CN102888382A - Cell model, construction method and application thereof for screening nervous disorder resistant medicines - Google Patents
Cell model, construction method and application thereof for screening nervous disorder resistant medicines Download PDFInfo
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Abstract
The invention discloses a cell model, a construction method and application of the model for screening nervous disorder resistant medicines, belonging to the technical field of genetic engineering and molecular pharmacology. The cell model is a mammalian cell carrying and expressing mGluR2 genes. The construction method of the cell model comprises the following steps: connecting the mGluR2 genes with expression vectors to construct recombinant plasmids, leading the recombinant plasmids into the mammalian cells, screening and culturing to obtain cell strains stably expressing the mGluR2 genes. The cell model is used for screening the nervous disorder resistant medicines, adding matters to be screened into cell model culture mediums and measuring the yield or the activated degree of responsive elements to judge whether the matters are screened. The cell model is capable of accurately screening the nervous disorder resistant medicines by high throughput and low cost and has the advantages of simple and controllable screening method steps, low cost, high throughput, good stability and reliability.
Description
Technical field
The present invention relates to genetically engineered and molecular pharmacology technical field, be specifically related to a kind of cell model and construction process thereof that can screen the anti-nervous system disorders medicine take mGluR2 as target spot, and the application of this cell model in the anti-nervous disorders disease medicament of screening.
Background technology
Modern society is along with environmental pollution and life stress increases the weight of day by day, and mental disease and nervous system disease, particularly nerve degenerative diseases are perplexing the mankind more and more." World Health Report " of up-to-date announcement pointed out: at present, nerve degenerative diseases and mental disease thereof have become the arch enemy of man.In the world, nerve degenerative diseases Parkinson's disease patient just has 5,000,000 people approximately, only is in China, and the patient has surpassed more than 200 ten thousand people.Therefore the new drug initiative for mental disease and nerve degenerative diseases has been put into the state key development strategy.Also become the focus that each large medicament research and development company of the whole world pays close attention to.
The GPCR member of C family is because its unique physiological function becomes the focus in mental disease and the nerve degenerative diseases medicament research and development focus.The GPCR member of C family comprises metabolic pattern gamma-aminobutyric acid receptor (GABABR), metabotropic glutamate receptor (mGluR1-8), calcium sensitivity acceptor (CaSR) and Taste Receptors, this family receptors participates in a lot of important physiology processes in vivo, and excessive activation or suppress can cause that all the pause pathology of the nervous disorders diseases such as disease, parkinsonism, drug dependence, pain of epilepsy, schizophrenia, prosperous front yard occurs.Importance on the function and structural easily handling so that this family member becomes important drug target, also becomes the emphasis of each large pharmaceutical companies research and development, and potential high scientific research and marketable value are arranged.By the end of 2005, according to the corresponding statistics that IMS Health makes, the antipsychotic drug relevant with GPCR in the medicine of selling now, sales volume has reached 47,600,000,000 dollars, and keeps staggering growth speed.
MGluR2 is an important acceptor in the central nervous system, belongs to the GPCR of C family, is classified as mGluR subtribe II(Group II mGluRs with mGluR3).MGluR2 is the transmembrane receptor that is positioned on the cytolemma, is a kind of important g protein coupled receptor.Its active state imbalance can cause that the downstream signal Pathway Activation is disorderly, thereby causes the generation of a series of abalienation diseases.MGluR2 is a homodimer, is distributed widely in pallium, anterior olfactory nucleus, and nucleus accumbens septi, hypothalamus is in the cell at the positions such as hippocampal gyrus.Each mGluR2 comprises VFT and two cross-film HD structural domains that two born of the same parents are outer.When L-glutamic acid is attached to positive structure site or allosteric agent is attached to allosteric site, mGluR2 is activated and is coupled mutually with Gi/o, can suppress adenylate cyclase after being activated and then suppress cAMP to produce.
Generally, mGluR2 mainly is positioned at presynaptic membrane, suppresses the release of neurotransmitter, and the affect the nerves excitability of unit and the transmission of cynapse are the desirable targets for the treatment of neurological disorder medicine.The at present research of this respect still is in the starting stage, and the medicine take mGluR2 as target spot also is not used to clinical.But a large amount of experimentation on animalies and preclinical study prove, mGluR2 knocks out model and other pharmacological research methods and find that mGluR2 plays a significant role in the physiology such as cognitive, drug habit and psychosis and pathologic process.Reach clinical study before clinical and show that mGluRs subtribe II is treatment anxiety and schizoid potential newtype drug target spot, this discovery has milestone significance in anxiety and treatment of schizophrenia history, might finally promote the agonist of mGluR subtribe II or the Primary Care scheme that PAMs becomes these two kinds of diseases for the treatment of.In addition, the agonist of mGluR2 and positive allosteric agent can also be treated other central nervous system diseases, comprise pain, habituation, depression and epilepsy etc.
High-flux medicaments sifting (high throughput screening based on the cell signal path, HTS) be to utilize the cell signal system as response element, detect signal event in the cell that acceptor and various albumen as drug target mediates by highly sensitive data collecting system and technique means, thereby whether the observation compound can act on drug target, and then finds and have optionally lead compound of target spot.Material usage is few, mechanism of drug action clearly waits characteristics and the medicaments sifting model of molecule and cell levels has, become present searching and find novel drugs main drug screening method (FONNUM F. A rapid radio chemical method for the determination of choline acetyltransferase [J]. J Neurochem, 1975,24 (2): 407-409.).With drug target import tool cell, and making up the cell screening model of stable restructuring high expression level drug target target spot, is the key link that realizes high-flux medicaments sifting.Medicaments sifting model is the experimental technique that has pharmacologically active (biological activity, therapeutic action) for certain material of proof, is to seek and find one of essential condition of novel drugs.
At present, relatively ripe based on the high-flux medicaments sifting methodology of GPCR downstream signal transduction, many companies can provide high throughput testing instrument and scheme.The discovery of medicaments sifting model and structure then become restriction and promote the important factor of this field development, and especially the model construction based on acceptor molecule mechanism becomes core technology and the difficult point of finding the pilot compound especially.Also do not have in the prior art can high flux screening neurological disorder disease medicament cell model.
Summary of the invention
For above-mentioned deficiency of the prior art, the present invention has set up a kind of screening model of anti-nervous disorders disease, it be a kind of can the stably express GPCR-mGluR2 relevant with nervous system disorders, the medicine take mGluR2 as target spot be can sensitive, special efficacy screen, agonist, inhibitor and allosteric agent etc. comprised.
A kind of cell model provided by the invention is the mammalian cell that carries and can express the mGluR2 gene.Namely this cell model is the mammalian cell of having cloned the vitro culture of expressing encoding human source mGluR2 gene order.
Preferably, the nucleotide sequence of described mGluR2 gene is shown in SEQ ID NO:1.
Preferably, described mammalian cell is Chinese hamster ovary celI or HEK293 cell.Other mammalian cell also can be realized the present invention, and with Chinese hamster ovary celI or HEK293 cell construction best results.
Preferably, the name of this cell model is called Chinese hamster ovary cell CHO-hmGluR2 (C12), is preserved in Chinese Typical Representative culture collection center on September 18th, 2012, and deposit number is CCTCC NO:C2012100.Preservation address: China. Wuhan. Wuhan University.
The present invention also provides a kind of construction process of above-mentioned cell model, and step is as follows:
(1) the mGluR2 gene is connected with expression vector, makes up the recombinant plasmid that contains the mGluR2 gene;
(2) with the Transfected Recombinant Plasmid mammalian cell, cultivate into the cell strain of energy stably express mGluR2 gene, this cell strain is described cell model.
The construction process of above-mentioned cell model, preferably, also comprise the step (3) of cell strain being carried out comprehensive assessment: in cell strain, add mGluR2 agonist or inhibitor, measure the downstream signal responsing reaction, according to this cell strain of responsing reaction outcome evaluation whether as cell model.
Described mensuration downstream signal responsing reaction comprises one or more that detect in mGluR2 and its ligand binding degree, mGluR2 downstream G albumen and [35S] GTP γ S combination degree, cyclic amp accumulation volume, InsP3 accumulation volume and the intracellular calcium stream variable quantity.Wherein detecting mGluR2 and its ligand binding degree and G albumen and [35S] GTP γ S combination degree can adopt radioactivity to be combined experiment.
Preferably, the described expression vector of step (1) is pcDNA5/FRT.
Preferably, the cell that uses of step (2) is Chinese hamster ovary celI.For example can use Invitrogen commercialization clone Flp-in Chinese hamster ovary celI, but Flp-In clone is expressed target protein through design rapid build stable expression cell line can adopt the Flp-In expression vector.These cells contain recombinase recognition sequence (FRT) site of a stable integration in the transcriptionally active district.The site-directed integration of Flp-In expression vector can be guaranteed the high expression level amount.With Flp-In expression vector and Flp recombinase carrier pOG44 cotransfection Flp-In clone so that expression vector by site-directed integration on the identical chromosomal foci of each cell.
The construction process of above-mentioned cell model, more preferably: step (1) also comprises the recombinant plasmid that the makes up evaluation of checking order; The described Transfected Recombinant Plasmid mammalian cell of step (2) is altogether to import in mammalian cell with the plasmid pOG44 that expresses recombinase recombinant plasmid, recycling Totomycin (Hygromycin) screens, acquisition utilizes Western blot after continuing to cultivate, surveys cAMP or Ca in the cell with the monoclonal cell of hygromycin resistance
2+The methods such as stream detect the positive colony that can express mGluR2, cultivate into the cell strain of energy stably express mGluR2 gene.
The present invention also provides the application of above-mentioned cell model in the anti-nervous disorders disease medicament of screening.
Preferably, described anti-nervous disorders disease medicament is the reticent allosteric agent of mGluR2 agonist, mGluR2 inhibitor, mGluR2 forward allosteric agent, the reverse allosteric agent of mGluR2 or mGluR2.
The present invention has following beneficial effect:
Cell model of the present invention is directly monitored the activity of mGluR2 by the acknowledge signal that detects cell signaling molecule and produce, can high-throughput, low cost, screen the medicine of nervous disorders disease exactly; The preparation method of cell model of the present invention is simply controlled, and is with low cost; Nervous disorders disease medicament screening method step of the present invention is simple, with low cost, and accuracy is high, can realize high-throughout screening, has good stability and reliability.For the screening of anti-nervous disorders disease medicament provides new thinking, have good market outlook.
Description of drawings
Fig. 1 is the fundamental diagram of cell model of the present invention.
Fig. 2 is the Western blot qualification result figure to positive colony, and wherein, CHO represents the CHO ghost, and CHO-mGluR2 C10, C11, C12, C13, C14 and C15 represent respectively the CHO-mGluR2 cell clone of different numberings.
Fig. 3 is that LY379268 flows the experimental result that affects that reaches cell viability on the CHO-mGluR2 intracellular Ca2+.A is the impact that different concns LY379268 flows intracellular Ca2+, and ordinate zou is the calcium release amount; B be different concns LY379268 on the impact of cell viability, ordinate zou is luminosity absorption value (be the light absorption value at 560nm place at wavelength), X-coordinate is the concentration of LY379268.
Fig. 4 is that LY341495 flows the result that affects who reaches cell viability to the CHO-mGluR2 intracellular Ca2+ that LY379268 induces.The impact of the A intracellular Ca2+ stream that to be different concns LY341495 induce LY379268, ordinate zou is the calcium release amount.B be different concns LY341495 on the impact of cell viability, ordinate zou is luminosity absorption value (be the light absorption value at 560nm place at wavelength), X-coordinate is the concentration of LY341495.
Fig. 5 is the impact that LY379268, DHPG flow intracellular Ca2+ in CHO ghost and the CHO-mGluR2 clone.A is different concns LY379268 on the impact of CHO ghost (CHO Mock) and CHO-mGluR2 clone (CHO-mGluR2-Clone12) cellular calcium stream, and B is that different concns medicine (LY379268 and DHPG) is on the impact of CHO-mGluR2 clone (CHO-mGluR2-Clone12) cellular calcium stream.
Fig. 6 is that different cells are inoculated numbers to replying the impact of element testing, and ordinate zou is CHO-mGluR2 calcium release amount, and X-coordinate is every porocyte number.
Fig. 7 be the different passage numbers of cell on replying the impact of element testing, A was the 7th generation, B was the 23rd generation, ordinate zou is CHO-mGluR2 calcium release amount, X-coordinate is LY379268 and L-Glutamate concentration.
Fig. 8 is the assessment result based on the drug screening method of this cell model, and ordinate zou is the calcium release amount, and X-coordinate is sample number.LY379268+ represents to add the experimental group of LY379268 drug treating, and LY379268-represents not add the control group that medicine is processed.
Embodiment
The invention will be further described below in conjunction with the drawings and specific embodiments so that those skilled in the art the present invention may be better understood and can be implemented, but illustrated embodiment is not as a limitation of the invention.
Material and the reagent used among the embodiment are as follows:
Flp-in Chinese hamster ovary celI (a kind of Chinese hamster ovary cell is called for short Chinese hamster ovary celI in following examples), expression vector pcDNA5/FRT and transfection reagent Lipofectin2000 are the commercially produced product of Invitrogen company; F12 substratum and foetal calf serum are available from Gibco company; Other reagent is import and domestic analytical reagent; LY379268, LY341495 are available from Tocris, L-Glutamate(L-L-glutamic acid) available from Sigma company.
Embodiment 1The structure of cell model
Take Chinese hamster ovary cell (CHO) as recipient cell, carry out the foundation of cell model.The Chinese hamster ovary celI cultivation is contained the F12 culture medium culturing of 10% foetal calf serum, placing 37 ℃, 5%CO
2Cultivate in the incubator.
(1) construction of recombinant plasmid
End user source mGluR2 cDNA(SEQ ID NO:1) sequence increases, and obtains goal gene mGluR2, the mGluR2 gene of amplification is connected ligase enzyme catalysis connects with the pcDNA5/FRT expression vector, transforms intestinal bacteria; Plasmid DNA is extracted in amplification, and extracting checks order to identify positive colony, identifies that correct positive colony is recombinant plasmid, called after pcDNA5/FRT-mGluR2.
(2) Transfected Recombinant Plasmid Chinese hamster ovary celI
The preparation (60mM culture dish) of DNA and liposome (Lipofectin2000) mixture
A solution:
Recombinant plasmid pcDNA5/FRT-mGluR2 0.8 μ g
Express the plasmid pOG44 7.2 μ g of recombinase (flp)
Substratum Opti MEM 500 μ L
A solution mixes, and room temperature is placed 5min;
B solution:
Liposome Lipofectamine2000 20 μ L
Substratum Opti MEM 500 μ L
B solution mixes, and room temperature is placed 5min;
A liquid is mixed with B liquid, and room temperature is placed 20min, then mixed solution is joined in the Chinese hamster ovary celI.At 37 ℃, 5%CO
2CO2gas incubator in cultivate 6h after, be changed to and contain serum and contain the microbiotic substratum.
(3) positive colony screening
Chinese hamster ovary celI behind the transfection 24h goes down to posterity by the density of 1:10 and cultivates 24h again, to contain 500 μ g/mL Hygromycin(Totomycin) the F12 selective medium carry out selective screening and cultivate, changed liquid once with selective medium in per 3 days, and continued 2 weeks visible positive colonies and occur.Adopt extreme dilution method that positive colony is chosen, be seeded to 96 orifice plates and select the nutrient solution amplification to go down to posterity with the F12 that contains 250 μ g/mL Hygromycin again, obtain the positive colony cell model of stable transfection.
(4) positive colony is identified
The cell model that step (3) obtains identifies that with western blot to obtain mGluR2 expression among the clone, the CHO ghost in contrast.Utilize the mGluR2 specific antibody, we find that the 10-15 number clone who selects has mGluR2 to express (C10 of CHO-mGluR2 clone, C11, C12, C13, C14, C15), and mGluR2 does not express (Fig. 2) in the ghost.We select 12 clones to do subsequent experimental.
No. 12 clone called after Chinese hamster ovary cell CHO-hmGluR2 (C12) are preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:C2012100.
Embodiment 2Cell model of the present invention is used for the specificity of drug screening
MGluR2 is the g protein coupled receptor of Gi/o coupling, produces by inhibition adenylate cyclase (AC) and then inhibition cAMP after it activates, thereby can detect mGluR2 by the generation that detects cAMP and activate situation; In addition can be in this clone transfection Gqi9 mosaic type G albumen, the signal that mGluR2 activates can be coupled to the PLC signal path and come, then promote intracellular calcium ion to discharge, thereby can be by detecting Ca
2+Release determines that mGluR2 activates situation, can the functional mGluR2 of stably express, all employing detection Ca for identification of cell system in the present embodiment
2+The method that discharges.
Be used for the specificity of drug screening for detecting this cell model, the response of guaranteeing response element is regulated and control by the activity change of mGluR2.Select the specific agonist LY379268 of Group II mGluRs, the specific inhibitor LY341495 of Group II mGluRs, process cell model and determine that by the response that molecule is replied in detection it induces activity ratio with different concentration respectively, determine the vigor of cell by mtt assay.
(1) LY379268 is to Ca in the cell
2+The dose-effect relationship of release and cell viability
1. cell is inoculated in 96 orifice plates and spends the night, after cell is fully adherent, change 1 μ M Fluo-4 AM(calcium fluorescent probe) and 0.02% pluronic acid hatch 1h.Then add respectively 10
-10, 10
-9, 10
-8, 10
-7, 10
-6, 10
-5, 10
-4The LY379268 of M stimulates, and establishes 3 repetitions for every group.Measure the Ca that each group is induced with FlexStation 3 multi-functional microplate reader workstations
2+Discharge.
Test-results is referring to Fig. 3 A, and LY379268 is as Group II mGluRs specific agonist, and is obvious to the activation effect of mGluR2.In this experiment, LY379268 is to Ca in the cell
2+Induce release rate and LY379268 concentration be dose-dependence.
2. cell is inoculated in 96 orifice plates and spends the night, after cell is fully adherent, changes fresh serum-free medium and cultivate 24h, adding respectively concentration is 10
-9, 10
-8, 3 * 10
-8, 10
-7, 3 * 10
-7, 10
-6, 10
-5, 10
-4The LY379268 of M stimulates, and establishes 3 repetitions for every group.Use the MTT(colorimetry after processing 24h) the detection cell viability.
Test-results detects as can be known by MTT referring to Fig. 3 B, and cell viability increases without significantly increasing (Fig. 3) with LY379268 concentration.LY379268 is to Ca in the cell in this explanation
2+The promotion that discharges and the vigor of cell proliferation are irrelevant.
(2) LY341495 induces the impact of interior calcium release on LY379268
1. cell is inoculated in 96 orifice plates and spends the night, after cell is fully adherent, changes 1 μ M Fluo-4 AM and 0.02% pluronic acid and hatch 1h.Use 10
-11, 10
-10, 10
-9, 10
-8, 10
-7, 10
-6, 10
-5, 10
-4M LY341495 preincubate 30min adds 10
-5The LY379268 mixed solution of M stimulates, and establishes 3 repetitions for every group.Measure the Ca that induces with FlexStation 3 multi-functional microplate reader workstations
2+Discharge.
Experimental result is seen Fig. 4 A, and LY341495 is as Group II mGluRs specific inhibitor, can establishment LY379268 to the activation of mGluR2.In this experiment, 10
-5M LY341495 is 10 to concentration
-5Calcium discharges and all can suppress in the clone that the LY379268 of M induces, and makes interior calcium releasing degree close to background.
2. cell is inoculated in 96 orifice plates and spends the night, after cell is fully adherent, changes fresh serum-free medium and cultivate 24h, adding respectively concentration is 10
-10, 10
-9, 10
-8, 3 * 10
-8, 10
-7, 10
-6, 3 * 10
-6, 10
-5The LY341495 of M processes, and establishes 3 repetitions for every group.Process 24h and detect cell viability with MTT.
Test-results is referring to Fig. 4 B, by test-results as seen: when LY341495 concentration 10
-10To 10
-5When M was upper, cell viability increased without significantly reducing with LY341495 concentration.The interior Ca that this explanation LY341495 induces LY379268
2+The inhibition that discharges and the vigor of cell proliferation are irrelevant.
(3) DHPG is to Ca in the cell
2+The dose-effect relationship of release and cell viability
1. cell is inoculated in 96 orifice plates and spends the night, after cell is fully adherent, change 1 μ M Fluo-4 AM(calcium fluorescent probe) and 0.02% pluronic acid hatch 1h.Then add respectively 10
-11, 10
-10, 10
-9, 10
-8, 10
-7, 10
-6, 10
-5, 10
-4, 10
-3The LY379268 mixed solution of M stimulates, and establishes 3 repetitions for every group, with the CHO ghost in contrast.Measure the Ca that each group is induced with FlexStation 3 multi-functional microplate reader workstations
2+Discharge.
2. cell is inoculated in 96 orifice plates and spends the night, after cell is fully adherent, changes fresh serum-free medium and continue to cultivate 24h, adding respectively concentration is 10
-11, 10
-10, 10
-9, 10
-8, 10
-7, 10
-6, 10
-5, 10
-4, 10
-3The DHPG of M stimulates, and establishes 3 repetitions for every group, with the CHO ghost in contrast.Measure the Ca that induces with the multi-functional microplate reader workstation of FlexStation3
2+Discharge.
Test-results is referring to Fig. 5, by test-results as seen: DHPG is as Group I mGluRs specific agonist, and without positive effect (Fig. 5 B), and Group II mGluRs specific agonist LY379268 can significantly activate Ca in the born of the same parents to the activation of CHO-mGluR2
2+Discharge and the CHO ghost is not had activation (Fig. 5 A), this ligands specific that other GPCR are described can not produce interference to this model.
The different cell inoculation of (4) 96 orifice plates number is to Ca in the cell
2+The impact that discharges
No matter be automatically or the artificial inoculation cell in 96 orifice plates, the cell number in every hole can be not identical.In order to check different cell numbers on the impact of the selection result, seek a cell inoculum density the suitableeest.Inoculate respectively number and be the cell in 5,000,10,000,20,000,40,000/ holes in 96 orifice plates, establish 3 repetitions for every group.After cell was fully adherent, the LY379268 that adds respectively 10 μ M stimulated, with the damping fluid that do not contain LY379268 as blank.Measure the Ca that induces with FlexStation 3 multi-functional microplate reader workstations
2+Discharge.
Find in this example (the results are shown in Figure 6) that when cell density was 5,000,10,000,20,000,40,000/hole, in this scope, the signal of fluorescence calcium current increased along with the increase of cell number.Consider each factor, all adopt in test the cell density in 40,000/ holes.
(5) cell stability detects
Along with the increase of passage number of times, the expression of external source goal gene may be lost, therefore, goal gene whether can be in host cell stably express, namely mitotic stability is the important indicator that detects the clone quality.We detect when the CHO-mGluR2 cell passes to the 7th generation and the 23rd generation for this reason.Cell is inoculated in 96 orifice plates spends the night, after cell is fully adherent, changes fresh serum-free medium and continue to cultivate 24h, the LY379268 and the L-Glutamate that add respectively different concns stimulate, and establish 3 repetitions for every group.Measure the Ca that induces with the multi-functional microplate reader workstation of FlexStation3
2+Discharge.
Experimental result is referring to Fig. 7, by experimental result as seen: LY379268 and L-Glutamate can obviously activate the Ca that mGluR2 induces
2+Discharge.Illustrate that this cell model has good mitotic stability.
Embodiment 3Assessment based on the drug screening method of cell model
Can purpose that set up screening model be screening active substances, reflects the biological activity that this material has, therefore, reflect exactly the biological activity of tested material to screening model, must estimate objectively.The quality of estimating a screening model has a lot of indexs, such as the stability of model, susceptibility, specificity etc.In addition, can reach the requirement of screening in order to make model, also need screening model is carried out quantitative evaluation, the activity of this model can reflection sample of Main Analysis.Estimate the quantitative technique parameter commonly used of medicaments sifting model, mainly as follows:
(1) signal background ratio (signal to background, S/B)
That the signal background ratio reflects is the data (M that medicaments sifting model obtains
Signal) and background data (M
Background) between distance.In general, this ratio is larger, and the distance of signal and background is larger, aspect the reflection sample effect larger scope being arranged, is easy to reflect sample effect.
Generally speaking, the numerical value of signal background ratio should be greater than 3, and is on duty less than 3 o'clock, just can not effectively reflect the biological activity of sample.
The calculation formula of signal background ratio is as follows:
S/B= M
Signal/M
Background
(2) signal to noise ratio (siganl to noise, S/N)
Signal to noise ratio is method evaluation parameter commonly used in the instrumental analysis, and noise typically refers under same condition determination, the recording signal that background produces.Generally, signal to noise ratio is greater than 10 and just can thinks adaptable method.The calculation formula of signal to noise ratio is as follows:
S/N=（M
Signal-M
Background）/SD
Background
SD wherein
BackgroundExpression background data standard deviation.
(3) the Z ‵ factor
Z ‵ factor integration has been considered variation and the fluctuation range of signal, it is only relevant with reliability with the circulation ratio of experiment and irrelevant with the content of experiment, thereby in the stability of estimating the Dominant Plat and reliability, be widely used, the Z ‵ factor is the statistics parameter that does not have unit, its value is between 0～1, the Z ‵ factor＜0.5 expression poor stability, the reliability of experiment is low, such model can not be used for drug screening, the Z ‵ factor was at 0.5～1 o'clock, model stability is high, can guarantee fully that the model of setting up is used for the screening of medicine.The calculation formula of the Z ‵ factor is as follows:
M
SignalData, M that the expression screening model obtains
BackgroundExpression background data, SD
BackgroundExpression background data standard deviation, SD
SignalThe data standard that screening model obtains is poor.
Whether stably be applicable to high flux screening in order to assess this screening method, in 60 holes choosing at random under optimizing resulting screening conditions in 96 orifice plates, 30 Kong Weiyi groups are take concentration as 10
-7The positive contrast of the LY379268 of M, do not add LY379268 as negative control, calculate respectively S/B according to formula, S/N and the Z ‵ factor are to carry out qualitative assessment to method.
Three evaluation index calculation result that adopt are S/B=7.48, and S/N=30.6, and Z ‵=0.75 illustrates that present method has good stability and reliability (Fig. 8).But the cell model Simultaneous Stabilization that efficiently expresses of the present invention is expressed the mGluR2 acceptor, stimulates the signaling molecule responsing reaction that causes so that detect mGluR2 being subject to forward.
By upper example as seen: cell model of the present invention can accurately screen anti-nervous system disease medicine in high-throughput ground, the control of the simple section of its preparation method, with low cost: drug screening method step of the present invention is simple, with low cost, accuracy is high, have good stability and reliability, can realize high-throughout screening, for the screening of anti-nervous system disease medicine provides new selection.
The above embodiment is the preferred embodiment that proves absolutely that the present invention lifts, and protection scope of the present invention is not limited to this.Being equal to that those skilled in the art do on basis of the present invention substitutes or conversion, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
SEQUENCE LISTING
＜110〉Ji Ruitai (Wuhan) bio tech ltd
＜120〉a kind of cell model and construction process thereof and the application in the anti-nervous disorders disease medicament of screening
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 2619
<212> DNA
＜213〉artificial sequence
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cccagtgccc gcgtggctgt cctgttcacc cgttctgagg atgcccggga gctgcttgct 840
gccagccagc gcctcaatgc cagcttcacc tgggtggcca gtgatggttg gggggccctg 900
gagagtgtgg tggcaggcag tgagggggct gctgagggtg ctatcaccat cgagctggcc 960
tcctacccca tcagtgactt tgcctcctac ttccagagcc tggacccttg gaacaacagc 1020
cggaacccct ggttccgtga attctgggag cagaggttcc gctgcagctt ccggcagcga 1080
gactgcgcag cccactctct ccgggctgtg ccctttgagc aggagtccaa gatcatgttt 1140
gtggtcaatg cagtgtacgc catggcccat gcgctccaca acatgcaccg tgccctctgc 1200
cccaacacca cccggctctg tgacgcgatg cggccagtta acgggcgccg cctctacaag 1260
gactttgtgc tcaacgtcaa gtttgatgcc ccctttcgcc cagctgacac ccacaatgag 1320
gtccgctttg accgctttgg tgatggtatt ggccgctaca acatcttcac ctatctgcgt 1380
gcaggcagtg ggcgctatcg ctaccagaag gtgggctact gggcagaagg cttgactctg 1440
gacaccagcc tcatcccatg ggcctcaccc tcagccggcc ccctgcccgc ctctcgctgc 1500
agtgagccct gcctccagaa tgaggtgaag agtgtgcagc cgggcgaagt ctgctgctgg 1560
ctctgcattc cgtgccagcc ctatgagtac cgattggacg aattcacttg cgctgattgt 1620
ggcctgggct actggcccaa tgccagcctg actggctgct tcgaactgcc ccaggagtac 1680
atccgctggg gcgatgcctg ggctgtggga cctgtcacca tcgcctgcct cggtgccctg 1740
gccaccctct ttgtgctggg tgtctttgtg cggcacaatg ccacaccagt ggtcaaggcc 1800
tcaggtcggg agctctgcta catcctgctg ggtggtgtct tcctctgcta ctgcatgacc 1860
ttcatcttca ttgccaagcc atccacggca gtgtgtacct tacggcgtct tggtttgggc 1920
actgccttct ctgtctgcta ctcagccctg ctcaccaaga ccaaccgcat tgcacgcatc 1980
ttcggtgggg cccgggaggg tgcccagcgg ccacgcttca tcagtcctgc ctcacaggtg 2040
gccatctgcc tggcacttat ctcgggccag ctgctcatcg tggtcgcctg gctggtggtg 2100
gaggcaccgg gcacaggcaa ggagacagcc cccgaacggc gggaggtggt gacactgcgc 2160
tgcaaccacc gcgatgcaag tatgttgggc tcgctggcct acaatgtgct cctcatcgcg 2220
ctctgcacgc tttatgcctt caagactcgc aagtgccccg aaaacttcaa cgaggccaag 2280
ttcattggct tcaccatgta caccacctgc atcatctggc tggcattcct gcccatcttc 2340
tatgtcacct ccagtgacta ccgggtacag accaccacca tgtgcgtgtc agtcagcctc 2400
agcggctccg tggtgcttgg ctgcctcttt gcgcccaagc tgcacatcat cctcttccag 2460
ccgcagaaga acgtggttag ccaccgggca cccaccagcc gctttggcag tgctgctgcc 2520
agggccagct ccagccttgg ccaagggtct ggctcccagt ttgtccccac tgtttgcaat 2580
ggccgtgagg tggtggactc gacaacgtca tcgctttga 2619
Claims (10)
1. a cell model is characterized in that, this cell model is the mammalian cell that carries and can express the mGluR2 gene.
2. cell model according to claim 1 is characterized in that, the nucleotide sequence of described mGluR2 gene is shown in SEQ ID NO:1.
3. cell model according to claim 1 is characterized in that, described mammalian cell is Chinese hamster ovary celI or HEK293 cell.
4. cell model according to claim 1 is characterized in that, name is called Chinese hamster ovary cell CHO-hmGluR2 (C12), is preserved in Chinese Typical Representative culture collection center on September 18th, 2012, and deposit number is CCTCC NO:C2012100.
5. the construction process of the arbitrary described cell model of claim 1 ~ 4 is characterized in that, step is as follows:
(1) the mGluR2 gene is connected with expression vector, makes up the recombinant plasmid that contains the mGluR2 gene;
(2) with the Transfected Recombinant Plasmid mammalian cell, cultivate into the cell strain of energy stably express mGluR2 gene, this cell strain is described cell model.
6. the construction process of cell model according to claim 5, it is characterized in that, also comprise the step (3) of cell strain being carried out comprehensive assessment: in cell strain, add mGluR2 agonist or inhibitor, measure the downstream signal responsing reaction, according to this cell strain of responsing reaction outcome evaluation whether as cell model.
7. the construction process of cell model according to claim 6 is characterized in that, the described expression vector of step (1) is pcDNA5/FRT.
8. the construction process of cell model according to claim 6 is characterized in that, the mammalian cell in the step (2) is Chinese hamster ovary celI.
9. the application of the arbitrary described cell model of claim 1 ~ 4 in the anti-nervous disorders disease medicament of screening.
10. the according to claim 9 application of described cell model in the anti-nervous disorders disease medicament of screening, it is characterized in that described anti-nervous disorders disease medicament is the reticent allosteric agent of mGluR2 agonist, mGluR2 inhibitor, mGluR2 forward allosteric agent, the reverse allosteric agent of mGluR2 or mGluR2.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108570450A (en) * | 2017-03-11 | 2018-09-25 | 华中科技大学鄂州工业技术研究院 | A kind of application of cell model and its construction and screening Serotonin receptor target active substance |
| CN108570451A (en) * | 2017-03-11 | 2018-09-25 | 华中科技大学鄂州工业技术研究院 | A kind of application of cell model and its construction method and screening HRH1 target drugs |
| CN114717195A (en) * | 2022-04-29 | 2022-07-08 | 武汉大学 | Cell model for screening exogenous compounds mediated by CYP3A7 and metabolized to toxicity, construction method and application thereof |
| CN114763560A (en) * | 2021-09-30 | 2022-07-19 | 生物岛实验室 | Cell line expressing metabotropic glutamate receptor 2 and G protein chimera |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108570450A (en) * | 2017-03-11 | 2018-09-25 | 华中科技大学鄂州工业技术研究院 | A kind of application of cell model and its construction and screening Serotonin receptor target active substance |
| CN108570451A (en) * | 2017-03-11 | 2018-09-25 | 华中科技大学鄂州工业技术研究院 | A kind of application of cell model and its construction method and screening HRH1 target drugs |
| CN114763560A (en) * | 2021-09-30 | 2022-07-19 | 生物岛实验室 | Cell line expressing metabotropic glutamate receptor 2 and G protein chimera |
| CN114717195A (en) * | 2022-04-29 | 2022-07-08 | 武汉大学 | Cell model for screening exogenous compounds mediated by CYP3A7 and metabolized to toxicity, construction method and application thereof |
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| Publication number | Publication date |
|---|---|
| CN102888382B (en) | 2014-04-09 |
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Inventor after: Liu Jianfeng Inventor after: Zhang Wenhua Inventor after: Meng Xia Inventor after: Pi Zhenjun Inventor after: Hu Ping Inventor after: Sun Bing Inventor before: Zhang Wenhua Inventor before: Pi Zhenjun Inventor before: Hu Ping Inventor before: Sun Bing Inventor before: Meng Xia Inventor before: Liu Jianfeng |
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