抗猪流行性腹泻病毒单克隆抗体的制备与鉴定

为了制备抗猪流行性腹泻病毒(PEDV)的单克隆抗体(MAb),本试验将Vero细胞中增殖的PEDV超速离心纯化,作为免疫原免疫BALB/c雌性小鼠,采用常规的淋巴细胞杂交瘤技术和PEDV重组S蛋白为基础的间接ELISA方法,制备并筛选能稳定分泌PEDV特异性抗体的杂交瘤细胞;分别采用间接ELISA、Western blot和间接免疫荧光(IFA)试验,对单克隆抗体的亚型、特异性等生物学特性进行鉴定。结果表明,本试验成功获得了1株稳定分泌PEDV特异性抗体的杂交瘤细胞株5C3。MAb亚类鉴定为IgM,轻链为k型。杂交瘤细胞连续传至20代,细胞培养上清液和小鼠腹水抗体效价分别稳定在1∶600和1∶10 000。特异性试验结果显示,MAb不与猪传染性胃肠炎病毒(TGEV)、猪伪狂犬病毒(PrV)、猪圆环病毒(PCV2)发生交叉反应。western-blot结果显示,MAb能特异性识别PEDV的重组S蛋白。IFA试验结果表明,MAb能与PEDV感染的vero细胞产生特异性荧光。说明本试验获得的MAb能特异性识别重组及天然的PEDV S蛋白,具有良好的抗原性和特异性。

Preparation and identification of monoclonal antibodies against porcine epidemic diarrhea virus

To prepare the monoclonal antibody(MAb)against porcine epidemic diarrhea virus (PEDV),BALB/c mice were immunized with purified PEDV for cell fusion.Hybridoma cell lines were screened by indirect ELISA based on PEDV recombined S protein.The subtype and specificity of MAb were identified by ELISA,Western blot and indirect immunofluorescent (IFA),respectively.A hybridoma cell line stably secreting MAb against PEDV S protein was obtained and designated as 5C3.The MAb was IgM subtype with k chain.The titers of 20rd passage hybridoma culture medium and ascetic were 1 ∶ 600 and 1 ∶ 10 000,respectively.There was no cross reaction between the MAb and pseudorabies virus(PrV),porcine circovirus 2 (PCV2),or transmissible gastroenteritis virus(TGEV).Western-blot and IFA showed that the MAb could specifically recognize recombinant and native S protein of PEDV.All these results indicated the high antigenicity and specificity of MAb.