猪流行性腹泻病毒S1蛋白B细胞表位原核表达及间接ELISA方法的建立
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摘要
应用DNAStar软件在猪流行性腹泻病毒S1蛋白70～280位氨基酸序列间预测出1个优势B细胞抗原表位,氨基酸序列为IARLRICQFP,命名为PEP3。借助基因工程技术成功对PEP3进行融合表达,经动物试验、Western blot和间接免疫荧光试验均表明,PEP3蛋白具有良好的抗原性。基于PEP3蛋白建立的ELISA检测方法,与用N蛋白建立的检测方法的符合率为95.94%;该方法具有较高的敏感性和良好的特异性,批内和批间变异系数分别为1.68%～4.65%和4.21%～9.32%。研究表明,本试验建立的基于PEP3蛋白的ELISA检测方法特异性强、敏感性好、重复性高,可以用于临床样本的检测,丰富了现有的猪流行性腹泻病的诊断防控技术。
A dominant B cell epitope on the porcine epidemic diarrhea virus(PEDV)was predicted by using DNAStar software in the 70-280 amino acid sequence of S1 protein,the amino acid sequence was IARLRICQFP,and named PEP3.The fusion expression of PEP3 was successfully carried out by gene engineering technology.The results of animal experiment,Western blot and indirect immunofluorescence showed that PEP3 protein had good antigenicity.Based on the PEP3 protein,an indirect ELISA method was established,which was compared with the ELISA method established by N protein,the coincidence rate with the established ELISA method were 95.94%.This method had high sensitivity and excellent specificity,respectively.Repeatability tests showed that the coefficient of variation of serum samples within and among runs were 1.68%-4.65% and 4.21%-9.32%,respectively.The results suggest that this method is simple to perform,and is a specific,sensitive and convenient method.The method can be used for the detection of clinical samples,and enrich the existing porcine epidemic diarrhea prevention and control technology.
A dominant B cell epitope on the porcine epidemic diarrhea virus(PEDV)was predicted by using DNAStar software in the 70-280 amino acid sequence of S1 protein,the amino acid sequence was IARLRICQFP,and named PEP3.The fusion expression of PEP3 was successfully carried out by gene engineering technology.The results of animal experiment,Western blot and indirect immunofluorescence showed that PEP3 protein had good antigenicity.Based on the PEP3 protein,an indirect ELISA method was established,which was compared with the ELISA method established by N protein,the coincidence rate with the established ELISA method were 95.94%.This method had high sensitivity and excellent specificity,respectively.Repeatability tests showed that the coefficient of variation of serum samples within and among runs were 1.68%-4.65% and 4.21%-9.32%,respectively.The results suggest that this method is simple to perform,and is a specific,sensitive and convenient method.The method can be used for the detection of clinical samples,and enrich the existing porcine epidemic diarrhea prevention and control technology.
引文
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[2]GERBER P F,GONG Q,HUANG Y W,et al.Detection of antibodies against porcine epidemic diarrhea virus in serum andcolostrum by indirect ELISA[J].Vet J,2014,202(1):33-36.
[3]王金良,祖立闯,唐娜,等.猪流行性腹泻病毒N蛋白表达与PPA-ELISA抗体检测方法的建立[J].中国兽医学报,2013,33(6):801-807.
[4]PENSAERT M B,DE B P.A new coronavirus-like particle associated with diarrhea in swine[J].Arch Virol,1978,58(3):243-247.
[5]PASICK J,BERHANE Y,OJKIC D,et al.Investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcineepidemic diarrhea in Canada[J].Transbound Emerg Dis,2014,61(5):397-410.
[6]SUO S,LI X L,LI P C,et al.Immune responses induced by DNA vaccines bearing spike gene of PEDV combined with porcine IL-18[J].Virus Res,2012,167(2):259-266.
[7]王伟,张双翔,金志强,等.猪流行性腹泻病毒RTLAMP检测方法的建立与应用[J].中国兽医学报,2017,37(7):1220-1224.
[8]白小磊,崔进,崔甜甜,等.广东部分地区猪流行性腹泻病毒流行病学调查[J].中国兽医学报,2016,36(11):1823-1828.
[9]王飞,陈小芬,苏丹萍,等.2014—2015年我国部分省份猪流行性腹泻病毒S基因的遗传变异分析[J].中国兽医学报,2016(9):1484-1488.
[10]雷喜梅,陈静,杨盼盼,等.分泌抗猪流行性腹泻病毒中国变异株S蛋白单克隆抗体杂交瘤细胞系的建立[J].中国兽医学报,2016,36(2):206-210.
[11]周如月,刘琪,甘振磊,等.1株猪流行性腹泻病毒S基因的克隆及序列分析[J].中国兽医学报,2017,37(12):2294-2299.
[12]王明月,鲁海冰,刘亚静,等.猪流行性腹泻病毒病S蛋白单抗制备及夹心ELISA检测PEDV方法的建立与应用[J].中国兽医学报,2017,37(11):2033-2037.
[13]乔涵,李辉,赵丽,等.猪流行性腹泻病毒河南流行株S基因的克隆与遗传进化分析[J].中国兽医学报,2017,37(5):781-786.
[14]李加飞,陈智,孙文博,等.山东地区猪流行性腹泻病毒遗传变异分析及其单克隆抗体的制备[J].中国兽医学报,2017,37(5):769-775.
[15]陈刚,李伟,张阳,等.猪流行性腹泻疫苗的研究进展[J].世界华人消化杂志,2013,21(1):28-32.
[16]REN X,SUO S,JANG Y S.Development of a porcine epidemic diarrhea virus M protein-based ELISA for virus detection[J].Biotechnol Lett,2011,33(2):215-220.