猪丁型冠状病毒重组N蛋白间接ELISA抗体检测方法的建立
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摘要
猪丁型冠状病毒(PDCoV)是2014年检测出的一种新型猪源肠道冠状病毒,对养殖业造成严重的经济损失。为建立快速检测PDCoV的血清学方法,本研究以原核表达的PDCoVN重组蛋白为包被抗原,通过反应条件优化,建立了一种PDCoV重组N蛋白的间接ELISA抗体检测方法。结果显示,原核表达的PDCoV重组N蛋白约为28ku,westernblot证实表达蛋白具有良好的反应原性;以其作为包被抗原建立的PDCoV抗体间接ELISA方法仅对PDCoV血清检测为阳性,与猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪繁殖与呼吸系统综合征病毒等7种主要猪源病毒阳性血清均无特异性反应,具有良好的特异性。该方法检测灵敏度较高、重复性好,批内和批间重复性变异系数均小于10%;采用该ELISA方法对我国2014年~2016年间采集的不同地区猪场的100份临床血清样品进行了检测,阳性率高于45.9%,表明我国猪群中存在PDCoV感染。本实验建立的ELISA方法可以用于检测临床样品中的PDCoV抗体,是PDCoV流行病学调查的一种有效工具,对该病的防控具有重要意义。
Porcine deltacoronavirus(PDCoV)is a new swine coronavirus identified in United States in 2014,causing significant economic loss in the swine industry.In this study,the prokaryotic expression of PDCoV N recombinant protein was used as the coated antigen,and an indirect ELISA antibody detection method of PDCoV was established by optimizing the reaction conditions.The results showed that the recombinant N protein was 28 ku,and had good reactivity by western blot.The ELISA was specific for PDCoV detection,which had no cross-reaction with other seven positive sera to PEDV,TGEV,PRRSV,etc.The sensitivity of this method was high.The ELISA was high reproducible with the coefficients of variation were less than 10%.100 clinical serum samples collected from different pig farms in different areas from 2014 to 2016 were detected by the assay.The result indicated that more than 45.9%positive samples were detected in different pig farms,and there was PDCoV infection in pig farms in China.The data showed that the developed ELISA was an useful tool for detecting antibodies against PDCoV and epidemiological investigation of PDCoV.
Porcine deltacoronavirus(PDCoV)is a new swine coronavirus identified in United States in 2014,causing significant economic loss in the swine industry.In this study,the prokaryotic expression of PDCoV N recombinant protein was used as the coated antigen,and an indirect ELISA antibody detection method of PDCoV was established by optimizing the reaction conditions.The results showed that the recombinant N protein was 28 ku,and had good reactivity by western blot.The ELISA was specific for PDCoV detection,which had no cross-reaction with other seven positive sera to PEDV,TGEV,PRRSV,etc.The sensitivity of this method was high.The ELISA was high reproducible with the coefficients of variation were less than 10%.100 clinical serum samples collected from different pig farms in different areas from 2014 to 2016 were detected by the assay.The result indicated that more than 45.9%positive samples were detected in different pig farms,and there was PDCoV infection in pig farms in China.The data showed that the developed ELISA was an useful tool for detecting antibodies against PDCoV and epidemiological investigation of PDCoV.
引文
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[2]Woo P C,Huang Yi,Lau S K,et al.Coronavirus genomics and bioinformatics analysis[J].Viruses,2010,2(8):1804-1820.
[3]Ma Yuan-mei,Zhang Yu,Liang Xue-ya,et al.Origin,evolution,and virulence of porcine deltacoronaviruses in the United States[J].M Bio,2015,6(2):e00064.
[4]Li Gan-wu,Chen Qi,Harmon K M,et al.Full-length genome sequence of porcine deltacoronavirus strain USA/IA/2014/8734[J].Genome Announc,2014,2(2):pii:e00278-14.
[5]Marthaler D,Jiang Yin,Collins J,et al.Complete genome sequence of strain SDCV/USA/Illinois121/2014,a porcine deltacoronavirus from the United States[J].Genome Announc,2014,2(2):e00218-14.
[6]Hu Hui,Jung K,Vlasova A N,et al.Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States[J].J Clin Microbiol,2015,53(5):1537-1548.
[7]逄凤娇,俞正玉,何孔旺,等.猪Deltacoronavirus RT-PCR检测方法的建立及其应用[J].中国预防兽医学报,2015,37(9):683-686.
[8]McB ride R,van Zyl M.The coronavirus nucleocapsid is a multifunctional protein[J].Viruses,2014,6(8):2991-3018.
[9]孙冰,李彬,何孔旺,等.猪流行性腹泻病毒变异株N基因的克隆及原核表达[J].畜牧与兽医,2014,46(5):72-76.
[10]Dong Nan,Fang Liu-rong.Porcine Deltacoronavirus in Mainland China[J].Emerg Infect Dis,2015,21(12):2254-2255.
[11]Wang Yi-Wen,Hua Yue,Fang Wei-huan,et al.Complete Genome Sequence of Porcine Deltacoronavirus Strain CH/Sichuan/S27/2012 from Mainland China[J].Genome Announc,2015,3(5):e00945-15.
[12]Song D,Zhou X,Peng Q,et al.Newly Emerged Porcine Deltacoronavirus Associated With Diarrhoea in Swine in China:Identification,Prevalence and Full-Length Genome Sequence Analysis[J].Transbound Emerg Dis,2015,62(6):575-580.
[13]Chen Fang-zhou,Zhu Yin-xing,Wu Mei-zhou,et al.FullLength Genome Characterization of Chinese Porcine Deltacoronavirus Strain CH/SXD1/2015[J].Genome Announc,2015,3(5):e01284-15.