Yousef Amini - Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
Mohsen Tafaghodi - Nanotechnology Research Center, Pharmaceutical Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
Saied Amel Jamehdar - Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
zahra Meshkat - Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
Bagher Moradi - Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
Mojtaba Sankian - Immunology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In this study, three immunogenic proteins of Mycobacterium tuberculosis, including HspX, Ppe۴۴, and EsxV as a new construction, were expressed alone and as a fusion protein to develop a new vaccine candidate against tuberculosis infection. Methods: To make the fusion protein, the three genes were linked together by AEAAAKEAAAKA linkers and inserted into pET۲۱b and pET۳۲b vectors. Escherichia coli (E. coli) Top۱۰ cells were transformed with the plasmid, and the purified plasmid was used to transform E. coli BL۲۱ cells. Protein expression was induced with IPTG. After optimizing protein expression, the recombinant proteins were purified by Ni-NTA chromatography. Protein purification was confirmed by SDS-PAGE and Western blotting with an anti-poly histidine-peroxidase monoclonal antibody against the ۶His–tags at the proteins’ C termini. Results: Directional cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme digestion, and sequencing. The highest expression of the tri-fusion protein and HspX were obtained by the addition of ۰.۲ mM of IPTG to E. coli BL-۲۱ cells at ۳۷ ˚C and ۱۸ h of incubation. For Ppe۴۴ and EsxV, the optimum expression conditions were ۱۸ ˚C and ۱۶ h of incubation. SDS-PAGE and Western blots confirmed that the desired proteins were produced. Conclusions: The three desired proteins and the fusion protein were successfully expressed and the conditions for optimum expression determined. These recombinant proteins will be evaluated as vaccine candidates against tuberculosis. Further studies are needed to evaluate the abilities of these proteins to induce strong immunological responses.

EsxV, Expression, HspX, Mycobacterium tuberculosis, Ppe۴۴, Purification