牛多杀性巴氏杆菌OmpA抗原间接ELISA方法的建立  

Establishment of an Indirect ELISA for OmpA Antigen of Pasteurella multocida bovis

作　　者：王润清 孙航 孙雨[1,2] 李歌 李琦 侯晓林[2] 阚威[3,4] Wang Runqing;Sun Hang;Sun Yu;Li Ge;Li Qi;Hou Xiaolin;Kan Wei(China Animal Disease Control Center,Beijing 102600,China;College of Veterinary Medicine,Beijing University of Agriculture,Beijing 100096,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,Gansu,China;Qinghai Center for Animal Disease Prevention and Control,Xining 810000,Qinghai,China)

机构地区：[1]中国动物疫病预防控制中心,北京102600 [2]北京农学院动物医学院,北京100096 [3]甘肃农业大学动物医学院,甘肃兰州730070 [4]青海省动物疫病预防控制中心,青海西宁810000

出　　处：《中国动物检疫》2025年第2期87-94,共8页China Animal Health Inspection

基　　金：国家现代农业产业技术体系建设专项(CARS39)。

摘　　要：为建立一种快速检测牛多杀性巴氏杆菌的方法,分析多杀性巴氏杆菌OmpA序列信号肽与跨膜疏水区序列,对选择的目的基因片段进行稀有密码子优化,将化学合成后的核苷酸序列连接至pET32a载体上,构建相应的重组质粒pET32a-OmpA;将重组质粒转化至大肠杆菌BL21(DE3)感受态细胞株中,经扩大培养成功诱导表达出了纯度较高的包涵体OmpA蛋白,将OmpA重组蛋白作为诊断抗原,经反应条件优化建立了间接ELISA抗体检测方法。经试验验证,该方法对其他相关的牛类病原无交叉反应,阳性血清稀释度为1:256时仍呈阳性,组内与组间变异系数均低于10%。结果表明,该方法特异性与敏感性较好,且具有良好的重复性与稳定性。本研究为进一步开发成熟的牛多杀性巴氏杆菌抗体检测试剂盒奠定基础。In order to develop a method for rapid detection of Pasteurella multocida bovis,the sequences of signal peptide and the transmembrane hydrophobic region of the OmpA gene were analyzed,the selected target gene fragment was optimized for rare codon,and the chemically synthesized nucleotide sequence was cloned into the pET32a vector to construct corresponding recombinant plasmid pET32a-OmpA;the recombinant plasmid was transformed into E.coli BL21(DE3)cells,and the inclusion-body protein OmpA with a high purity was successfully induced and expressed by expanded culture,then an indirect ELISA was established through optimising reaction conditions using the recombinant OmpA protein as a diagnostic antigen.It was validated that the method failed to react with other related bovine pathogens,the positive serum remained the same at a dilution of 1:256,and its intra-and intergroup coefficients of variation were less than 10%.In conclusion,the method was with good specificity,sensitivity,reproducibility and stability.A foundation was laid for further development of a mature test kit for antibodies against Pasteurella multocida bovis.

关 键 词：牛多杀性巴氏杆菌 OmpA蛋白 原核表达 蛋白纯化 ELISA 

分 类 号：S851.3[农业科学—预防兽医学]