嗜肺军团菌巨噬细胞感染增强蛋白的表达和纯化及其在血清学诊断中的应用  

Expression and purification of Legionella pneumophila MIP protein and its application in serological diagnosis

作　　者：刘黎[1] 曹秀琴[2] 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 [2]生育力保持省部级共建教育部重点实验室,宁夏银川750004

出　　处：《细胞与分子免疫学杂志》2013年第6期577-580,共4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：目的表达和纯化出嗜肺军团菌巨噬细胞感染增强(MIP)蛋白,研究其在军团菌肺炎血清学诊断中应用价值。方法将已构建成功的重组质粒pET-mip转化E.coli BL21感受态细胞,诱导MIP蛋白表达,运用SDS-PAGE分析和亲和层析法纯化。用DRG军团菌IgG/IgM/IgA的ELISA检测试剂盒筛出40份阳性血清和30份阴性血清,用纯化的MIP蛋白建立间接ELISA,同时与R&D的ELISA IgG、IgM、IgA试剂盒分别检测已筛选血清中的IgG、IgM、IgA抗体,然后对这两种方法进行比较,通过敏感性、特异性以及不同检测结果的一致性,来评价该方法的应用价值。结果诱导相对分子质量(Mr)大约40 000的MIP融合蛋白在E.coli BL21中表达并纯化。纯化蛋白建立的间接ELISA分别检测筛选血清中IgG、IgM、IgA抗体与国外ELISA试剂盒(R&D)进行比较,IgG抗体的灵敏度88.5%,特异度95.5%,一致性Kappa值0.846(P<0.05),ROC曲线下面积0.927。IgM抗体的灵敏度89.3%,特异度97.6%,一致性Kappa值0.88(P<0.05),ROC曲线下面积0.947。IgA抗体的灵敏度90%,特异度95.2%,一致性Kappa值0.856(P<0.05),ROC曲线下面积0.931。结论成功诱导了嗜肺军团菌MIP蛋白稳定表达并纯化,运用MIP作为包被抗原的军团菌肺炎的血清学诊断方法具有较高的诊断价值。Objective To express and purify macrophage infectivity potentiator （MIP） protein of Legionella pneumophila （ Lp）, and explore its value in the serological diagnosis of Lp. Methods The recombinant plasmid pET-mip was transformed into E. coli BL21 competent cells. The expression of MIP protein was induced, and then analyzed by SDS-PAGE electropho- resis, purified by affinity chromatography. We screened out 40 positive blood serum and 30 negative blood serum using the DRG （ Germany, IgG/IgM/IgA） Lp kit. The blood serum samples were detected for IgG, IgM, IgA antibody levels by indirect ELISA that we had established with the purified MIP protein as the coating antigen, as well as by R＆D （LISA, IgG/IgM/IgA） Lp kit. The two methods were compared in the sensitivity, specificity and consistency of the test results. Results The recombinant MIP protein was successfully expressed and purified with Mr being 40 000 in E. coli BL2I. In comparison of the indirect ELISA we developed with the R＆D Lp kit for detecting Lp antibody IgG, IgM and IgA in blood serum, the specificity of IgG was 88.5% and the sensitivity was 95.5%, the Kappa value was 0.846 （ P 〈 0.05）, the area under the ROC curve was 0.927; the specificity of IgM was 89.3% and the sensitivity was 97.6%, the Kappa value was 0.88 （ P〈0.05）, the area under the ROC curve was 0.947; the specificity of IgA was 90% and the sensitivity was 95.2%, the Kappa value was 0. 856 （ P 〈 0.05）, the area under the ROC curve was 0.931. Conclusion MIP proteins of L. pneumophila was expressed and purified successfully, and MIP protein can be used as a coating antigen in serological diagnosis of L. pneumophila.

关 键 词：嗜肺军团菌 巨噬细胞感染增强蛋白 蛋白表达 血清学诊断 

分 类 号：R392-33[医药卫生—免疫学] R517.9[医药卫生—基础医学]