绵羊肺炎支原体EF-Tu蛋白原核表达及ELISA方法建立  

Development of an Indirect ELISA Method for Antibody Detection of Mycoplasma ovnipneumoniae Based on EF-Tu Protein

作　　者：陈思宇 张梦洁 田睿 陈益 刘镝钺 李文良[2] 毛立[2] 程子龙 杨蕾蕾[2] 孙敏 张纹纹[2] 杜改梅[3] 储岳峰 王金泉[1] 刘茂军[1,2,4,5] CHEN Siyu;ZHANG Mengjie;TIAN Rui;CHEN Yi;LIU Diyue;LI Wenliang;MAO Li;CHENG Zilong;YANG Leilei;SUN Min;ZHANG Wenwen;DU Gaimei;CHU Yuefeng;WANG Jinquan;LIU Maojun(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture and Rural Affairs,Nanjing 210014,China;College of Animal Science and food Engineering,Jinling Institute of Technology,Nanjing 211169,China;State Key Laboratory for Animal Disease Control and Prevention,College of Veterinary Medicine,Lanzhou University,Lanzhou Veterinary Research Institute,CAAS,Lanzhou 730000,China;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou,225300,China)

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052 [2]江苏省农业科学院兽医研究所农业农村部兽用生物制品工程技术重点实验室,南京210014 [3]金陵科技学院动物科学与食品工程学院,南京211169 [4]中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室,兰州730000 [5]兽用生物制品(泰州)国泰技术创新中心,泰州225300

出　　处：《中国动物传染病学报》2025年第1期97-105,共9页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金项目(32202809);动物疫病防控全国重点实验室开放基金项目(SKLVEB-KFKT-04)。

摘　　要：绵羊肺炎支原体(Mo)是引起规模化羊场呼吸道病的主要病原,危害严重;快速特异诊断是防控该病的保障。本文旨在建立一种检测Mo血清抗体的间接ELISA方法,利用大肠杆菌的密码子偏好性优化Mo EF-Tu基因序列进行原核表达,纯化出高纯度重组rEF-Tu蛋白,以纯化的rEF-Tu为包被抗原,建立Mo间接ELISA抗体检测方法。结果显示,原核表达的EF-Tu蛋白分子质量约为45.4 kDa,与预期相符;Western blot证实具有良好的反应原性;建立的间接ELISA方法最佳优化条件显示,包被抗原浓度为1.25 mg/L,37℃孵育2 h;封闭条件为30 g/L BSA,37℃孵育1 h;待检血清1∶100稀释,37℃孵育45 min;酶标二抗1∶20000稀释,37℃孵育30 min;底物TMB 37℃避光反应15 min;样品OD450值≥0.296时判定为阳性,OD450值≤0.265时判定为阴性,OD 450值0.265~0.296则判为可疑;组内和组间变异系数均低于10%;用该方法与实验室所建Mo全菌蛋白间接ELISA检测方法对233份血清进行检测,两者特异性为90.70%,敏感性为82.69%,符合率约为87.12%。上述结果表明,本研究建立的间接ELISA方法具有良好的敏感性、重复性及特异性,为Mo的临床诊断、抗体监测等提供简单快速的血清学诊断方法,也为Mo抗体检测试剂盒的开发奠定了基础。Mycoplasma ovipneumoniae(Mo)is the main pathogen causing harmful respiratory diseases on large-scale sheep farms.Rapid and specific diagnosis methods are the guarantee for preventing and controlling the disease.The aim of this study was to develop an indirect ELISA method for detecting serum antibodies to Mo.In this study,the gene sequence of Mo EF-Tu was optimized and synthetized for prokaryotic expression in E.coli and the recombinant EF-Tu protein was purified and used as the coating antigen for development of an indirect ELISA method.The results showed that the recombinant EF-Tu protein had molecular weight of 45.4 kDa and good reactivity in Western blot.The optimal reaction conditions included the coating antigen at 1.25 mg/L,incubation condition at 37℃for 2 h,blocking with 30 g/L BSA PBS at 37℃for 1 h,serum dilution at 1:100 and incubation at 37℃for 45 min,dilution of the HRP conjugated rabbit anti-goat IgG at 1:20000 and incubation at 37℃for 30 min,and substrate color development at 37℃for 15 min in dark.The criterion for judgement of a serum sample included the positive reaction at more than or equal to 0.296 OD450,negative at less than or equal to 0.265 OD450,and suspicious between these OD450 values.Intra-group and inter-group coefficients of variation were below 10%.Finally,233 serum samples were using this method and compared with the indirect ELISA method based on Mo whole bacterial proteins developed in our laboratory.The results showed that these two methods had specificity at 90.70%,sensitivity at 82.69%and coincidence at about 87.12%.The above results showed that the indirect ELISA method developed in this study had good sensitivity,reproducibility and specificity,which could be used for simple and rapid serological diagnosis for Mo disease and antibody monitoring.

关 键 词：绵羊肺炎支原体 EF-Tu蛋白 原核表达 间接ELISA 

分 类 号：S852.62[农业科学—基础兽医学]