基于绵羊肺炎支原体EF-Tu蛋白间接ELISA方法的建立  

Establishment of an Indirect ELISA Method Based on Mycoplasma Ovipneumoniae Elongation Factor Tu Protein

作　　者：张洁[1] 曹军军 祝明松 杨亚军 王小凤 王璐 石峰[1] ZHANG Jie;CAO Jun-jun;ZHU Ming-song;YANG Ya-jun;WANG Xiao-feng;WANG Lu;SHI Feng(College of Life Sciences,Shihezi University,Shihezi,Xinjiang,832000,China;College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang,832000,China;Hangzhou Beken Leber Biotechnology Co.,Ltd,Hangzhou,Zhejiang,310000,China)

机构地区：[1]石河子大学生命科学学院,新疆石河子832000 [2]石河子大学动物科技学院,新疆石河子832000 [3]杭州毕肯莱博生物技术有限公司,浙江杭州310000

出　　处：《动物医学进展》2019年第11期6-13,共8页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31360226)

摘　　要：为解决绵羊肺炎支原体感染的预防问题,建立一种简单、快速、成本低的诊断方法,构建绵羊肺炎支原体EF-Tu基因原核表达载体,诱导纯化重组蛋白并用Western blot分析其反应原性,以重组蛋白为诊断抗原,建立绵羊肺炎支原体间接ELISA检测方法,评估其特异性和灵敏性。结果表明,构建的重组蛋白纯化后,经Western blot证实具有良好的反应原性;间接ELISA反应条件优化显示,抗原稀释度为0.25μg/mL,一抗最佳稀释浓度为1∶50,最佳孵育时间为90 min,封闭液为50 g/L脱脂奶粉,二抗最佳稀释倍数为1∶8 000,最佳孵育时间为60 min,特异性与灵敏性良好;该方法与间接血凝法对100份血清进行检测,两者符合率为88.7%。说明试验建立的绵羊肺炎支原体间接ELISA检测方法可以推广使用。In order to solve the problem of prevention of Mycoplasma ovipneumoniae infection,we explored a simpler,faster and less costly diagnostic method.The prokaryotic expression vector of the elongation factor Tu gene of Mycoplasma ovipneumoniae was induced,the recombinant protein was purified,and its reactogenicity was analyzed by Western blot.Using recombinant protein as a diagnostic antigen,an indirect ELISA method for detection of Mycoplasma ovipneumoniae was established to evaluate its specificity and sensitivity.The results showed that the purity of the constructed recombinant protein was confirmed by Western blot.The indirect ELISA reaction conditions showed that the antigen dilution was 0.25μg/mL,and the optimal dilution of the primary antibody was 1∶50.The incubation time is 90 min,the blocking solution is 5%skim milk powder,the optimal dilution ratio of the second antibody is 1∶8 000,and the optimal incubation time is 60 min.The specificity and sensitivity are good;the method and indirect hemagglutination method were tested using 100 sera,and the coincidence rate was 88.7%.It indicated that the indirect ELISA for Mycoplasma pneumoniae established by the test can be widely used.

关 键 词：绵羊肺炎支原体 原核表达 EF-Tu蛋白 Western BLOT 间接ELISA 

分 类 号：S852.62[农业科学—基础兽医学]