Background: Tuberculosis (TB) is a contagious disease caused by
Mycobacterium tuberculosis. Development of a new vaccine for tuberculosis requires immunogenic antigens capable of inducing suitable and long-lasting T cell immunity. The emergence of multidrugs and extensively drug-resistant strains of
M. tuberculosis has made it a global public health concern. Objectives: DNA vaccine is a straightforward method to introduce antigens to the host. In the present study, two immunodominant mycobacterial antigens (
Mtb32C and
HBHA) were isolated and cloned into pcDNA3.1 (+) to design and construct a new DNA vaccine. This vector is capable of producing new potent chimeric protein. Materials and Methods:
Mtb32C (Rv0125) and
heparin-binding haemagglutinin adhesion (
HBHA) genes were amplified using polymerase chain reaction (PCR) of
M. tuberculosis H37Rv genome and ligated into pcDNA3.1 (+). Colony-PCR and restriction enzyme analysis were used to confirm the accuracy of the cloning procedure. Results: In the current study, recombinant pcDNA3.1 (+) vector containing
Mtb32C and
HBHA genes was successfully constructed. The desired size of DNA fragment was observed using single and double digestion methods. Conclusions:
Mtb32C and
HBHA genes were successfully isolated from H37Rv genome and cloned into an eukaryotic expression vector. This vector can be considered as a vaccine to evaluate immune responses in animal models. Keywords: Vaccines, DNA;
HBHA protein; Mtb32 antigen; Mycobacterium tuberculosis