OBJECTIVE: To screen and construct recombinant BCG strains which express the Ag85B-ESAT6 fusion protein. METHODS: The heat shock protein 60 (Hsp60) and the alpha-ss signal peptide encoding sequence were amplified by PCR from Mycobacterium tuberculosis H(37)Rv and cloned into E. coli/Mycobacteria shuttle vector-pOLYG. The resulting expression vector was named pDE22, and then ag85b and esat6 genes were cloned into pDE22 at different sites. The resulting recombinant plasmids Ag85B-ESAT6 and ESAT6-Ag85 were electroporated into BCG. Positive clones were screened by hygromycin resistance and confirmed by PCR. Recombinant BCG culture supernatants were collected and analyzed by SDS-PAGE and Western blot. RESULTS: Two recombinant BCG strains were obtained, which secreted the 37,000 fusion protein in their culture supernatant, which was confirmed by Western blot with specific immune serum against Ag85B and ESAT6. CONCLUSIONS: Recombinant BCG strains expressing Ag85B and ESAT6 fusion proteins of Mycobacterium tuberculosis were constructed. They may serve as new vaccine candidates for preventing tuberculosis.
OBJECTIVE: To screen and construct recombinant BCG strains which express the Ag85B-ESAT6 fusion protein. METHODS: The heat shock protein 60 (Hsp60) and the alpha-ss signal peptide encoding sequence were amplified by PCR from Mycobacterium tuberculosis H(37)Rv and cloned into E. coli/Mycobacteria shuttle vector-pOLYG. The resulting expression vector was named pDE22, and then ag85b and esat6 genes were cloned into pDE22 at different sites. The resulting recombinant plasmids Ag85B-ESAT6 and ESAT6-Ag85 were electroporated into BCG. Positive clones were screened by hygromycin resistance and confirmed by PCR. Recombinant BCG culture supernatants were collected and analyzed by SDS-PAGE and Western blot. RESULTS: Two recombinant BCG strains were obtained, which secreted the 37,000 fusion protein in their culture supernatant, which was confirmed by Western blot with specific immune serum against Ag85B and ESAT6. CONCLUSIONS: Recombinant BCG strains expressing Ag85B and ESAT6 fusion proteins of Mycobacterium tuberculosis were constructed. They may serve as new vaccine candidates for preventing tuberculosis.