Oncotarget, Vol. 7, No. 26
www.impactjournals.com/oncotarget/
Research Paper: Immunology
Immunoproteomic identification of MbovP579, a promising diagnostic biomarker for serological detection of Mycoplasma bovis infection Farhan Anwar Khan1,2,3,*, Muhammad Faisal1,2,*, Jin Chao1,2, Kai Liu1,2, Xi Chen2, Gang Zhao1,2, Harish Menghwar1,2, Hui Zhang1,2, Xifang Zhu1,2, Muhammad Asif Rasheed1,2, Chenfei He1,2, Changmin Hu2, Yingyu Chen1,4, Eric Baranowski6,7, Huanchun Chen1,2,4 and Aizhen Guo1,2,4,5 1
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China
2
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
3
Department of Animal Health, The University of Agriculture, Peshawar, Pakistan
4
Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, International Joint Research and Training Centre for Veterinary Epidemiology, Huazhong Agricultural University, Wuhan, China 5
International Joint Research Centre for Veterinary Epidemiology, Huazhong Agricultural University, Wuhan, China
6
INRA, UMR 1225, IHAP, Toulouse, France
7
INP-ENVT, UMR 1225, IHAP, Université de Toulouse, Toulouse, France
*
These authors have contributed equally to this work
Correspondence to: Aizhen Guo, email: [email protected] Keywords: diagnostic biomarker, ELISA, immunoproteomics, immunoinformatics, Mycoplasma bovis, Immunology and Microbiology Section, Immune response, Immunity Received: April 27, 2016
Accepted: May 23, 2016
Published: June 02, 2016
ABSTRACT 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 antisera were 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
www.impactjournals.com/oncotarget/
Research Paper: Immunology
Immunoproteomic identification of MbovP579, a promising diagnostic biomarker for serological detection of Mycoplasma bovis infection Farhan Anwar Khan1,2,3,*, Muhammad Faisal1,2,*, Jin Chao1,2, Kai Liu1,2, Xi Chen2, Gang Zhao1,2, Harish Menghwar1,2, Hui Zhang1,2, Xifang Zhu1,2, Muhammad Asif Rasheed1,2, Chenfei He1,2, Changmin Hu2, Yingyu Chen1,4, Eric Baranowski6,7, Huanchun Chen1,2,4 and Aizhen Guo1,2,4,5 1
The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China
2
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China
3
Department of Animal Health, The University of Agriculture, Peshawar, Pakistan
4
Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, International Joint Research and Training Centre for Veterinary Epidemiology, Huazhong Agricultural University, Wuhan, China 5
International Joint Research Centre for Veterinary Epidemiology, Huazhong Agricultural University, Wuhan, China
6
INRA, UMR 1225, IHAP, Toulouse, France
7
INP-ENVT, UMR 1225, IHAP, Université de Toulouse, Toulouse, France
*
These authors have contributed equally to this work
Correspondence to: Aizhen Guo, email: [email protected] Keywords: diagnostic biomarker, ELISA, immunoproteomics, immunoinformatics, Mycoplasma bovis, Immunology and Microbiology Section, Immune response, Immunity Received: April 27, 2016
Accepted: May 23, 2016
Published: June 02, 2016
ABSTRACT 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 antisera were 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
