Probe reagent for measuring oxidative stress

摘要

The present invention relates to fluorescent or luminescent probe reagents for measuring oxidative stress in a cell or an organism. Examples of the probe reagents include: a fluorescent or luminescent protein and a marker protein; a fluorescent or luminescent protein, a marker protein, and a regulatory factor; or a fluorescent or luminescent protein, a marker protein, a cleavage sequence, and a regulatory factor. In the probe reagents, the marker protein makes it possible to detect the oxidative stress caused by reactive oxygen species and comprises a regulatory factor-binding site and a ubiquitin-binding site; and the regulatory factor is a protein making it possible to regulate degradation of the marker protein in response to the reactive oxygen species. The present invention also relates to a method of measuring oxidative stress in a cell or an organism, or a method of screening a substance which suppresses or promotes the oxidative stress in a cell or an organism by using the probe reagent.

主权项

1. A fluorescent or luminescent probe reagent for measuring oxidative stress in a cell or an organism, the reagent being a fusion protein comprising:
a fluorescent or luminescent protein; a marker protein; a cleavage sequence; and a regulatory factor,wherein
the marker protein makes it possible to detect the oxidative stress caused by reactive oxygen species, and comprises a regulatory factor-binding site and a ubiquitin-binding site; the regulatory factor is a protein capable of regulating degradation of the marker protein in response to the reactive oxygen species; the fluorescent or luminescent protein and the marker protein are adjacent to each other in any order so as to form a fusion molecule; the cleavage sequence is positioned between the fusion molecule and the regulatory factor and comprises a cleavable sequence that is cleaved in the cell or the organism, so that the reagent comprises a protein containing the fusion molecule and a protein containing the regulatory factor separated at the cleavage sequence; and binding or dissociation between the regulatory factor and the marker protein promotes or reduces the degradation of the marker protein, andwherein the fusion protein comprises the components positioned from N-terminus to C-terminus in one of the following orders:
(1) the fluorescent or luminescent protein, the marker protein, the cleavage sequence, and the regulatory factor; (2) the marker protein, the fluorescent or luminescent protein, the cleavage sequence, and the regulatory factor; (3) the regulatory factor, the cleavage sequence, the fluorescent or luminescent protein, and the marker protein; and (4) the regulatory factor, the cleavage sequence, the marker protein, and the fluorescent or luminescent protein.