Generating Recombinant BCG Strains Expressing HIV-1 p24

To generate three different types of rBCG strains expressing HIV-1 p24 Gag, BCG with the pMyong2-p24 plasmid (designated rBCG-pMyong2-p24), BCG with the pAL-p24 plasmid (designated rBCG-pAL-p24), and BCG with the pMV306-p24 plasmid (designated rBCG-pMV306-p24), the three constructed plasmids, i.e., pMV306-p24, pAL-p24, and pMyong2-p24 (40 (link)), were electroporated into the competent BCG strain (Tokyo 172) using the Gene Pulser II electroporation apparatus (Bio-Rad, Hercules, CA, USA) (58 (link)). Transformants were selected on Middlebrook 7H10 medium (Difco Laboratories, Detroit, MI, USA) supplemented with OADC containing 100 µg/ml of kanamycin. Typically, colonies of transformants were selected from the plates, transferred into 7H9 broth medium (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% glycerol, 0.05% Tween-80, 10% ADC and kanamycin and cultured for 3~4 weeks. The growth rate of the rBCG strains was determined by optical density (OD) at 600 nm.