Mycoplasma synoviae surface protein MSPB as a recombinant antigen in an indirect ELISA

Noormohammadi, Amir H.; Markham, Philip F.; Markham, Jillian F.; Whithear, Kevin G.; Browning, Glenn F.

doi:10.1099/13500872-145-8-2087

Cited by 21 publications

(7 citation statements)

References 5 publications

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“…Although ELISA based on purified native antigens are sensitive, the production of sufficient and consistent antigens is expensive. Alternatively, an iELISA developed using recombinant MSPB protein expressed in E. coli showed good performance in the detection of anti- M. synoviae antibodies ( Noormohammadi et al, 1999 ). In this study, we established and evaluated rLP78-based iELISA.…”

Section: Discussionmentioning

confidence: 99%

“…However, the cross-reactivity of these ELISAs with M. gallisepticum and non-specific reactions impede the development of specific serodiagnostic tests ( Opitz et al, 1983 ; Higgins and Whithear, 1986 ; Opitz and Cyr, 1986 ). ELISA based on MSPB, the amino-terminal end of vlhA, has demonstrated good correlation with SPA without any cross-reactivity with sera against M. gallisepticum ( Noormohammadi et al, 1999 ). However, a high degree of amino acid variations is observed in MSPB across strains, which affects the sensitivity of ELISA ( Noormohammadi et al, 1998 , 2002 ).…”

Section: Introductionmentioning

confidence: 99%

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Mycoplasma synoviae LP78 is a fibronectin/plasminogen binding protein, putative adhesion, and potential diagnostic antigen

Han,

Wang,

Wang

et al. 2024

Front. Microbiol.

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Mycoplasma synoviae (M. synoviae) is one of the major poultry pathogens causing infectious synovitis, airsacculitis, a high incidence of shell breakage, and egg production loss. However, the pathogenesis of M. synoviae remains unclear. Adhesion of mycoplasmas to host cells is a crucial step in infection and colonization. The purpose of this study was to determine the adhesive function of a putative P80 family lipoprotein (LP78) and evaluate its application in the detection of antibodies against M. synoviae. Recombinant LP78 (rLP78) was expressed in the supernatant component of Escherichia coli and mouse anti-rLP78 serum was prepared. Bioinformatic analysis and western blotting results revealed that LP78 was conservative among M. synoviae strains. It was distributed not only in the cytoplasm but also on the membrane of M. synoviae through western blotting and indirect immunofluorescence (IFA). The adherence of M. synoviae to DF-1 cells was significantly inhibited by mouse anti-rLP78 serum (p < 0.01). IFA revealed that rLP78 adhered to DF-1 cells, and this adherence was prevented by mouse anti-rLP78 serum. Furthermore, rLP78 was found to bind to the DF-1 cells membrane proteins in a dose-dependent manner by enzyme-linked immunosorbent assay (ELISA). Screening of DF-1 cells membrane proteins by western blotting showed that proteins with molecular weight of 35–40 kDa and 55–70 kDa bound to rLP78. Moreover, rLP78 was identified to be a fibronectin/plasminogen binding protein. The sensitivity and specificity of rLP78-based iELISA were 85.7 and 94.1%, respectively. The maximum dilution of positive serum (HI titer, 1:128) detected via rLP78-based iELISA was 1:6,400, whereas that detected using a commercial ELISA kit was 1:12,800–1:25,600. Both rLP78-based iELISA and the commercial ELISA kit detected seroconversion after 7 days of challenge and immunization. No cross-reactivity with positive sera against other avian pathogens was observed in rLP78-based iELISA. Collectively, these results indicate that LP78 is a fibronectin/plasminogen-binding adhesion protein of M. synoviae and a potential diagnostic antigen. The present study will facilitate a better understanding of the pathogenesis of M. synoviae and the development of new diagnostic.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mycoplasma synoviae LP78 is a fibronectin/plasminogen binding protein, putative adhesion, and potential diagnostic antigen

Han,

Wang,

Wang

et al. 2024

Front. Microbiol.

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“…Owing to the widespread prevalence of MS infection and its substantial economic effects on the chicken industry, establishing an accurate and effective diagnostic method is particularly important. For detection of MS antibodies, the major membrane protein MSPB has been used as a coating antigen, and has been thought to be a specific and sensitive diagnostic antigen [ 48 , 49 ]. However, MSPB contains a proline-rich repeat region that is prone to insertion or deletion mutations, thus resulting in antigenic variation [ 50 , 51 ].…”

Section: Discussionmentioning

confidence: 99%

NADH oxidase of Mycoplasma synoviae is a potential diagnostic antigen, plasminogen/fibronectin binding protein and a putative adhesin

Zhao

et al. 2022

BMC Vet Res

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Background Mycoplasma synoviae (MS) is an important pathogen causing respiratory diseases and arthritis in chickens and turkeys, thus, resulting in serious economic losses to the poultry industry. Membrane-associated proteins are thought to play important roles in cytoadherence and pathogenesis. NADH oxidase (NOX) is an oxidoreductase involved in glycolysis, which is thought to be a multifunctional protein and potential virulence factor in some pathogens. However, little is known regarding the NOX of MS (MSNOX). We previously demonstrated that MSNOX was a metabolic enzyme distributed in not only the cytoplasm but also the MS membrane. This study was aimed at exploring NOX’s potential as a diagnostic antigen and its role in MS cytoadherence. Results Western blots and ELISAs indicated that recombinant MSNOX (rMSNOX) protein reacted with sera positive for various MS isolates, but not MG isolates or other avian pathogens, thus, suggesting that rMSNOX is a potential diagnostic antigen. In addition, rabbit anti-rMSNOX serum showed substantial complement-dependent mycoplasmacidal activity toward various MS isolates and MG Rlow. MSNOX protein was found not only in the cytoplasm but also on the membrane of MS through suspension immunofluorescence and immunogold electron microscopy assays. Indirect immunofluorescence assays indicated that rMSNOX adhered to DF-1 cells, and this adherence was inhibited by rabbit anti-rMSNOX, but not anti-MG serum. Furthermore, indirect immunofluorescence and colony counting assays confirmed that the rabbit anti-rMSNOX serum inhibited the adherence of various MS isolates but not MG Rlow to DF-1 cells. Moreover, plasminogen (Plg)- and fibronectin (Fn)-binding assays demonstrated that rMSNOX bound Plg and Fn in a dose-dependent manner, thereby further confirming that MSNOX may be a putative adhesin. Conclusion MSNOX was identified to be a surface immunogenic protein that has good immunoreactivity and specificity in Western blot and ELISA, and therefore, may be used as a potential diagnostic antigen in the future. In addition, rMSNOX adhered to DF-1 cells, an effect inhibited by rabbit anti-rMSNOX, but not anti-MG serum, and anti-rMSNOX serum inhibited the adherence of various MS isolates, but not MG Rlow, to DF-1 cells, thus indicating that the inhibition of adherence by anti-MSNOX serum was MS specific. Moreover, rMSNOX adhered to extracellular matrix proteins including Plg and Fn, thus suggesting that NOX may play important roles in MS cytoadherence and pathogenesis. Besides, rabbit anti-rMSNOX serum presented complement-dependent mycoplasmacidal activity toward both MS and MG, indicating the MSNOX may be further studied as a potential protective vaccine candidate.

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“…The following research was designed chimeric protein to develop a recombinant antigen applicable for indirect ELISA to prepare a rapid, accurate, time-saving, and cheap kit for diagnosis of avian mycoplasmosis. The creation of improved serodiagnostic assays that offer optimal specificity in the detection of the highly cross-reactive avian Mycoplasma species envisaged with the advent of recombinant DNA technology [ 5 , 42 ]. Recombinant technology could significantly help to reduce defects, allowing the generation of unlimited amounts of multiple specific antigens [ 6 ].…”

Section: Discussionmentioning

confidence: 99%

Designing of novel chimeric PvpA-pMGA protein of Mycoplasma gallisepticum, applicable for indirect ELISA

Fatideh

Esmaelizad

Kargar

et al. 2022

Journal of Genetic Engineering and Biotechnology

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Background Mycoplasma gallisepticum is the primary agent of chronic respiratory disease in chickens creating important economic losses in poultry industry. pMGA and pvpA genes encode major surface proteins in M. gallisepticum containing pathogenic, antigenic, and immune evasion characteristics. The objective of the present study was to design, express, and purify the recombinant chimeric PvpA-pMGA protein from M.gallisepticum for using in serological diagnostic test. Methods Antigenic regions of PvpA and pMGA proteins were predicted for designing chimeric pvpA-pMGA gene construct. The codon optimized sequence was cloned into the expression vector pET32a+ and transformed into the Escherichia coli strain BL21 (DE3). The pET32a-PvpA-pMGA recombinant plasmid was expressed and confirmed by SDS-PAGE and immunoblotting. PvpA-pMGA recombinant protein (20μg and 50μg), ts-11 vaccine strain, and S6 strain that formulated by montanide adjuvant and two control groups (PBS and adjuvant) were injected subcutaneously to six groups of chickens. Results High yield of protein was purified amount 138 mg/L by affinity batch formation method. Indirect ELISA showed the levels of antibodies in rPvpA-pMGA was significantly higher than ts-11 and S6 groups (p<0.05). The results indicated that antigen-specific response was successfully elicited by the rpMGA-PvpA in chickens. The result of the ELISA with sera collected from ts-11 and S6 groups showed that indirect PvpA-pMGA-ELISA is appropriate candidate for detection of specific antibodies against M. gallisepticum with 100% sensitivity and specificity. Conclusions The rPvpA-pMGA is a highly candidate immunogenic protein which induced high amount of humoral immune response. Novel rPvpA-pMGA protein could be useful for evaluation of antibody level in vaccinated poultry flocks.

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Mycoplasma synoviae surface protein MSPB as a recombinant antigen in an indirect ELISA

Cited by 21 publications

References 5 publications

Mycoplasma synoviae LP78 is a fibronectin/plasminogen binding protein, putative adhesion, and potential diagnostic antigen

Mycoplasma synoviae LP78 is a fibronectin/plasminogen binding protein, putative adhesion, and potential diagnostic antigen

NADH oxidase of Mycoplasma synoviae is a potential diagnostic antigen, plasminogen/fibronectin binding protein and a putative adhesin

Designing of novel chimeric PvpA-pMGA protein of Mycoplasma gallisepticum, applicable for indirect ELISA

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