PCR-MEDIATED CLONING A ND EXPRESSION OF THE GENE FOR THE B-SUBUNIT OF VIBRIO CHOLERAE TOXIN ISOLATED RECENTLY IN IRAN

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 Bsubunit (CTB) was reamplified and cloned between the BamH1 and EcoR1 sites of the same vector to create a recombinant plasmid pR18CTB. Temperaturecontrolled 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 a n 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 OM 1- ELISA.