Volume 12, Number 2 (8-1998)                   Med J Islam Repub Iran 1998 | Back to browse issues page


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SHAH-HOSSEINY M, AKBARI M, TABARRAI B, RECHINSKY V. PCR-MEDIATED CLONING A ND EXPRESSION OF THE GENE FOR THE B-SUBUNIT OF VIBRIO CHOLERAE TOXIN ISOLATED RECENTLY IN IRAN. Med J Islam Repub Iran. 1998; 12 (2) :123-128
URL: http://mjiri.iums.ac.ir/article-1-1017-en.html

From the Biology Unit, Faculty of Science, Imam Hossein University, Tehran
Abstract:   (1770 Views)
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.
Keywords: V. cholerae, toxin, cloning, PCR
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