Melanocortin receptor 1 drug screening model based on fluorescence resonance energy transferring technology

A technology for corticosteroids and receptors, applied in the field of pharmacology, can solve the problems of time-consuming, labor-intensive, and difficult to apply high-throughput screening

Inactive Publication Date: 2016-01-27
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, the existing screening methods for melanocortin receptor 1 agonists / antagonists mostly use the luciferase reporter gene method and enzyme-linked immunosorbent assay, but these two methods are time-consuming and laborious, and are difficult to apply to high-throughput filter

Method used

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  • Melanocortin receptor 1 drug screening model based on fluorescence resonance energy transferring technology
  • Melanocortin receptor 1 drug screening model based on fluorescence resonance energy transferring technology
  • Melanocortin receptor 1 drug screening model based on fluorescence resonance energy transferring technology

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Experimental program
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specific Embodiment approach

[0016] Cell line: CHO-MC1

[0017] Reagents: Ham's F12, 10% FBS, 100 IU / ml penicillin, 100 μg / ml streptomycin, 400 μg / ml G418, Hank's balanced salt solution (HBSS), StimulationBufferA, StimulationBufferB.

[0018] Instruments: carbon dioxide incubator; 384-well plate; Multidrop sampler; EnVision2014MultilabelReader.

[0019] 1. Cell culture

[0020] (1) The adherence degree of the cells should reach 80%, reaching the logarithmic growth phase.

[0021] (2) Rinse the cells once with mild PBS.

[0022] (3) Digest the cells with 0.05% trysin-lm MEDTA.

[0023] (4) Centrifuge at 1500 rpm for 2 minutes.

[0024] (5) Wash once with complete medium after stopping.

[0025] (6) Count the cells, put the cell suspension into 15ml culture medium, and make the cell concentration reach 7.5×10 5 cells / ml, add this cell suspension into T75 culture flask, let the cells in 37℃ / 5%CO 2 overnight for incubation.

[0026] Second, the determination of the standard curve

[0027] (1) Prepara...

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Abstract

The invention provides a drug screening method for screening a melanocortin receptor 1 agonist. Based on a fluorescence resonance energy transferring principle, cAMP produced by complete cells and cAMP (b-cAMP) marked by biotins in a reaction system are competitively combined with an anti-cAMP antibody and a fluorescence resonance energy transferring process is interrupted to cause weakening of fluorescence signals, so that whether a compound has an excitement effect of a melanocortin receptor 1 or not is reflected. The drug screening method comprises the following steps: (1) culturing cells; (2) determining a standard curve; and (3) identifying a model: identifying that a positive drug IC50 is consistent to references. The method is simple and rapid; a fluorescence detection value has high sensitivity and the detection accuracy is improved; and the result is stable and reliable and the reproducibility is good.

Description

technical field [0001] The invention belongs to the field of pharmacology. A drug screening model of melanocortin receptor is constructed by using fluorescence detection technology, which is used for drug screening of melanocortin receptor 1 activity of test samples. Background technique [0002] The MC1 receptor is the melanocortin receptor 1 (melanocortin1 receptor, MC1R), the gene of this receptor is an important gene that controls the synthesis of melanin in animals, and it shows different functions in melanocytes, adrenal cortex cells, nervous system and immune system. Features. By controlling the amount of eumelanin to determine the coat color of animals, and affect the occurrence of diseases in animals. Mutations in the MC1R gene have been linked to variations in melanin traits and diseases such as skin cancer. Therefore, the establishment of MC1R agonist screening model is of great significance for the treatment of skin cancer and other diseases. [0003] The Lanc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533
CPCG01N33/533
Inventor 严明王翔高鹏张陆勇
Owner CHINA PHARM UNIV
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