Detection method for compound specificity through G protein-coupled receptor (GPCR)

Abstract

The invention discloses a detection method for compound specificity through a G protein-coupled receptor (GPCR). The method comprises following steps: preparing recombination HEK-293 cell suspension; adding coelenterazine to the cell suspension to obtain recombination cell sap; dividing the recombination cell sap into a control group and a to-be-detested group, adding a to-be-detested compound solution to the to-be-detested group, and adding a Hank's balanced salt solution to the control group; if the strength of fluorescence signals emitted by the to-be-detested group is higher than the strength of the fluorescence signals emitted by the control group, significant differences are provided, then a to-be-detested compound acts activation on the GPCR of recombination HEK-293 cells, and otherwise the activation does not exist; continually adding agonist corresponding to the GPCR to the to-be-detested group to detect the fluorescence signals; and if the strength of the fluorescence signals of the to-be-detested group is significantly lower than the strength of the fluorescence signals of the control group, then the to-be-detested compound acts an inbibitional effect on the GPCR of the recombination HEK-293 cells, and otherwise the inbibitional effect does not exist. The method is a flexible and changeable detection mode, an unknown compound can be detected by means of the GPCR, and meanwhile reverse detection for the GPCR in specific binding with a known compound can be achieved by means of the known compound.

Description

technical field

[0001] The invention relates to a method for detecting the specificity of G protein-coupled receptors to compounds. Background technique

[0002] G protein-coupled receptors (GPCRs) are a class of membrane proteins containing seven transmembrane α-helices, and are the largest family of receptors on the surface of human cell membranes. There are approximately 800 genes encoding GPCR proteins in the human genome. When an extramembrane ligand acts on a GPCR receptor, the inner portion of the receptor binds to the G protein, thereby activating the G protein. G proteins transmit extracellular signals into cells through a variety of signal transduction pathways, and play an important role in the regulation of growth and development, reproduction, immunity, nerve conduction, metabolism and behavior.

[0003] The types of GPCR ligands are very wide, covering taste, smell, light, metal ions, biogenic amines, fatty acids, steroids and other categories. About 80% of ...

Examples

Embodiment 1

[0056] Substance P activates tachykinin receptors (NK 1 ) dose effect detection

[0057] Substance P is a neuropeptide widely distributed among nerve fiber cells, which is NK 1 A natural agonist of receptors, its main function is to transmit pain information, participate in the regulation of sensation, movement, emotion, etc., and is also related to the pathogenesis of anxiety, depression, and schizophrenia.

[0058] Express NK 1 A method for preparing recombinant HEK-293 cells, comprising the following steps:

[0059] (1) Preparation of HEK-293 cell suspension: HEK-293 cells (purchased from ATCC; the same below) were mixed with 6×10 5 The concentration of cells / mL was suspended in Free Style 293 medium (purchased from Life Technologies; the same below) containing 0.05% (mass volume ratio; the same below) F-68 (purchased from Sigma-Aldrich; the same below), At 37°C, 5% CO 2 Environment, 160rpm suspension shaking culture for 24 hours;

[0060] (2) Preparation of plasmid m...

Embodiment 2

[0073] Detection of GPCRs that specifically bind to Ritanserin

[0074]Ritanserin is a blocker of 5-hydroxytryptamine receptor (5-HTR, a G protein-coupled receptor), and has a selective blocking effect on 5-HTR. In peripheral tissues, 5-THR mediates vasoconstriction and smooth muscle contraction, and is an effective antagonist in response to 5-hydroxytryptamine (5-HT)-induced vasoconstriction and bronchospasm, and has the effect of reducing hypertension.

[0075] A method for preparing recombinant HEK-293 cells expressing different types of GPCRs, comprising the following steps:

[0076] (1) Preparation of HEK-293 cell suspension: HEK-293 cells were mixed with 6×10 5 Suspension culture at the concentration of cells / mL in Free Style 293 medium containing 0.05% F-68, at 37°C, 5% CO 2 Environment, 160rpm suspension shaking culture for 24 hours;

[0077] (2) Preparation of plasmid mixture: different GPCR plasmids (total 40 kinds, respectively GAL2, LTD4R2, LTD4R1, HRH2, GCGR, S...

Embodiment 3

[0095] Detection of Dose Effect of Ritanserin Inhibiting 5HTR Receptor

[0096] The preparation steps of recombinant HEK-293HEK-293 cells expressing HTR4 and HTR2A are as follows:

[0097] (1) Preparation of HEK-293 cell suspension: HEK-293 cells were mixed with 6×10 5 Suspension and dispersion culture at the concentration of cells / ml in Free Style 293 medium containing 0.05% F-68, 37°C, 5% CO 2 , 160rpm suspension shaking culture for 24 hours.

[0098](2) Preparation of plasmid mixture: Add HTR4 plasmid, G protein mutant plasmid, and aequorin plasmid at a mass ratio of 3 / 1 / 1 (unit: 0.1 μg / mL cell suspension) to Free In Style293 medium, the plasmid mixture of the HTR4 group was obtained; the preparation method of the plasmid mixture of the HTR2A group was the same as that of the HTR4 group;

[0099] (3) Prepare the transfection complex: Add polyethyleneimine to 50 μL serum-free medium in an amount twice the mass of the luminescent protein particle to obtain PEI reagent; mix...

technical field

[0001] The invention relates to a method for detecting the specificity of G protein-coupled receptors to compounds. Background technique

[0002] G protein-coupled receptors (GPCRs) are a class of membrane proteins containing seven transmembrane α-helices, and are the largest family of receptors on the surface of human cell membranes. There are approximately 800 genes encoding GPCR proteins in the human genome. When an extramembrane ligand acts on a GPCR receptor, the inner portion of the receptor binds to the G protein, thereby activating the G protein. G proteins transmit extracellular signals into cells through a variety of signal transduction pathways, and play an important role in the regulation of growth and development, reproduction, immunity, nerve conduction, metabolism and behavior.

[0003] The types of GPCR ligands are very wide, covering taste, smell, light, metal ions, biogenic amines, fatty acids, steroids and other categories. About 80% of ...

Examples

Embodiment 1

[0056] Substance P activates tachykinin receptors (NK 1 ) dose effect detection

[0057] Substance P is a neuropeptide widely distributed among nerve fiber cells, which is NK 1 A natural agonist of receptors, its main function is to transmit pain information, participate in the regulation of sensation, movement, emotion, etc., and is also related to the pathogenesis of anxiety, depression, and schizophrenia.

[0058] Express NK 1 A method for preparing recombinant HEK-293 cells, comprising the following steps:

[0059] (1) Preparation of HEK-293 cell suspension: HEK-293 cells (purchased from ATCC; the same below) were mixed with 6×10 5 The concentration of cells / mL was suspended in Free Style 293 medium (purchased from Life Technologies; the same below) containing 0.05% (mass volume ratio; the same below) F-68 (purchased from Sigma-Aldrich; the same below), At 37°C, 5% CO 2 Environment, 160rpm suspension shaking culture for 24 hours;

[0060] (2) Preparation of plasmid m...

Embodiment 2

[0073] Detection of GPCRs that specifically bind to Ritanserin

[0074]Ritanserin is a blocker of 5-hydroxytryptamine receptor (5-HTR, a G protein-coupled receptor), and has a selective blocking effect on 5-HTR. In peripheral tissues, 5-THR mediates vasoconstriction and smooth muscle contraction, and is an effective antagonist in response to 5-hydroxytryptamine (5-HT)-induced vasoconstriction and bronchospasm, and has the effect of reducing hypertension.

[0075] A method for preparing recombinant HEK-293 cells expressing different types of GPCRs, comprising the following steps:

[0076] (1) Preparation of HEK-293 cell suspension: HEK-293 cells were mixed with 6×10 5 Suspension culture at the concentration of cells / mL in Free Style 293 medium containing 0.05% F-68, at 37°C, 5% CO 2 Environment, 160rpm suspension shaking culture for 24 hours;

[0077] (2) Preparation of plasmid mixture: different GPCR plasmids (total 40 kinds, respectively GAL2, LTD4R2, LTD4R1, HRH2, GCGR, S...

Embodiment 3

[0095] Detection of Dose Effect of Ritanserin Inhibiting 5HTR Receptor

[0096] The preparation steps of recombinant HEK-293HEK-293 cells expressing HTR4 and HTR2A are as follows:

[0097] (1) Preparation of HEK-293 cell suspension: HEK-293 cells were mixed with 6×10 5 Suspension and dispersion culture at the concentration of cells / ml in Free Style 293 medium containing 0.05% F-68, 37°C, 5% CO 2 , 160rpm suspension shaking culture for 24 hours.

[0098](2) Preparation of plasmid mixture: Add HTR4 plasmid, G protein mutant plasmid, and aequorin plasmid at a mass ratio of 3 / 1 / 1 (unit: 0.1 μg / mL cell suspension) to Free In Style293 medium, the plasmid mixture of the HTR4 group was obtained; the preparation method of the plasmid mixture of the HTR2A group was the same as that of the HTR4 group;

[0099] (3) Prepare the transfection complex: Add polyethyleneimine to 50 μL serum-free medium in an amount twice the mass of the luminescent protein particle to obtain PEI reagent; mix...

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