Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors
Figure 4
Expression of HIV-1gp120 protein in rBCG:HIV-1gp120 (pMV261) and rBCG:HIV-1gp120 (pJH222) strains by Western blot analysis of whole-cell BCG lysates. (a) A band of 28 kDa was detected when analizing the rBCG:HIV-1gp120 (pMV261) colonies. (b) A 67 kDa protein was detected in lysates of rBCG:HIV-1gp120 (pJH222) bacterial cells containing the 1.82 kilobase (kb) HIV-1gp120 DNA coding sequence into pJH222 vector. The gp120 expression was determined using anti-HA MAb. Not transformed BCG (BCG wild type) were utilized as negative controls. (c) The HIV-1gp120 (HXBc2) gene was cloned into the E.coli/mycobacteria shuttle plasmids pMV261 and pJH222 (both multicopy and episomal). The gp120 env in the plasmids is under the control of BCG hsp60 promoter (P hsp60) and M.Tuberculosis -antigen promoter (P -Ag). Both plasmids contained kanamycin resistance gene (aph) and an E.coli origin of replication (ori E) and a mycobacterial origin of replication (oriM). In addition, pJH222 contained the complementing lysA gene. The HIV-1gp120 gene was fused to the first six codons of BCG hsp60 (pMV261) or fused to M. tuberculosis 19-kDa lipoprotein signal sequence (pJH222). MWM, molecular weight markers.
