Immunoproteomic identification of MbovP579, a promising diagnostic biomarker for serological detection of Mycoplasma bovis infection

• Published in: Oncotarget Vol. 7; no. 26; pp. 39376 - 39395
• Main Authors: Khan, Farhan Anwar, Faisal, Muhammad, Chao, Jin, Liu, Kai, Chen, Xi, Zhao, Gang, Menghwar, Harish, Zhang, Hui, Zhu, Xifang, Rasheed, Muhammad Asif, He, Chenfei, Hu, Changmin, Chen, Yingyu, Baranowski, Eric, Chen, Huanchun, Guo, Aizhen
• Format: Journal Article
• Language: English
• Published: United States Impact journals 28.06.2016; Impact Journals LLC
• Subjects: Animals; Antibodies, Bacterial - blood; Antibodies, Monoclonal - chemistry; Antigens, Bacterial - immunology; Biomarkers - metabolism; Cattle; Computational Biology; Electrophoresis, Gel, Two-Dimensional; Enzyme-Linked Immunosorbent Assay; Female; Life Sciences; Lipoproteins - chemistry; Mice; Mice, Inbred BALB C; Mycoplasma agalactiae; Mycoplasma bovis; Mycoplasma Infections - blood; Mycoplasma Infections - diagnosis; Mycoplasma Infections - microbiology; Proteomics - methods; Recombinant Proteins - immunology; Research Paper: Immunology; ROC Curve; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; immunoproteomics; Immune response; Immunology and Microbiology Section; immunoinformatics; Immunity; Mycoplasma bovis; diagnostic biomarker; ELISA; mycoplasma bovis
• ISSN: 1949-2553; 1949-2553
• DOI: 10.18632/oncotarget.9799

A lack of knowledge regarding the antigenic properties of Mycoplasma bovis proteins prevents the effective control of bovine infections using immunological approaches. In this study, we detected and characterized a specific and sensitive M. bovis diagnostic biomarker. After M. bovis total proteins and membrane fractions were separated with two dimensional gel electrophoresis, proteins reacting with antiserawere detected using MALDI-TOF MS. Thirty-nine proteins were identified, 32 of which were previously unreported. Among them, immunoinformatics predicted eight antigens, encoded by Mbov_0106, 0116, 0126, 0212, 0275, 0579, 0739, and 0789, to have high immunological value. These genes were expressed in E. coli after mutagenesis of UGA to UGG using overlap extension PCR. A lipoprotein, MbovP579, encoded by a functionally unknown gene, was a sensitive and specific antigen for detection of antibodies in sera from both M. bovis-infected and vaccinated cattle. The specificity of MbovP579 was confirmed by its lack of cross-reactivity with other mycoplasmas, including Mycoplasma agalactiae. An iELISA based on rMbovP579 detected seroconversion 7 days post-infection (dpi). The ELISA had sensitivity of 90.2% (95% CI: 83.7%, 94.3%) and a specificity of 97.8% (95% CI: 88.7%, 99.6%) with clinical samples. Additional comparative studies showed that both diagnostic and analytic sensitivities of the ELISA were higher than those of a commercially available kit (p<0.01). We have thus detected and characterized the novel antigen, MbovP579, and established an rMbovP579-based ELISA as a highly sensitive and specific method for the early diagnosis of M. bovis infection.