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[Expression and purification of Legionella pneumophila MIP protein and its application in serological diagnosis]
Li Liu1, Xiuqin Cao, Zhiwei Yang
1Department of Pathogenic Biology and Immunology, School of Basic Medicine, Ningxia Medical University, Ministry of Education, Yinchuan 750004, China.
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 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.
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.
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.
Insights
Legionella pneumophila macrophage infectivity potentiator (MIP) protein was successfully expressed and purified. This MIP protein shows high sensitivity and specificity, making it valuable for serological diagnosis of Legionella pneumophila infections.
Area of Science:
- Microbiology
- Immunology
- Protein Expression
Context:
- Legionella pneumophila (Lp) is a significant human pathogen.
- Accurate serological diagnostic methods for Lp are crucial for timely treatment.
- The macrophage infectivity potentiator (MIP) protein is a key virulence factor of Lp.
Purpose:
- To express and purify the recombinant MIP protein of Legionella pneumophila.
- To evaluate the diagnostic value of the purified MIP protein in serological detection of Lp.
Summary:
- Recombinant MIP protein was successfully expressed and purified from E. coli BL21.
- An indirect ELISA using MIP protein as coating antigen was developed.
- The developed ELISA demonstrated high sensitivity (95.2–97.6%) and specificity (88.5–90%) for detecting Lp-specific IgG, IgM, and IgA antibodies.
Impact:
- The study successfully produced a purified MIP protein for diagnostic use.
- The developed indirect ELISA shows potential as a reliable tool for Lp serological diagnosis.
- This advancement can improve the accuracy and efficiency of Legionella infection diagnosis.
