Construction of Mtb۷۲F Plasmid as a DNA Vaccine Candidate for Mycobacterium tuberculosis

Background:  With one-third of the world’s population infected, tuberculosis (TB) is one of the most common infectious diseases and a major public health problem, especially in developing countries. The efficacy of the BCG vaccine for controlling the disease in adults is poor. The development of an effective TB vaccine is a global objective. An effective tuberculosis vaccine should stimulate cellular immunity. DNA vaccines are a new generation of vaccines with the potential to achieve this goal. The aim of this study was to produce a DNA vaccine of Mtb۷۲F.   Methods: mtb۳۲C, mtb۳۹, and mtb۳۲N were cloned into pcDNA۳.۱ using restriction enzyme digestion and T۴ DNA ligase. Colony-PCR and restriction enzyme digestion were performed to detect transformed bacteria. DNA sequencing confirmed the desired gene insertion into the vector. A Chinese hamster ovary (CHO) cell line was transfected with the recombinant plasmid and RT-PCR was performed to assess gene expression. Results: Gel electrophoresis showed the expected amplified gene fragments of ۴۲۹, ۶۱۴, and ۱۲۰۰ base pairs (bps) for mtb۳۲C, mtb۳۲N, and mtb۳۹, respectively. Enzyme digestion and gel electrophoresis showed the expected fragments, indicating the desired gene position and orientation in the recombinant plasmid. This finding was verified by DNA sequencing, and RT-PCR demonstrated gene expression in the CHO cell line. Conclusions: An Mtb۷۲F DNA plasmid was successfully constructed. This plasmid may be a candidate for animal immunizations.

Keywords:

Cloning , DNA vaccine , mtb۳۲C , mtb۳۲N , mtb۳۹ , Mycobacterium tuberculosis.