Proteomics identification and characterization of MbovP730 as a potential DIVA antigen of Mycoplasma bovis
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Farhan Anwar Khan1,2,3,*, Gang Zhao1,2,*, Yusi Guo1,2, Muhammad Faisal1,2, Jin Chao1,2, Xi Chen2, Chenfei He1,2, Harish Menghwar1,2, Rahim Dad2,6, Muhammad Zubair1,2, Changmin Hu2, Yingyu Chen1,4, Huanchun Chen1,2,4, Zhang Rui1,2 and Aizhen Guo1,2,4,5
1The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China
2College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China
3Department of Animal Health, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Khyber Pakhtunkhwa 25120, Pakistan
4Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, Wuhan 430070, People’s Republic of China
5International Joint Research and Training Centre for Veterinary Epidemiology, Hubei Province, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China
6Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China
*These authors contributed equally to this work
Aizhen Guo, email: firstname.lastname@example.org
Keywords: bovine; mycoplasma bovis; membrane proteins; immunoproteomics; qPCR
Received: September 05, 2017 Accepted: October 17, 2017 Epub: November 02, 2017 Published: June 19, 2018
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.
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