Liu Li, Cao Xiuqin, Yang Zhiwei
Department of Pathogenic Biology and Immunology, School of Basic Medicine, Ningxia Medical University, Ministry of Education, Yinchuan 750004, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Jun;29(6):577-80.
To express and purify macrophage infectivity potentiator (MIP) protein of Legionella pneumophila(Lp), and explore its value in the serological diagnosis of Lp.
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 electrophoresis, 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 (USA, IgG/IgM/IgA) Lp kit. The two methods were compared in the sensitivity, specificity and consistency of the test results.
The recombinant MIP protein was successfully expressed and purified with Mr; being 40 000 in E.coli BL21. 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.
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.
表达并纯化嗜肺军团菌（Lp）的巨噬细胞感染增强因子（MIP）蛋白，探讨其在Lp血清学诊断中的价值。
将重组质粒pET-mip转化至大肠杆菌BL21感受态细胞中。诱导MIP蛋白表达，然后通过SDS-PAGE电泳进行分析，采用亲和层析法进行纯化。使用德国DRG公司的Lp试剂盒筛选出40份阳性血清和30份阴性血清。以纯化的MIP蛋白为包被抗原，通过我们建立的间接ELISA法以及美国R&D公司的Lp试剂盒检测血清样本中的IgG、IgM、IgA抗体水平。比较两种方法检测结果的敏感性、特异性和一致性。
重组MIP蛋白在大肠杆菌BL21中成功表达并纯化，其相对分子质量为40 000。将我们建立的间接ELISA法与R&D公司的Lp试剂盒用于检测血清中Lp抗体IgG、IgM和IgA相比较，IgG的特异性为88.5%，敏感性为95.5%，Kappa值为0.846（P<0.05），ROC曲线下面积为0.927；IgM的特异性为89.3%，敏感性为97.6%，Kappa值为0.88（P<0.05），ROC曲线下面积为0.947；IgA的特异性为90%，敏感性为95.2%，Kappa值为0.856（P<0.05），ROC曲线下面积为0.931。
成功表达并纯化了嗜肺军团菌的MIP蛋白，MIP蛋白可作为嗜肺军团菌血清学诊断的包被抗原。
