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Active pharmaceutical ingredient screening method based on melatonin receptor 2 molecular probe

A technology of molecular probes and screening methods, applied in the field of molecular biology, to achieve high sensitivity and low noise

Inactive Publication Date: 2016-06-08
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no high-sensitivity screening method that can directly and real-time monitor the receptor's response to drugs by taking the conformational change of the receptor as a pointer

Method used

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  • Active pharmaceutical ingredient screening method based on melatonin receptor 2 molecular probe
  • Active pharmaceutical ingredient screening method based on melatonin receptor 2 molecular probe

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Embodiment 1

[0023] like figure 1 As shown, the present invention discloses a method for screening drug active ingredients based on melatonin receptor 2 molecular probe, and the screening method includes the following steps:

[0024] Step 1: The melatonin receptor 2 molecular probe of construction:

[0025] A. Introduce the VSV-Gtag and restriction endonuclease sites into the 5' end of the cDNA of human melatonin by PCR method, and connect the cDNA of eCFP to the 3' end of the acceptor cDNA by OverlappingPCR method, insert " Receptor-eCFP" is connected to pcDNA5 / FRT / TO vector, thereby obtaining pcDNA5 / FRT / TO-VSV-receptor-eCFP plasmid;

[0026] B. Insert the cDNA of the eYFP fluorescent group into the third inner loop of the acceptor in the plasmid by the mutation method, so as to obtain the original molecular probe plasmid;

[0027] C. Transfect HEK293 cells with the probe plasmid for 48 hours to express the receptor molecular probe, activate the probe with a commercially available recep...

Embodiment 2

[0034] Experimental purpose and method: In order to verify the feasibility and effectiveness of the molecular probe prepared by the present invention, this embodiment uses physiological saline, the positive control drug Ramelteon and the crude extract of ginseng to study the effects of the three on the melatonin receptor 2 molecular probe respectively. For the change of fluorescence resonance energy transfer, the specific experimental method is as described in Example 1, and will not be repeated here.

[0035] Experimental results: if figure 2 As shown, the cells expressing the melatonin receptor 2 molecular probe were placed under a high-speed fluorescence microscope, and the cells were treated with normal saline, the positive control drug Ramelteon, and the crude extract of ginseng to monitor the melatonin receptor 2 molecular probe Experiments have proved that the melatonin receptor 2 molecular probe has no obvious response to normal saline, but has a highly sensitive resp...

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Abstract

The invention relates to the field of molecular biology and discloses an active pharmaceutical ingredient screening method based on a melatonin receptor 2 molecular probe. The active pharmaceutical ingredient screening method includes constructing the melatonin receptor 2 molecular probe and establishing an Flp-In T-Rex HEK293 cell line for induced expression of a G-protein-coupled receptor molecular probe. The melatonin receptor 2 molecular probe is established by a fluorescence resonance energy transfer method, and is high in sensitivity, low in noise, high in throughput, capable of screening not only receptor agonist but also antagonist / inverse agonist and immune to interference of downstream signals. The reasonably designed molecular probe can screen traditional Chinese medicine mixtures and screen lead compounds specific in molecular action target at the receptor level in a one-step manner.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for screening active ingredients of medicines based on melatonin receptor 2 molecular probes. Background technique [0002] Conventional cell-level and animal-level drug screening methods are not only time-consuming and labor-intensive, but also susceptible to interference from multiple factors, with high false positive and false negative rates, and it is difficult to determine the target of drug action, and requires a large number of compounds to be screened . In the past ten years, with the rapid development of biological detection technology and computer technology, drug screening methods at the molecular level have emerged, such as surface plasmon resonance, nuclear magnetic resonance, downstream signal molecule detection, fluorescence polarization, and virtual screening. Etc., each method has its own advantages and disadvantages, all belong to the indirect screenin...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12N15/62C12N15/85C12N5/10
CPCG01N33/5008G01N33/5044C07K14/705C07K2319/60C12N2503/02
Inventor 徐天瑞杨洋刘莹王珍
Owner KUNMING UNIV OF SCI & TECH
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