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Proteomics identification and characterization of MbovP730 as a potential DIVA antigen of Mycoplasma bovis
Mycoplasma bovis (M. bovis) is an important pathogen of cattle. An attenuated live vaccine has recently been developed by this laboratory. However, an effective assay for the differentiation of infected from vaccinated animals (DIVA) is still lacking. Therefore, a comparative immunoproteomics study...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Impact Journals LLC
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033335/ https://www.ncbi.nlm.nih.gov/pubmed/29983863 http://dx.doi.org/10.18632/oncotarget.22265 |
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author | Khan, Farhan Anwar Zhao, Gang Guo, Yusi Faisal, Muhammad Chao, Jin Chen, Xi He, Chenfei Menghwar, Harish Dad, Rahim Zubair, Muhammad Hu, Changmin Chen, Yingyu Chen, Huanchun Rui, Zhang Guo, Aizhen |
author_facet | Khan, Farhan Anwar Zhao, Gang Guo, Yusi Faisal, Muhammad Chao, Jin Chen, Xi He, Chenfei Menghwar, Harish Dad, Rahim Zubair, Muhammad Hu, Changmin Chen, Yingyu Chen, Huanchun Rui, Zhang Guo, Aizhen |
author_sort | Khan, Farhan Anwar |
collection | PubMed |
description | Mycoplasma bovis (M. bovis) is an important pathogen of cattle. An attenuated live vaccine has recently been developed by this laboratory. However, an effective assay for the differentiation of infected from vaccinated animals (DIVA) is still lacking. Therefore, a comparative immunoproteomics study of the membrane and membrane associated proteins (MAPs) of M. bovis HB0801 and its attenuated strain (M. bovis-150) was aimed to identify potential antigens for DIVA assay. Triton-X-114 fractionated liposoluble proteins of both the virulent and attenuated strains were separated with 2-DE and proteins reacting with sera against the virulent M. bovis strain were detected by MS. A total of 19 differently expressed proteins were identified by MS, among them twelve proteins were detected by MALDI-TOF MS and seven antigenic proteins were identified by short-gun LC-MS/MS. Furthermore, these findings were confirmed at mRNA level by qRT-PCR. The results demonstrated that a putative lipoprotein encoded by functionally unknown gene Mbov_0730 (MbovP730) is a sensitive and specific antigen for DIVA assay. MbovP730 is absent in M. bovis-150 confirmed with Western blot assay and also didn’t cross-react with other antisera against common pathogens including infectious bovine rhinotracheitis virus and bovine viral diarrhea virus by iELISA. Thereby rMbovP730-based iELISA was established. For clinical samples, this ELISA provided a sensitivity of 95.7% (95% CI: 90.4%, 98.2%) and specificity was 97.8% (95% CI: 88.4%, 99.6%). Antisera from vaccinated calves (n = 44) were found negative with rMbovP730 based iELISA, while positive with assays based on whole cell proteins of M. bovis-150 and M. bovis HB0801, respectively. In conclusion, this study identified the differential antigen MbovP730 between virulent and attenuated strains and established rMbovP730-based iELISA as a new DIVA method. |
format | Online Article Text |
id | pubmed-6033335 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Impact Journals LLC |
record_format | MEDLINE/PubMed |
spelling | pubmed-60333352018-07-08 Proteomics identification and characterization of MbovP730 as a potential DIVA antigen of Mycoplasma bovis Khan, Farhan Anwar Zhao, Gang Guo, Yusi Faisal, Muhammad Chao, Jin Chen, Xi He, Chenfei Menghwar, Harish Dad, Rahim Zubair, Muhammad Hu, Changmin Chen, Yingyu Chen, Huanchun Rui, Zhang Guo, Aizhen Oncotarget Research Paper Mycoplasma bovis (M. bovis) is an important pathogen of cattle. An attenuated live vaccine has recently been developed by this laboratory. However, an effective assay for the differentiation of infected from vaccinated animals (DIVA) is still lacking. Therefore, a comparative immunoproteomics study of the membrane and membrane associated proteins (MAPs) of M. bovis HB0801 and its attenuated strain (M. bovis-150) was aimed to identify potential antigens for DIVA assay. Triton-X-114 fractionated liposoluble proteins of both the virulent and attenuated strains were separated with 2-DE and proteins reacting with sera against the virulent M. bovis strain were detected by MS. A total of 19 differently expressed proteins were identified by MS, among them twelve proteins were detected by MALDI-TOF MS and seven antigenic proteins were identified by short-gun LC-MS/MS. Furthermore, these findings were confirmed at mRNA level by qRT-PCR. The results demonstrated that a putative lipoprotein encoded by functionally unknown gene Mbov_0730 (MbovP730) is a sensitive and specific antigen for DIVA assay. MbovP730 is absent in M. bovis-150 confirmed with Western blot assay and also didn’t cross-react with other antisera against common pathogens including infectious bovine rhinotracheitis virus and bovine viral diarrhea virus by iELISA. Thereby rMbovP730-based iELISA was established. For clinical samples, this ELISA provided a sensitivity of 95.7% (95% CI: 90.4%, 98.2%) and specificity was 97.8% (95% CI: 88.4%, 99.6%). Antisera from vaccinated calves (n = 44) were found negative with rMbovP730 based iELISA, while positive with assays based on whole cell proteins of M. bovis-150 and M. bovis HB0801, respectively. In conclusion, this study identified the differential antigen MbovP730 between virulent and attenuated strains and established rMbovP730-based iELISA as a new DIVA method. Impact Journals LLC 2017-11-02 /pmc/articles/PMC6033335/ /pubmed/29983863 http://dx.doi.org/10.18632/oncotarget.22265 Text en Copyright: © 2018 Khan et al. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Paper Khan, Farhan Anwar Zhao, Gang Guo, Yusi Faisal, Muhammad Chao, Jin Chen, Xi He, Chenfei Menghwar, Harish Dad, Rahim Zubair, Muhammad Hu, Changmin Chen, Yingyu Chen, Huanchun Rui, Zhang Guo, Aizhen Proteomics identification and characterization of MbovP730 as a potential DIVA antigen of Mycoplasma bovis |
title | Proteomics identification and characterization of MbovP730 as a potential DIVA antigen of Mycoplasma bovis |
title_full | Proteomics identification and characterization of MbovP730 as a potential DIVA antigen of Mycoplasma bovis |
title_fullStr | Proteomics identification and characterization of MbovP730 as a potential DIVA antigen of Mycoplasma bovis |
title_full_unstemmed | Proteomics identification and characterization of MbovP730 as a potential DIVA antigen of Mycoplasma bovis |
title_short | Proteomics identification and characterization of MbovP730 as a potential DIVA antigen of Mycoplasma bovis |
title_sort | proteomics identification and characterization of mbovp730 as a potential diva antigen of mycoplasma bovis |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033335/ https://www.ncbi.nlm.nih.gov/pubmed/29983863 http://dx.doi.org/10.18632/oncotarget.22265 |
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