Cloning, expression, and antigenic characterization of recombinant protein ofMycoplasma gallisepticum expressed inEscherichia 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.

Cloning and expression of P67 protein of Mycoplasma leachii

“…Moreover, recombinant protein based serological tests show high sensitivity and specificity, owing to high concentration of immunoreactive antigens and the lack of non-specific moieties present in whole cell preparations [ 29 ]. Thus, recombinant antigen based diagnostic tests have effectively replaced whole cell antigen based immunodiagnostic assays in the diagnosis of various Mycoplasma infections pertaining to veterinary field [ 30 - 32 ]. Moreover, several authors have recently claimed that recombinant antigen based immunodiagnostics, especially ELISA have proven to be highly sensitive and specific method for the early diagnosis of bovine mycoplasmas such as M. bovis infection [ 33 - 35 ].…”