猪肺炎支原体P36蛋白间接ELISA检测方法的建立  

Establishment of Indirect ELISA for Detection of Antibodies against My coplasma hyopneumoniae P36 Protein

作　　者：祝永琴[1,2] 冯志新[1] 刘茂军[1] 吴叙苏[1] 邵国青[1] 

机构地区：[1]江苏省农业科学院兽医研究所,农业部动物疫病诊断与免疫重点开放实验室,国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京农业大学,江苏南京210095

出　　处：《江苏农业学报》2010年第5期987-992,共6页Jiangsu Journal of Agricultural Sciences

基　　金：农业科技成果转化基金项目(2009GB2C100129);公益性行业(农业)科研专项(200803020);江苏省农业科技自主创新基金项目[CX(09)118]

摘　　要：将已构建的含有猪肺炎支原体P36基因的酵母表达质粒pPICZα-A-P36在酵母菌X-33里进行表达,并对表达的蛋白进行Western-blotting鉴定。将其作为包被抗原包被酶标板,用羊抗猪IgG-HRP作为酶标二抗,并分别对抗原浓度、血清稀释度、酶标二抗稀释度以及作用时间等进行了优化,最终建立了稳定的抗猪肺炎支原体IgG的间接ELISA检测方法。应用该方法检测猪鼻支原体、猪絮状支原体、猪圆环病毒阳性血清等都是阴性,说明该方法具有良好的特异性。批内重复性试验和批间重复性试验的变异系数均小于8%,说明该方法具有良好的重复性。利用该方法检测猪肺炎支原体活疫苗(168株)免疫猪血清中IgG的动态变化,结果表明,免疫猪血清中的IgG在免疫后分泌逐渐增加,直至免疫后8周,而经强毒攻毒后抗体滴度明显增高。The P36 protein of Mycoplasma hyopneumoniae（Mhp） was expressed in P.pastoris X-33 with the recombinant plasmid pPICZα-A-P36.The results of SDS-PAGE and Western blotting analysis revealed that the P36 protein which have good reacting ability was successfully secreted into the supernatant of medium.The recombinant protein was used as coating antigen for ELISA,and goat anti-pig IgG as the second antibody.The concentrations of coating antigen,the dilutions of sera samples and second antibody were optimized,as well as their working time.As a result,a stable and specific indirect ELISA for detecting anti-Mhp IgG was developed.The assay showed that there was no cross-reaction between the P36 protein and the positive sera of Mycoplasma hyorhinitis,Mycoplasma flocculare and porcine circovirus,and it was highly reproducible with a less than 8% coefficient of variation in intro-batch and inter-batch tests.With the assay,the anti-Mhp IgG secretion kinesis in the sera of pigs immunized with attenuated vaccine was observed.The result showed that the level of specific IgG continually rose until 8 weeks post-immunization,and the level of specific IgG rose significantly after challenged with the field strain of Mhp.

关 键 词：猪肺炎支原体 P36蛋白 IGG 间接ELISA 

分 类 号：S858.282.43[农业科学—临床兽医学]