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A polypeptide for dpp-4 detection and a fluorescent probe comprising the polypeptide

A DPP-4, fluorescent probe technology, applied in the field of fluorescent probes

Active Publication Date: 2018-06-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there are no related reports on AIE fluorescent probes that can specifically recognize DPP-4 and detect its activity

Method used

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  • A polypeptide for dpp-4 detection and a fluorescent probe comprising the polypeptide
  • A polypeptide for dpp-4 detection and a fluorescent probe comprising the polypeptide
  • A polypeptide for dpp-4 detection and a fluorescent probe comprising the polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 has the fluorescent probe synthesis of AIE characteristic

[0048] A kind of synthetic method of the fluorescent probe with AIE characteristic of this embodiment, its synthetic procedure is as follows figure 1 shown, including:

[0049] (1) Separately synthesize a polypeptide chain through a solid-phase polypeptide synthesis reaction, and the sequence of the polypeptide chain is glutamic acid-proline-phenylalanine-lysine;

[0050](2) Add 4-hydroxybenzophenone (1.9g, 10mmol), benzophenone (2.2g, 12mmol) and zinc powder (2.9g, 44mmol) into a 250ml three-necked flask, pump air, nitrogen, and repeat three times; Add 80ml of THF (tetrahydrofuran), 0 ℃ ice-water bath for 30min; dropwise add titanium tetrachloride (2.4ml, 22mmol) under ice-water bath, reflux overnight, spin dry; add appropriate amount of dichloromethane and dilute hydrochloric acid for extraction, remove the organic layer, dried with anhydrous magnesium sulfate, filtered, spin-dried, and subject...

Embodiment 2

[0055] Example 2 Application of Fluorescent Probe TPE-KFPE in DPP-4 Activity Detection

[0056] Detection of DPP-4 activity with fluorescent probe TPE-KFPE

[0057] Sample group 1: add 5 μL TPE-KFPE (1mM);

[0058] Sample group 2: add 25μL DPP-4 (100mU / mL), 5μL TPE-KFPE (1mM);

[0059] Sample group 3: Add 25μL DPP-4 (100mU / mL), 5μL TPE-KFPE (1mM), 2.5μL inhibitor Diprotin A (1mM);

[0060] The three sample groups were incubated at 37°C for 30min. After the reaction, a JASCO FP-6500 spectrophotometer was used to measure the fluorescence spectrum from 400 nm to 600 nm under 320 nm excitation.

[0061] Test results such as image 3 shown.

[0062] Depend on image 3 It can be seen that before the addition of DPP-4, the system only contained fluorescent probes, and the fluorescence intensity was low; after the addition of DPP-4, the fluorescence intensity of the system increased rapidly; when the DPP-4 inhibitor Diprotin A was added to the system, the fluorescence intensity ...

Embodiment 3

[0064] Example 3 Application of Fluorescent Probe TPE-KFPE in Screening DPP-4 Inhibitors

[0065] (1) Application of fluorescent probe TPE-KFPE in screening DPP-4 inhibitors

[0066] Take 1 μL fluorescent probe TPE-KFPE (1 mM), add 5 μL DPP-4 (100 mU / mL) and a certain volume of Diprotin A mother solution with different concentrations (making the final concentration of Diprotin A 0.005, 0.025, 0.05, 0.1, 0.2, 0.5, 5 , 10, 20, 50, 100 μM), make up to 100 μL with buffer solution HEPES pH 7.0, incubate at 37°C for 30 minutes and measure with a Tecan microplate reader, set E x 320nm (±25nm), E m 450nm (±25nm).

[0067] See the test results Figure 5 .

[0068] Depend on Figure 5 It can be seen that with the increase of the concentration of Diprotin A, the inhibitory effect of Diprotin A on DPP-4 also gradually increases; indicating that the fluorescent probe TPE-KFPE can better reflect the inhibitory effect of the inhibitor on DPP-4, and can be used for DPP-4 Inhibitor scree...

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Abstract

The invention discloses a polypeptide for DPP-4 detection and a fluorescence probe containing the polypeptide. The fluorescence probe is formed by connecting the polypeptide for DPP-4 specific recognition and an aggregation-induced emission molecule; the amino acid sequence of the polypeptide is glutamic acid-proline-phenylalanine-lysine, and the aggregation-induced emission molecule is connected with lysine in the polypeptide; proline is contained in the second position at the N terminal of the polypeptide, so that the DPP-4 specific recognition on the polypeptide in the fluorescence probe can be achieved, and the polypeptide can be cut off; the fluorescence probe is free from fluorescence absorption basically, but obvious fluorescence absorption is achieved when a dipeptide at the N terminal of the fluorescence probe is lost, so that real-time specific detection can be conducted on the DPP-4 activity.

Description

technical field [0001] The invention belongs to the field of drug screening and evaluation methods, and in particular relates to a polypeptide used for DPP-4 detection and a fluorescent probe containing the polypeptide. Background technique [0002] Incretin is a kind of polypeptide hormone produced in the intestinal tract with the effect of stimulating insulin secretion. It can not only stimulate insulin secretion, but also inhibit postprandial glucagon secretion, delay intestinal emptying, suppress appetite, Enhance insulin sensitivity and other effects. Incretins include glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), both of which show many advantages in promoting insulin release and regulating insulin levels outside the pancreas. However, GLP-1 and GIP are quickly hydrolyzed by DPP-4 in vivo, and the hydrolyzed products affect their physiological functions, making them unable to be well applied clinically. [0003] DPP-4 is a se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K5/113C09K11/06G01N21/64
Inventor 程翼宇王毅赵筱萍
Owner ZHEJIANG UNIV
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