免疫磁珠技术分离猪胸膜肺炎放线杆菌  

Isolation of Actinobacillus pleuropneumoniae by Immunomagnetic Beads Technique

作　　者：张蓉蓉[1] 梁望旺 方兵兵[1] 邵华斌 徐涤平 何启盖[1] 

机构地区：[1]华中农业大学农业微生物国家重点实验室,武汉430070 [2]湖北省动物胚胎工程及分子育种重点实验室,武汉430064

出　　处：《畜牧兽医学报》2008年第3期343-348,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技支撑计划(2006BAD06A12);湖北省科技攻关项目(2006AA20205)

摘　　要：利用表达并纯化的胸膜肺炎放线杆菌保守外膜蛋白作为抗原,免疫家兔制备抗血清,并利用纯化后的IgG包被免疫微球,确定了包被磁珠所需的IgG浓度及包被时间,建立了利用免疫磁珠技术检测胸膜肺炎放线杆菌的操作流程。该方法能检测本室保存的胸膜肺炎放线杆菌的14个标准血清型,敏感性高于直接涂布平皿法,用此方法分离送检的20个疑似菌株,分离出10个菌株,与apxⅣ-PCR检测结果相比,符合率为100%。该方法也能从人工感染猪的肺和扁桃体回收到病原。用建立的免疫磁珠技术检测了从河南、湖南和湖北等地送检的病料,成功分离了5株可疑胸膜肺炎放线杆菌,经apxⅣ-PCR鉴定为胸膜肺炎放线杆菌。上述结果表明,建立的免疫磁珠技术可用于胸膜肺炎放线杆菌的分离。The conservative outer membrane lipoprotein of Actinobacillus pleuropneumoniae （APP） was expressed, purified and used to immunize the rabbit for production of antiserum, from which IgG was purified and used to sensitize the immunomagnetic beads. The optimal conditions for coating immunomagnetic beads with IgG, including IgG concentration and time, were determined. The protocol for detection of App by coated immunomagnetic bead was developed. All of the 14 APP reference strains could be detected by the new method. The analytical sensitivity of the assay was higher than that of the traditional procedure. Ten strains of twenty suspected strains were identified as APP,and consistently confirmed by ApxⅣ-based PCR. The coincidence rate between ApxⅣ-based PCR and developed immunomagnetic beads separation was 100%. The challenged APP serotype 1 and serotype 7 could be recovered by the novel method. Finally, this method was clinically applied, and five APP strains were isolated from the samples that collected from Henan, Hunan and Hubei province. These isolates were confirmed by ApxⅣ-based PCR. The results indicated that the developed immunomagnetic bead-based method can be used to separate APP.

关 键 词：胸膜肺炎放线杆菌 免疫磁珠技术 外膜蛋白 分离 

分 类 号：S852.619[农业科学—基础兽医学]