CN105671119A - Drug activity ingredient screening method based on histamine receptor 3 molecular probe - Google Patents
Drug activity ingredient screening method based on histamine receptor 3 molecular probe Download PDFInfo
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- CN105671119A CN105671119A CN201610118510.8A CN201610118510A CN105671119A CN 105671119 A CN105671119 A CN 105671119A CN 201610118510 A CN201610118510 A CN 201610118510A CN 105671119 A CN105671119 A CN 105671119A
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- histamine receptor
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- 239000003068 molecular probe Substances 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 33
- 102000000543 Histamine Receptors Human genes 0.000 title claims abstract description 23
- 108010002059 Histamine Receptors Proteins 0.000 title claims abstract description 23
- 238000012216 screening Methods 0.000 title claims abstract description 18
- 239000003814 drug Substances 0.000 title claims abstract description 16
- 229940079593 drug Drugs 0.000 title abstract description 4
- 230000000694 effects Effects 0.000 title abstract description 3
- 239000004615 ingredient Substances 0.000 title abstract 2
- 102000005962 receptors Human genes 0.000 claims abstract description 22
- 108020003175 receptors Proteins 0.000 claims abstract description 22
- 238000002866 fluorescence resonance energy transfer Methods 0.000 claims abstract description 10
- 230000035945 sensitivity Effects 0.000 claims abstract description 8
- 230000008569 process Effects 0.000 claims abstract description 6
- 230000001939 inductive effect Effects 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 29
- 239000013612 plasmid Substances 0.000 claims description 12
- 238000005457 optimization Methods 0.000 claims description 11
- 239000000556 agonist Substances 0.000 claims description 10
- 238000012360 testing method Methods 0.000 claims description 9
- 239000000470 constituent Substances 0.000 claims description 7
- 238000012544 monitoring process Methods 0.000 claims description 7
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 claims description 6
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 6
- 239000002299 complementary DNA Substances 0.000 claims description 6
- 239000002269 analeptic agent Substances 0.000 claims description 4
- 235000001014 amino acid Nutrition 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 229960001340 histamine Drugs 0.000 claims description 3
- 235000018102 proteins Nutrition 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000001890 transfection Methods 0.000 claims description 3
- 239000005557 antagonist Substances 0.000 abstract description 2
- 150000002611 lead compounds Chemical class 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 108091006027 G proteins Proteins 0.000 abstract 1
- 102000030782 GTP binding Human genes 0.000 abstract 1
- 108091000058 GTP-Binding Proteins 0.000 abstract 1
- 230000008878 coupling Effects 0.000 abstract 1
- 238000010168 coupling process Methods 0.000 abstract 1
- 238000005859 coupling reaction Methods 0.000 abstract 1
- 230000004907 flux Effects 0.000 abstract 1
- 229940125425 inverse agonist Drugs 0.000 abstract 1
- 239000000018 receptor agonist Substances 0.000 abstract 1
- 229940044601 receptor agonist Drugs 0.000 abstract 1
- HEEACTTWORLLPM-UHFFFAOYSA-N 2-(1h-imidazol-5-yl)ethanol Chemical compound OCCC1=CNC=N1 HEEACTTWORLLPM-UHFFFAOYSA-N 0.000 description 4
- 239000000287 crude extract Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000007877 drug screening Methods 0.000 description 2
- 239000003390 Chinese drug Substances 0.000 description 1
- 108091005942 ECFP Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 238000002875 fluorescence polarization Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000001225 nuclear magnetic resonance method Methods 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000003041 virtual screening Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
Abstract
The invention relates to the field of molecular biology, and discloses a drug activity ingredient screening method based on a histamine receptor 3 molecular probe. The method comprises constructing a histamine receptor 3 molecular probe and establishing a Flp-InT-Rex HEK293 cell line for inducible expression of a G protein coupling receptor molecular probe. According to the method, a fluorescent resonance energy transfer method is used to establish the histamine receptor 3 molecular probe; the molecular probe has the characteristics of being high in sensitivity, low in noise and high in flux, not only be the receptor agonist can screened, but also the antagonist/ inverse agonist can be screened, and the screening process is free from the interference of the downstream signal; the reasonably designed molecular probe can be used for screening the traditional Chinese medicine mixture so as to screen the lead compound with obvious molecular target on the receptor level in one step.
Description
Technical field
The present invention relates to biology field, particularly a kind of active constituents of medicine screening technique based on histamine receptor 3 molecular probe.
Background technology
Conventional cellular level, the drug screening method of animal level not only time-consuming, effort, and it is easily subject to the interference of multiple factors, false positive, false negative rate are higher, it is also difficult to determine pharmaceutically-active target spot, and need substantial amounts of screened compound. Nearly ten years, fast development along with Measurement for Biotechnique and computer technology, the drug screening method of molecular level continues to bring out, as: surface plasmon resonance, nuclear magnetic resonance method, downstream signaling molecule detection method, fluorescence polarization method, virtual screening method etc., every kind of method has the pluses and minuses of oneself, broadly falls into indirectly screening technique. So far it is changed to pointer but without with receptor space conformation, it is possible to monitor the receptor highly sensitive screening method to drug reaction directly, in real time.
Summary of the invention
It is an object of the invention to provide a kind of active constituents of medicine screening technique based on histamine receptor 3 molecular probe, be changed to pointer according to receptor space conformation, it is possible to monitor the receptor highly sensitive screening method to drug reaction directly, in real time.
For realizing above-mentioned technical purpose, reaching above-mentioned technique effect, the invention discloses a kind of active constituents of medicine screening technique based on histamine receptor 3 molecular probe, screening technique comprises the steps that
Step 1: build histamine receptor 3 molecular probe:
A. adopting Clontech test kit, the cDNA of people's histamine receptor 3 being connected on the existing carrier pcDNA5/FRT/TO-VSV-G-eCFP of laboratory, thus obtaining pcDNA5/FRT/TO-VSV-histamine receptor 3-eCFP plasmid;
B. with Clontech test kit, the cDNA of eYFP is connected in the middle part of acceptor control three internal ring, thus obtaining original molecular probe plasmid;
C. probe plasmid transfection HEK293 cell is used 48 hours, with expressed receptor molecular probe, and with commercially available agonist activation probe, with the sensitivity of Olympus high speed fluorescence microscope monitoring probe;
D. the monitoring result according to probe sensitivity, carries out structure optimization to probe;
Step 2: set up the Flp-InT-RexHEK293 cell line of abduction delivering g protein coupled receptor molecular probe:
A. by the molecular probe plasmid after optimization and pOG44 carrier cotransfection Flp-InT-RexHEK293 cell, and positive cell clone is screened with 200ug/mlHygromycin;
B. express corresponding g protein coupled receptor molecular probe with 1ug/mlDoxycycline inducing cell, with the expression of VSV antibody test probe proteins, monitor the cell location of molecular probe with fluorescence microscope;
C. process, with receptor stimulating agent, the acceptor molecule probe Flp-InT-RexHEK293 cell expressed, obtain the FRET curve of the receptor activation of different agonist dose, and after stopping administration continuation normal saline eluting agonist.
Wherein, probe is carried out structure optimization by step 1 specific as follows:
Adjust eYFP in the position of acceptor control three internal ring: acceptor control three internal ring is truncated to two ends respectively and respectively retains 12 aminoacid, before optimizing, the eYFP of probe is positioned at the centre position of acceptor control three internal ring, after optimization, the eYFP of probe is positioned at the 230-347 position of acceptor control three internal ring, and eCFP is positioned at 445 positions of receptor C-end.
The method have the advantages that
1. the present invention adopts the method for FRET (fluorescence resonance energy transfer) to establish histamine receptor 3 molecular probe, this molecular probe has high sensitivity, low noise, high-throughout feature, receptor stimulating agent can not only be screened and also can screen antagonists/inverse agonists, and screening process is not by the interference of downstream signal.
2. can Traditional Chinese drug mixture being screened through the molecular probe of appropriate design, what can settle at one go screens the lead compound that molecular action target spot is clear and definite on acceptor levels.
Accompanying drawing explanation
Fig. 1 is the structural representation of molecular probe of the present invention.
Fig. 2 is the result schematic diagram of the embodiment of the present invention 2.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.
Embodiment 1
As it is shown in figure 1, the invention discloses a kind of active constituents of medicine screening technique based on histamine receptor 3 molecular probe, screening technique comprises the steps that
Step 1: build histamine receptor 3 molecular probe:
A. adopting Clontech test kit, the cDNA of people's histamine receptor 3 being connected on the existing carrier pcDNA5/FRT/TO-VSV-G-eCFP of laboratory, thus obtaining pcDNA5/FRT/TO-VSV-histamine receptor 3-eCFP plasmid;
B. with Clontech test kit, the cDNA of eYFP is connected in the middle part of acceptor control three internal ring, thus obtaining original molecular probe plasmid;
C. probe plasmid transfection HEK293 cell is used 48 hours, with expressed receptor molecular probe, and with commercially available agonist activation probe, with the sensitivity of Olympus high speed fluorescence microscope monitoring probe;
D. the monitoring result according to probe sensitivity, carries out structure optimization to probe; Adjust eYFP in the position of acceptor control three internal ring: acceptor control three internal ring is truncated to two ends respectively and respectively retains 12 aminoacid, before optimizing, the eYFP of probe is positioned at the centre position of acceptor control three internal ring, and after optimization, the eYFP of probe is positioned at the 230-347 position of acceptor control three internal ring. ECFP is positioned at 445 positions of receptor C-end.
Step 2: set up the Flp-InT-RexHEK293 cell line of abduction delivering g protein coupled receptor molecular probe:
A. by the molecular probe plasmid after optimization and pOG44 carrier cotransfection Flp-InT-RexHEK293 cell, and positive cell clone is screened with 200ug/mlHygromycin;
B. express corresponding g protein coupled receptor molecular probe with 1ug/mlDoxycycline inducing cell, with the expression of VSV antibody test probe proteins, monitor the cell location of molecular probe with fluorescence microscope;
C. process, with receptor stimulating agent, the acceptor molecule probe Flp-InT-RexHEK293 cell expressed, obtain the FRET curve of the receptor activation of different agonist dose, and after stopping administration continuation normal saline eluting agonist.
Embodiment 2
Experiment purpose and method: in order to verify that the present invention prepares feasibility and the effectiveness of molecular probe, the present embodiment is studied three respectively with normal saline, positive control medicine IMET and Rhizoma Acori Graminei crude extract respectively and histamine receptor 3 molecular probe FRET (fluorescence resonance energy transfer) is changed, concrete experimental technique as described in Example 1, repeats no more herein.
Experimental result: as shown in Figure 2, the cell expressing histamine receptor 3 molecular probe is placed under high speed fluorescence microscope, respectively with normal saline, positive control medicine IMET and Rhizoma Acori Graminei crude extract process cell, the FRET (fluorescence resonance energy transfer) change of monitoring histamine receptor 3 molecular probe, experiment proves that normal saline can be there is no significant reaction by histamine receptor 3 molecular probe, there is highly sensitive reaction in positive control medicine IMET, Rhizoma Acori Graminei crude extract is also had the reaction similar to positive control medicine IMET, the active component activating histamine receptor 3 molecular probe can be contained by preliminary proof Rhizoma Acori Graminei crude extract.
The above; being only the present invention preferably detailed description of the invention, but protection scope of the present invention is not limited thereto, any those familiar with the art is in the technical scope that the invention discloses; the change that can readily occur in or replacement, all should be encompassed within protection scope of the present invention.
Claims (2)
1. the active constituents of medicine screening technique based on histamine receptor 3 molecular probe, it is characterised in that described screening technique comprises the steps that
Step 1: build histamine receptor 3 molecular probe:
A. adopting Clontech test kit, the cDNA of people's histamine receptor 3 being connected on the existing carrier pcDNA5/FRT/TO-VSV-G-eCFP of laboratory, thus obtaining pcDNA5/FRT/TO-VSV-histamine receptor 3-eCFP plasmid;
B. with Clontech test kit, the cDNA of eYFP is connected in the middle part of acceptor control three internal ring, thus obtaining original molecular probe plasmid;
C. probe plasmid transfection HEK293 cell is used 48 hours, with expressed receptor molecular probe, and with commercially available agonist activation probe, with the sensitivity of Olympus high speed fluorescence microscope monitoring probe;
D. the monitoring result according to probe sensitivity, carries out structure optimization to probe;
Step 2: set up the Flp-InT-RexHEK293 cell line of abduction delivering g protein coupled receptor molecular probe:
A. by the molecular probe plasmid after optimization and pOG44 carrier cotransfection Flp-InT-RexHEK293 cell, and positive cell clone is screened with 200ug/mlHygromycin;
B. express corresponding g protein coupled receptor molecular probe with 1ug/mlDoxycycline inducing cell, with the expression of VSV antibody test probe proteins, monitor the cell location of molecular probe with fluorescence microscope;
C. process, with receptor stimulating agent, the acceptor molecule probe Flp-InT-RexHEK293 cell expressed, obtain the FRET curve of the receptor activation of different agonist dose, and after stopping administration continuation normal saline eluting agonist.
2. a kind of active constituents of medicine screening technique based on histamine receptor 3 molecular probe as claimed in claim 1, it is characterised in that: probe is carried out structure optimization by described step 1 specific as follows:
Adjust eYFP in the position of acceptor control three internal ring: acceptor control three internal ring is truncated to two ends respectively and respectively retains 12 aminoacid, before optimizing, the eYFP of probe is positioned at the centre position of acceptor control three internal ring, after optimization, the eYFP of probe is positioned at the 230-347 position of acceptor control three internal ring, and eCFP is positioned at 445 positions of receptor C-end.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610118510.8A CN105671119A (en) | 2016-03-02 | 2016-03-02 | Drug activity ingredient screening method based on histamine receptor 3 molecular probe |
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| CN201610118510.8A CN105671119A (en) | 2016-03-02 | 2016-03-02 | Drug activity ingredient screening method based on histamine receptor 3 molecular probe |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023010329A1 (en) * | 2021-08-04 | 2023-02-09 | 杭州浙大迪迅生物基因工程有限公司 | Reagent for detecting expression level of human histamine receptor hrh4 mrna, reagent kit, and detection method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102439444A (en) * | 2009-01-29 | 2012-05-02 | 联邦科学技术研究组织 | Measuring g protein coupled receptor activation |
-
2016
- 2016-03-02 CN CN201610118510.8A patent/CN105671119A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102439444A (en) * | 2009-01-29 | 2012-05-02 | 联邦科学技术研究组织 | Measuring g protein coupled receptor activation |
Non-Patent Citations (5)
| Title |
|---|
| CARSTEN HOFFMANN等: "A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells", 《NATURE METHODS》 * |
| JEAN-PIERRE VILARDAGA等: "A millisecond activation switch for G protein coupled receptors in living cells", 《NATURE BIOTECHNOLOGY》 * |
| TIAN-RUI XU等: "Intramolecular Fluorescence Resonance Energy Transfer (FRET) Sensors of the Orexin OX1 and OX2 Receptors Identify Slow Kinetics of Agonist Activation", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| 尹琪: "基于天然产物的药物开发", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 梁菊等: "荧光共振能量转移新技术及其在药物筛选中的应用", 《中国药学杂质》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023010329A1 (en) * | 2021-08-04 | 2023-02-09 | 杭州浙大迪迅生物基因工程有限公司 | Reagent for detecting expression level of human histamine receptor hrh4 mrna, reagent kit, and detection method |
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Application publication date: 20160615 |