Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins

Simionatto, Simone; Marchioro, Silvana Beutinger; Galli, Vanessa; Brum, Clarice Brink; Klein, Cátia Silene; Rebelatto, Raquel; Silva, Éverton F.; Borsuk, Sibele; Conceição, Fabrı́cio R.; Dellagostin, Odir A.

doi:10.1016/j.cimid.2012.01.007

Cited by 26 publications

(19 citation statements)

References 27 publications

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“…To assess the antigenicity of M. hyopneumoniae proteins expressed in E. coli, Western blotting and enzyme-linked immunosorbent assays (ELISAs) were performed as previously described (18). For the Western blot analysis, two pools containing five convalescent-phase pig sera and one pool with five SPF pig sera were tested.…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant plasmids were transformed into E. coli BL21(DE3)-RIL expression-competent cells. Recombinant protein expression and solubility testing were performed as previously described (16,18). The purity of the purified recombinant proteins was analyzed on a 12% SDS-PAGE gel, and the protein concentrations were determined using the BCA protein assay kit (Pierce) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…In serum from immunized mice, specific antibodies against the recombinant proteins were quantified by ELISA as previously described (18,19), with some modifications. Microtiter plates were coated with 100 ng of each recombinant protein (indirect ELISA).…”

Section: Methodsmentioning

confidence: 99%

“…The sera from immunized mice (diluted 1:20) were incubated with 1 g of crude extract of M. hyopneumoniae 7448, in order to verify if the antibodies induced by the recombinant vaccines recognized the native proteins of M. hyopneumoniae. The ELISA was performed as previously described (18,19). Sera from mice were assayed individually and all reactions were performed in triplicate.…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

Galli

Simionatto

Marchioro

et al. 2013

Clin Vaccine Immunol

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bMycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), which is a respiratory disease responsible for huge economic losses in the pig industry worldwide. The commercially available vaccines provide only partial protection and are expensive. Thus, the development of alternatives for the prophylaxis of EP is critical for improving pig health. The use of multiple antigens in the same immunization may represent a promising alternative. In the present study, seven secreted proteins of M. hyopneumoniae were cloned, expressed in Escherichia coli, and evaluated for antigenicity using serum from naturally and experimentally infected pigs. In addition, the immunogenicity of the seven recombinant proteins delivered individually or in protein cocktail vaccines was evaluated in mice. In Western blot assays and enzyme-linked immunosorbent assays, most of the recombinant proteins evaluated were recognized by convalescent-phase serum from the animals, indicating that they are expressed during the infectious process. The recombinant proteins were also immunogenic, and most induced a mixed IgG1/ IgG2a humoral immune response. The use of these proteins in a cocktail vaccine formulation enhanced the immune response compared to their use as antigens delivered individually, providing evidence of the efficacy of the multiple-antigen administration strategy for the induction of an immune response against M. hyopneumoniae. P orcine enzootic pneumonia (EP) is a chronic respiratory disease with a high incidence in pig farms worldwide. Mycoplasma hyopneumoniae, the etiological agent of this disease, colonizes and destroys the main mechanism of innate immunity of the pig respiratory system-the respiratory ciliated epithelium-predisposing the pigs to secondary and opportunistic infections (1). EP prophylaxis comprises the use of antibiotics, management procedures, and vaccination, which is considered the most efficient control measure (2). The commercially available vaccines consist of inactivated whole cells (bacterins) and have high production costs, mainly due to the fastidious growth of this microorganism. In addition, these vaccines do not eliminate M. hyopneumoniae from infected pig herds (1, 3, 4). Thus, efforts have been directed toward the search for new prophylaxis strategies against EP that do not include the use of bacterins.M. hyopneumoniae does not penetrate host cells, and thus it is believed that the pathogenesis of this microorganism is mediated by a complex and multifactorial process that involves components of the cell membrane and secreted proteins, many of which are still unidentified (5). Data generated from the sequencing of four isolates of M. hyopneumoniae (7448, 168, J, and 232) and the comparative genomic and proteomic analyses of pathogenic strains (7448 and 232) and a nonpathogenic strain (J) of M. hyopneumoniae (6,7,8,9) have allowed the identification of coding sequences (CDS) from secreted antigenic proteins and/or proteins involved in the pathogenicity of the microorganism. Som...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

Galli

Simionatto

Marchioro

et al. 2013

Clin Vaccine Immunol

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“…M. hyopneumoniae culture is laborious, time-consuming, and requires several weeks to produce results; frequently, the culture media can become overgrown with M. hyorhinis or M. flocculare as well (DorigoZetsma et al, 1999). Serological methods are insufficiently sensitive and cannot distinguish between vaccinated and M. hyopneumoniae-infected pigs in field screening (Moitinho-Silva et al, 2012;Simionatto et al, 2012). More rapid, higher sensitivity methods were therefore developed, one of them being the PCR technique for M. hyopneumoniae DNA detection (Dorigo-Zetsma et al, 1999;Lu et al, 2010;Shen et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

A rapid and sensitive loop-mediated isothermal amplification procedure (LAMP) for Mycoplasma hyopneumoniae detection based on the p36 gene

Mj¹,

Gm²,

Ff³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. The aim of this study was to establish a method for sensitive and rapid diagnosis of Mycoplasma hyopneumoniae in clinical specimens. To this effect, we employed three sets of primers specifically designed for amplification of nucleic acids under isothermal conditions. After optimization of reaction conditions, M. hyopneumoniae could be successfully detected at 63°C in 45 min through use of the loop- (2015) mediated isothermal amplification (LAMP) assay. A positive reaction was identified visually as white precipitate and confirmed by gel electrophoresis. The detection limit for this assay was 10 copies/µL, as observed by electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease digestion as well as by direct sequencing of the amplified product. This method can specifically detect M. hyopneumoniae; other species with high homology and other bacterial and virus strains gave negative results. To test the utility of this procedure, the LAMP assay was applied to 40 clinical samples collected from swine lung tissues experimentally challenged with M. hyopneumoniae isolates, and compared to the results from a real-time polymerase chain reaction (PCR) assay. A concordance of 100% was observed between the two assays. In conclusion, the results from our study demonstrated that the LAMP assay provided a rapid reaction and was inexpensive to perform, with no need of complex instruments or systems such as Geneamp PCR. The LAMP assay may therefore be applied in routine diagnosis in the clinical laboratory and for in-field detection of M. hyopneumoniae infection.

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Porcine antibody profiles of 33 Mycoplasma hyopneumoniae fusion proteins from M. hyopneumoniae natural infection but not vaccination

Ning

Yang

Tian

et al. 2022

Veterinary Medicine & Sci

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Background Mycoplasma hyopneumoniae , the primary pathogen responsible for porcine enzootic pneumonia, reduces average daily weight gain and causes substantial economic losses to the pig industry worldwide. Vaccination is the most common strategy to control this disease but offers partial protection. Therefore, developing next‐generation vaccines by screening protective antigens is crucial. Objectives The aim of this study was to evaluate the antibody response to 33 recombinant proteins in pigs naturally infected with M. hyopneumoniae . Methods The genes encoding 33 (hypothetical) membrane proteins or secretory proteins were ligated into pGEX‐6P‐1, pGEX‐6P‐2, pGEX‐5X‐3 or pGEX‐4T‐3 vectors and transformed into Escherichia coli BL21(DE3) or E. coli XL‐1 Blue to construct recombinant bacteria and to express the recombinant proteins. The recombinant bacteria expressing the target proteins reacted with porcine convalescent sera and negative sera to screen immunodominant proteins by ELISA. Then, recombinant bacteria expressing immunodominant proteins were used to identify the discriminating immunodominant proteins that were recognised by convalescent sera nut not hyperimmune sera. Results All recombinant bacteria could express the target recombinant proteins in soluble form. Twenty‐one proteins were shown to present immunodominant antigens, and four proteins were not recognised by convalescent sera. Moreover, six proteins were considered discriminating and reacted with convalescent sera but not with hyperimmune sera. Conclusions The identified immunodominant proteins were antigenic and expressed during bacterial infection, suggesting that these proteins, especially those capable of discriminating between sera, can be used to identify protective antigens with the view to develop more effective vaccines against M. hyopneumoniae infection.

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Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins

Cited by 26 publications

References 27 publications

Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

A rapid and sensitive loop-mediated isothermal amplification procedure (LAMP) for Mycoplasma hyopneumoniae detection based on the p36 gene

Porcine antibody profiles of 33 Mycoplasma hyopneumoniae fusion proteins from M. hyopneumoniae natural infection but not vaccination

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