1. Cloning and expression of a secretory protein Esat-6 of Mycobacterium tuberculosis in E. coli
Ahangarzadeh, Sh. *1 Bandehpour, M.1, Kazemi, B.1 , Yarian, F.1
1- Shahid Beheshti University of Medical Sciences, Medical college, Biotechnology Department *
Results:
The desired gene is 300 nucleotides and it was Successfully PCR and cloned in pQE30 vector with enzymatic digestion. The construct was confirmed by colony PCR, digestion and sequencing. The expressed protein was analyzed on SDS-PAGE and confirmed by western blotting using specific antibody to His tag.
Introduction:
Tuberculosis remains a major infectious disease with over eight million new cases and two million deaths annually, and improved diagnostic methods are needed urgently. Diagnosis of latent Mycobacterium tuberculosis infection is considered essential for tuberculosis control but is hampered by the lack of specific reagents. The development of improved diagnostic tests requires detailed understanding of the immune responses generated and the antigens recognized during the disease. An immunogenic antigen, 6-kDa early secretory antigenic target (Esat-6), is highly specific to M. tuberculosis complex but is absent from Mycobacterium bovis BCG. This study was designed for cloning and expression of ESAT-6 as a potent antigen of M. tuberculosis.
Material and methods:
In this study, A DNA encoding the Esat-6 of Mycobacterium tuberculosis was amplified by PCR using specific primers with appropriate restriction enzyme sites in their ends. The gene was cloned in the pQE30 vector. Extracted recombinant plasmids were transformed into E. coli BL21 by heat shock and were plated on an LB agar containing
ampicillin at 37°C overnight. Overnight incubated colonies were inoculated into an LB broth and was grown to OD , then IPTG added and growth was continued on shaker incubator at 37°C. Then, the total broth media was centrifuged and the bacteria cell pellet was collected. Bacteria was disrupted at 0º C using a sonicator. This extract was centrifuged for 20 minutes at rpm. Fraction containing ESAT-6 was analyzed by SDS-PAGE electrophoresis. Result of SDS-PAGE was confirmed by western blotting using anti His Tag antibody.
cut uncut
10 KDa
Conclusion:
We well cloned and expressed Esat-6 protein of M. tuberculosis in E. coli. As Esat-6 is a specific antigen and produce in early stage of infection, it can be valuable for diagnosis of both active and latent tuberculosis in future.
References:
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2-Wu, X., et al., Comparison of antibody responses to seventeen antigens from Mycobacterium tuberculosis. Clin Chim Acta, (19-20): p
3-Kashyap, R.S., et al., Diagnostic value of early secreted antigenic target-6 for the diagnosis of tuberculous meningitis patients. Infection, (6): p
4- Abebe, F., et al., Progress in serodiagnosis of Mycobacterium tuberculosis infection. Scand J Immunol, (2-3): p
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