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Fluorescent probe for detecting activity of proline isomerase and preparation method and application thereof

A technology of fluorescent probes and isomerases, applied in peptide preparation methods, biochemical equipment and methods, luminescent materials, etc., can solve problems such as limited application range, inability to use living cells and in vivo detection, and achieve detection methods Simple, good probe stability

Active Publication Date: 2017-11-03
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no detection reagents for the activity and content of proline isomerase on the market and clinically, and the detection of proline isomerase recombinant protein activity is generally detected by the double-enzyme-linked enzyme digestion method. Protease cleavage and proline isomerase activity of recombinant protein. At the same time, this method is only suitable for in vitro detection and cannot be used for living cells and in vivo detection, which limits the scope of application of this method

Method used

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  • Fluorescent probe for detecting activity of proline isomerase and preparation method and application thereof
  • Fluorescent probe for detecting activity of proline isomerase and preparation method and application thereof
  • Fluorescent probe for detecting activity of proline isomerase and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1: the synthesis of probe molecule Ia

[0022]

[0023] "N-Fmoc-phenylalanyl-p-nitroaniline" was removed from the Fmoc protecting group and dissolved in dry dichloromethane methane solution, and an equivalent amount of "N,N-dimethylnaphthoic acid-labeled alanine -Phenylalanine-proline tripeptide chain, and equivalent EDC, HOBt condensation, the product is separated by preparative liquid phase to obtain the compound shown in formula Ia. MS: 798.3550.

Embodiment 2

[0024] Embodiment 2: the synthesis of probe molecule Ib

[0025]

[0026] "N-Fmoc-phenylalanyl-p-nitroaniline" was removed from the Fmoc protecting group and dissolved in dry dichloromethane methane solution, and an equivalent amount of "N,N-dimethylnaphthoic acid-labeled alanine - Glycine-proline tripeptide chain, and equivalent EDC, HOBt condensation, the product is separated by preparative liquid phase to obtain the compound shown in formula Ia. MS: 722.3333.

Embodiment 3

[0027] Embodiment 3: Probe molecule Ia detects the proline isomerase activity in the HBVP cell protein extract

[0028] Preparation of cell protein extract: Prepare cell lysate (10mM Tris, 150mM NaCl, 1mM EDTA, pH7.5), add 100μl lysate to each bottle of cells, and add corresponding protease inhibitor cocktail at the same time, lyse on ice for 30min, and centrifuge at 12000g After 10 minutes, pipette the supernatant into a new EP tube, use a kit (Bio-Rad, Cat. No. 5000111) for quantitative analysis of the protein extract, and finally supplement the lysate until the final concentration of the protein extract is 1 μg / μl.

[0029] Add Ia to the phosphate buffered saline solution to make the final concentration 10 micromoles, add 20 microliters of protein extract, and record the fluorescence emission intensity at 460nm with time under the excitation condition of 363nm with a fluorescence spectrophotometer. The experimental results show that the addition of protein extract can incre...

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Abstract

The invention provides a fluorescent probe for detecting the activity of proline isomerase. The probe produces weaker fluorescence in a physiological environment, can specifically produce configuration inversion under the catalytic action of the proline isomerase to generate a fluorescent signal enhanced product. Therefore, the probe can be used for active evaluation of the proline isomerase, can be applied to detection of the activity of proline isomerase in a cell or tissue protein extraction solution and detection of the activity of proline isomerase in living cells. The fluorescent probe has the following advantages that the probe is good in stability and can be saved and used for a long time; a detection method is simple, the probe is added in a detection system, incubation is performed for 10 minutes, fluorescence intensity is measured, and a result can be given out without assistance of other reagents; in-vivo detection of the activity of the proline isomerase in the living cells can be achieved, other reported methods can be only used for a cell homogenization solution. The specific structure of the probe is shown in Figures (shown in the description).

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a fluorescent probe for detecting the activity of proline isomerase, a preparation method thereof and an application in detecting the activity of proline isomerase in biological samples. Background technique [0002] Proline is a special kind of amino acid, and the amino group in its structure is a disubstituted secondary amine. In the protein secondary structure, due to the presence of proline, the normal α-helical skeleton is disintegrated, and a β-turn is formed at proline. Proline isomerase is a highly conserved family of immunophilins, which consists of three subfamilies, FK-506-binding proteins (FKBPs) and cyclophilins. And parvulins family, in prokaryotic and eukaryotic and other microorganisms, animals and plants are expressed. It is also abundantly expressed in multiple tissue sites in the human body and can regulate diverse cellular functions such as regulation of ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/103C07K1/04C07K1/06C09K11/06C12Q1/533
CPCC07K5/1008C09K11/06C09K2211/1416C12Q1/533Y02P20/55
Inventor 李新蒋权卢应梅邓亚萍蒋国军韩峰胡永洲
Owner ZHEJIANG UNIV
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