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.