猪流行性腹泻病毒单克隆抗体胶体金免疫层析检测方法的建立

发布时间：2018-04-03 22:28

  本文选题：猪流行性腹泻病毒　切入点：N蛋白　出处：《河北农业大学》2015年硕士论文

【摘要】：猪流行性腹泻(Porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(PEDV)引起的以腹泻、呕吐、脱水和哺乳仔猪高致死率为主要特征的一种高度接触性肠道传染病。其临床症状与猪传染性胃肠炎、轮状病毒感染等其他肠道传染病极易混淆,制备PEDV特异性诊断试剂,并建立快速检测方法具有重要的实践意义。核蛋白(nucleoprotein,N蛋白)是PEDV的主要结构蛋白,保守性强,是病毒感染早期诊断和疫病监测的主要靶蛋白。本论文应用杂交瘤技术,制备针对N蛋白的特异性单克隆抗体(monoclonal antibody,Mc Ab),旨在以Mc Ab为材料建立检测PEDV的胶体金免疫层析检测方法,为进一步研发特异、便捷的诊断工具搭建必要的技术平台。本研究以纯化的重组N蛋白作为抗原,免疫4只雌性BALB/c小鼠,利用杂交瘤技术,经过3~5次亚克隆和筛选,获得了4株分泌抗PEDV Mc Ab的杂交瘤细胞,分别命名为A7、E10、F7、C1株杂交瘤细胞,其染色体数目在97~103之间。经亚类鉴定,4株杂交瘤细胞分泌的Mc Ab均为Ig M类,其轻链均为κ链,分别识别4个不同的抗原表位。Western blot和免疫组化鉴定显示,4株Mc Ab均能够与PEDV N蛋白,或与PEDV感染猪小肠绒毛上皮细胞内的PEDV特异结合。A7、E10、F7和C1株杂交瘤细胞的培养上清抗体效价分别为1:1600、1:100、1:100和1:100,腹水抗体效价分别为1:10~6、1:10~5、1:10~3和1:10~2。将4株杂交瘤细胞连续传代30代,具有良好的抗体分泌稳定性。应用柠檬酸三钠还原法制备粒径为20 nm的酒红色胶体金悬液,用其标记纯化的Mc Ab A7。将40μg/m L p H 8.0的胶体金标记抗体Mc Ab A7吸附于玻璃纤维素膜上,制备金标垫;以1 mg/m L的Mc Ab F7和羊抗鼠Ig M,按照5μL/cm的包被量,印迹于硝酸纤维膜,分别作为检测线与质控线,组装试纸条。该试纸条能够特异地与PEDV感染猪小肠组织中的病毒反应,在检测线与质控线处分别出现一条红色线。同时用组装的试纸条和RT-PCR检测13份来自不同猪场的腹泻仔猪小肠内容物,试纸条与RT-PCR的阳性检出率分别为38.46%(5/13)和46.15%(6/13),两者的总符合率为83.3%,表明利用抗PEDV N蛋白单克隆抗体建立的PEDV胶体金免疫层析试纸条能够快速、特异地检测PEDV,为进一步开发应用PEDV胶体金免疫层析试纸条检测试剂盒搭建了必要的技术平台。
[Abstract]:Porcine epidemic diarrhea (PED) is a highly contagious intestinal infection caused by porcine epidemic diarrhea virus (PEDVV), which is characterized by high mortality of diarrhea, vomiting, dehydration and breast-feeding piglets.Its clinical symptoms are easily confused with other enteric diseases such as porcine infectious gastroenteritis rotavirus infection and so on. It is of great practical significance to prepare PEDV specific diagnostic reagent and establish a rapid detection method.Nucleoprotein N (nucleoprotein N) is the main structural protein of PEDV, and is the main target protein for the early diagnosis of virus infection and the surveillance of epidemic disease.In this paper, a monoclonal antibody (McAb) against N protein was prepared by using hybridoma technique. The aim of this study was to establish a colloidal gold immunochromatographic assay for the detection of PEDV using McAb as a material, so as to develop a specific method for further development.Convenient diagnostic tools to build the necessary technical platform.In this study, 4 female BALB/c mice were immunized with purified recombinant N protein as antigen. Four hybridoma cells secreting anti PEDV McAb were obtained by using hybridoma technique.Its chromosome number is between 97 and 103.The McAb secreted by four hybridoma strains were identified as Ig M, and the light chain was 魏 chain, which recognized four different epitopes. Western blot and immunohistochemical analysis showed that Mc-Ab of the four strains could be associated with PEDV N protein.鎴栦笌PEDV鎰熸煋鐚�灏忚偁缁掓瘺涓婄毊缁嗚優鍐呯殑PEDV鐗瑰紓缁撳悎.A7,E10,F7鍜孋1鏍�鏉備氦鐦ょ粏鑳炵殑鍩瑰吇涓婃竻鎶椾綋鏁堜环鍒嗗埆涓�1:1600,1:100,1:100鍜，

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