胸膜肺炎放线杆菌抗体ApxⅣ-Dot-PPA-ELISA检测方法的建立  

Development of Dot-PPA-ELISA for detection of Actinobacillus pleuropneumoniae antibody using ApxⅣ antigen

作　　者：姜宏泽[1] 李英[1] 文心田[1] 黄小波[1] 伍锐[1] 文翼平[1] 张俊[1] 杨瑞晨 曹三杰[1] 

机构地区：[1]四川农业大学动物医学院猪病研究中心,四川成都611130

出　　处：《中国兽医科学》2015年第6期633-638,共6页Chinese Veterinary Science

基　　金：四川省科技支撑计划项目(2013NZ0056)

摘　　要：本研究构建了胸膜肺炎放线杆菌溶血外毒素ⅣA(ApxⅣA)的原核表达质粒pET-28a(+)-ApxⅣ,并在大肠杆菌BL21(DE3)中成功表达出分子质量约为48ku的ApxⅣ蛋白,经Western-blot分析表明该重组ApxⅣ蛋白具有反应原性。试验进一步用纯化的重组ApxⅣ蛋白作为抗原,建立了胸膜肺炎放线杆菌抗体的Dot-PPA-ELISA检测方法,该方法诊断膜片上点样ApxⅣ抗原质量为0.737μg,该方法检测灵敏度高,可检测到胸膜肺炎放线杆菌标准阳性血清IgG的最低含量为8.374×10-9 g;检测特异性好,不与副猪嗜血杆菌、大肠杆菌、猪链球菌2型、多杀性巴氏杆菌、日本脑炎病毒的阳性血清发生交叉反应。应用建立的诊断方法对446份临床血清样品进行检测,胸膜肺炎放线杆菌抗体的阳性检出率为25.37%。结果表明,该方法具有操作方便、灵敏、结果易于判读等优点,可应用于猪场猪传染性胸膜肺炎的血清学诊断。The recombinant expression plasmid pET-28a(+)-ApxⅣ A was constructed,and ApxⅣ A of Actinobacillus pleuropneumoniae was expressed successfully in Escherichia coli BL21(DE3).The molecular weight and reactionogenicity of the recombinant ApxⅣ were confirmed by SDS-PAGE and Western-blot analyses,respectively.A Dot-PPA-ELISA method was developed on basis of the purified recombinant protein ApxⅣ to detect antibodies against A.pleuropneumoniae in the serum.There are 0.737μg antigen of ApxⅣ fixed on each diagnostic membrane.The Dot-PPA-ELISA is sensitive and specific,with the minimum detection content of IgG is 8.374×10-9 g in positive serum and no cross reaction with positive sera against Haemophilus parasuis,E.coli,Streptococcus suis,Pasteurella multocida and Japanese encephalitis virus,respectively.A total of 446 clinical serum samples were detected by the Dot-PPA-ELISA,of which positive rate was 25.37%.Results showed that the Dot-PPA-ELISA is simple,sensitive and easy to be determined,which could be applied to serological diagnosis of A.pleuropneumoniainfection on pig farms.

关 键 词：胸膜肺炎放线杆菌 ApxⅣ 表达 DOT-PPA-ELISA 

分 类 号：S852.61[农业科学—基础兽医学]