Production of toxoid and monoclonal antibody by mutation of toxin gene from Escherichia coli O157: H7 for detection of low levels of the toxin I. Expression of toxoid by mutagenesis of verotoxin gene

대장균 O157:H7의 독소 생성 유전자의 변이에 의한 변성독소 생산 및 미량독소 검출을 위한 단클론성 항체생산 I. 독소 생성 유전자의 변이에 의한 변성독소의 발현

Single base substitution and deletion mutation have been introducted 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 duoble 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 toxid from mutant did not show cytotoxicity to vero cells.