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A gene-encoded hydrogen peroxide fluorescent probe and its preparation method and application

A fluorescent probe, hydrogen peroxide technology, applied in chemical instruments and methods, fluorescence/phosphorescence, introduction of foreign genetic material using carriers, etc., can solve the problems of limited dynamic changes and small fluorescence

Active Publication Date: 2019-03-29
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
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  • Claims
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Problems solved by technology

Both are derivatives of GFP, but their fluorescence is much smaller than that of chemical probes, the fluorescence intensity is only 5% to 7% of EGFP, and only weak fluorescence is observed in zebrafish embryos, which is a great limitation H in living animals or subcellular organelles 2 o 2 Detection of Dynamic Changes

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  • A gene-encoded hydrogen peroxide fluorescent probe and its preparation method and application
  • A gene-encoded hydrogen peroxide fluorescent probe and its preparation method and application
  • A gene-encoded hydrogen peroxide fluorescent probe and its preparation method and application

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Embodiment Construction

[0044] I. Definition:

[0045] As used herein, the term "about" when a value or range is given means that the value or range is within 20%, within 10%, and within 5% of the given value or range.

[0046] As used herein, the terms "comprising", "comprising" and their equivalents include the meanings of "containing" and "consisting of", for example a composition "comprising" X may consist solely of X or may contain other substances, such as X+ Y.

[0047] In the present invention, the term "HyPer" refers to a genetically encoded hydrogen peroxide fluorescent probe that has been reported to insert the cpYFP protein into OxyR-RD, which is connected by a short chain of amino acids in the middle.

[0048] The term "HyPer protein mutant" or "HyPer mutant" or "rationally designed HyPer mutant" as used herein refers to introducing a mutant site respectively at the corresponding site on the HyPer probe cpYFP element, and then increasing the fluorescence The mutants were continuously i...

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Abstract

The present invention provides a gene encoding hydrogen peroxide fluorescent probe, a preparation method and applications thereof, wherein the gene encoding hydrogen peroxide fluorescent probe comprises a H2O2-sensitive polypeptide OxyR and a fluorescent protein cpYFP showing through H2O2, and the fluorescent protein cpYFP is inserted into the H2O2-sensitive polypeptide OxyR to form the fluorescent probe. The gene encoding hydrogen peroxide fluorescent probe is characterized in that the gene encoding hydrogen peroxide fluorescent probe is the mutant obtained by carrying out mutation on the sequence of the fluorescent protein cpYFP in the fluorescent probe. According to the present invention, the gene encoding hydrogen peroxide fluorescent probe can detect H2O2 at a live bacterial level and a subcellular level, and has good specificity and H2O2 selectivity, and the gene encoding hydrogen peroxide fluorescent probe on the H2O2 dynamic response change is not affected by the introduction of the mutation site.

Description

technical field [0001] The invention belongs to the fields of biochemistry, protein chemistry and molecular biology, and in particular relates to a mutant protein capable of increasing the fluorescence intensity of a hydrogen peroxide probe HyPer and a gene-encoded nucleotide sequence, and also relates to the mutant protein and its nucleotide sequence The preparation method and its detection of H 2 o 2 in the application. Background technique [0002] Reactive oxygen species (ROS) are a series of reactive molecules derived from molecular oxygen (such as H 2 o 2 ), the general term for free radicals. During redox metabolism, ROS are key mediators of different physiological and pathological processes. Free oxygen molecules are harmful to aerobic organisms. These molecules are mainly by-products of electron transfer during mitochondrial respiration, and by-products of oxidoreductase and metal-catalyzed oxidation. Previous reports have suggested that only phagocytes are i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K14/00C12N15/11C12N15/63G01N21/64
Inventor 杨弋赵玉政邓蕾陶荣坤
Owner EAST CHINA UNIV OF SCI & TECH
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