Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) ofEscherichia coliand Characterization of Purified Mutant Toxins

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Abstract
The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide‐directed site‐specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q‐R170L (glutamic acid at position 167 and arginine at position 170 from N‐terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q‐R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild‐type VT2vp1. Since the antigenic property of the E167Q‐R170L was demonstrated to be similar to that of the wild‐type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q‐R170L, as a toxoid is discussed.