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Cloning, expression and purification of Mycobacterium tuberculosis ESAT-6 and CFP-10 antigens

Shima Mahmoudi, Setareh Mamishi, Mona Ghazi, Reihaneh Hosseinpour Sadeghi, Babak Pourakbari
Iranian Journal of Microbiology 2013, 5 (4): 374-8

BACKGROUND AND OBJECTIVES: ESAT-6 (6-kDaearly secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein) have been described as dominant antigens recognized by T-cells and considered as virulence factors in Mycobacterium tuberculosis. The aim of this study was to clone, express and purify recombinant ESAT-6 andCFP-10 proteins of M. tuberculosis in soluble form.

MATERIALS AND METHODS: ESAT-6 andCFP-10 genes were amplified by PCR, cloned into pET32a (+) vector, and overexpress-ed using isopropyl-beta-D-thiogalactopyranoside in E. coli BL21 (DE3). ESAT-6 andCFP-10 proteins were purified by Ni-NTA affinity chromatography and were detected by anti- ESAT-6 and anti -CFP-10 antibodies.

RESULTS: ESAT-6 andCFP-10 genes were successfully expressed and purified. Anti- ESAT-6 and anti-CFP-10 antibodies were produced after induction of immunization against purified ESAT-6 andCFP-10 proteins in rabbit.

CONCLUSION: In this study, we cloned, expressed and purified sufficient amounts of ESAT-6 andCFP-10 and it would be tested for the development of diagnostic kit for M. tuberculosis in future.


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