Detection of Botulinum Neurotoxin Type A by In Vitro Bioassay Based on Endopeptidase Activity
Journal of Bacteriology and Virology
; : 29-37, 2010.
Article
Dans Ko
| WPRIM
| ID: wpr-136973
Responsable en Bibliothèque :
WPRO
ABSTRACT
Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that cleaves SNAP-25 (synaptosome-associated protein of 25 kDa), a specific cellular protein essential for neurotransmitter release. As well as mouse bioassay to detect BoNT/A, various assay methods based on its endopeptidase activity have been developed. In this study, we tried to develop a BoNT/A assay system using recombinant SNAP-25 with glutathione S-transferase (GST) tags at both termini as substrate. The recombinant GST-SNAP-25-GST with 70 kDa was expressed and purified in E. coli and synthesized N-terminal 50 kDa and C-terminal 25 kDa fragment after cleavage at the Gln(197)-Arg(198) bond by BoNT/A. To detect both fragments, we obtained rabbit antisera against peptides corresponding to the cleaved ends of each fragment. In the western blotting, the N-terminal fragment was detected by the antibody specifically recognizing the newly exposed C-terminus (corresponding to amino acid residue 191-197). This assay system was able to detect until 3.125 ng of BoNT/A, which corresponded to about 90 fold LD50 in mice. These results suggest that the in vitro endopeptidase assay developed in this study would replace others to detect BoNT/A.
Texte intégral:
1
Indice:
WPRIM
Sujet Principal:
Peptides
/
Dosage biologique
/
Technique de Western
/
Agents neuromédiateurs
/
Glutathione transferase
/
Sérums immuns
/
Dose létale 50
Type d'étude:
Diagnostic_studies
Limites du sujet:
Animals
langue:
Ko
Texte intégral:
Journal of Bacteriology and Virology
Année:
2010
Type:
Article