Method and compositions for detecting botulinum neurotoxin

C - Chemistry – Metallurgy – 12 – N

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C12N 15/62 (2006.01) C07K 14/33 (2006.01) C07K 19/00 (2006.01) C12N 1/21 (2006.01) C12N 5/10 (2006.01) C12N 15/12 (2006.01) C12N 15/31 (2006.01) C12N 15/63 (2006.01) C12N 15/85 (2006.01) C12Q 1/04 (2006.01) C12Q 1/37 (2006.01) G01N 33/58 (2006.01)

Patent

CA 2550401

A molecular construct capable of fluorescent resonance energy transfer (FRET), comprising a linker peptide, a donor fluorophore moiety and an acceptor fluorophore moiety, wherein the linker peptide is a substrate of a botulinum neurotoxin selected from the group consisting of synaptobrevin, syntaxin and SNAP-25, or a fragment thereof capable being cleaved by the botulinum neurotoxin, and separates the donor and acceptor fluorophores by a distance of not more than 10 nm, and wherein emission spectrum of the donor fluorophore moiety overlaps with the excitation spectrum of the acceptor fluorophore moiety; or wherein the emission spectra of the fluorophores are detectably different. Also provided are isolated nucleic acid expressing the construct, kits comprising said construct and cell lines comprising said nucleic acid. Further provided are methods of detecting a BoNT using the above described construct via FRET, and methods for detecting a BoNT using surface plasmon resonance imaging.

L'invention concerne une construction moléculaire capable de réaliser un transfert d'énergie de fluorescence par résonance (FRET) comprenant un peptide de liaison, une fraction fluorophore donneur et une fraction fluorophore accepteur. Le peptide de liaison est un substrat d'une neurotoxine botulique choisi dans le groupe constitué de la synaptobrévine, la syntaxine et la SNAP-25 ou un fragment de celles-ci pouvant être clivé par la neurotoxine botulique et sépare le fluorophore donneur et le fluorophore accepteur d'une distance qui ne dépasse pas 10 nm. Le spectre d'émission de la fraction fluorophore donneur chevauche le spectre d'excitation de la fraction fluorophore accepteur ou les spectres d'émission des fluorophores présentent une différence détectable. L'invention concerne également un acide nucléique isolé exprimant ladite construction, des trousses comprenant ladite construction et des lignées cellulaires comprenant ledit acide nucléique. L'invention concerne en outre des méthodes qui permettent de détecter une neurotoxine botulique au moyen de ladite construction par FRET ainsi que des méthodes qui permettent de détecter une neurotoxine botulique à l'aide de l'imagerie par résonance plasmonique de surface.

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