嗜肺军团菌重组MOMP蛋白间接ELISA方法的建立及其在血清学诊断中的应用  

An indirect ELISA using Legionella pneumophila recombinant MOMP protein and its application in serological diagnosis

作　　者：王涛[1] 张彩霞[2] 曹秀琴[3] 杨志伟[1] 

机构地区：[1]宁夏医科大学基础医学院病原生物学与免疫学系,宁夏银川750004 [2]山东省滨州医学院附属医院药学部,山东滨州256603 [3]宁夏医科大学生育力保持省部级共建教育部重点实验室

出　　处：《细胞与分子免疫学杂志》2013年第12期1322-1326,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30860264);宁夏自然科学基金(NZ0992)

摘　　要：目的表达及纯化出嗜肺军团菌主要外膜蛋白(MOMP)作为诊断抗原,研究其在嗜肺军团菌感染血清学诊断中的实用价值。方法将已成功构建的重组质粒pET-momp转化到大肠杆菌E.coli BL21感受态细胞中,诱导重组MOMP表达,运用SDS-PAGE和亲和层析法纯化。用DRG军团菌IgG、IgM、IgA的ELISA试剂盒筛选出58份阳性血清和32份阴性血清,用纯化的MOMP蛋白作为诊断抗原建立间接ELISA,同时与R&D公司军团菌的IgG、IgM、IgA ELISA试剂盒分别检测已筛选血清中的IgG、IgM、IgA抗体,然后用受试者工作特征(ROC)曲线对这2种方法进行比较,通过敏感性、特异性以及检测结果的一致性,来评价重组MOMP间接ELISA的应用价值。结果成功诱导表达并纯化出相对分子质量(M r)大约为45 000的重组MOMP蛋白。重组MOMP建立的间接ELISA分别检测筛选血清中IgG、IgM、IgA抗体与R&D公司的ELISA试剂盒进行比较,IgG抗体灵敏度90.9%,特异度91.7%,一致性Kappa值0.784(P<0.05),ROC曲线下的面积0.913;IgM抗体灵敏度91.4%,特异度90.6%,一致性Kappa值0.809(P<0.05),ROC曲线下的面积为0.910;IgA抗体灵敏度92.1%,特异度88.9%,一致性Kappa值0.793(P<0.05),ROC曲线下的面积0.905。结论成功诱导表达及纯化出重组MOMP蛋白,将其作为诊断抗原所建立的间接ELISA表现出了较好的特异性,灵敏度和一致性,为军团病的血清学诊断试剂盒的研制奠定了基础。Objective To express and purify the recombinant major outer membrane protein （MOMP） of Legionellapneumophila （Lp） as diagnostic antigen, and explore its practical value in the serological diagnosis of Lp infection. MethodsThe recombinant plasmid pET-momp was transformed into the E. coli BL21 competent cells. The recombinant MOMP wasinduced to express, and then analyzed by SDS-PAGE electrophoresis, purified by affinity chromatography. We screened andobtained 58 positive blood serum and 32 negative blood serum using the DRG （Germany, IgG/IgM/IgA） Lp kit. The bloodserum samples were detected for IgG, IgM, IgA antibody levels by indirect ELISA that we had established with the purifiedMOMP as the coating antigen, as well as by R＆D （ USA, IgG/lgM/lgA） Lp kit. Then using the receiver operatingcharacteristic （ROC） curve, we compared these two methods in the sensitivity, specificity and consistency of the testresults, for evaluating the application value of the indirect ELISA of recombinant MOMP. Results The approximately 45 000recombinant MOMP was successfully expressed and purified. Compared with the indirect ELISA we established with the R＆DLp kit for detecting Lp antibody IgG, IgM and IgA in blood serum, the sensitivity of the indirect ELISA of recombinant MOMPto IgG was 90.9%,the specificity was 91.7%,the Kappa value was 0.784 （户 〈 0_ 05）,and the area under the ROC curvewas 0.913 ; the sensitivity to IgM was 91.4% and the specificity was 90.6%,the Kappa value was 0.809 （P 〈 0.05）,and the area under the ROC curve was 0. 910; the sensitivity to IgA was 92.1% and the specificity was 88.9%, the Kappa value was 0.793 （P 〈 0.05）, and the area under the ROC curve was 0. 905. Conclusion The recombinant MOMP was successfully induced to express and purified. The indirect ELISA we established with the recombinant MOMP protein as a diagnostic antigen showed good specificity, sensitivity and consistency, which laid a foundation for the development of serological diagnosis kit of Legionn

关 键 词：嗜肺军团菌 表达 重组蛋白MOMP ELISA 

分 类 号：R392.11[医药卫生—免疫学] R446.61[医药卫生—基础医学]