摘要
为了建立一种猪肺炎支原体抗体间接ELISA检测方法,构建pCAGGS-P46真核表达载体,转染至HEK293F细胞中进行真核表达,纯化后采用SDS-PAGE鉴定蛋白纯度及分子质量大小,经Western blot检测重组p46蛋白特异性和反应原性。以重组蛋白p46为包被抗原,优化反应条件,确定阴性、阳性临界值,建立猪肺炎支原体抗体间接ELISA检测方法,并对方法的敏感性、稳定性、特异性进行评价。结果显示,p46重组蛋白分子质量大小为48.0 ku,纯度在90%以上,特异性和反应性良好。建立的间接ELISA检测方法最优检测条件为:抗原包被浓度为5μg/mL,采用本实验室封闭液37℃封闭120 min,待检血清1∶50稀释,37℃孵育30 min,二抗稀释度为1∶10000,37℃孵育30 min,TMB室温显色20 min。结果判定标准为:OD 450>0.2887为阳性,OD 450<0.2487为阴性,0.2487≤OD 450≤0.2887为可疑。建立的基于猪肺炎支原体p46蛋白的猪肺炎支原体抗体间接ELISA检测方法具有良好的敏感性、特异性及稳定性,与IDEXX的间接ELISA检测试剂盒检测的阴性、阳性血清符合率分别为100%、93.3%,研究结果为猪肺炎支原体抗体检测和免疫评价提供了技术支持。
To establish an indirect ELISA method for detecting antibodies against Mycoplasma hyopneumoniae and provide a method for assessing Mhp infection and the efficacy of vaccines,the pCAGGS-p46 eukaryotic expression plasmid was constructed and transfected into HEK293F cells for eukaryotic expression.The results of purification of the recombinant P46 protein were verified by SDS-PAGE.The specificity and reactivity of the recombinant p46 protein were detected by Western blotting.For the establishment of indirect ELISA antibody detection method,the recombinant p46 protein was coated,and the optimization of reaction conditions and determination of the positive and negative cut-off values were performed.The sensitivity,stability,and specificity of this new method were detected.The results showed that high purity recombinant p46 protein was purified with the molecular weight of approximately 48.0 ku,which has good specificity and reactivity.The optimized conditions were as follows:antigen coating concentration of 5μg/mL,incubation with blocking solution from this laboratory at 37℃for 120 min,serum dilution at 1∶50,incubation at 37℃for 30 min,secondary antibody dilution at 1∶10000,incubation at 37℃for 30 min,and TMB color development at room temperature for 20 min.The criterion for result determination was OD 450>0.2887 for positive,OD 450<0.2487 for negative and 0.2487≤OD 450≤0.2887 for suspicious.Basing on Mhp p46 protein,the indirect ELISA antibody detection method with good sensitivity,specificity,and stability were established.The agreement rates of positive and negative sera with the IDEXX indirect ELISA antibody detection kit were 93.3%and 100%,respectively,which could contribute to effective detection and immune evaluation of Mhp.
作者
许世萱
孙亚宁
邢云瑞
杨苏珍
郭军庆
乔松林
陈鑫鑫
张改平
XU Shi-xuan;SUN Ya-ning;XING Yun-rui;YANG Su-zhen;GUO Jun-qing;QIAO Song-lin;CHEN Xin-xin;ZHANG Gai-ping(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China;Institute for Animal Health,Henan Academy of Agricultural Sciences,Zhengzhou,Henan,450003,China)
出处
《动物医学进展》
北大核心
2025年第6期77-83,共7页
Progress In Veterinary Medicine
基金
河南省自然科学基金项目(222300420467)
河南省重大科技专项项目(221100110600-03)
国家生猪产业技术体系项目(CARS-35)
河南省生猪产业技术体系项目(HARS-22-12-S)。
