FIG. 2.

(A) Construction of integrative rBCG(pBlaF*nef-gag) strains by means of mycobacteriophage Ms6-derived vector pAV6950. The pBlaF*-nef-gag operon was isolated from pJLN29 and was cloned into the pAV6950 vector carrying the attP site and integrase gene from the Ms6 cassette. This gave rise to pOIP4 or pOIP12, according to the orientation of the expression cassette compared to the Ms6 int gene. In pOIP33, the pBlaF*-nef-gag operon was flanked by two T4 transcription terminators to prevent potential readthrough from outside promoters. (B) Western blot analysis of integrative rBCG pBlaF*-nef-gag strains. Western blotting was performed on 20 μg of total protein from rBCG strains cultured in vitro with anti-Gag(p26) monoclonal antibody (left panel) or with a serum from an SIVmac251-infected macaque reacting against the SIVmac251 Nef protein (right panel). On both gels positive controls were loaded on lane 1 and were either recombinant purified Gag(p26) (1 μg) or 10 μl of extract of insect cells infected with recombinant SIVmac251 nef baculovirus. Lanes for left and right panels: 2, rBCG(pJN30) (negative control); 3, rBCG(pJLN29); 4, rBCG::pOIP4; 5, rBCG::pOIP12; 6, rBCG::pOIP33. Comparable levels of Gag and Nef were produced by rBCG(pJLN29) and rBCG::pOIP4 strains. Approximately 10 times less Nef and Gag were produced by rBCG::pOIP33 than by rBCG::pOIP4. Nef and Gag proteins were barely detectable in rBCG::pOIP12.