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

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

Centro de Biotecnologia, Universidade Federal de Pelotas, Campus Universitário, Pelotas, Brazil.
Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil.
Instituto Oswaldo Cruz, Funda??o Oswaldo Cruz, Rio de Janeiro, Brazil.

DOI: 10.4236/ajmb.2013.32011                

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 P_AN 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.

Keywords

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

Conflicts of Interest

The authors declare no conflicts of interest.

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