1887

Abstract

Recombinant mycobacteria expressing extracellular proteins are leading candidates for new vaccines against tuberculosis and other mycobacterial diseases, and important tools both in antimycobacterial drug development and basic research in mycobacterial pathogenesis. Recombinant mycobacteria that stably overexpress and secrete major extracellular proteins of in native form on plasmids pSMT3 and pNBV1 were previously constructed by the authors. To enhance the versatility of this plasmid-based approach for mycobacterial protein expression, the /mycobacteria shuttle plasmid pGB9 was modified to accommodate mycobacterial genes expressed from their endogenous promoters. Previous studies showed that the modified plasmid, designated pGB9.2, derived from the cryptic plasmid pMF1, was present at a low copy number in both and mycobacteria, and expression of recombinant proteins was found to be at levels paralleling its copy number, that is, approximating their endogenous levels. Plasmid pGB9.2 was compatible with the shuttle vectors pSMT3 and pNBV1 and in combination with them it simultaneously expressed the 30 kDa extracellular protein FbpB. Plasmid pGB9.2 was stably maintained in the absence of selective pressure in three mycobacterial species: BCG, and . Plasmid pGB9.2 was found to be self-transmissible between both fast- and slow-growing mycobacteria, but not from mycobacteria to or between strains. The combination of two compatible plasmids in one BCG strain allows expression of recombinant mycobacterial proteins at different levels, a potentially important factor in optimizing vaccine potency.

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2004-07-01
2021-10-24
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