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

Background Mycoplasma hyorhinis (Mhr) is the etiologic agent of lameness and polyserositis in swine. Mhr P37 is a membrane protein that may play a critical role in immunity. It is a potential target for diagnostic development, but there is little information concerning its B cell epitopes. To investigate the epitopes of Mhr P37, a recombinant protein was developed in a baculovirus system using monoclonal antibodies (mAbs) prepared against P37 protein. Results Western 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 mAbs were found to be positive for Mhr. 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. This study identified mAbs that could provide useful tools for investigating the antigenic structure and function of the Mhr P37 protein as well as its application to diagnostic techniques.


Abstract Background
Mycoplasma hyorhinis (Mhr) is the etiologic agent of lameness and polyserositis in swine.
Mhr P37 is a membrane protein that may play a critical role in immunity. It is a potential target for diagnostic development, but there is little information concerning its B cell epitopes. To investigate the epitopes of Mhr P37, a recombinant protein was developed in a baculovirus system using monoclonal antibodies (mAbs) prepared against P37 protein.

Results
Western 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 mAbs were found to be positive for Mhr. 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.

Conclusions
This study identified mAbs that could provide useful tools for investigating the antigenic structure and function of the Mhr P37 protein as well as its application to diagnostic techniques.

Background
Mycoplasma hyorhinis (Mhr) is first isolated in 1953 and found to lack a cell wall [1]. It is a commensal microorganism that inhabits the upper respiratory tract of swine [2]. Mhr infections in pigs can cause lameness and polyserositis, and severe infections can cause pneumonia [3]. Systemic infection caused by Mhr is found on pig farms worldwide and is characterized by high morbidity and low mortality rates [4,5]. At present, Mhr infection detection mainly depends on pathogen isolation and culture and polymerase chain reaction (PCR) methods, and there is no commercially available kit for serological detection [6]. Although Mhr is easily isolated from porcine alveolar lavage fluid and nasal swabs, the process of isolation and identification of Mhr is often time consuming [7]. Mhr has also been proven to be a zoonotic disease that promotes the malignancy of tumor cells, such as in gastric cancer, and antibodies against Mhr P37 protein can inhibit the migration of infected cells [8,9]. Mycoplasma surface lipoproteins have been shown to perform a variety of functions during infection and interactions with the host [10]. P37 is an important membrane protein of Mhr and is part of the periplasmic binding protein-dependent transport system [11,12]. P37 may play a role in tumor invasion, and detection of antibodies against P37 in human serum may help diagnose cancer [13,14].
Previously, the P37 protein was used as a coating antigen to measure the immunoglobulin G (IgG) responses in swine vaccinated with an inactivated Mhr vaccine [15]. However, the mechanism by which host antibodies to P37 are produced, their function in the process of infection, and the precise epitope of P37 is still unknown.
In this study, the P37 protein was expressed using a baculovirus expression system. Specific antibodies were obtained from the expressed P37 protein, and these antibodies were used to analyze the key amino acids and core epitope of the antigens. Analysis of the antigenic epitope of the P37 protein will facilitate the development of effective tools for serological diagnostic techniques.

Identification of recombinant plasmid and shuttle plasmid
The recombinant plasmid pFastBac™1-His-P37 was identified by dual-restriction endonuclease digestion with BamH I and Xho I, and the 4693 bp vector fragment and the 1140 bp target gene fragment were visualized by 1% agarose gel electrophoresis ( Fig.   2A). pFastBac™1-His-P37 was specifically amplified using M13 primers, and a 3440 bp band was obtained on a 1% agarose gel. The negative control pFastBac™1 was observed as a 2300 bp fragment (Fig. 2B).
Detection and purification of recombinant protein Using fluorescence microscopy, strong fluorescence was observed in insect cells infected with pFastBac™1-His-P37 (Fig. 3A), whereas no fluorescence was observed in uninfected cells (Fig. 3B), indicating that the P37 protein was successfully expressed in insect cells.
Western blot analysis showed that the protein reacted with the prepared anti-Mhr mouse positive serum, and a specific reaction band appeared at approximately 43.3 kDa (Fig. 4).
We speculate that the P37 protein was modified in eukaryotic cells.

Characterization of P37 protein-specific mAbs
The four positive mAbs were designated C6, C8, E1, and E6. Subtype identification results showed that the heavy chain subtype of all four mAbs was IgG1, and the light chain subtype of all mAbs was kappa ( Table 2). Reactivity analysis of mAbs to Mhr showed that the Mhr strain reacted specifically with the four mAbs, and a specific reaction band appeared at a position of 43.3 kDa (Fig. 5).
Identification of P37 protein B cell linear epitopes using P37-specific mAbs To determine the epitopes of the four generated mAbs, a series of overlapping peptides were analyzed by Western blot analysis. The results showed the presence of specific bands at aa128-254, aa171-254, and aa199-226 (Fig. 6). Because the four mAbs were identical in reactivity, only one result is shown here.
To confirm the epitopes of the mAbs, Western blot analysis was performed after mutating each aa one by one. No specific bands were detected after deletion of aa 206 Lys (Fig. 7A) and aa 222 Phe (Fig. 7B). This indicates that aa 206 Lys and aa 222 Phe are key amino acids of the core epitope of the P37 protein.

Analysis of P37 protein from different Mhr strains
Analysis of the p37 sequence of seven Mhr strains demonstrated that the epitope 206 KIKKAWNDKDWNTFRNF 222 was highly conserved (100% aa identity, Fig. 8).
Homology modeling of the P37 protein epitope The spatial structure of the P37 protein from aa1-379 was predicted using threedimensional homology modeling. Model analysis showed that the overall shape of P37 is an irregular prolate ellipsoid, and the core epitope domain consists of two α-helices and a nonregular coil (Figs. 9A and 9B). The epitope region where the antigen-antibody reaction was detected was located on the surface of the P37 protein (Fig. 9C).

Discussion
Mhr is the most common mycoplasma in clinical farms [16]. Typical lesions found in infected animals include serum fibrotic pleurisy, otitis media, pericarditis and peritonitis, which may cause fibrous adhesions during the chronic phase [17,18]. Most pigs infected with Mhr do not show significant clinical symptoms, which may contribute to several swine disease complexes. Therefore, accurate diagnosis is critical to establishing effective treatment and prevention measures in affected herds. A previous study reported an ELISA method using Tween-20-extracted membrane protein as an antigen for detecting serum antibodies in pigs after Mhr challenge; however, this method showed cross-reactivity between mycoplasmas [19]. The high-resolution structure of Mh-p37 solved using the new heavy-atom derivative I3C has contributed to better understanding of the biological significance of P37 in tumorigenesis and cancer progression [20]. P37 is the major antigenic protein in Mhr, it is an ideal target for the development of diagnostic techniques. Therefore, we expressed the P37 protein in a baculovirus system. Western blot analysis revealed that the P37 protein was modified during expression; however, the mechanism of P37 protein modification is unknown and requires further study. A linear B cell epitope (aa206-222, 206 KIKKAWNDKDWNTFRNF 222 ) was identified as specific to the P37 protein and was highly conserved among Mhr strains. Structural predictions revealed that the epitope consists of an α-helix, a nonregular coil, and a second α-helix. The functional part of the enzyme was often in the conformational region of the nonregular coil, although its specific function requires further study. Analysis of P37 protein sequence revealed that this epitope region may be one of the predominant domains of the P37 protein. Therefore, these mAbs could provide useful tools for investigating the antigenic structure and function of Mhr P37 protein as well as its application in diagnostic procedures.

Conclusions
This study identified the core epitope of P37 protein using a eukaryotic expression system.
The epitope of P37 protein was 206 KIKKAWNDKDWNTFRNF 222 , and homology modeling analysis showed that this epitope consists of two α-helices and a nonregular coil. The The plate was incubated for 1 h at room temperature in the dark, washed three times with PBST, and analyzed using the Odyssey infrared imaging system (Licor Odyssey).

Precise localization of the P37 protein B cell epitope
To define the minimal linear epitope of the P37 protein, aa199-226 was deleted one by one (listed in Table 1) and their corresponding peptides were assessed by Western blot as

Consent for publication
Not applicable.

Availability of data and material
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.        Western blot analysis of mAbs that reacted specifically with the P37 protein fragment.
Western blot analysis showed that mAbs reacted specifically with aa128-254, aa171-254, and aa199-226, and the results were consistent for the four mAbs.

Figure 7
Western blot analysis verification of key amino acids.
In order to identify key amino acids, peptides were deleted one by one. B1-B8 indicates deleted peptides aa209-206, respectively. B9-B16 indicates deleted peptides aa226-219, respectively. (a) No specific band was detected when 206Lys was deleted. (b) No specific band was detected when 222Phe was deleted.

Figure 8
Alignment of sequences with Mhr P37 epitopes.
A total of seven Mhr strains were analyzed. The sequence motif recognized by mAbs is shown in the red box.

Figure 9
Structural analysis of the P37 protein.
Structural analysis and identification of B cell epitopes in the predicted threedimensional structure of the P37 protein. Red areas represent α-helix regions, blue areas represent nonregular coil regions, and yellow areas represent strand regions.
(a) The location of epitope 206KIKKAWNDKDWNTFRNF222 is shown in a gray box.
(b) Areas where antigen reacted to antibodies. The epitope was located on the surface of the P37 protein (c).

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