title: Cloning, expression, and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli.
abstract: Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and leading to important economic losses in the poultry industry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in particular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma-free breeder flocks. In this study, we identified a component of the pyruvate dehydrogenase dihydrolipoamide acetyltransferase (i.e., E2) protein by 2-dimensional electrophoresis (2-DE), characterized it in immunoblotting assays, and analyzed its recombinant (r-E2) in a rec-ELISA test. For full-length protein expression in Escherichia coli (EC) a point mutation was introduced. A rabbit antiserum produced against r-E2 was tested in a Western Blot using different samples of Mycoplasma species. The results showed the applicability of site-directed mutagenesis, with a good yield of the r-E2 after purification. Also, anti-E2 serum reacted with all the tested MG strains showing no cross reaction with other mycoplasmas. The developed E2 ELISA test was capable of detecting MG antibodies in the sera examined. Those results demonstrate the antigenic stability of the E2 protein which could represent a recombinant antigen with potential diagnostic applications.
authors: University of Turin (UNITO), Department of Veterinary Science. 10100 Torino, Italy ticirocha@yahoo.com.br.
authors: T S
authors: TS
authors: Rocha
authors: University of Turin (UNITO), Department of Veterinary Science. 10100 Torino, Italy.
authors: C
authors: C
authors: Tramuta
authors: Istituto Zooprofilattico Sperimentale delle Venezie, Diagnostic Service Avian Medicine Laboratory-Mycoplasma Unit, 35020 Legnaro, Padova, Italy.
authors: S
authors: S
authors: Catania
authors: Istituto Zooprofilattico Sperimentale delle Venezie, Diagnostic Service Avian Medicine Laboratory-Mycoplasma Unit, 35020 Legnaro, Padova, Italy.
authors: A
authors: A
authors: Matucci
authors: Istituto di Scienze delle Produzioni Alimentari-Consiglio Nazionale delle Ricerche (ISPA-CNR), Bioindustry Park S. Fumero, 10100 Giacosa, Torino, Italy.
authors: M G
authors: MG
authors: Giuffrida
authors: Istituto di Scienze delle Produzioni Alimentari-Consiglio Nazionale delle Ricerche (ISPA-CNR), Bioindustry Park S. Fumero, 10100 Giacosa, Torino, Italy.
authors: C
authors: C
authors: Baro
authors: University of Turin (UNITO), Department of Veterinary Science. 10100 Torino, Italy.
authors: M
authors: M
authors: Profiti
authors: University of Turin (UNITO), Department of Veterinary Science. 10100 Torino, Italy.
authors: L
authors: L
authors: Bertolotti
authors: University of Turin (UNITO), Department of Veterinary Science. 10100 Torino, Italy.
authors: S
authors: S
authors: Rosati
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D002645:Chickens / Q000382:microbiology*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D045686:Mycoplasma gallisepticum / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins
keywords: E2; Mycoplasma gallisepticum; recombinant protein