Monoclonal antibodies against P46 and P65 rec. proteins for Mycoplasma hyopneumoniae detection by Indirect Immunofluorescence (IIF) and Immunoperoxydase (IP).
Abstract Text
Mycoplasma hyopneumoniae is the causative agent of enzootic pig pneumonia. Its genome encodes for several immunodominant proteins, among which the P46, P65 and P74 membranous lipoproteins, the P36 cytosolic protein and the P97 adhesin carrying species-specific antigenic determinants. The P46 and P65 lipoproteins are major immunogens which trigger early humoral immune response in pigs, the C-terminal region of P65 corresponding to the most immunogenic domain. As for several Mycoplasma genes, TGA codons are translated as tryptophan residues rather than corresponding to translational stop signals as in mammalian and other bacterial cells. For M. hyopneumoniae, the ORFs encoding for P46 (1260 bp) and P65 (1803 bp) possess 3 and 1 TGA codons, respectively. In the present study, the entire P46 and P65 genes of the reference ATCC-25934 strain of M. hyopneumoniae were amplified by PCR and oligonucleotide primers were also designed such as to permit directed mutagenesis of the TGA codons in TGG. The mutated genes were ligated into the procaryotic pGEX-4T1 vector and used to transform competent E. coli, strain BL21 cells to produce recombinant (rec) proteins fused to glutathione S-transferase (GST). The affinity-purified GST-P65c and GST-P46 rec fusion proteins were used to immunize SPF piglets, as well as, Balb/c mice for production of monoclonal antibodies. Specific humoral immune responses were induced in both animal species, high antibody titers being detected by indirect ELISA. The specificities of MAbs towards the rec proteins and the native membranous proteins were confirmed by western blotting and ELISA. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in SPF pigs experimentally-infected with a virulent Quebec field strain (IAF-DM9827). M. hyopneumoniae could be recovered by both PCR and cultivation procedures from lung homogenates of pigs that were killed after the 3-week observation period. MAbs against both proteins permitted effective detection of M. hyopneumoniae in lungs from infected pigs by indirect immunofluorescence or immunoperoxydase
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