沙门菌H:t抗原表位单克隆抗体的制备及初步应用  

Preparation and preliminary application of monoclonal antibody against Salmonella H:t antigen epitope

作　　者：尚月月 丁睿清 曹李妍 徐双媛 胡凌芸 宋丽 康喜龙[1,2,3] 焦新安 孟闯[1,2,3] 潘志明 SHANG Yueyue;DING Ruiqing;CAO Liyan;XU Shuangyuan;HU Lingyun;SONG Li;KANG Xilong;JIAO Xinan;MENG Chuang;PAN Zhiming(Jiangsu Key Laboratory of Zoonoses,Yangzhou University,Yangzhou,Jiangsu 225009,China;Jiangsu Key Laboratory of Prevention and Control of Biological Hazard Factors(Animal Origin)for Agrifood Safety and Quality,Yangzhou University,Yangzhou,Jiangsu 225009 China;Jiangsu Co-Innovation Center for Prevention and Control of Im portant Animal Infectious Diseases and Zoonoses of Universities,Yangzhou University,Yangzhou,Jiangsu 225009,China)

机构地区：[1]扬州大学,江苏省人兽共患病学重点实验室,江苏扬州225009 [2]扬州大学,农业农村部农产品质量安全生物性危害因子(动物源)控制重点实验室,江苏扬州225009 [3]扬州大学,江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医学报》2023年第7期1442-1449,共8页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32002295,31902278);江苏省重点研发计划(现代农业)资助项目(BE2021354);江苏省政策引导类计划(国际科技合作港澳台科技合作)资助项目(BZ2020013);扬州大学科技创新团队(2016)资助项目。

摘　　要：山夫顿堡沙门菌、加利福尼亚沙门菌、奥拉宁堡沙门菌等含有H:t表位的血清型沙门菌具有较广泛的流行性和致病性,研制特异性识别H:t表位的单克隆抗体可为这些血清型沙门菌的快速、高效检测提供重要生物材料。本研究选用奥拉宁堡沙门菌鞭毛蛋白作为免疫原,通过经典B细胞杂交瘤技术筛选制备特异性识别沙门菌H:t抗原表位单克隆抗体,利用间接ELISA、Western blot方法对其亚型、效价、亲和力、特异性等生物学特性进行分析。结果显示,筛选获得2株单抗并命名为4H4和5G10,其亚型均为IgG1,其中4H4单抗的细胞培养上清效价达2.0×10^(5),腹水效价达1.0×10^(7),亲和力为2.04×10^(10)mol/L。Western blot分析显示4H4单抗与含H:t抗原表位的奥拉宁堡沙门菌、山夫顿堡沙门菌、加利福尼亚沙门菌特异性反应,而与不含H:t抗原表位的沙门菌和非沙门菌不反应。将该单抗用于鸡肉模拟污染样品中沙门菌的ELISA检测,能够检测出含H:t表位的血清型沙门菌,而不含H:t表位的沙门菌和非沙门菌均为阴性。本研究制备了沙门菌H:t抗原表位特异性单抗,可应用于ELISA检测含H:t表位的沙门菌造成的食品污染,为建立特定血清型沙门菌相关免疫学检测方法提供了重要材料基础。The pathogenicity of different serotypes of Salmonella infection is quite different.Serotypes of Salmonella containing H:t epitopes,such as Salmonella Senftenberg,Salmonella California,and Salmonella Oranienburg also have widespread prevalence and pathogenicity.The development of monoclonal antibodies(MAbs)that specifically recognize the H:t epitope can provide important biological materials for the rapid and efficient detection of Salmonella serotypes containing H:t epitope.In this study,Salmonella Oranienburg flagellin was selected as the immunogen,and the MAbs that specifically recognized the H:t epitope of Salmonella was prepared by B cell hybridoma technology,and their biological characteristics such as subtype,potency,affinity and specificity were analyzed.Two MAbs named 4H4 and 5G10 were obtained with subtypes of IgG1.The cell culture supernatant and ascites titer of 4H4 was 2.0×10^(5) and 1.0×10^(7) respectively,and the affinity was 2.04×10^(10) mol/L.Specificity analysis showed that the MAb of 4H4 could reacted with Salmonella strains containing H:t epitopes including Senftenberg,California,and Oranienburg,but did not react with non-Salmonella strains and Salmonella without H:t epitopes.Then the monoclonal antibody was used for Salmonella detection in simulated contaminated chicken samples by ELISA.Salmonellaserotypes containing H:t epitope were detected,while both Salmonella without H:t epitope and non-Salmonella were negative.In this study,H:t epitope specific monoclonal antibodies were prepared,which could be used for ELISA to detect food contamination caused by Salmonella containing H:t epitopes.It provides an important material basis for the establishment of specific serotype Salmonella related immunological detection methods.

关 键 词：沙门菌 鞭毛蛋白 H:t抗原表位 单克隆抗体 ELISA检测 

分 类 号：S852.61[农业科学—基础兽医学] R535[农业科学—兽医学]