PEDV N蛋白单克隆抗体的制备及间接免疫荧光检测方法的建立

    Preparation of monoclonal antibody against N protein of porcine epidemic diarrhea virus and establishment of indirect immuno-fluorescence assay

    • 摘要:
      目的  制备猪流行性腹泻病毒(PEDV) N蛋白单克隆抗体,并建立检测PEDV的间接免疫荧光试验方法。
      方法  以重组表达的PEDV N蛋白为免疫原,免疫8周龄雌性BALB/c小鼠,分离高抗体效价小鼠的脾细胞,与SP2/0细胞融合。筛选分泌抗PEDV N蛋白单克隆抗体的杂交瘤细胞株。在已经感染PEDV的Vero细胞中,以抗PEDV N蛋白的单克隆抗体为一抗,FITC−羊抗鼠IgG为二抗,建立PEDV的间接免疫荧光检测方法。
      结果  制备的杂交瘤细胞株可以稳定分泌抗PEDV N蛋白抗体,细胞上清液的ELISA抗体效价在1∶3 200以上,而诱导的小鼠腹水抗体效价在1∶1 000 000以上。将单克隆抗体应用在间接免疫荧光试验时,最适条件为−20 ℃ 80%(φ)丙酮溶液中固定30 min;一抗用PBS缓冲液按体积比1∶1 000稀释,4 ℃条件下过夜孵育;二抗用PBS缓冲液按体积比1∶100稀释,37 ℃条件下孵育1 h。以建立的间接免疫荧光试验方法检测细胞中的猪传染性胃肠炎病毒(TGEV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病毒(PPRV)、猪肠道α冠状病毒(PEAV)、猪轮状病毒(PoRV)和PEDV,只有PEDV显示阳性,其他病毒均为阴性。
      结论  制备了抗PEDV N蛋白单克隆抗体,以该抗体为一抗建立检测PEDV的间接免疫荧光试验方法具有良好的特异性,为PEDV的实验室检测及PEDV在培养细胞中的定位和动态分布提供了有效的手段。

       

      Abstract:
      Objective  To prepare monoclonal antibodies against porcine epidemic diarrhea virus (PEDV) N protein, and develop an indirect immuno-fluorescence assay method used for detecting PEDV.
      Method  The expressed recombinantly PEDV N protein was used as an immunogen and 8-week-old female BALB/c mice were immunized. Then their spleen cells with high antibody titer were isolated and fused with SP2/0 cells. The hybridoma cell lines secreting monoclonal antibodies against PEDV N protein were screened. In Vero cells infected with PEDV, monoclonal antibody of anti-PEDV N protein was used as the primary antibody and FITC-goat-anti-mouse IgG was used as the secondary antibody to develop indirect immuno-fluorescence assay method used for detecting PEDV.
      Result  The prepared hybridoma cell lines could stably secrete anti-PEDV N protein antibodies, ELISA antibody titer in cell supernatant was above 1∶3 200, and in mouse ascites above 1∶1 000 000. While monoclonal antibodies were applied in established indirect immuno-fluorescence assay, the optimal conditions were that cells were fixed with 80% (φ) acetone at −20 ℃ for 30 min; The primary antibody was diluted 1 000 times by PBS buffer solution and incubated at 4 ℃ overnight; The secondary antibody was diluted 100 times by PBS buffer solution and incubated at 37 ℃ for 1 h. Transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine reproductive virus (PRV), porcine enteric α corone virus (PEAV), porcine rotavirus (PoRV) and PEDV were detected by established indirect immuno-fluorescence assay method, only PEDV showed positive, all the else viruses showed negative.
      Conclusion  An anti-PEDV N protein monoclonal antibody is prepared, and the indirect immuno-fluorescence assay method used for detecting PEDV is established with high specificity. It provides an effective method for laboratory detection of PEDV and for localization and dynamic distribution of PEDV in infected cells.

       

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