Expression and antigenic characterization of recombinant Mycoplasma agalactiae P48 major surface protein.
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.
Rosati
S
S
Dipartimento di Produzioni Animali, Epidemiologia ed Ecologia, Università di Torino, Italy. rosati@veter.unito.it
Robino
P
P
Fadda
M
M
Pozzi
S
S
Mannelli
A
A
Pittau
M
M
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D015964:Gene Expression Regulation, Bacterial; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009174:Mycoplasma / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D009175:Mycoplasma Infections / Q000382:microbiology / Q000662:veterinary*; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D012756:Sheep; D012757:Sheep Diseases / Q000382:microbiology*
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins