Vasoactive intestinal peptide I type receptor inhibitor high-throughput screening method based on fluorescence resonance energy transfer technology

A technology of vasoactive intestinal peptide and receptor inhibitors, which is applied in the field of pharmacology and can solve problems such as screening methods for less vasoactive intestinal peptide type I receptor inhibitors

Inactive Publication Date: 2016-04-06
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few screening methods for vasoactive intestinal peptide type I receptor inhibitors. Therefore, the establishment of convenient, fast and accurate detection methods, especially in vitro functional detection, has received more and more attention in drug screening.

Method used

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  • Vasoactive intestinal peptide I type receptor inhibitor high-throughput screening method based on fluorescence resonance energy transfer technology
  • Vasoactive intestinal peptide I type receptor inhibitor high-throughput screening method based on fluorescence resonance energy transfer technology
  • Vasoactive intestinal peptide I type receptor inhibitor high-throughput screening method based on fluorescence resonance energy transfer technology

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Experimental program
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Effect test

specific Embodiment approach

[0016] 1. Establishment of screening method for vasoactive intestinal peptide type I receptor inhibitors

[0017] 1. Experimental materials

[0018] d2-cAMP detection kit (Cisbio, France ), VIP (Sigma-aldrich, U.S.), VIP(1-7)-GRF(8-27) (Sigma-aldrich, U.S.), DMEM, FBS (Gibco, U.S.) 384-well polypropylene microplate Cat#3573 (Corning, U.S.), disposable gun tip (Axygen, U.S.)

[0019] 2. Experimental steps

[0020] 1) Determination of the optimal concentration of vasoactive intestinal peptide type I receptor cells

[0021] 1) Different concentrations of vasoactive intestinal peptide type I receptor cells were prepared, and 5 μl was added to each well.

[0022] 2) Add 5 μl 1× reaction buffer containing VIP to each well.

[0023] 4) React at room temperature for 45 minutes.

[0024] 5) Add 10 μl × stop buffer and incubate at room temperature for 1 hour. Detect the fluorescence intensity to determine the optimal concentration of vasoactive intestinal peptide type I receptor ...

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Abstract

The invention provides a high-throughput screening method for screening a vasoactive intestinal peptide I type receptor inhibitor; in experiments, with use of coupling of a vasoactive intestinal peptide type I receptor and a Gs protein, AC is activated, the cAMP content is increased, a product with fluorescence can be produced, the role of the vasoactive intestinal peptide type I receptor is judged by the fluorescence intensity, and thus whether the obtained compound has an inhibitory effect of the vasoactive intestinal peptide type 1 receptor is obtained. The method comprises the following steps: 1) establishment and optimization of a screening model of the vasoactive intestinal peptide I type receptor inhibitor: determining the most suitable concentration for a reaction of VIP and vasoactive intestinal peptide I type receptor cells; and 2) model reliability verification with positive drug: verifying that a positive drug IC50 is in accordance with that of a reference. The method has the advantages that the method is simple and fast, sensitivity of the fluorescence detection value is high, detection accuracy is increased, the result is stable and reliable, and the reappearance is good.

Description

technical field [0001] The invention belongs to the field of pharmacology, and uses fluorescence detection technology to construct a high-throughput screening model for vasoactive intestinal peptide type I receptor inhibitors, which is used for high-throughput screening of the inhibitory activity of test samples on vasoactive intestinal peptide type I receptors. volume detection. technical background [0002] Vasoactive intestinal peptide type I receptor is a co-receptor of neuropeptide and vasoactive intestinal peptide, mediating a variety of important biological functions, such as vasoactive intestinal peptide type I receptor mediating neuropeptide and vasoactive intestinal peptide inhibition Biological functions of appetite and anti-inflammation. Vasoactive intestinal peptide type I receptors are abundantly expressed in the brain and T cells and can regulate neuronal differentiation and T cell activation. Vasoactive intestinal peptide type I receptor, as one of the subt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 严明沈灵孙丹丹何玲张陆勇
Owner CHINA PHARM UNIV
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