Molecular cloning and expression of shiga-like toxin II gene (slt-II) form an isolate of healthy Korean native bovine feces, Escherichia coli KSC109

By PCR amplification using the sequence of the previously cloned shiga-like toxin n DNA, a gene encoding it has been cloned from an isolate of healthy Korean native bovine feces, Escherichia coli KSC109. The nucleotide sequences included two open reading frames coding for 319 and 89 amino acids corresponding to A and B subunits, respectively. Comparsion of the nucleotide and predicted amino acid sequences of newly cloned gene (sft-II) with those of others in the SLT-II family revealed completely identical homology with SLT-II cloned previously from bacteriophage DNA of E. coli 933 derived from a patient with hemorrhagic colitis. In addition, the sequence homology of SLT-II with SLT-II variant from bovine was more than 95% at both the nucleotide and protein levels. Overexpression of SLT-II recombinant gene by induction with IPTG using an E. coli host-vector system was conducted and the correctly processed products with active mature form exhibited 1000-fold higher cytotoxicity for Vero cells than that from original strain.ope