Cloning, expression, and antigenic characterization of recombinant protein ofMycoplasma gallisepticumexpressed inEscherichia coli
Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and lead ing to important economic losses in the poultry in dustry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in par ticular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma--free breeder flocks. In this study, we
... ied a component of the pyruvate dehydrogenase dihydrolipoamide acetyltrans ferase (i.e., E2) protein by 2--dimensional electrophore sis (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 rab bit 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 anti genic stability of the E2 protein which could repre sent a recombinant antigen with potential diagnostic applications.