畜牧与兽医2026，Vol.58Issue(10)：43-49,7.
猪肺炎支原体截短融合蛋白P46-P97-P65的表达及间接ELISA抗体检测方法的建立
Expression of truncated fusion protein P46-P97-P65 from Mycoplasma hyopneumoniae and development of an indirect ELISA antibody detection method
摘要
Abstract
To establish a method for the detection of Mycoplasma pneumoniae(Mhp)antibody,the P46,P97 and P65 proteins of Mhp were selected as antigens,and the three fragments were ligated with flexible ligation peptide Linker(GGGGS)between different antigens,and the recombinant plasmid pET28-Mhp-P46-P97-P65 was constructed,which was correctly identified by double digestion and trans-formed into BL21(DE3)competent cells.Solubly expressed Mhp-P46-P97-P65 truncated fusion protein was obtained after induction of ex-pression.The purified Mhp-P46-P97-P65 truncated fusion protein was used as the encapsulated antigen,and the reaction conditions were optimized to establish a preliminary indirect ELISA antibody detection method.The optimization results showed that:the antigen encapsulation concentration of the ELISA method was 1.0 μg/mL,and the serum to be examined was diluted 1∶50;and the concentration of the sheep anti-swine IgG-HRP antibody used was 1∶1 000,with a critical value of 0.250.This method exhibited high specificity,with no cross-reactivity observed against antibodies to pathogens such as Erysipelothrix rhusiopathiae(Er),pseudorabies virus(PRV),and clas-sical swine fever virus(CSFV).The intra-and inter-batch coefficients of variation were lower than 10%,showing good reproducibility.Comparison of 217 serum samples using the method established in this study and IDEXX ELISA kit for Mhp antibody showed that the positive rate of Mhp ELISA was 47%(102/217)and the negative rate was 53%(115/217);and the positive rate of IDEXX ELISA was 54.84%(119/217)and the negative rate was 45.16%(98/217).The positive compliance rate of the two was 80.67%,the negative compliance rate was 93.88%,and the total compliance rate was 86.64%.The above results indicated that the Mhp ELISA test established here was accurate and reliable,with a promising application prospect.关键词
猪肺炎支原体/融合蛋白/P46/P97/P65/间接ELISAKey words
Mycoplasma hyopneumoniae/fusion protein/P46/P97/P65/indirect ELISA分类
农业科技引用本文复制引用
朱艳丽,葛猛,刘甜甜,何瑜,陈智,陈志雄,李欣,戴伶,张震萍,刘增再..猪肺炎支原体截短融合蛋白P46-P97-P65的表达及间接ELISA抗体检测方法的建立[J].畜牧与兽医,2026,58(10):43-49,7.基金项目
湖南省重点研发计划项目(2023NK2017) （2023NK2017）
