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Novel polypeptide exhibiting fluorescent properties and use thereof UPDATE 実績あり

外国特許コード F190009896
整理番号 08118-EP
掲載日 2019年8月26日
出願国 欧州特許庁(EPO)
出願番号 14757001
公報番号 2963115
出願日 平成26年2月28日(2014.2.28)
公報発行日 平成28年1月6日(2016.1.6)
国際出願番号 JP2014055160
国際公開番号 WO2014133158
国際出願日 平成26年2月28日(2014.2.28)
国際公開日 平成26年9月4日(2014.9.4)
優先権データ
  • 特願2013-040097 (2013.2.28) JP
  • 2014JP55160 (2014.2.28) WO
発明の名称 (英語) Novel polypeptide exhibiting fluorescent properties and use thereof UPDATE 実績あり
発明の概要(英語) In order to provide a novel fluorescent protein and use thereof, the polypeptide according to the present invention has fluorescent properties in the presence of bilirubin and includes (1) the amino acid sequence of SEQ ID NO: 1, (2) an amino acid sequence having, for example, substitution of 1 to 21 amino acids in the amino acid sequence of SEQ ID NO: 1, (3) an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 1, or (4) the amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide consisting of a sequence complementary to the polynucleotide encoding the polypeptide according to the amino acid sequence in (1) under a stringent condition.
従来技術、競合技術の概要(英語) Background Art
Fluorescent proteins, such as green fluorescent protein (GFP), are indispensable as a tool for visualizing cells, tissue, biological individuals, and so on.
Most of fluorescent proteins have been isolated from invertebrates such as coral, sea anemones, and arthropods. However, for example, Non Patent Literatures 1 and 2 and Patent Literatures 1 and 2 report that a vertebrate, Japanese eel (Anguilla japonica), has a fluorescent protein.
Citation List
Patent Literature
Patent Literature 1: JP Patent Publication (Kokai) No. 2007-254371 A (Date of publication: October 4, 2007)
Patent Literature 2: JP Patent Publication (Kokai) No. 2008-141988 A (Date of publication: June 26, 2008)

Non Patent Literature
Non Patent Literature 1: Masao HONDA, Jinsuke KISHINO, Miyuki IMAMURA, Seiichi HAYASHI: Abstracts of the Fiscal Year Heisei 16 meeting of the Japanese Society of Fisheries Science (April 2, 2004, p. 203, 1101)
Non Patent Literature 2: Hayashi et al., Fisheries Science, 75, 1461-1469, 2009
特許請求の範囲(英語) [claim1]
1. A polypeptide shown in any of the following (1) to (4) and having fluorescent properties in the presence of bilirubin:
(1) a polypeptide including the amino acid sequence of SEQ ID NO: 1;
(2) a polypeptide including an amino acid sequence having substitution, deletion, insertion, and/or addition of 1 to 21 amino acids in the amino acid sequence of SEQ ID NO: 1;
(3) a polypeptide having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 1; and
(4) a polypeptide including an amino acid sequence encoded by a polynucleotide that hybridizes with a polynucleotide consisting of the sequence complementary to a polynucleotide encoding the polypeptide according to the above (1) under a stringent condition.

[claim2]
2. A polynucleotide shown in any of the following (1) to (4):
(1) a polynucleotide encoding a polypeptide including the amino acid sequence of SEQ ID NO: 1;
(2) a polynucleotide encoding a polypeptide including an amino acid sequence having substitution, deletion, insertion, and/or addition of 1 to 21 amino acids in the amino acid sequence of SEQ ID NO: 1 and having fluorescent properties in the presence of bilirubin;
(3) a polynucleotide encoding a polypeptide having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 and having fluorescent properties in the presence of bilirubin; and
(4) a polynucleotide hybridizing with a polynucleotide consisting of a sequence complementary to the polynucleotide according to the above (1) under a stringent condition and encoding a polypeptide having fluorescent properties in the presence of bilirubin.

[claim3]
3. The polypeptide according to claim 1, wherein the polypeptide has a dissociation constant for bilirubin of 0.1 nM or more and 100 nM or less.

[claim4]
4. A recombinant vector comprising the polynucleotide according to claim 2.

[claim5]
5. A transformant comprising the polynucleotide according to claim 2 or the recombinant vector according to claim 4.

[claim6]
6. A fusion polypeptide comprising the polypeptide according to claim 1 or 3 and another polypeptide.

[claim7]
7. A polypeptide-bilirubin complex constituted by bringing bilirubin into contact with the polypeptide according to claim 1, 3, or 6 in a form free from bilirubin.

[claim8]
8. A method for detecting bilirubin in a subject, the method comprising:
a contact step of bringing the polypeptide according to claim 1, the polypeptide according to claim 3, or the fusion polypeptide according to claim 6 into contact with the subject; and
a detection step of detecting fluorescence emitted by the polypeptide or the fusion polypeptide, after the contact step.

[claim9]
9. The method according to claim 8, wherein
the subject is a sample derived from blood or urine collected from a living body.

[claim10]
10. The method according to claim 8 or 9, further comprising:
a testing step of inspecting the presence or absence of a predisposing factor or onset of liver disease or hemolytic disease based on a detection result in the detection step.

[claim11]
11. A bilirubin detecting kit comprising at least one selected from the polypeptide according to claim 1, the polynucleotide according to claim 2, the polypeptide according to claim 3, the recombinant vector according to claim 4, the transformant according to claim 5, and the fusion polypeptide according to claim 6.
  • 出願人(英語)
  • RIKEN
  • 発明者(英語)
  • MIYAWAKI, Atsushi
  • KUMAGAI, Akiko
国際特許分類(IPC)
指定国 Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

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