Résumé
Knowing the nucleotide sequence of the cholera toxin operon, we designed oligonucleotide primers for its-PCR amplification from local clinical isolates of V. cholerae. The resulting amplification product was cloned in a common pUC18 vector. Subsequently, a part of this operon encoding the cholera toxin B-subunit [CTB] was reamplified and cloned between the BamH 1 and EcoR1 sites of the same vector to create a recombinant plasmid pRI8CTB. Temperature-controlled expression of the target protein was achieved by supplementing pR18CTB with a DNA fragment which contained a strong promoter PR and the gene for a heat-sensitive repressor cI857 of bacteriophage lambda from an expression vector pCQV2. When induced, the constructed plasmid pSCTB18 provided for the production of recombinant CTB secreted into the periplasmic space in a yield of about 3mg per liter of bacterial culture, as revealed by GM 1-ELISA