Iranian Red Crescent Medical Journal ORIGINAL ARTICLE Expression and Purification of Recombinant Mycobacterium Tuberculosis (TB) Antigens, ESAT-6, CFP-10 and ESAT- 6/CFP-10 and Their Diagnosis Potential for Detection of TB Patients M Hemmati1, A Seghatoleslam1, M 1 Rasti , S M Taheri 4 , AR Sardarian 5 , Z Mostafavi-Pour1,6 Ebadat1, N 2 Mosavari , M Habibagahi3, 1Department of Biochemistry, Recombinant Protein Laboratory, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran 2Department of PPD Tuberculin, Razi Vaccine and Serum Research In- stitute, Tehran, Iran 3Department of Immunology, Medical School, Shiraz University of Medical Scienc- es, Shiraz, Iran 4Department of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran 5Dental School, Shiraz University of Medical Sciences, Shiraz, Iran 6Department of Biomedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran Abstract Background: One of the most widely used methods to detect tuberculosis (TB) infection is the tuberculin skin test (TST). The completion of Mycobacterium tuberculosis (M. tuberculosis) genome sequence has led to identi- fication of several antigens that can be utilized for accurate diagnosis and control of TB. The aim of this study was to purify the recombinant M. tuberculosis antigens for the evaluation of their potential in TB diagnosis. Methods: The recombinant secretory antigens, ESAT-6, CFP-10 and ESAT-6/CFP-10 were produced by PCR and cloning methods. To investigate antigen specific responses of these recombinant antigens in detection of TB, ex vivo enzyme linked immunospot (ELISPOT) test in 30 clinically diagnosed TB patients was evaluated. Results: The selected M. tuberculosis antigens were cloned, expressed and purified in Escherichia coli (BL21). ELISPOT assay for detection of TB showed the sensitivity of 93, 90 and 100% for recombinant ESAT-6, CFP-10 and ESAT-6/CFP-10 proteins respectively, which is significantly higher than conventional TST. Conclusion: The recombinant antigens of ESAT-6, CFP-10 and ESAT-6/CFP-10 can be used as an accurate means of detecting TB in Iran. Keywords: Mycobacterium tuberculosis; ELISPOT; CFP-10; ESAT-6; Tuberculosis; Diagnosis Introduction of Tuberculosis Despite more than a century of research on detection and treatment (TB) infection throughout the world, the disease remains a major cause of morbidity and mortality. TB accounts for approximately three million deaths annually with nine million new cases of the disease diagnosed each *Correspondence: Zohreh Mostafavi-Pour, PhD, Associate profes- sor of Biochemistry, Shiraz University of Medical Sciences, Recom- binant Proteins Lab, Faculty for advanced Biomedical Sciences, PO Box: 71345-1167, Shiraz, Iran. Tel: +98-711-2303029, Fax: +98- 711-2303029, e-mail: zmostafavipour88@yahoo.co.uk Received: November 25, 2010 Accepted: April 11, 2011 year.1,2 In 2008, the World Health Organization (WHO) estimated that yearly about 1.7 million people die because of TB.3 The incidence of all forms of TB in Eastern Mediterranean sub region countries such as Iran is 104 per 100000 and the incidence of cases which were smear positive is 24 per 100000 popula- tions.4 In Iran, most of the new TB cases occur among immigrants especially in Afghan immigrants. The major cause of TB is Mycobacterium tubercu- losis (M. tuberculosis) which is an intracellular path- ogen. Alveolar macrophages are the main target of invasion by M. tuberculosis bacilli that have the po- tential to survive and grow in this cells.5 Fast and ac- curate diagnosis of TB is an important element in Iran Red Crescent Med J 2011; 13(8):556-563 ©Iranian Red Crescent Medical Journal