Volume 14, Issue 4 (2012)                   mjms 2012, 14(4): 1-12 | Back to browse issues page

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Aghababa H, Mohabbati Mobarez A, Behmanesh M, Khoramabadi N, Nejati M. Cloning, Expression and Purification of Mycolyltransferase C of Mycobacterium tuberculosis in prokaryotic host. mjms. 2012; 14 (4) :1-12
URL: http://mjms.modares.ac.ir/article-30-11841-en.html
1- Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2- Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
3- Head Department of Bacterial Vaccines, Production and Research Complex, Pasteur Institute of Iran, Alborz, Iran
Abstract:   (4553 Views)
Objective: Antigen 85 complex of Mycobacterium tuberculosis includes three immunogenic proteins which are important TB vaccine candidates. The use of these protein as a component of subunit vaccines, as a part of DNA vaccines or recombinant BCG boosters, enhances their recombinant production. Recombinant production of these mycobacterial proteins located in the cell wall somehow differs from the other proteins, as they are partially apolar. Therefore, this study aims to produce recombinant Ag85C as a vaccine candidate. Methods: Ag85C gene was cloned in pJET1.2 and subsequently in pET32a(+). Both recombinant plasmids were sequenced. Expression of the recombinant protein was induced with 1mM IPTG. Recombinant Ag85C was purified through dissolving the inclusions in 8M urea buffer, absorbed to Ni-NTA resins, washed by buffers with decreasing urea concentrations, and finally eluted in aqueous solution. Western blot analysis was performed using anti-6His tag antibody, rabbit anti-M. tuberculosis polyclonal antibody, and the serum from hospitalized TB patients. Results: Ag85C successfully cloned in both plasmid vectors. The recombinant Ag85C expressed in the E. coli host and was purified with significant yield. Conclusion: Western blot results along with that of sequencing ensure accurate production of recombinant Ag85C, retaining its partial epitopes
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Article Type: Original Manuscipt | Subject: Molecular|Bacteriology
Received: 2011/05/25 | Accepted: 2011/12/7

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