Construction of mutant genes for a non-toxic verotoxin 2 variant (VT2vp1) of Escherichia coli and characterization of purified mutant toxins.
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.
Department of Microbiology, Faculty of Medicine, Kyoto University, Japan.
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Cao
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Kurazono
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Yamasaki
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Kashiwagi
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Igarashi
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Takeda
D000818:Animals; D001427:Bacterial Toxins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D005779:Immunodiffusion; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D017354:Point Mutation; D022641:Shiga Toxin 2; D014709:Vero Cells / Q000382:microbiology
D001427:Bacterial Toxins; D004768:Enterotoxins; D009841:Oligonucleotides; D022641:Shiga Toxin 2