抗猪流行性腹泻病毒单克隆抗体的制备与鉴定

发布时间：2018-06-30 02:41

本文选题：猪流行性腹泻病毒 + 单克隆抗体　；参考：《河北农业大学学报》2017年04期

【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。
[Abstract]:In order to prepare monoclonal antibody (MAB) against porcine epidemic diarrhea virus (PEDV), the proliferating PEDV in Vero cells was purified by ultracentrifugation. Using routine lymphocyte hybridoma technique and indirect Elisa based on PEDV recombinant S protein, hybridoma cells secreting specific antibodies to PEDV were prepared and screened, and indirect ELISAV Western blot and indirect immunofluorescence (IFA) tests were used, respectively. The subtype and specificity of monoclonal antibody were identified. The results showed that a hybridoma cell line 5C3. MAb which stably secreted PEDV specific antibody was successfully identified as IgM and light chain as k type. The antibody titers of supernatant of cell culture and ascites of mice were stable at 1: 600 and 1:10 000, respectively. The results of specific test showed that MAB did not cross react with porcine transmissible gastroenteritis virus (TGEV), porcine pseudorabies virus (PRV) and porcine circovirus (PCV2). PEDV-infected vero cells produced specific fluorescence. The results showed that MAB could specifically recognize recombinant and natural PEDV S protein, and had good antigenicity and specificity.
【作者单位】：河北农业大学动物医学院;河北省动物疫病预防控制中心;
【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
【分类号】：S852.651

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