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Medeiros, M.A., Dellagostin, O.A., Armôa, G.R., Degrave, W.M., De Mendonça-Lima, L., Lopes, M.Q., Costa, J.F., Mcfadden, J. and McIntosh, D. (2002) Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics. Microbiology, 148, 1999-2009.

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• TITLE: Plasmid instability when the hsp60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

  AUTHORS: Dilzamar V. Nascimento, Odir A. Dellagostin, Raphael Hirata Junior, Geraldo M. B. Pereira, Ana Luiza Mattos-Guaraldi, Geraldo R. Garcia Armôa

  KEYWORDS: BCG Vaccine; Recombinant BCG; Fragment B of Diphtheria Toxin; Anti-Diphtheria Vaccine

  JOURNAL NAME: American Journal of Molecular Biology, Vol.3 No.2, April 30, 2013

  ABSTRACT: The genetic modification of the live attenuated Mycobacterium bovis BCG to deliver a protective Corynebacterium 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 transformation 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 PAN promoter from M. paratuberculosis stably expressed the dtb gene. Conversely, rBCGs transformed with the construct carrying the strong mycobacterium hsp60 promoter were unstable and consequently unfit for the expression of the C. diphtheriae gene.