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Probe reagent for measuring oxidative stress

外国特許コード F110005410
整理番号 E08801WO
掲載日 2011年9月5日
出願国 アメリカ合衆国
出願番号 92028309
公報番号 20110206615
公報番号 8865124
出願日 平成21年2月27日(2009.2.27)
公報発行日 平成23年8月25日(2011.8.25)
公報発行日 平成26年10月21日(2014.10.21)
国際出願番号 JP2009054236
国際公開番号 WO2009107876
国際出願日 平成21年2月27日(2009.2.27)
国際公開日 平成21年9月3日(2009.9.3)
優先権データ
  • 特願2008-049895 (2008.2.29) JP
  • 2009JP054236 (2009.2.27) WO
発明の名称 (英語) Probe reagent for measuring oxidative stress
発明の概要(英語) 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.
特許請求の範囲(英語) [claim1]
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, and
wherein 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.
[claim2]
2. The probe reagent according to claim 1, wherein the fluorescent or luminescent protein is selected from coral or Aequorea victoria.
[claim3]
3. The probe reagent according to claim 1, wherein the luminescent protein is luciferase.
[claim4]
4. The probe reagent according to claim 1, wherein the marker protein and the regulatory factor are human Nrf2 (Accession No. AAB32188) and human Keap1 (Accession No. AAH21957), respectively.
[claim5]
5. A DNA or RNA encoding the probe reagent according to claim 1.
[claim6]
6. The DNA according to claim 5, wherein the DNA comprises the nucleotide sequence shown in SEQ ID NO: 7.
[claim7]
7. A vector comprising the DNA according to claim 5.
[claim8]
8. The vector according to claim 7, further comprising a regulatory sequence.
[claim9]
9. A method of measuring oxidative stress in a cell, the method comprising: introducing the probe reagent according to claim 1 or the vector according to claim 7 into a cell or an organism excluding human; and
measuring the oxidative stress based on fluorescent or luminescent intensity in the cell or the organism.
[claim10]
10. The method according to claim 9, wherein the measurement is performed in real-time.
[claim11]
11. A method of screening for an oxidative stress-regulating substance, the method comprising: introducing a candidate substance and the probe reagent according to claim 1 or the vector according to claim 7 into a cell or organism, excluding human, which has been loaded with oxidative stress; and
screening for a substance which decreases or increases a fluorescent or luminescent intensity in the cell or organism.
[claim12]
12. A kit for measuring oxidative stress or screening an oxidative stress-regulating substance, the kit comprising 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, and
wherein the fusion protein comprises the components positioned from N-terminus to C-terminus in the following order: (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; or
(4) the regulatory factor, the cleavage sequence, the marker protein, and the fluorescent or luminescent protein.
  • 発明者/出願人(英語)
  • MIYAWAKI ATSUSHI
  • SASAKI KAZUKI
  • RIKEN
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
国際特許分類(IPC)
米国特許分類/主・副
  • 424/1.69
  • 424/1.11
  • 424/1.65
  • 424/9.1
  • 424/9.6
参考情報 (研究プロジェクト等) ERATO MIYAWAKI Life Function Dynamics AREA
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