摘要
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。
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 20 rd passage hybridoma culture medium and ascetic were 1∶600and 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 thatthe MAb could specifically recognize recombinant and native S protein of PEDV.All these results indicated the high antigenicity and specificity of MAb.
出处
《河北农业大学学报》
CAS
CSCD
北大核心
2017年第4期96-100,共5页
Journal of Hebei Agricultural University
基金
河北省自然科学基金项目(C2015204121)
河北农业大学百名青年学术带头人培养计划
关键词
猪流行性腹泻病毒
单克隆抗体
S蛋白
porcine epidemic diarrhea virus
monoclonal antibody
S protein
