Construction of Mtb72F Plasmid as a DNA Vaccine Candidate for .

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 Mtb72F.

Methods: , and were cloned into pcDNA3.1 using restriction enzyme digestion and 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 429, 614, and 1200 base pairs (bps) for , and , 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.

Conclusion: An Mtb72F DNA plasmid was successfully constructed. This plasmid may be a candidate for animal immunizations.