Plasmid instability when the hsp 60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

Abstract

The genetic modification of the live attenuated My-cobacterium bovis BCG to deliver a protective Cory-nebacterium diphtheriae antigen in vivo could be a safer and less costly alternative to the new and more expensive DTP vaccines available today, in particular to third world-countries. The stability of expression of heterologous antigens in BCG, however, is a major challenge to the use of live recombinant bacteria in vaccine development and appears to be dependent to a certain extent, on a genetic compatibility between the expression cassette within the plasmid construct and the mycobacterium host. In the quest for the best recombinant BCG transformant to express the dtb gene of C. diphtheriae we generated two new rBCG strains by transforming the Moreau substrain of BCG with the mycobacterial expression vectors pUS973 and pUS977, each one carrying a different promoter to drive the expression of the target antigen. After trans-formation recombinant BCG clones were selected on Middlebrook 7H10 kanamycin Agar plates, expanded in Middlebrook 7H9 kanamycin Broth and analyzed by agarose gel electrophoresis and immunoblotting. rBCGs transformed with the construct carrying the weak P AN promoter from M. paratuberculosis stably expressed the dtb gene. Conversely, rBCGs trans-formed with the construct carrying the strong myco-bacterium hsp60 promoter were unstable and conse-quently unfit for the expression of the C. diphtheriae gene.