Introduction: Tuberculosis which has killed many people and caused many financial damages since many years ago is one of the major health problems. In spite of serious efforts to prevent disease, it is one of the important causes of death throughout world with two million death annually. One of the most important immunizing proteins of Mycobacterium tuberculosis is ESAT–6 which is a 9.8KDa polypeptide and is recognized by T cells and stimulates strong cell immunity responses and produces high amount of IFN–γ. Objects and methods: This research was done in order to clone and sequence the ESAT–6 protein from Mycobacterium tuberculosis into a eukaryotic expression vector named pEGFP– N1.Characterizing of this gene by PCR cloning, restriction enzyme analysis and sequencing the gene can be the basis to produce a type of Stable Cell Line to be used for evaluation of ESAT-6 immune responses. Results: Comparison of the nucleotide sequence of this gene and sequence of amino acid product of that with registered sequence in gene bank shows that this gene with isolated type (Rv3875) has high identity. This recombinant plasmid will be express on the CT-26 cell line which is a tumor cell from the BaIb/C mouse.
