intra-and inter-assay,respectively,showing that the detection method was stable.The established indirect ELISA method was used to detect 50 different clinical porcine sera,and the positive rate was 12%.In conclusion,the establishment of the indirect ELISA method based on E2 protein provides a reliable tool for the detection of APPV infections,and also offers basic materials for developing APPV-related work.
Atypical porcine pestivirus(APPV) is a newly discovered pathogen which cause the congenital tremor of piglets,and the serological detection method needs to be established urgently.E2 protein is the major antigen of neutralizing antibodies against APPV and one of the most significant target of serological detection.In this study,the recombinant E2 protein of prokaryotic expression was coated as the antigen to establish an indirect ELISA method.In result,the optimal antigen coating concentration was 10 g/mL,and the optimal serum dilution was 1/40.The positive and negative critical value was D450=0.243 and the sensitivity of the indirect ELISA was 1/64.There were no cross reactions with the positive sera with classical swine fever virus,porcine pseudorabies virus,porcine reproductive and respiratory syndrome virus,swine influenza virus and porcinecircovirus virus,respectively,showing that it had a good specificity of detection.The coefficient of maximum variation was 3.797% and 5.425% in intra-and inter-assay,respectively,showing that the detection method was stable.The established indirect ELISA method was used to detect 50 different clinical porcine sera,and the positive rate was 12%.In conclusion,the establishment of the indirect ELISA method based on E2 protein provides a reliable tool for the detection of APPV infections,and also offers basic materials for developing APPV-related work.
引文
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[17] TARRADAS J,MONSOM,MUNOZ M,et al.Partial protection against classical swine fever virus elicited by dendrimeric vaccine-candidate peptides in domestic pigs[J].Vaccine,2011,29(26):4422-4429.
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[20]鲍家桢,张洪让,杜念兴,等.仔猪先天性震颤病的研究概况[J].中国兽医杂志,1988(3):23-24.BAO Jia-zhen,ZHANG Hong-rang,DU Nian-xing,et al.A survey of congenital tremor disease in piglets[J].Chinese Journal of Veterinary Medicine,1988(3):23-24.(in Chinese)
[21] NUC P,NUC K.Recombinant protein production in Escherichia coli[J].Postepy Biochemii,2006,52(4):448-456.
[2] DE GROOF A,DEIJS M,GUELEN L,et al.Atypical porcine pestivirus:A possible cause of congenital tremor type A-II in newborn piglets[J].Viruses,2016,8(10):e271.
[3] HAUSE B M,COLLIN E A,PEDDIREDDI L,et al.Discovery of a novel putative atypical porcine pestivirus in pigs in the USA[J].Journal of General Virology,2015, 96(10):2994-2998.
[4] ZHANG K,WU K,LIU J,et al.Identification of atypical porcine pestivirus infection in swine herds in China[J]. Transboundary and Emerging Diseases,2017,64(4):1020-1023.
[5]许古明,葛士坤,张凯照,等.猪非典型瘟病毒的基因组特征分析及荧光定量RT-PCR检测方法的建立[J].中国兽医科学,2017,47(11):1357-1362.XU Gu-ming,GE Shi-kun,ZHANG Kai-zhao,et al.Genomic characterization and development of real-time RT-PCR for the detection of atypical porine pestivirus[J].Chinese Veterinary Science,2017,47(11):1357-1362.(in Chinese)
[6] POSTEL A,MEYER D,CAGATAY G N,et al.High abundance and genetic variability of atypical porcine pestivirus in pigs from Europe and Asia[J].Emerging Infectious Diseases,2017,23(12):2104-2107.
[7] BEER M,WERNIKE K,DRAGER C,et al.High prevalence of highly variable atypical porcine pestiviruses found in Germany[J].Transboundary and Emerging Diseases,2017,64(5):e22-e26.
[8] BECHER P,AVALOS RAMIREZ R,ORLICH M,et al.Genetic and antigenic characterization of novel pestivirus genotypes:Implications for classification[J].Virology,2003,311:96-104.
[9] MEYERS G,THIEL H J.Molecular characterization of pestiviruses[J].Advances in Virus Research,1996,47:53-118.
[10] KREY T,BONTEMS F,VONRHEIN C,et al.Crystal structure of the pestivirus envelope glycoprotein Erns and mechanistic analysis of its ribonuclease activity[J].Structure,2012,20:862-873.
[11] WEILAND E,AHL R,STARK R,et al.A second envelope glycoprotein mediates neutralization of a pestivirus,hog cholera virus[J].Journal of Virology,1992,66(6):3677-3682.
[12] WEN G,XUE J,SHEN Y,et al.Characterization of classical swine fever virus(CSFV)nonstructural protein 3(NS3)helicase activity and its modulation by CSFV RNA-dependent RNA polymerase[J].Virus Research,2009,141(1):63-70.
[13] KALAIYARASU S,MISHRA N,RAJUKUMAR K,et al.Development and evaluation of a truncated recombinant NS3 antigen-based indirect ELISA for detection of pestivirus antibodies in sheep and goats[J].Journal of Immunoassay and Immunochemistry,2015,36(3):312-323.
[14] CHANG C Y,HUANG C C,LIN Y J,et al.Antigenic domains analysis of classical swine fever virus E2glycoprotein by mutagenesis and conformation dependent monoclonal antibodies[J].Virus Research,2010,149(2):183-189.
[15] CHANG C Y,HUANG C C,DENG M C,et al.Identification of conformational epitopes and antigen specific residues at the D/A domains and the extramembrane C-terminal region of E2 glycoprotein of classical swine fever virus[J].Virus Research,2012,168(1-2):56-63.
[16] LIN M,LIN F,MALLORY M,et al.Deletions of structural glycoprotein E2 of classical swine fever virus strain alfort/187 resolve a linear epitope of monoclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum[J].Journal of Virology,2000,74(24):11619-11625.
[17] TARRADAS J,MONSOM,MUNOZ M,et al.Partial protection against classical swine fever virus elicited by dendrimeric vaccine-candidate peptides in domestic pigs[J].Vaccine,2011,29(26):4422-4429.
[18] QI Y,ZHANG B Q,SHEN Z,et al.Antigens containing TAVSPTTLR tandem repeats could be used in assaying antibodies to classical swine fever virus[J].Acta Virologica,2009,53(4):241.
[19] YUAN J,HAN Z Y,LI J,et al.Atypical porcine pestivirus as a novel type of pestivirus in pigs in China[J].Frontiers in Microbiology,2017,8:862.
[20]鲍家桢,张洪让,杜念兴,等.仔猪先天性震颤病的研究概况[J].中国兽医杂志,1988(3):23-24.BAO Jia-zhen,ZHANG Hong-rang,DU Nian-xing,et al.A survey of congenital tremor disease in piglets[J].Chinese Journal of Veterinary Medicine,1988(3):23-24.(in Chinese)
[21] NUC P,NUC K.Recombinant protein production in Escherichia coli[J].Postepy Biochemii,2006,52(4):448-456.