CN107201342A - It is a kind of to be used to screen cell line of peptides and the non-analogs of peptides GLP 1 and preparation method and application - Google Patents
It is a kind of to be used to screen cell line of peptides and the non-analogs of peptides GLP 1 and preparation method and application Download PDFInfo
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- CN107201342A CN107201342A CN201710568912.2A CN201710568912A CN107201342A CN 107201342 A CN107201342 A CN 107201342A CN 201710568912 A CN201710568912 A CN 201710568912A CN 107201342 A CN107201342 A CN 107201342A
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Abstract
A kind of cell line and its application for being used to screen peptides and the non-analogs of peptides GLP 1, belongs to medicament high flux screening detection technique field.The present invention relates to a kind of stable acceptor (GLP 1R) albuminous cell strain GLP 1R/U2OS of expression fluorescence labeling human glucagon-like-peptide 1 foundation, and intracellular formation fluorescence spot after being stimulated based on GLP 1R/U2OS cell lines by the analog Exendin 4 of GLP 1, the detection method of the similar microbic activities of GLP 1 is determined with high intension system observation analysis metamorphosis.The detection method is easily standardized, reproducible, low, accurate and the characteristics of facilitate with the receptor-specifics of GLP 1, cost, is had a good application prospect.
Description
The application be submit on May 29th, 2014, it is entitled " one kind be used for screen peptides and non-peptides GLP-1 analogs
Cell line and preparation method and application " China's application 201410234121.2 divisional application.
Technical field
The invention belongs to medicament high flux screening detection technique field, in particular it relates to a kind of expression fluorescence labeling people's pancreas
The acceptor of glucagon-like peptide -1 (GLP-1R) albuminous cell strain GLP-1R/U2OS cell lines and its method for building up, and based on this
The metamorphosis of cell line intracellular generation fluorescence spot after being stimulated by glucagon-like peptide-1 analogs is high to determine pancreas
The detection method of blood glucose element sample peptide -1 similar microbic activity and its receptor stimulating agent, antagonist and conditioning agent.
Background technology
Incretin is the hormone that intestines and stomach discharge when Nutrient Absorption is to increase insulin secretion.Account for incretin
Two kinds of enteron aisle peptides of most of effects are：1) GLP-1 (Glucagon-like peptide, glucagon-like peptide 1)：Be by
Proglucagon gene code, under nutriment stimulation by Intestinal L cells (Langerhans cell) secrete containing 30 or
The hormone of 31 amino acid, GLP-1 plays the secretion for promoting insulin and suppression pancreas hyperglycaemia by acting on pancreatic beta cell
The effect of element secretion, GLP-1 and its acceptor are distributed widely in central nervous system, pancreas and gastrointestinal system.2)GIP
(Gastric inhibition polypeptide or glucose-dependent insulinotropic peptide,
Glucose-dependent-insulinotropic polypeptide)：Secreted by duodenum and proximal jejunum K cells.The a few minutes discharged from small intestine site
In clock, GLP-1 and GIP carries out rapid metabolization (proteolytic cleavage) into inactive metabolism by dipeptidyl peptidase-IV (DPP-IV)
Thing.The acceptor site that a small amount of active hormones arrival pancreas are acted on beta- cells stimulates pancreas islet with glucose-dependent manner
Element secretion；And GLP-1 acts on the secretion of alpha cells and glucagon suppression.It is currently based on controlling for incretin
Treatment is broadly divided into two major classes i.e. DPP-IV inhibitor (such as Xi Gelieting, BI 1356) and GLP-1 receptor stimulating agents (are based on
Exendin-4 is treated, such as Exenatide；People's GLP-1 analogs, such as Liraglutide).
GLP-1 is presently believed to be most important incretin, and its incretin for playing about 70-80% is lived
Property, all these physiological characteristics show that GLP-1 will play a significant role in the treatment of diabetes B.Newly-developed hypoglycemic agent
GLP-1 receptor stimulating agents can significantly, persistently reduce glycated hemoglobin levels, and with it is rapid, efficiently reduce blood glucose, improve pancreas
The effects such as island β cell functions, losing weight.Either animal level or cellular level, GLP-1 receptor stimulating agents are shown
The physiological action of antihyperglycemic；Promote the release of insulin, the secretion of glucagon suppression postpones gastric emptying, promotes β cells
Propagation and regeneration, suppress β cells apoptosis.
Although GLP-1 and the like correlative study has been achieved for good progress, and many analogues are also entered
Clinical experimental stage, the drawbacks of having one jointly yet with polypeptide drug, that is, cannot be administered orally, can only be by subcutaneous
Injection, therefore its compliance is not still high for type-II diabetes (T2DM) patient of long-term prescription.Due to polypeptide medicine
The characteristic of thing, its stability dependency is in time, temperature and pH value, and storage conditions require higher.Further, since these activators are all
Foreign protein is introduced to human body, the immune response of body may be triggered.The GLP-1 analogs listed at present are expensive,
The financial burden of meeting making patients.Screening small molecule GLP-1 analogs turn into study hotspot at present.
The assay method of the similar microbic activities of effective GLP-1 is set up, is to evaluate one of key measure of new drug drug effect.
The content of the invention
It is an object of the invention to provide a kind of peptides and non-peptides of new in-vitro screening glucagon-like peptide-1 receptor
There is provided one kind expression fluorescence labeling human glucagon-like-peptide-1 acceptor (GLP- for the method for activator, conditioning agent and antagonist
1R) albuminous cell strain GLP-1R/U2OS cell lines and its method for building up, XFP directly marks GLP-1R C-terminal, and is based on
This cell line after being stimulated by glucagon-like peptide-1 analogs the intracellular metamorphosis for producing fluorescence spot determine pancreas
The detection method of the similar microbic activity of glucagon-like peptide -1.
The present invention is achieved the above-mentioned purpose of the present invention with following technical schemes：
One plant of GLP-1R/U2OS cell line, is obtained by following methods：Build human glucagon-like-peptide-1 acceptor (GLP-
1R) fluorescin (XFP) carrier for expression of eukaryon GLP-1R/pCMV6, and stable transfection human osteosarcoma U2OS cells, obtain one plant
GLP-1R/U2OS cell lines.
The fluorescin (XFP) that described GLP-1R/U2OS cell lines contain, is GFP (green fluorescence
Protein), YFP (yelow fluorescence protein), RFP (red fluorescence protein) or any
Albumen with photoluminescent property.
Described GLP-1R/U2OS cell lines or other applicable cell such as HEK293 (human embroynic
Kidney 293) cell, obtained by following methods：First build human glucagon-like-peptide-1 acceptor (GLP-1R) green fluorescence egg
(GFP) carrier for expression of eukaryon GLP-1R/pCMV6, then in vain thin by Lipofectamine2000 operational manuals progress transfection U2OS
Born of the same parents.G418 (400 μ g/ml) pressurizations screening 2 weeks, obtains the positive cell clone with antibiotic resistance, amplification cultivation is steady
Surely expression people GLP-1R U2OS cell lines, are named as GLP-1R/U2OS cell lines.
The method that the similar microbic activities of GLP-1 are determined with described GLP-1R/U2OS cell lines, has transfected XFP marks
GLP-1R genes under conditions of, receptor stimulating agent stimulation under induce the intracellular generation fluorescence spots of GLP-1R/U2OS, transfection
GLP-1R can stablize express.
Method as mentioned, wherein described receptor stimulating agent is GLP-1 analogs, after GLP-1 binding of receptor and ligand
Intracellular generation fluorescence spot intensity and the activity of receptor stimulating agent are proportionate, and GLP- is determined with high intension system observation analysis
1R/U2OS intracellular Fluorescence spot intensities, determine the bioactivity of receptor stimulating agent.
Method as mentioned, wherein described GLP-1 analogs are insulin secretion accelerating peptide Exendin-4.
Method, GLP-1R expression result is observed with high intension system as mentioned, intracellular with GLP-1R/U2OS
The change of fluorescence spot mean intensity, which determines GLP-1 analogs and GLP-1R biological active matters, includes GLP-1 receptor stimulating agents, antagonism
Agent and conditioning agent etc..
The invention provides stable expression fluorescence labeling human glucagon-like-peptide-1 acceptor (GLP-1R) albuminous cell strain
GLP-1R/U2OS foundation, and based on GLP-1R/U2OS cell lines by cell after GLP-1 analogs Exendin-4 stimulations
Interior formation fluorescence spot, the detection side of the similar microbic activities of GLP-1 is determined with high intension system observation analysis metamorphosis
Method.The detection method is easily standardized, reproducible, low, accurate and the characteristics of facilitate with GLP-1 receptor-specifics, cost, tool
There is good application prospect.
The method of domestic screening GLP-1 receptor stimulating agents is by the gene of GLP-1 acceptors and reporter gene cotransfection at present
Activator is carried out by intracellular cAMP levels change after stimulation in cell surface to intracellular, and by GLP-1 expression of receptor
Screening.The invention provides fluorescence labeling human glucagon-like-peptide-1 acceptor (GLP-1R) albuminous cell strain GLP-1R/U2OS,
Have the advantages that quick, sensitive, selectivity is high compared to domestic screening technique, and the generation of false positive can be substantially reduced, and
And can be screened for mixture, while can also reduce testing cost.
More specifically, the application is related to：
1st, one plant of GLP-1R/U2OS cell line, it is characterised in that obtained by following methods：Build human glucagon-like
Peptide-1 receptor fluorescin carrier for expression of eukaryon GLP-1R/pCMV6, and stable transfection human osteosarcoma U2OS cells, obtain one plant
GLP-1R/U2OS cell lines.
2nd, the fluorescin XFP that the GLP-1R/U2OS cell lines according to item 1 contain is GFP, YFP, RFP or any
Albumen with photoluminescent property.
3rd, according to item 1 GLP-1R/U2OS cell lines or other applicable cells such as HEK293 cells, its feature exist
Obtained in by following methods：Human glucagon-like-peptide-1 acceptor GLP-1R green fluorescent protein GFP eukaryotic expressions are first built to carry
Body GLP-1R/pCMV6, then carry out transfection U2OS cells, G418 pressurizations screening 2 by Lipofectamine2000 operational manuals
In week, the positive cell clone with antibiotic resistance is obtained, amplification cultivation is stable expression people GLP-1R U2OS cell lines,
It is named as GLP-1R/U2OS cell lines.
4th, the method that the similar microbic activities of GLP-1 are determined with the GLP-1R/U2OS cell lines described in item 1, its feature exists
In：Under the conditions of the GLP-1R genes for having transfected XFP marks, the intracellular productions of GLP-1R/U2OS are induced under receptor stimulating agent stimulation
Raw fluorescence spot, the GLP-1R of transfection is stable to express.
5th, the method as described in item 4, it is characterised in that described receptor stimulating agent be GLP-1 analogs, GLP-1 acceptors with
Intracellular generation fluorescence spot intensity and the activity of receptor stimulating agent are proportionate after ligand binding, with the observation point of high intension system
Analysis determines GLP-1R/U2OS intracellular Fluorescence spot intensities, determines the bioactivity of receptor stimulating agent.
6th, the method as described in item 4, it is characterised in that described GLP-1 analogs are insulin secretion accelerating peptide Exendin-
4。
7th, the method as described in item 4, it is characterised in that GLP-1R expression result is observed with high intension system, is used
The change of GLP-1R/U2OS intracellular Fluorescence spots mean intensity, which determines GLP-1 analogs and GLP-1R biological active matters, to be included
GLP-1 receptor stimulating agents, antagonist and conditioning agent etc..
Brief description of the drawings
Fig. 1 XFP mark GLP-1R schematic diagrames；
Fig. 2 recombinant eukaryon expression vector GLP-1R/pCMV6 schematic diagrames；
The high intension system observation analysis cell surface GLP-1R of Fig. 3 expression result；
Intracellular formation fluorescence spot after the high intension system observation analysis of Fig. 4 is stimulated；
Active reaction Dependence Results after Fig. 5 GLP-1R/U2OS cells are activated through Exendin-4.
Embodiment
Below in conjunction with the accompanying drawings, further illustrated with embodiments of the invention the present invention essentiality content, but not with
This limits the present invention.
The GFP fluorescence labeling people's GLP-1R carrier for expression of eukaryon GLP-1R/pCMV6 of embodiment 1 structure, GLP-1R/U2OS
The foundation of cell line, and the change of GLP-1R/U2OS intracellular Fluorescence spots mean intensity determine GLP-1 analogs biology and lived
Property.
First, GFP fluorescence labelings people GLP-1R carrier for expression of eukaryon GLP-1R/pCMV6 structure：
People GLP-1R cDNA are expanded：Using people GLP-1R cDNA as template, total length people GLP-1R is expanded by the way of PCR
CDNA code areas, total length 1392bp.
Its sense primer:5’-gaggcgatcgccATGGCCGGCGCCCCCGGC-3’；
Anti-sense primer:5’-gcgacgcgtGCTGCAGGAGGCCTGG CAAG-3’.
2. recovery, purified pcr product, are connected on pMD18-T Vector, recombinant plasmid GLP-1R/pMD18-T is obtained,
Digestion is carried out to plasmid using restriction enzyme Sgf I and Mlu I.
3. with restriction enzyme Sgf I and Mlu I digested plasmids G-D (pCMV6-AC-GFP-DR5).
4. reclaiming digestion products, connected 8 hours at 16 DEG C, obtain recombinant plasmid GLP-1R/pCMV6, gone according to OMEGA interior
The a large amount of extracts kit specifications of toxin plasmid extract GLP-1R/pCMV6 plasmids.
The clone PCR reaction system of gene order is 50 μ L, by 5 μ 10 × Ex-Taq of L buffer, 1 μ L Ex-Taq
Archaeal dna polymerase 5U/ μ L, 3 μ L2.5mM dNTP, 1 μ L sense primer 10pmol/ μ L, 1 μ L anti-sense primer 10pmol/ μ L, 1 μ L matter
Grain (BC113493) 100ng/ μ L and 38 μ L sterilized waters composition；PCR reaction conditions：95 DEG C of pre-degenerations 5min, 95 DEG C of denaturation 1min,
65 DEG C of annealing 0.5min, 72 DEG C of extension 1min, totally 30 circulations, 72 DEG C of extension 10min；10 × Ex-Taq buffer used
In contain Mg2+。
Reclaim PCR primer and be connected to pMD18-T Vector reaction systems for 10 μ L, PCR primer, 4.5 μ L are reclaimed by 5 μ L
SolutionI, 0.5 μ L pMD18-T Vector are constituted；Reaction condition：Connected 4 hours at 16 DEG C.
Double digestion plasmid GLP-1R/pMD18-T reaction systems are 50 μ L, by 2 μ L Sgf I (5U/ μ L), 2 μ L Mlu I
(5U/ μ L), 5 μ L10 × Tango Buffer, 25 μ LGLP-1R/pMD18-T and 16 μ L sterilized waters composition；Reaction condition：37℃
Incubate 2 hours.
Double digestion plasmid G-D (pCMV6-AC-GFP-DR5) reaction system is 50 μ L, by 2 μ L Sgf I (5U/ μ L), 2 μ L
Mlu I (5U/ μ L), 5 μ L10 × Tango Buffer, 10 μ LG-D and 31 μ L sterilized waters composition；Reaction condition：37 DEG C of incubations 2 are small
When.
Construction recombination plasmid GLP-1R/pCMV6 is to use the restricted digestion products in step (2) and (3), in 16 DEG C of bar
Connected 8 hours under part；Reaction system is 11 μ L, the plasmid pCMV6 after restricted digestion in 5 μ L SolutionI, step (4)
Fragment and GLP-1R gene order fragments are respectively 1 μ L and 5 μ L compositions；Reaction condition：Connected 8 hours at 16 DEG C.
The plasmid obtained in step (4) further can carry out preservation amplification by Transformed E .coli DH5 α.
2nd, the foundation of stable transfection people GLP-1R U2OS cell lines：
U2OS cells are in the DMEM culture mediums containing 10% (volume fraction) calf serum, in 37 DEG C, 5% (volume fraction)
CO2Saturated humidity under cultivate.Carried out when cell fusion is to 70%-80% by Lipofectamine2000 operational manuals
Recombinant expression carrier GLP-1R/pCMV6 transfection.Transfect after 48h, with trypsin digestion and cell, containing G418 (400 μ g/
Ml pressurization screening 2 weeks, obtain the positive cell clone with antibiotic resistance, amplification cultivation is steady in Selective agar medium)
Surely expression people GLP-1R U2OS cell lines, are named as GLP-1R/U2OS cell lines.GLP-1R table is observed with high intension system
Up to situation result.
3rd, the change of GLP-1R/U2OS intracellular Fluorescences spot mean intensity determines the similar microbic activities of GLP-1：
The GLP-1R/U2OS cells of above-mentioned acquisition are incubated at 96 well culture plates by 100 μ L/ holes (1000 cells/well), used
DMEM culture mediums containing 10% (volume fraction) calf serum, 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours.Remove within second day
Full culture medium, is cleaned with PBS, adds various concentrations sample to be tested Exendin-4, and remove after 20 minutes molten in hole
Liquid, 30 minutes are fixed with 4% paraformaldehyde room temperature, are washed with PBS 3 times, are added DAPI dyestuffs lucifuge and are dyed 15 minutes, use
PBS is washed 5 times, and 100 μ LPBS buffer solutions are stayed in hole, is observed analysis of fluorescence spot with high intension system, is used in combination
Transfluor module analysis calculates spot mean intensity, and standard curve is made with Excel.
GLP-1R expression on the GLP-1R/U2OS cell membranes of embodiment 2
GLP-1R/U2OS cells are incubated at 96 well culture plates by 100 μ L/ holes (1000 cells/well), with containing 10% (volume
Fraction) calf serum DMEM culture mediums, 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours.Remove complete medium within second day, use
PBS is cleaned, and is fixed 30 minutes with 4% paraformaldehyde room temperature, is washed with PBS 3 times, adds DAPI dyestuffs lucifuge dye
Color 15 minutes, is washed 5 times with PBS, and 100 μ LPBS buffer solutions are stayed in hole, and fluorescence is observed with high intension system.
The GLP-1R/U2OS cells of embodiment 3 express GLP-1 receptor activity assays
GLP-1R/U2OS cells are incubated at 96 well culture plates by 100 μ L/ holes (1000 cells/well), with containing 10% (volume
Fraction) calf serum DMEM culture mediums, 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours.Remove complete medium within second day, use
PBS is cleaned, and adds sample to be tested Exendin-4, and room temperature is placed 20 minutes, removes solution in hole, use 4% paraformaldehyde
Room temperature fixes 30 minutes, is washed with PBS 3 times, adds DAPI dyestuffs lucifuge and dyes 15 minutes, is washed with PBS 5 times,
100 μ LPBS buffer solutions are stayed in hole, change in fluorescence is observed with high intension system.As a result intracellular generation fluorescence spot is shown, and
The U2OS cells of untransfected GLP-1R genes do not have any change.Above experimental result confirms restructuring GLP-1R/U2OS cell tables
The GLP-1R albumen reached has the similar physiologically active of natural GLP-1R albumen, with specificity.
The insulin secretion accelerating peptide Determination of biological activity of embodiment 4
The insulin secretion accelerating peptide Exendin-4 of various concentrations is taken, with the GLP-1R/ in the method stimulating growth of embodiment 3
U2OS cells, high each sample of intension system observation analysis stimulates the fluorescence spot mean intensity after GLP-1R/U2OS cells.With sample
This concentration is Transverse coordinates, using spot mean intensity as ordinate, makees curve.As a result represent, spot is put down after being stimulated through Exendin-4
Equal intensity curve is in typical anti-S types, and the Exendin-4 of various concentrations, which is stimulated, forms the flat of spot after GLP-1R/U2OS cells
There is dosage effect correlation in equal intensity.
Claims (8)
1. the U2OS cell lines of one kind expression human glucagon-like-peptide-1 acceptor (GLP-1R), the C-terminal band of the GLP-1R
There is fluorescence labeling.
2. the cell line of claim 1, fluorescence labeling therein is the albumen with photoluminescent property.
3. the cell line of claim 2, the albumen therein with photoluminescent property is green fluorescent protein (GFP).
4. the preparation method of the cell line of any one of claims 1 to 3, comprises the following steps：
(1) carrier for expression of eukaryon of people's GLP-1R- fluorescins is built,
(2) with the carrier transfection human osteosarcoma U2OS cells of step (1).
5. the method for in-vitro screening GLP-1R peptides and non-peptide excitomotor, conditioning agent and antagonist, be with claim 1~
Any one of 3 cell line is screened.
6. the method for claim 5, fluorescence is observed with high intension system.
It is with any one of claims 1 to 3 7. determining the method for the bioactivity of GLP-1R activators, conditioning agent and antagonist
Cell line be measured.
8. the method for claim 7, fluorescence is observed with high intension system.
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| CN110129279A (en) * | 2019-04-24 | 2019-08-16 | 昆明理工大学 | A kind of enterococcus faecalis bacteriophage and its separation, purifying, enrichment and application |
Families Citing this family (5)
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| CN104877966A (en) * | 2014-12-23 | 2015-09-02 | 昆明理工大学 | P2Y1/U2OS cell strain and preparation method and application thereof |
| CN107462559B (en) * | 2017-08-16 | 2020-10-13 | 中国食品药品检定研究院 | In vitro assay method for biological activity of GLP-1-like compounds |
| CN108220383A (en) * | 2018-01-05 | 2018-06-29 | 北京博康健基因科技有限公司 | The detection kit and detection method of a kind of rExendin-4 pharmaceutical activity and application |
| CN113755442B (en) * | 2020-06-03 | 2023-07-14 | 珠海联邦制药股份有限公司 | Cell strain for measuring pharmaceutical activity and preparation method and application thereof |
| CN114763562B (en) * | 2021-05-28 | 2023-04-11 | 生物岛实验室 | Glucagon-like peptide-1 receptor stable expression cell strain |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333555A (en) * | 2007-12-25 | 2008-12-31 | 浙江大学 | Novel horizontal screening system construct by recipient cell G protein coupling and applications |
| CN103374591A (en) * | 2012-04-13 | 2013-10-30 | 天津拓飞生物科技有限公司 | Establishment and application of stable and high-expression cell line of GLP-1 receptor |
| US20160313251A1 (en) * | 2015-04-22 | 2016-10-27 | Academia Sinica | BIOSENSOR FOR DETECTING INTRACELLULAR CYCLIC ADENOSINE MONOPHOSPHATE (cAMP) AND USES THEREOF |
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| EP2494062B1 (en) * | 2009-10-28 | 2016-12-28 | Janssen Biotech, Inc. | Anti-glp-1r antibodies and their uses |
| CN102649947A (en) * | 2012-04-20 | 2012-08-29 | 无锡和邦生物科技有限公司 | Cell strain for measuring bioactivity of GLP-1 and functional analogue thereof and application of cell strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101333555A (en) * | 2007-12-25 | 2008-12-31 | 浙江大学 | Novel horizontal screening system construct by recipient cell G protein coupling and applications |
| CN103374591A (en) * | 2012-04-13 | 2013-10-30 | 天津拓飞生物科技有限公司 | Establishment and application of stable and high-expression cell line of GLP-1 receptor |
| US20160313251A1 (en) * | 2015-04-22 | 2016-10-27 | Academia Sinica | BIOSENSOR FOR DETECTING INTRACELLULAR CYCLIC ADENOSINE MONOPHOSPHATE (cAMP) AND USES THEREOF |
Non-Patent Citations (1)
| Title |
|---|
| 殷承慧等: "针对G蛋白偶联受体的药物筛选新方法", 《世界临床药物》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129279A (en) * | 2019-04-24 | 2019-08-16 | 昆明理工大学 | A kind of enterococcus faecalis bacteriophage and its separation, purifying, enrichment and application |
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