Document Type : Research Paper

Authors

• Mitra Ashayeripanah ¹
• fereshteh eftekhar ²
• Bahram Kazemi ³
• Mohammad Mehdi Feizabadi ⁴

¹ Department of Microbiology, Faculty of Biological Sciences and Technology, Shahid Beheshti University

² Associate Professor Department of Microbiology, Faculty of Biological Sciences and Technology, Shahid Beheshti University

³ Professor Department of Biotechnology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences

⁴ Professor Department of Microbiology, Faculty of Medicine, Tehran University of Medical Sciences

Abstract

Due to the large population latently infected with Mycobacterium tuberculosis worldwide, vaccines against latent tuberculosis can make a dramatic impact on the global tuberculosis problem. In this study, bioinformatics analysis of a latency antigen from M. tuberculosis, Rv1733c, predicted that Rv1733c is an integral-membrane protein with a large number of B-cell and T-cell epitopes, making it a potential vaccine candidate. Therefore, rv1733c gene from M. tuberculosis strain H37Rv was chemically synthesized, inserted into pTG19-T plasmid and the recombinant plasmid was transformed into E. coli Top10. After confirming the presence of the DNA fragment by digestion with restriction endonucleases and sequencing, it was subcloned into pET-23a (+) and expressed in E. coli strain BL21 (DE3). The recombinant protein was observed as a band of ~ 24 kDa on SDS-PAGE gel and western blot analysis using an antibody against the hexa-histidine-tag of the protein revealed a band with the same size. Finally, the protein was successfully purified using a histidine-tag purification kit.

Keywords

• Rv1733c
• Mycobacterium tuberculosis
• Escherichia coli
• Cloning and Expression
• Protein purification