禽呼肠孤病毒σA蛋白单克隆抗体的制备及夹心ELISA检测方法的建立  

Preparation of monoclonal antibody againstσA protein of avian reovirus and establishment of sandwich ELISA method for detection

作　　者：杨冰怡 谢芝勋 谢志勤 任红玉 韦悠 谢丽基 黄娇玲 王盛 YANG Bingyi;XIE Zhixun;XIE Zhiqin;REN Hongyu;WEI You;XIE Liji;HUANG Jiaoling;WANG Sheng(College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nanning530001,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]广西壮族自治区兽医研究所广西兽医生物技术重点实验室,广西南宁530001

出　　处：《中国兽医学报》2024年第7期1373-1379,共7页Chinese Journal of Veterinary Science

基　　金：广西科技重大专项资助项目(桂科AA23062050);“广西八桂学者”专项资助项目(2019A50)。

摘　　要：旨在制备禽呼肠孤病毒(ARV)σA蛋白单克隆抗体及建立一种夹心ELISA方法检测ARV病原。以原核表达ARVσA蛋白作为抗原,免疫BALB/c小鼠,筛选稳定的杂交瘤细胞株制备单克隆抗体,建立基于单克隆抗体的σA-夹心ELISA检测方法,并进行敏感性、特异性、重复性和准确性检验。结果显示,成功构建了重组质粒pET-32a-σA,并在大肠杆菌中良好表达。通过杂交瘤细胞筛选,获得了2株能稳定分泌单克隆抗体的杂交瘤细胞株,分别命名为6B3和8E11,2株杂交瘤细胞分泌的单克隆抗体均能与天然ARV反应。选择其中1株6B3分泌的单克隆抗体作为捕获抗体,ARV阳性鸡多克隆抗体作为检测抗体,通过优化反应条件,建立了检测ARV的σA-夹心ELISA方法。特异性检测显示该法只检出ARV病原,没有检出其他常见鸡病病原;敏感性试验显示其能检出7.72×10^(2)EID50/mL的ARV;重复性试验显示批内和批间试验的变异系数(Cv)均小于5.0%,重复性好。用σA-夹心ELISA方法与PCR方法同时对30份样品进行检测,两者检测结果相符。结果表明成功建立基于ARVσA蛋白单克隆抗体的夹心ELISA方法用于ARV的鉴别检测,为准确快速检测ARV提供技术手段。In order to prepare monoclonal antibody toσA protein of avian reovirus(ARV)and establish a sandwich ELISA method for the detection of ARV pathogens.In this study,theσA protein of ARV was expressed as antigen by prokaryotic expression and used to immunize BALB/c mice.Then,stable hybridoma cell lines were screened,and monoclonal antibodies were prepared.A sandwich ELISA detection method based on monoclonal antibody ofσA protein was established,and the sensitivity,specificity,repeatability,and accuracy were tested.The results showed that the recombinant plasmid pET-32a-σA was successfully constructed and well expressed in Escherichia coli.After immunizing mice,two hybridoma cell lines 6B3and 8E11,which could secrete monoclonal antibodies stably,were successfully prepared.Both monoclonal antibodies could react with natural ARV.One of the monoclonal antibodies secreted by 6B3was selected as the capture antibody and the ARV-positive chicken polyclonal antibody was used as the detection antibody.A sandwich ELISA method was established to detect ARV by optimizing the reaction conditions.The specific test showed that the method only detected ARV pathogens and no other common chicken viral pathogens were detected.The detection limit was 7.72×102 EID50/mL of ARV antigen.The coefficient of variation of the intra-and inter-assay tests were less than 5.0%and the reproducibility was good.Thirty samples were tested simultaneously byσA-sandwich ELISA and PCR,and the results were consistent with each other.In conclusion,a sandwich ELISA method based on the monoclonal antibody ofσA protein was successfully established for the identification and detection of ARV,which provided a technical means for the accurate and rapid detection of ARV.

关 键 词：禽呼肠孤病毒 σA蛋白 单克隆抗体 夹心ELISA 

分 类 号：S852.65[农业科学—基础兽医学]