动物医学进展2025，Vol.46Issue(6)：77-83,7.
猪肺炎支原体重组蛋白p46真核表达及间接ELISA抗体检测方法的建立
Eukaryotic Expression of Recombinant Protein P46 of Mycoplasma hyopneumoniae and Establishmentof Indirect ELISA Antibody Detection Method
摘要
Abstract
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 plas-mid was constructed and transfected into HEK293F cells for eukaryotic expression.The results of purification of the re-combinant 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 5 μg/mL,in-cubation 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∶10 000,incubation at 37 ℃ for 30 min,and TMB color development at room temperature for 20 min.The criterion for result determination was OD450＞0.288 7 for positive,OD450＜0.248 7 for negative and 0.248 7≤OD450 ≤0.288 7 for suspicious.Basing on Mhp p46 protein,the indirect ELISA antibody de-tection method with good sensitivity,specificity,and stability were established.The agreement rates of positive and neg-ative sera with the IDEXX indirect ELISA antibody detection kit were 93.3％and 100％,respectively,which could con-tribute to effective detection and immune evaluation of Mhp.关键词
猪肺炎支原体/p46蛋白/间接酶联免疫吸附试验Key words
Mycoplasma hyopneumoniae/p46 protein/Indirect ELISA分类
农业科技引用本文复制引用
许世萱,孙亚宁,邢云瑞,杨苏珍,郭军庆,乔松林,陈鑫鑫,张改平..猪肺炎支原体重组蛋白p46真核表达及间接ELISA抗体检测方法的建立[J].动物医学进展,2025,46(6):77-83,7.基金项目
河南省自然科学基金项目(222300420467) （222300420467）
河南省重大科技专项项目(221100110600-03) （221100110600-03）
国家生猪产业技术体系项目(CARS-35) （CARS-35）
河南省生猪产业技术体系项目(HARS-22-12-S) （HARS-22-12-S）
