Construction of pcDNA3.1 Vector Encoding rpfD Gene of Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). TB is still a major health problem. The Bacillus Calmette-Guérin (BCG) vaccine is the only one available for TB and is known to confer variable levels of protection. Because of this variability, a new vaccine is needed to control TB. Proteins secreted by M. tuberculosis are known to induce protective immunity. Within the genome of M. tuberculosis, there is a family of proteins called resuscitation promoting factor (Rpf), which plays a role in the reactivation of M. tuberculosis. RpfD is a member of the Rpf family that has been shown to be immunogenic, making it suitable for use as a TB vaccine. The rpfD gene of the M. tuberculosis Beijing strain from the bacterial stock of the Department of Microbiology at the Medical Faculty of the Universitas Indonesia was amplified using polymerase chain reaction (PCR) and then inserted into the mammalian expression vector pcDNA3.1(+). Then, the pcDNA3.1(+)-rpfD vector was transformed to Escherichia coli DH5α. A 465-bp target fragment was obtained, and the accuracy of the cloning was confirmed using colony PCR, restriction enzyme digestion, and sequencing. We expect that this recombinant plasmid will induce immunity in future animal models and thus will prove itself to be a candidate for an M. tuberculosis vaccine.

Keywords

• Tuberculosis
• M. tuberculosis
• Mammalian expression vector pcDNA3.1(+)
• Resuscitation promoting factor D (rpfD)