Production of a non-toxic site-directed mutant of Clostridium perfringens epsilon-toxin which induces protective immunity in mice.
A panel of ten site-directed mutants of Clostridium perfringens epsilon-toxin was generated. All of the mutated proteins expressed in Escherichia coli were recognized in immunoblots by a neutralizing mAb raised against wild-type native epsilon-toxin. The cytotoxicity of the site-directed mutated toxins was assayed in vitro against MDCK cells. One mutation resulting in loss of activity in the assay was identified. This non-toxic protein was derived by substituting a proline for the histidine at residue 106 of the toxin. Immunization of mice with the non-toxic mutated epsilon-toxin resulted in the induction of a specific antibody response and immunized mice were protected against 1000 LD50 doses of wild-type recombinant epsilon-toxin.
Oyston
Petra C F
PCF
Defence Evaluation and Research Agency, CBD Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
Payne
Dean W
DW
Defence Evaluation and Research Agency, CBD Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
Havard
Helen L
HL
Defence Evaluation and Research Agency, CBD Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
Williamson
E Diane
ED
Defence Evaluation and Research Agency, CBD Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
Titball
Richard W
RW
Defence Evaluation and Research Agency, CBD Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
D019943:Amino Acid Substitution; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism*; D001681:Biological Assay; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003015:Clostridium Infections / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D003016:Clostridium perfringens / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000235:genetics; D015151:Immunoblotting; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D010957:Plasmids; D011392:Proline / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology; D014611:Vaccination; D014774:Virulence / Q000235:genetics
D001427:Bacterial Toxins; C039811:Clostridium perfringens epsilon-toxin; D017931:DNA Primers; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006639:Histidine; D011392:Proline