Yousef Amini1, Mohsen Tafaghodi2, Saeid Amel Jamehdar1, Zahra Meshkat1, Bagher Moradi1, Mojtaba Sankian3. 1. Microbiology & Virology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran. 2. Nanotechnology Research Center, Pharmaceutical Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. 3. Immunology Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
Abstract
BACKGROUND: Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In this study, three immunogenic proteins of Mycobacterium tuberculosis, including HspX, Ppe44, 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 pET21b and pET32b vectors. Escherichia coli (E. coli) Top10 cells were transformed with the plasmid, and the purified plasmid was used to transform E. coli BL21 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 6His-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 0.2 mM of IPTG to E. coli BL-21 cells at 37 °C and 18 h of incubation. For Ppe44 and EsxV, the optimum expression conditions were 18 °C and 16 h of incubation. SDS-PAGE and Western blots confirmed that the desired proteins were produced. CONCLUSION: 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.
BACKGROUND: Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In this study, three immunogenic proteins of Mycobacterium tuberculosis, including HspX, Ppe44, 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 pET21b and pET32b vectors. Escherichia coli (E. coli) Top10 cells were transformed with the plasmid, and the purified plasmid was used to transform E. coli BL21 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 6His-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 0.2 mM of IPTG to E. coli BL-21 cells at 37 °C and 18 h of incubation. For Ppe44 and EsxV, the optimum expression conditions were 18 °C and 16 h of incubation. SDS-PAGE and Western blots confirmed that the desired proteins were produced. CONCLUSION: 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.