Design and construction of an expression plasmid containing mycobacterial ESAT-6, CFP-10 fusion protein cassette

Mycobacterium tuberculosis, the causative agent of tuberculosis disease, has infected roughly one third of the world's population. Based on World Health Organization (WHO) reports, around 2 million people die due to this disease annually, necessitating development of an effective, rapid and precise diagnostic strategies to detect tuberculosis. Quantification of interferon-γ which has been secreted by lymphocytes exposed to mycobacterial ESAT-6 and CFP-10 proteins is the basis of a specific diagnostic test to detect latent tuberculosis infection (LTBI). This method is an appropriate replacement for tuberculin skin test (TST) without limitations related to BCG vaccinated individuals. The aim of our study was to create an expression construct which contains genes encoding two specific proteins from M. tuberculosis: ESAT-6 and CFP-10. Expressed fusion protein could be applied to design tuberculosis diagnostic kits.Methods and Materials: Genomic DNA from Mycobacterium tuberculosis (H37Rv) was extracted and its concentration was determined. The primers were designed to amplify ESAT-6, CFP-10 and overlap segment of these two genes. PCR1, 2 for ESAT-6 and CFP-10 and finally Overlap PCR were performed. ESAT6-CFP10 fusion segment was cloned in pTZ57T/A cloning vector. PT-ESAT-CFP and PQE-60 plasmids were digested with BamHI and NcoI and ESAT-CFP DNA fragment and back bone of pQE60 were extracted from agarose gel. Then ESAT-CFP was sub-cloned into pQE60.The resulted colonies were analyzed by PCR, restriction endonuclease digestion and sequencing. The results of PCR, restriction enzyme digestion and sequencing for PCR products,pTZ clone and final plasmid, confirmed the authenticity of them.The purified fusion protein that resulted from expression of this construct can be used in diagnostic kit for detecting latent tuberculosis infection (LTBI).