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Expression and antigenic characterization of recombinant Mycoplasma agalactiae P48 major surface protein
1Dipartimento di Produzioni Animali, Epidemiologia ed Ecologia, Università di Torino, Italy. rosati@veter.unito.it
Abstract:
The gene encoding the P48 major surface lipoprotein of M. agalactiae has been recently characterised. Since its product plays an important role in the immune response of infected animals, in this study we analysed a recombinant P48 expressed in E. coli. Multiple point mutations were introduced by site directed mutagenesis in order to convert four tryptophan TGA codons, which are a typical feature of the mycoplasma genetic code, into the standard TGG. The mutated p48 gene was subcloned into pGex-2T and expressed in fusion with glutathione-S transferase. Following purification steps, P48 was eluted from carrier protein by thrombin digestion and used in Western blot and indirect ELISA using well-characterised sheep sera. Results demonstrate that specific antibodies against P48 are detected 3 weeks after onset of clinical disease and the recombinant P48 is a diagnostically relevant marker of M. agalactiae infection.
Insights
A recombinant P48 protein from Mycoplasma agalactiae was developed to detect infections. Specific antibodies against P48 are detectable three weeks after disease onset, marking it as a relevant diagnostic tool for M. agalactiae.
Area of Science:
- Molecular biology
- Immunology
- Veterinary microbiology
Background:
- Mycoplasma agalactiae causes significant economic losses in the sheep and goat industry.
- The P48 major surface lipoprotein is crucial for the immune response in animals infected with M. agalactiae.
- The mycoplasma genetic code presents challenges for recombinant protein expression.
Purpose of the Study:
- To produce a recombinant P48 protein for diagnostic purposes.
- To investigate the diagnostic relevance of P48 as a marker for M. agalactiae infection.
- To overcome challenges associated with mycoplasma genetic code for protein expression.
Main Methods:
- Site-directed mutagenesis was used to alter TGA codons to TGG in the p48 gene.
- The mutated p48 gene was expressed in E. coli as a fusion protein with glutathione-S transferase.
- Recombinant P48 was purified and used in Western blot and indirect ELISA with sheep sera.
Main Results:
- Specific antibodies against P48 were detected in sheep sera three weeks post-infection.
- The recombinant P48 protein demonstrated diagnostic relevance for M. agalactiae infection.
- Successful expression and purification of the modified P48 protein were achieved.
Conclusions:
- Recombinant P48 is a valuable diagnostic marker for Mycoplasma agalactiae infections in animals.
- The developed recombinant protein facilitates serological detection of M. agalactiae.
- This study provides a foundation for improved diagnostics in veterinary mycoplasmosis.