The Nucleotide and Deduced Amino Acid Sequences of EcoRI Fragment Containing the 5′-Terminal Region of Clostridium botulinum Type E Toxin Gene Cloned from Mashike, Iwanai and Otaru Strains

Chromosomal DNAs were extracted from toxigenic three Clostridium botulinum type E strains isolated from food-borne botulism. After digestion by EcoRI, the fragments were cloned into Escherichia coli by using bacteriophage lambda gt11 and screened with monoclonal antibody recognizing the light chain component of botulinum type E toxin. The fragments (about 1kbp size) cloned from each strain were recloned into a plasmid vector pUC118. The E. coli cells transformed with the recombinant plasmids produced 33kDa protein with or without IPTG (isopropyl-β-D-thiogalactopyranoside) which reacted with the monoclonal antibody. The nucleotide sequences of the cloned EcoRI fragments from the three type E strains were identical and contain the 5'-terminal region of the type E toxin gene. It was also found that there exist several highly homologous nucleotide sequences among the botulinum types A, C and E, and tetanus toxin genes in both translated and untranslated regions.