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KMID : 0359320010410020189
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Korean Journal of Veterinary Research
2001 Volume.41 No. 2 p.189 ~ p.195
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Production of toxoid and monoclonal antibody by mutation of toxin gene from Escherichia coli O157 : H7 for detection of low levels of the toxin
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김상현/Kim, Sang Hyun
김용환/이은주/강호조/차인호/이우원/Kim, Yong Hwan/Lee, Eun Joo/Kang, Ho Jo/Cha, In Ho/Lee, Woo Won
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Abstract
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Single base substitution and deletion mutation have been introduced into the verotoxin 2 (VT2)A subunit gene from O157:H7 isolates to reduce cytotoxicity of VT2 and the cytotoxicity between wild type toxin and mutant toxoid were compared. A M13-derived recombinant plasmid pEP19RF containing a 940bp EcoRI-PstI fragment of VT2A gene was constructed for oligonucleotide-directed mutagenesis. The double mutant pDOEX was constructed by point and deletion mutation of two different highly conserved regions of VT2A encoding active site cleft of enzymatic domain. The key residue, Glu 167(GAA) and the pentamer (WGRIS) consisting of the enzymatic domain were replaced by ASP(GAC) and completely deleted in nucleotide sequence analysis of mutant, respectively. In the comparision of vero cell cytotoxicity between wide type toxin and toxoid from mutant, the wild type toxin expressed cytotoxicity in dilution of 10^(-6), but the toxoid from mutant did not show cytotoxicity to vero cells.
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