Identification of specific B cell linear epitopes of mycoplasma hyorhinis P37 protein using monoclonal antibodies against baculovirus-expressed P37 protein

Hongzhen Zhu,Yanwu Wei,Dan Liu,Deli Xia,Liping Huang,Yongxing Xie,Changming Liu,Li Feng,Haiqiao Bian

doi:10.1186/s12866-019-1614-4

Hongzhen Zhu, Yanwu Wei + Show 7 more

Open Access

https://doi.org/10.1186/s12866-019-1614-4

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Abstract

BackgroundMycoplasma hyorhinis (Mhr) is the etiologic agent of lameness and polyserositis in swine. P37 is a membrane protein of Mhr that may be an important immunogen and is a potential target for diagnostic development. However, there is little information concerning Mhr P37 protein epitopes. A precise analysis of the P37 protein epitopes should extend our understanding of the antigenic composition of the P37 protein and the humoral immune responses to Mhr infection. Investigating the epitopes of Mhr P37 will help to establish a detection method for Mhr in tissue and provide an effective tool for detecting Mhr infection.ResultsWestern blot and indirect immunofluorescence assays (IFA) confirmed that the expressed P37 protein was recognized by Mhr-positive porcine and mouse sera. Furthermore, the P37 protein was purified using affinity chromatography and used to immunize mice for hybridoma cell fusion. Four monoclonal antibodies (mAbs) found to be positive for Mhr were detected in infected lung tissue. A panel of truncated P37 proteins was used to identify the minimal B cell linear epitopes of the protein based on these mAbs. The core epitope was determined to be 206KIKKAWNDKDWNTFRNF222.ConclusionsIn this study, we identified 17 critical amino acids that determine the epitope of the P37 protein of Mhr. This study identified mAbs that could provide useful tools for investigating the Mhr P37 antigenic core epitope (amino acids 206–222) and detecting Mhr-specific antigens in infected tissue.

Highlights

Mycoplasma hyorhinis (Mhr) is the etiologic agent of lameness and polyserositis in swine
Detection and purification of recombinant protein Using fluorescence microscopy, strong fluorescence was observed in insect cells infected with pFastBacTM1-HisP37, whereas no fluorescence was observed in uninfected cells (Fig. 2), indicating that the P37 protein was successfully expressed in insect cells
Western blot analysis showed that the protein reacted with the prepared antiMhr mouse positive serum, and a specific reaction band appeared at approximately 43.3 kDa (Fig. 3)

Summary

Introduction

Mycoplasma hyorhinis (Mhr) is the etiologic agent of lameness and polyserositis in swine. P37 is a membrane protein of Mhr that may be an important immunogen and is a potential target for diagnostic development. There is little information concerning Mhr P37 protein epitopes. A precise analysis of the P37 protein epitopes should extend our understanding of the antigenic composition of the P37 protein and the humoral immune responses to Mhr infection. Mhr infections in pigs can cause lameness and polyserositis, and severe infections can cause pneumonia [3, 4]. Systemic infection caused by Mhr is found on pig farms worldwide and is characterized by high. P37 is an important membrane protein of Mhr and is part of the periplasmic binding protein-dependent transport system [15, 16]. P37 may play a role in tumor invasion, and detection of antibodies against P37 in human serum may help diagnose cancer [17, 18]

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