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Cloning, expression and purification of early secretory antigenic target 6kDa protein (ESAT-6) of Mycobacterium tuberculosis

Author(s): Zahra Farshadzadeh | Mojtaba Sankian | Forough Yousefi | Aida Gholobi | Reza Zarif | Mahboobeh Naderi nasab | Tahereh Rashed | Abdolreza Varasteh

Journal: Jundishapur Journal of Microbiology
ISSN 2008-3645

Volume: 3;
Issue: 2;
Start page: 53;
Date: 2010;
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Keywords: ESAT6 antigen | Cloning | Mycobacterium tuberculosis

Introduction and objective: The early secretory antigenic target 6kDa protein (ESAT-6) antigen from Mycobacterium tuberculosis is a dominant target for cell-mediated immunity in the early phase of tuberculosis (TB) in TB patients. ESAT-6 was found to distinguish TB patients from BCG-vaccinated donors. The aim of this study was cloning and expression of ESAT-6 of M. tuberculosis.Materials and methods: DNA was extracted from M. tuberculosis H37Rv. PCR was performed using gene-specific oligonucleotide primers and the PCR products were inserted into the pET102/D vector and transferred into Escherichia coli strain TOPO10. The recombinant plasmids transferred into E. coli strain BL21. Results: The transformed plasmid into E. coli strain BL21 was effectively expressed. The expressed fusion protein (23kDa on SDS-PAGE) was found almost entirely in the soluble form and the recombinant protein was purified by Ni-NTA column.Conclusion: We successfully cloned and expressed ESAT-6 protein of M. tuberculosis in E. coli. As a specific antigen, it can be useful for diagnosis of both active and latent tuberculosis with ELISA in future.
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