Escherichia coli heat-labile toxin B subunit: Construction and evaluation of plasmids providing controlled high level production of the protein

ABSTRACT

With the plasmid DNA from a clinical isolate of enterotoxigenic Escherichia coli [ETEC] H10407 as template, PCR -mediated cloning of the sequence encoding the heat-labile toxin B subunit [L T -B] has been carried out Then this sequence was recloned into the pTrc 99A and pET23a expression vectors to give the plasmids pTRCLTB and pETLTB, respectively. After induction, the former plasmid provides for the production of rL T -Bin a yield of up to 15 mg per liter of bacterial culture. The recombinant protein was shown to be structurally and immunologically identical with the native L TB. High titer antibodies capable of neutralizing the native toxin were raised in mice by oral administration of the rL T - B. Hence the constructed plasmids provide the basis for an oral ETEC vaccine, as well as for genetic fusion of foreign antigens with the aim of developing polyvalent vaccines