鉴别猪流行性腹泻病毒变异与经典毒株的单克隆抗体的制备与鉴定  

Preparation and Identification of Monoclonal Antibodies Distinguishing Porcine epidemic diarrhea virus Variants from Classical Strains

作　　者：王亚文 赵雪[1] 李潭清[1] 米建华 苗云艳 孙立丹 张义明[1] 宋勤叶[1] WANG Ya-Wen;ZHAO Xue;LI Tan-Qing;MI Jian-Hua;MIAO Yun-Yan;SUN Li-Dan;ZHANG Yi-Ming;SONG Qin-Ye(Research Center of Veterinary Biologicals Engineering and Technology of Hebei/College of Veterinary Medicine,Hebei Agricultural University,Baoding 071000,China;Hebei Laishui County Animal Husbandry and Aquaculture Bureau,Baoding 074199,China)

机构地区：[1]河北农业大学动物医学院/河北省兽用生物制品工程技术研究中心,保定071000 [2]河北涞水县畜牧局,保定074199

出　　处：《农业生物技术学报》2020年第6期1123-1131,共9页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2016YFD0500703);国家自然基金(31772738);河北省科技计划(17226613D-2);河北省研究生创新资助项目(CXZZSS2018056)。

摘　　要：猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引起的猪流行性腹泻是危害养猪(Sus scrofa)生产的具有高度接触传染性的重要肠道传染病,血清学检测技术是临床调查PEDV感染状态及该病毒相关研究的重要手段,然而目前尚无鉴别PEDV变异毒株与经典毒株的血清学检测技术。单克隆抗体(monoclonal antibody,McAb)是血清学检测或诊断技术研究的重要实验材料,为了制备鉴别PEDV变异毒株与经典毒株的单克隆抗体,应用DNAMAN和DNAStar7.1软件比较分析了PEDV变异毒株与经典毒株的纤突(spike,S)蛋白核苷酸序列与氨基酸序列,筛选并合成了变异毒株与经典毒株S蛋白氨基酸序列之间的差异序列55IGENQGVNSTWYCAGRHPTAS75。将该多肽偶联钥孔戚血蓝蛋白(keyhole limpet haemocyanin,KLH)后免疫BALB/c小鼠(Mus musculus),利用细胞融合技术,经过3次克隆筛选和间接酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测,获得了1株分泌抗PEDV McAb的杂交瘤细胞(命名为3#),其分泌的抗体(命名为McAb 3#)类型为IgG2bκ。通过ELISA和Western blot证明,该单克隆抗体可与PEDV S蛋白特异性结合,与PEDV N蛋白不发生反应。间接免疫荧光实验(indirect immunofluorescence assay,IFA)结果显示,Vero-81细胞内的PEDV变异毒株能够与McAb 3#特异性结合。获得的杂交瘤细胞株诱生的小鼠腹水ELISA抗体效价达1∶10~6。进一步以McAb 3#为检测抗体,通过IFA检测PEDV变异毒株HBQY2016与经典弱毒株CV777分别感染的Vero-81细胞,结果只在PEDV变异毒株感染的Vero-81细胞浆内出现绿色荧光信号,表明McAb 3#能够鉴别PEDV变异毒株和经典毒株。上述实验结果表明,制备的单克隆抗体能够与PEDV变异毒株的S蛋白特异性结合,并可以鉴别PEDV变异毒株与经典毒株。该单克隆抗体的制备为PEDV的感染免疫以及PEDV变异毒株与经典毒株抗原或抗体鉴别检测方法的研究提供了重要材料基础。Porcine epidemic diarrhea(PED)caused by Porcine epidemic diarrhea virus(PEDV)is a highly contagious and devastating enteric infectious disease to pig(Sus scrofa)production.Serological techniques are absent for distinguishing PEDV variants from classical strains.Monoclonal antibody(McAb)is a kind of important and basic experimental materials for serological testing or diagnosing technique researches.In order to prepare McAb for distinguishing PEDV variants from classical strains,this study compared and analyzed the nucleotide and amino acid sequences of spike(S)protein of PEDV virulent variants and classical strains by the software of DNAMAN and DANStar7.1.The polypeptide,55IGENQGVNSTWYCAGRHPTAS75 being different between the PEDV variants and the classical ones,was screened out and synthesized.BALB/c mice(Mus musculus)were immunized 3 times at an interval of 2 weeks with the polypeptide conjugated keyhole limpet haemocyanin(KLH),and then one strain of hybridoma(named 3#)which secreted IgG2bκmonoclonal antibody(named McAb 3#)was obtained through cell fusion technique,3 rounds of clone selecting as well as enzyme-linked immunosorbent assay(ELISA)detection.The McAb could specifically react with PEDV-S protein whereas it did not combine with PEDV nucleocapsid(N)protein by ELISA and Western blot,meanwhile,PEDV variants could specifically bind to the McAb 3#in Vero-81 cells using the indirect immunofluorescence assay(IFA).The specific ELISA antibody titer reached to 1∶106 in mouse ascitic fluid induced by 3#hybridoma cells.Moreover,IFA based on the McAb 3#was performed in antigen detection of PEDV variant HBQY2016 and the classical attenuated strain CV777 in Vero-81 cells,respectively.Green fluorescence signals were observed only in the cytoplasm of Vero-81 cells infected with PEDV variant HBQY2016,but not in the cells infected with the classical attenuated strain CV777,which indicated the McAb 3#could distinguish the PEDV variant from the classical strain.The results showed that the McAb prepared in this study speci

关 键 词：猪流行性腹泻病毒(PEDV) 变异毒株 经典毒株 多肽 单克隆抗体 鉴别 

分 类 号：S855.3[农业科学—临床兽医学]