猪传染性胃肠炎病毒单克隆抗体的制备及其在检测组织中病毒的初步应用  

Development of monoclonal antibodies against transmissible gastroenteritis virus in pigs and its preliminary application in virus detection in tissues

作　　者：徐明丽 师一鸣 张柳 王晓娜 周晗[1,2] 单智夫 崔文[1,2] 姜艳平 王丽[1,2] 乔薪瑗 李一经[1,2] 唐丽杰 XU Mingli;SHI Yiming;ZHANG Liu;WANG Xiaona;ZHOU Han;SHAN Zhifu;CUI Wen;JIANG Yanping;WANG Li;QIAO Xinyuan;LI Yijing;TANG Lijie(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;Heilongjiang Province Key Laboratory of Technology and Preparations Creation for Animal Disease Control and Prevention,Harbin 150030,China)

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]黑龙江省动物疾病防控技术与制剂创制重点实验室,黑龙江哈尔滨150030

出　　处：《畜牧与兽医》2022年第10期74-80,共7页Animal Husbandry & Veterinary Medicine

基　　金：“十三五”国家重点研发计划(2017YFD0501100)。

摘　　要：旨在建立一种在感染初期对猪传染性胃肠炎(TGE)进行快速准确诊断的方法。本试验使用猪传染性胃肠炎病毒(TGEV)经蔗糖梯度离心后用于免疫BALB/c小鼠制备单克隆抗体,经4次亚克隆及Western blot、间接免疫荧光(IFA)鉴定后筛选出1株能够稳定分泌抗体的杂交瘤细胞株3G11。通过试剂盒将3G11单抗与辣根过氧化物酶(HRP)偶联,然后以HRP-3G11作为一抗,利用免疫酶组织化学法检测小肠组织病料中的TGEV。结果表明:HRP-3G11作为一抗经镜检可见特异性红棕色沉淀,即HRP-3G11单抗可以与小肠组织中病毒发生特异结合,试验过程耗时少且可视化显色结果优于未标记一抗使用酶标二抗组,证实了标记后单抗HRP-3G11可以作为直接检测抗体应用于免疫酶组织化学法中检测用小肠组织中的TGEV。The transmissible gastroenteritis virus of swine(TGEV)is a highly contagious gastrointestinal disease.Infected piglets mainly suffer from diarrhea and vomiting,with a mortality rate of more than 70%,causing severe economic losses to the pig breeding industry.Therefore,rapid and accurate diagnosis of TGE in the early stage of infection is crucial.In this study,TGEV was used to immunize BALB/C mice after sucrose gradient centrifugation to prepare monoclonal antibodies.Hybridma cell line 3G11 with stable antibody secretion was obtained after 4 sub-clonal screening and identification by western blot and IFA.After coupling the 3G11 monoclonal antibody with HRP through the kit,based on HRP-3G11 as a primary antibody,the TGEV in the small intestine tissues was detected by immunohistochemistry.The results showed that the specific red-brown precipitate of the HRP-3G11 monoclonal antibody reacting with the TGEV antigen was observed by microscopic examination using HRP-3G11 as a primary antibody.That is,the HRP-3G11 monoclonal antibody could bind to TGEV in the small intestinal tissues specifically.The experimental process took less time and the visual color rendering result was better than that of the unlabeled 3G11 and a secondary antibody group,which confirmed that the labeled monoclonal antibody HRP-3G11 could be used as a direct detection antibody for the detection of TGEV in small intestine tissues by immunohistochemistry.

关 键 词：猪传染性胃肠炎病毒 单克隆抗体 免疫酶组织化学法 

分 类 号：S852.6[农业科学—基础兽医学]