中国兽医科学2025，Vol.55Issue(11)：1479-1486,8.DOI:10.16656/j.issn.1673-4696.2025.0204
基于山羊支原体山羊肺炎亚种HP65蛋白的间接ELISA抗体检测方法的建立与应用
Establishment and application of an indirect ELISA antibody detection method based on HP65 protein of Mycoplasma capricolum subsp.capripneumoniae
摘要
Abstract
To establish an antibody detection method for Mycoplasma capricolum subsp.capripneumoniae(Mccp),a potential diagnostic target-the hypothetical membrane protein HP65 was screened.The purified rHP65 protein was used as the coating antigen,and an indirect ELISA antibody detection method was established through conditions optimization.Its specificity,sensitivity,repeatability and application were evaluated and applied clinically.The results showed that the size of rHP65 protein was approximately 72 kDa.Western-blot analysis demonstrated that it showed good reactivity with positive serum of Mccp.The optimal conditions for the established ELISA method were as follows:coating the antigen of 180 ng/well at 37 ℃for 2 h and then overnight at 4 ℃,blocking with 10 g/L BSA at 37 ℃for 90 min,diluting the sera at 1∶200 and incubated at 37 ℃for 1 h,and diluting the enzyme-labeled secondary antibody at 1∶10 000 and incubated at 37 ℃ for 1 h.The substrate was incubated at 37 ℃ for 10 min.There was no cross-reactivity with the positive sera of Mycoplasma ovipneumoniae,Mycoplasma capricolum subsp.capricolum,Mycoplasma mycoides subsp.capricolum,peste des petits ruminants virus,and Chlamydia pecorum,indi-cating good specificity,and the sensitivity reached 1∶10 240.The intra-and inter-batch coefficients of variation were both less than 10％.The method was used to detect the sera of Mccp-immunized,infected and clinical animals.Serum antibodies could be detected 7 days after vaccination and positive sera could be detected 7 to 14 days after artificial infection.Among 187 clinical sera,102 were positive and 85 were negative.These results indicate that the established ELISA method based on the HP65 protein could be used for the detection of antibodies in contagious caprine pleuropneumonia vaccine immunization and laid a foundation for the development of clinical diagnostic methods for this disease.关键词
山羊支原体山羊肺炎亚种/山羊传染性胸膜肺炎/HP65蛋白/原核表达/间接ELISAKey words
Mycoplasma capricolum subsp.capripneumoniae/contagious caprine pleuropneumonia/HP65 protein/prokaryotic expression/indirect ELISA分类
农业科技引用本文复制引用
张一帆,郝华芳,陈胜利,金祥瑞,金珊宇,梁晋嘉,兰仕梅,李章程,储岳峰,史耀旭..基于山羊支原体山羊肺炎亚种HP65蛋白的间接ELISA抗体检测方法的建立与应用[J].中国兽医科学,2025,55(11):1479-1486,8.基金项目
甘肃省科技重大专项(23ZDNA007) （23ZDNA007）
甘肃省2024年度重点人才项目(2024RCXM36) （2024RCXM36）
中国农业科学院创新工程项目(CAAS-ASTIP-2021-LVRI) （CAAS-ASTIP-2021-LVRI）
