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

authors:

avatar Zahra Farshadzadeh 1 , * , avatar Mojtaba Sankian 2 , avatar Forough Yousefi 2 , avatar Aida Gholobi 2 , avatar Reza Zarif 2 , avatar Mahboobeh Naderi nasab 2 , avatar Tahereh Rashed 2 , avatar Abdolreza Varasteh 2

Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Zahra.farshadzadeh@gmail.com, Iran
Booali Immunology Research Center Mashhad University of Medical Sciences, Iran
Jundishapur Journal of Microbiology: Vol.3, issue 2; 53-60
article type: Research Article
received: August 1, 2009
accepted: February 1, 2010

how to cite: Farshadzadeh Z, Sankian M, Yousefi F, Gholobi A, Zarif R, et al. Cloning, expression and purification of early secretory antigenic target 6kDa protein (ESAT-6) of Mycobacterium tuberculosis. Jundishapur J Microbiol. 2010;3(2): 53-60. 

Abstract

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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