Isolation and purification of ESAT6/CFP10 complex antigen secreted by Mycobacterium tuberculosis strains C

Introduction and Objectives:Tuberculosis (TB), caused by Mycobacterium tuberculosis,is one of the most prevalent infectious diseases. The pathogenesis of Mycobacterium tuberculosis is related to its proteins; the early secretory antigenic target (ESAT6) and culture filtrateprotein-10 (CFP10), expressed from the region of deletion-1 (RD1). These proteins are highly specific and potentially useful for the diagnosis of tuberculosis.This research focused on purification of low molecular weight proteins from M.tuberculosis C vaccinal strain by ammonium sulphate precipitation. Materials and Methods: M.tuberculosis C vaccinal strain grown in Löwenstein-Jensen medium and Dorset Henley liquidmediumwere inactivated by subjecting to 68ºC for 90 min, filtered, and the proteins in the supernatant fluid precipitated with ammonium sulphate at 4ºC. The obtained precipitates were dialyzed and subjected to gel chromatography (G-50) and the obtained fractions analyzed for protein concentrations by Lowry method. The approximate molecular weight in the purified fractions were determined by SDS-PAGE. Finally, ESAT6 and CFP10 protein complex in the purified fractions were confirmed by western blot analysis using ESAT6 specific antibody. Results: Maximum amount of the proteins were precipitated in the presence of 20 %ammonium sulphate. During SDS-PAGE analysis, protein bands of approximately 10-15 kDa were observed. Purity of the proteins preparations was ≥95%, as judged by sodium dodecyl sulfate (SDS)-PAGE and subsequent staining with Coomassie blue. The presence of ESAT-6/CFP10 complex in the partially purified fractions were confirmed by Western blot analysis. Conclusion: Sufficient amounts of ESAT-6/CFP-10 purified by the mentioned method might be suitable for the development of diagnostic kit for Mycobacterium tuberculosisin future.

کلیدواژه ها:

Mycobacterium tuberculosis ، ESAT-6/CFP10 ، purification ، chromatography ، SDS-PAGE ، Western blotting