To confirm the in vitro expression of Mtb32C-HBHA fusion genes, the plasmid was transfected into HeLa cells (American Type Culture Collection Manassas, VA, USA) using lipofectamine transfection reagent according to the manufacturer’s instructions (Invitrogen, USA). Then, 72 hr after transfection, the cells were treated with 0.5 ml trypsin (Invitrogen, USA) and were incubated for 10 min. Cell suspension was harvested and total RNA was extracted using RNX-Plus (SinaClon, Iran), as described previously (16 (link), 17 (link)). Purified RNA was used for cDNA synthesis using cDNA synthesis kit (Pars Tous, Iran) and was amplified by PCR. To detect the presence of His-Tag marker in chimeric Mtb32C-HBHA protein (a marker of constructed rather than natural protein), Western blot method was performed using mouse anti-His Tag antibody as the primary antibody and peroxidase conjugated rabbit anti-mouse IgG as the secondary antibody (AbD SeroTec, USA).