Community Research and Development Information Service - CORDIS



Project ID: ICA4-CT-2000-30032
Funded under: FP5-INCO 2
Country: Brazil

generation of leuD/BCG vector-host system for expressing recombinant proteins in BCG

Mycobacterium bovis BCG has been studied as a live vaccine vector with the aim of developing an efficient multivalent vaccine. Most mycobacterial cloning vectors rely on antibiotic resistance genes as selectable markers. We report the use of auxotrophic complementation as a selectable marker. BCG auxotrophic for the amino acid leucine was constructed by knocking out the leuD gene by unmarked homologous recombination. Expression of leuD in a plasmid not only allowed complementation, but also acted as a selectable marker. Removal of the kanamycin resistance gene, still necessary for plasmid manipulation in E. coli, was accomplished by two different methods: restriction enzyme digestion followed by re-ligation before BCG transformation, or by Cre-loxP in vitro recombination mediated by the bacteriophage P1 Cre recombinase. Both methods worked well, however digestion and re-ligation was more efficient resulting in higher transformation efficiency. Stability of the plasmid was evaluated during in vitro and in vivo growth of the recombinant BCG in comparison to selection by antibiotic resistance. The new system was highly stable even during in vivo growth, as the selective pressure is maintained, whereas the conventional vector was unstable in the absence of selective pressure.

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Campus Universitario
96010-900 PELOTAS
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