FIG. 1.

Expression of HIV-1 HXBc2 gp120 envelope in nonpathogenic Mycobacterium smegmatis. (A) Codon-optimized HXBc2 gp120 env was cloned into the E. coli/mycobacteria shuttle plasmids pJH222 (multicopy) and pJH223 (integrative). The gp120 env in the plasmids is under the control of the M. tuberculosis α-Ag promoter (P α-Ag). A fusion protein was created in which the M. tuberculosis 19-kDa signal sequence was at the N terminus and an influenza hemagglutinin (HA) epitope tag was at the C terminus of the gp120 Env. Both plasmids contained the Tn903-derived aph gene conferring kanamycin resistance as a selectable marker and an E. coli origin of replication (oriE). The origin of replication (oriM) was inserted into the pJH222 plasmid, while the attP site and the int gene of mycobacteriophage L5 were included in pJH223. (B) Western blot analysis showed expression of the gp120 protein in recombinant M. smegmatis MC²155. The gp120 expression of three independent clones of mycobacteria transformed with either pJH222-gp120 (lanes 1 to 3) or pJH223-gp120 (lanes 4 to 6) was determined using an anti-HA MAb (clone 3F10). Mycobacteria transformed with either mock pJH222 or pJH223 containing an irrelevant gene (malaria msp1) were utilized as negative controls (lanes 7 and 8). MW, molecular mass.