Cao C, Kurazono H, Yamasaki S, Kashiwagi K, Igarashi K, Takeda Y
Department of Microbiology, Faculty of Medicine, Kyoto University, Japan.
Microbiol Immunol. 1994;38(6):441-7. doi: 10.1111/j.1348-0421.1994.tb01805.x.
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.
编码维罗毒素2变体VTvp1的基因通过寡核苷酸定向位点特异性诱变进行了突变。在由突变基因编码的6种突变毒素中，发现E167Q-R170L（A亚基N端第167位的谷氨酸和第170位的精氨酸分别被谷氨酰胺和亮氨酸取代）的活性显著降低；纯化的E167Q-R170L对蛋白质合成的抑制、对Vero细胞的细胞毒性以及对小鼠的致死率分别是纯化的野生型VT2vp1的1/1900、1/125000和1/2000。由于通过Ouchterlony双凝胶扩散试验以及对VT2vp1的Vero细胞细胞毒性进行中和试验证明E167Q-R170L的抗原特性与野生型VT2vp1相似，因此讨论了将突变体VT2vp1 E167Q-R170L用作类毒素的可能性。
