摘要
研究旨在制备沙门氏菌SifA蛋白的单克隆抗体,分析其亚类、腹水效价、亲和力和反应性,并初步应用于检测沙门氏菌感染巨噬细胞后SifA分泌的生物学特性。试验通过构建重组表达大肠杆菌BL21(DE3)/pET-28a-sifA和BL21(DE3)/pGEX-6P-1-sifA,表达和纯化得到rHis-SifA和rGST-SifA蛋白,以rHis-SifA作为免疫抗原,rGST-SifA作为检测抗原,采用B细胞杂交瘤技术制备SifA单克隆抗体,并使用鼠源亚类鉴定试剂盒确定其亚类,间接ELISA方法测定其腹水效价和亲和力,Western blot分析其反应性、与肠杆菌科其他细菌交叉反应的特异性和巨噬细胞中沙门氏菌SifA的分泌特性,评价其效果或应用潜力。结果显示:成功制备10株单克隆抗体,其中单克隆抗体10G6亚型为IgG1,其效价达1.0×10^(6)以上,亲和常数为3.66×10^(9)/M,能特异性结合rHis-SifA和rGST-SifA蛋白,Western blot结果显示该单克隆抗体能特异性识别8种不同血清型沙门氏菌,并能确定沙门氏菌感染巨噬细胞后SifA的分泌特性。研究表明,成功制备10株单克隆抗体,其中单克隆抗体10G6具有良好的特性,为沙门氏菌的研究和检测提供生物材料。
In order to prepare monoclonal antibody(McAb)against Salmonella SifA protein,its subclass,ascitic titer,affinity and reactivity were analyzed and it was applied to detect SifA secretion in Salmonella-infected macrophages.Recombinant E.coli BL21(DE3)/pET-28a-sifA and BL21(DE3)/pGEX-6P-1-sifA were constructed and expressed rHis-SifA and rGST-SifA proteins were purified,rHis-SifA was served as immunizing antigen and rGST-SifA was used as detecting antigen to prepare McAb by B-cell hybridoma technology.The subclasses of McAbs were determined by kit,ascitic titer and affinity were identified through indirect ELISA,while reactivity,cross-reactivity for specificity with other Enterobacteriaceae bacterias and the secretion of SifA in macophages were analyzed by Western blot.The results showed that 10 strains of McAbs were successly prepared,10G6 of which belonged to IgG1,its ascitic titer was over 1.0×10^(6) and an affinity constant was 3.66×10^(9)/M,and it was capable to specifically bind to rHis-SifA and rGST-SifA proteins and specifically recognize 8 serotypes of Salmonella and detect secreted SifA in Salmonella-infected macrophages.All results indicated that the 10G6 among 10 screened McAbs possessed good properties and severed as biological materials for Salmonella study and diagnostics in the future.
作者
苗苏南
何嘉文
王静
夏泽淼
廖宗杰
赵一丁
焦新安
耿士忠
MIAO Su'nan;HE Jiawen;WANG Jing;XIA Zemiao;LIAO Zongjie;ZHAO Yiding;JIAO Xin'an;GENG Shizhong(Key Laboratory of Zoonoses of Jiangsu Province,Yangzhou University,Yangzhou,Jiangsu 225009;Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses of Jiangsu Province,Yangzhou,Jiangsu 225009;Key Laboratory of Prevention and Control of Biological Hazard Factors(Animal Origin)for Agrifood Safety and Quality,Yangzhou University,Yangzhou,Jiangsu 225009;Joint International Research Laboratory of Agriculture&Agri-Product Safety,Ministry of Education,Yangzhou University,Yangzhou,Jiangsu 225009)
出处
《中国家禽》
北大核心
2025年第6期64-71,共8页
China Poultry
基金
国家重点研发项目(2016YFD0501607)
苏北科技专项(SZ-SQ2021046)。
