鸭疫里默氏杆菌Porin蛋白的原核表达及其间接ELISA检测方法的建立与应用  

Prokaryotic expression of Porin protein of Riemerella anatipestifer and establishment and application of ELISA detection method

作　　者：包涛涛 鲜思美[1,3] 顾庆林 杨倩 梁倩 杨先富 吴通奎 王正文 廖飞 BAO Taotao;XIAN Simeil;GU Qinglin;YANG Qian;LIANG Qian;YANG Xianfu;WU Tongkui;WANG Zhengwen;LIAO Fei(College of Animal Science,Guizhou Universily,Gruiyang 550025,China;Agricultural and Rural Bureau of Qiandongnan Prefecture,Qiandongnan,Guizhou 556000,China;Guizhou Institute of Animal Diseases,Guiyang 550025,China;Guizhou Vocational College of Agriculture,Guiyang 550000,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省黔东南州农业农村局,贵州黔东南56000 [3]贵州省动物疫病研究所,贵州贵阳550025 [4]贵州农业职业学院,贵州贵阳550000

出　　处：《中国兽医学报》2022年第9期1830-1837,共8页Chinese Journal of Veterinary Science

基　　金：贵州省农业攻关资助项目(黔科合支撑[2016]2506号)。

摘　　要：旨在建立一种新型可适用于鸭疫里默氏杆菌(RA)病抗体检测的间接ELISA方法。以血清2型RA贵州分离株为研究对象,将其微孔蛋白Proin编码的基因克隆至pET-32a载体,构建重组表达质粒pET-32a-porin,在E.coli BL21(DE3)中使用IPTG诱导后表达。以纯化后的Porin蛋白作为包被抗原,通过一系列条件的优化建立一种检测RA抗体的间接ELISA方法,对其性能进行综合评价。结果显示,该方法具有较好的敏感性、重复性及特异性,阳性血清经1∶6 400稀释后检测结果仍为阳性,批内变异系数(CV)在1.27%~4.90%之间,批间CV在2.59%~5.43%之间,该方法可特异性地检测出抗RA抗体,与禽流感病毒(AIV)H5亚型、AIV H7亚型、新城疫病毒(NDV)、鸭圆环病毒(DuCV)、鸭坦布苏病毒(DTMUV)及E.coli阳性血清均无交叉反应,与商品化ELISA抗体试剂盒检测相比,两者符合率为97.26%。使用建立的ELISA方法对临床采集的801份样品血清开展抗体检测分析(626份为已免疫鸭传染性浆膜炎二价灭活疫苗“血清1型+血清2型”的样品血清,175份为未免疫任何鸭传染性浆膜炎疫苗的样品血清),有564份为免疫抗体阳性,检出阳性率为90.01%(564/626),有36份为感染抗体阳性,检出阳性率为20.99%(36/175)。结果表明,本研究建立了一种新型可用于RA抗体检测的间接ELISA方法,为RA的血清学流行病学调查提供了新的检测方法。The aim of this study was to establish a new type of indirect ELISA method suitable for the detection of antibodies against Riemerella anatipestifer(RA) disease.Taking the Guizhou isolate of RA serotype 2 as the research object, the gene encoding Porin was cloned into the pET-32 a vector, and the recombinant expression plasmid pET-32 a-porin was constructed, then the Proin was expressed in E.coli BL21(DE3) after induction with IPTG.Using the purified Porin protein as the coating antigen, an indirect ELISA method for detecting RA antibodies was established by optimizing a series of conditions, and its performance was comprehensively evaluated.The results showed that the method had good sensitivity, reproducibility and specificity.After the positive serum was diluted by 1∶6 400,the test result was still positive, and the intra-assay coefficient of variation was between 1.27% and 4.90%.The coefficient of variation was between 2.59% and5.43%.This method could specifically detect antibodies against RA and there was no cross reaction with AIV H5subtype,AIV H7subtype,NDV,DuCV,DTMUV and E.coli.Compared with the commercial ELISA antibody kit,the coincidence rate of the two methods was 97.26%.The established ELISA method was used to carry out antibody detection and analysis on 801samples of sera collected clinically(626samples were the sera samples of ducks immunized with the duck infectious serositis bivalent inactivated vaccine“serotype 1+serotype 2”,and 175samples were collected from ducks which were not immunized with the duck infectious serositis vaccine),564samples were immuno-antibody positive,and the detection rate was 90.01%(564/626),36samples were infection antibody-positive,and the detection rate was 20.99%(36/175).The results show that this study has established a new indirect ELISA method that can be used for the detection of RA antibodies.The establishment of this method provides a new detection method for the serological epidemiological investigation of RA.

关 键 词：鸭疫里默氏杆菌 Porin蛋白 ELISA 

分 类 号：S852.61[农业科学—基础兽医学]