Species-Specific Monoclonal Antibodies to Escherichia coli -Expressed p36 Cytosolic Protein of Mycoplasma hyopneumoniae

Abstract

The p36 protein of Mycoplasma hyopneumoniae is a cytosolic protein carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the p36-encoding gene (948 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species and pathogenic bacteria that commonly colonize the porcine respiratory tract. The amplified p36 gene was subcloned into the pGEX-4T-1 vector to be expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The GST-p36 recombinant fusion protein was purified by affinity chromatography and cut by thrombin, and the enriched p36 protein was used to immunize female BALB/c mice for the production of anti-p36 monoclonal antibodies (MAbs). The polypeptide specificity of the nine MAbs obtained was confirmed by Western immunoblotting with cell lysates prepared from the homologous strain. Cross-reactivity studies of the anti-p36 MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture suggested that these anti-p36 MAbs were directed against a highly conserved epitope, or closely located epitopes, of the p36 protein. No reactivity was demonstrated against other mycoplasma species tested. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs infected experimentally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. The bacteria could be recovered from lung homogenates of pigs that were killed after the 3-week observation period by both PCR and cultivation procedures. Furthermore, the anti-p36 MAbs permitted effective detection by indirect immunofluorescence of M. hyopneumoniae in frozen lung sections from experimentally infected pigs. However, attempts to use the recombinant p36 protein as an antigen in an indirect enzyme-linked immunosorbent assay for the detection of antibodies in sera from convalescent pigs showed no correlation with clinical and pathological findings.Mycoplasma hyopneumoniae is the causative agent of enzootic pig pneumonia (15, 21), a disease found on pig farms worldwide which is characterized by high morbidity and low mortality rates (19,25). Coughing is the principal clinical sign, and retarded growth and poor food conversion may result in considerable economic losses. Furthermore, this agent predisposes the pigs to secondary pulmonary infections, hence, increasing the mortality among the herds and the financial problems associated with such losses (19,25).M. hyopneumoniae encodes several characterized immunodominant proteins, among which are the p36 cytosolic protein (28, 29), the p46, p65, and p74 membranous proteins (3,17,18,22), and the p97 adhesin (31). The functions of these proteins have not been yet elucidated, but specific reactants may eventually be useful tools for the diagnosis of M. hyopneumoniae.The cytosolic p36 prote...

Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic 168 strain and its high-passaged attenuated strain

“…The functions of these proteins have not been well elucidated, but specific reactants may eventually be useful tools to diagnose M. hyopneumoniae [48]. …”

Monoclonal Antibodies to Escherichia coli -Expressed P46 and P65 Membranous Proteins for Specific Immunodetection of Mycoplasma hyopneumoniae in Lungs of Infected Pigs

“…Intraperitoneal injections were given at 2-week intervals, followed by an intraperitoneal dose of the antigen without adjuvant 3 days prior to the fusion experiment. The fusion protocol for sensitized splenocytes with SP2/O-Ag14 myeloma cells was essentially similar to that previously described (7). Hybrid cells were cultured in hypoxanthine-aminopterine-thymidine medium containing l5% fetal calf serum.…”

“…Hybrid cells were cultured in hypoxanthine-aminopterine-thymidine medium containing l5% fetal calf serum. Hybridoma supernatants were screened for the presence of anti-P46 and anti-P65c antibodies by an indirect ELISA as described elsewhere (7). The use of GST-coated plates permitted the elimination of all clones directed against GST.…”

“…Each strip was incubated for 1 h at room temperature in the presence of either a 1:200 to 1:1,000 dilution of porcine hyperimmune serum, or a 1:1,000 dilution of mouse ascitic fluid in the blocking buffer. The immune reactions were revealed following a 30-min incubation into a 1:2,000 dilution of the appropriate peroxidase-labeled anti-IgG conjugate, followed by a final incubation period in the enzyme substrate solution, as previously described (7).…”

“…Six crossbred F 1 (Landrace ϫ Yorkshire) castrated SPF pigs (5 to 6 weeks old) were obtained from the aforementioned breeding farm. Experimental inoculation of pigs by the intratracheal route was conducted as described previously (7). The animals were separated in two groups, consisting of two control pigs that received only fresh Friis medium whereas the remaining four pigs received an identical volume (8 ml) of a culture of the virulent IAF-DM9827 field strain of M. hyopneumoniae, corresponding to an infectious dose of 10 7 color-changing units/ml.…”