CN115927202A - TRPC5 mutant cell strain and construction method and application thereof - Google Patents
TRPC5 mutant cell strain and construction method and application thereof Download PDFInfo
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of biology, in particular to a TRPC5 mutant cell strain and a construction method and application thereof, wherein the TRPC5 mutant cell strain is TRPC5-478T>The specific construction method of the C mutant cell strain comprises the steps of mutating the 478 th T of a basic group sequence of a TRPC5 coding region into C, constructing the mutated sequence into a carrier, transferring the carrier into cells by a liposome transfection method, and screening to obtain the TRPC5 mutant cell strain. TRPC5-478T of the present invention>C mutant cell line capable of increasing monovalent cation Na after opening TRPC5 channel by using agonist + And K + And divalent cation Ca 2+ Enter cells, improve the problem of small detection current of whole-cell patch clamp, and increase FLIPR detectionThe medium calcium current/membrane potential signal intensity further improves the TRPC5 small molecular inhibitor drug screening method and lays a foundation for rapidly determining the screening of small molecular inhibitor candidate drugs for focal segmental glomerulosclerosis diseases.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a TRPC5 mutant cell strain and a construction method and application thereof.
Background
TRPC5 is a receptor-activated non-selective cation channel belonging to the classical subfamily (TRPC) of the transient receptor potential channel (TRP) family, which is a non-selective channel on the cell membrane that can pass calcium ions, and activation of TRPC5 channel will cause depolarization of the cell membrane and an increase in the cytosolic calcium concentration.
The TRPC5 channel is mainly expressed in brain tissue and also has a certain degree of distribution in organs such as liver and kidney. In addition, TRPC5 mediates a variety of physiological processes, and is closely related to the generation of emotions such as fear, anxiety, depression, and kidney diseases. Recent studies have found that TRPC5 channels are potential drug targets for the treatment of anxiety, depression and renal disease.
At present, most of TRPC5 stable cell lines are constructed by TRPC5-WT type, a monoclonal antibody with high TRPC5 protein expression quantity is screened out through a western blot experiment, the monoclonal antibody with high expression quantity is used subsequently, the construction is carried out by an electrophysiology method based on a manual patch clamp technology, and the sizes of inward current and outward current are recorded. However, the detection method based on the manual patch clamp technology has low throughput and high cost, and cannot realize high-throughput and low-cost drug screening work.
In addition, a FLIPR calcium current or membrane potential non-electrophysiological screening method based on fluorescence high-throughput detection is not reported more mature. For example, FLIPR detection using transient transfection of TRPC5 wild-type overexpression plasmids can detect changes in membrane potential, but calcium flux and membrane potential signals are extremely low, and drug screening is not possible.
Aiming at the problems that the current of a wild type TRPC5 overexpression cell strain detected by a whole-cell patch clamp is too small and the screening difficulty of related drugs is high, the invention designs and constructs a TRPC5 synonymous mutant cell strain stably overexpressing 478T > -C, so that the ion channel current recorded by the whole-cell patch clamp is enhanced, and a high-flux small-molecule drug screening technology aiming at a TRPC5 target spot is established.
Disclosure of Invention
The invention aims to provide a TRPC5 mutant cell strain and a construction method and application thereof, wherein the mutated TRPC5 channel gene can allow more cations to enter cells through a TRPC5 channel, so that the current intensity of the TRPC5 channel is enhanced, the problem of small detection current of a whole-cell patch clamp is effectively solved, the signal intensity of calcium current/membrane potential in FLIPR detection is increased, and the method for screening TRPC5 small-molecule inhibitor drugs is further perfected.
In a first aspect, the invention provides a TRPC5 mutant cell line, wherein the TRPC5 mutant cell line is a TRPC5-478T >.
Aiming at the problems that the wild type TRPC5 stable cell strain in the prior art uses high-flux FLIPR calcium flow and a membrane potential kit to test extremely low signals and can not realize drug screening. The invention provides a novel TRPC5-478T >. Based on the reason why the TRPC5-478T > C mutant cell strain mutated at the site can allow more cations to enter cells through TRPC5 channels, research and exploration stages are still carried out.
In a second aspect, the invention further specifically discloses a method for constructing the TRPC5 mutant cell strain, which comprises the following steps:
and (2) mutating the 478 th T of the basic group sequence of the TRPC5 coding region into C, constructing the mutated sequence into a vector, transferring the vector into cells by a liposome transfection method, and screening to obtain the TRPC5 mutant cell strain.
According to the coding region (CDS) base sequence of TRPC5, base T at position 478 is mutated into C. Due to the degeneracy of codons, the mutation of the site is synonymous mutation, and does not affect the coded amino acid sequence, but researches show that the mutated TRPC5-478T >C mutant cell strain can allow more cations to enter cells through TRPC5 channels, so that the whole-cell current is enhanced.
In the process of constructing mutant cell strain, the vector used in the present invention is not limited strictly, and may be pcDNA3.1/Zeo (+), and the vector may realize high level and constitutive expression in various mammal cell lines and contains Zeocin selectable marker and forward multiple cloning site.
After the mutated sequence is constructed into a vector, the mutated sequence needs to be transferred into a cell by a liposome transfection method, and when the transfection is carried out specifically, the constructed TRPC5-478T >C-pcDNA3.1/Zeo (+) plasmid is transferred into a HEK293 cell by the liposome transfection method, and after the plasmid is cultured for 1 to 3 days under certain conditions, a polyclonal cell pool can be obtained.
The polyclonal cells obtained by culturing need to be screened for monoclonal cells that are highly expressed and are capable of stimulating whole-cell current, and therefore, in the screening, methods used include antibiotic screening and monoclonal screening.
Preferably, in the technical scheme, during the antibiotic screening, bleomycin with the final concentration of 80-120 mu g/mL is added into a culture medium of a polyclonal cell pool for screening for 4-6 days, and the screened polyclonal cells are diluted and distributed into a 96-well plate to select a single clonal cell strain for amplification culture.
Considering that a monoclonal gene with high expression level and capable of exciting whole-cell current is required to be used when the FLIPR calcium current or membrane potential method is established, monoclonal screening is required after antibiotic screening, and the monoclonal screening comprises qRT-PCR screening and whole-cell patch clamp screening. The qRT-PCR screening is mainly used for screening high-expression monoclonal cells through the expression level of genes, and the whole-cell patch clamp screening is mainly used for screening monoclonal cells capable of exciting whole-cell current. And finally, carrying out mass amplification on the monoclonal cells subjected to antibiotic screening and monoclonal screening, and freezing and storing.
In a third aspect, the invention also discloses an application of the TRPC5 mutant cell strain, and therefore, the application of the TRPC5 mutant cell strain in drug screening also belongs to the protection scope of the invention.
The specific application of the TRPC5 mutant cell strain in drug screening is to establish a high-flux small-molecule drug screening technology aiming at a TRPC5 target spot by a whole-cell patch clamp electrophysiological method and a FLIPR calcium flow or membrane potential non-electrophysiological method based on the TRPC5 mutant cell strain, thereby perfecting a drug screening platform of the TRPC5 small-molecule inhibitor.
Further preferably, in the technical scheme, when the FLIPR calcium current or membrane potential detection method is established, firstly, the TRPC5 channel agonist EC50 and inhibitor IC50 are verified by a whole-cell patch clamp method, and monoclonal cells with verification results consistent with literature are screened, wherein the consistency of the literature means that the EC50 and IC50 are within three times of the data of the literature, which indicates that the monoclonal cells can be used for drug screening, and then the FLIPR calcium current or membrane potential method can be established based on the monoclonal cells.
The TRPC5-478T >:
1. according to the coding sequence (CDS) base sequence of the TRPC5, the 478 th base T is mutated into C, and the site is mutated into the same sense mutation due to the degeneracy of the codon, so that the amino acid sequence is not influenced. However, the mutated TRPC5 channel gene can allow more cations to enter cells through the TRPC5 channel, thereby enhancing the current intensity passing through the TRPC5 channel and improving the protein expression level;
2. TRPC5-478T of the present invention>C mutant cell line capable of increasing monovalent cation Na after opening TRPC5 channel by using agonist + And K + And divalent cation Ca 2+ The compound enters cells, the problem of small detection current of a whole-cell patch clamp is solved, the calcium current/membrane potential signal intensity in FLIPR detection is increased, and the TRPC5 small-molecule inhibitor drug screening method is further perfected;
3. the invention lays a foundation for rapidly determining the screening of small molecule inhibitors for focal segmental glomerulosclerosis diseases based on the establishment of a screening method of TRPC5-478T >C mutant cell strain high-flux TRPC5 small molecule inhibitors.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a flow chart of TRPC5-478T >;
FIG. 2 is a TRPC5-WT cell line ML204 inhibition curve of the present invention;
FIG. 3 shows the current intensity of the TRPC5-WT cell line of the present invention at different concentrations of the inhibitor;
FIG. 4 is a TRPC5-478T > -C mutant cell line ML204 inhibition curve of the present invention;
FIG. 5 is the current strength of TRPC5-478T >;
fig. 6 shows the qRT-PCR assay of TRPC5 transcript levels of the present invention, where x represents P <0.001 with very significant statistical differences;
FIG. 7 shows that FLIPR membrane potential of the present invention detects TRPC5-WT and TRPC5-478T > -C cell line agonist riluzole (riluzole) EC50;
FIG. 8 shows that FLIPR calcium flux detects TRPC5-WT and TRPC5-478T > -C cell line agonist riluzole (riluzole) EC50 in accordance with the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Construction of TRPC5-478T >
Firstly, mutating a 478 th site T into C according to a coding sequence (CDS) base sequence of a TRPC5, and constructing the mutated sequence into a pcDNA3.1/Zeo (+) vector;
transferring the constructed TRPC5-478T >C-pcDNA3.1/Zeo (+) plasmid into HEK293 cells by a liposome transfection method, and culturing for two days to form a polyclonal cell pool;
adding bleomycin with the final concentration of 100 mu g/mL into a culture medium, screening for 5 days, diluting screened polyclonal cells, and distributing the cells into a 96-well plate to culture monoclonals;
screening monoclonals with high gene expression level and capable of exciting whole-cell current by using a qRT-PCR and whole-cell patch clamp method, carrying out large-scale amplification and freezing;
verifying a channel agonist EC50 and an inhibitor IC50 by a whole-cell patch clamp technology, screening out monoclonals with IC50 less than or equal to 3, and establishing a FLIPR calcium current or membrane potential method.
Example 2
qRT-PCR detection
Collecting a TRPC5-478T >;
designing a qRT-PCR primer sequence of TRPC5, wherein a forward primer is as follows: 5' TGAACTCCCTCT ACCTGGCA AC-; the reverse primer is: 5 'AGTTGGCTGTGAACAGGGGA T-doped 3';
the mRNA transcript level of TRPC5 was determined by relative quantification of 2-DELTA. Ct using a SYBR dye qPCR kit (cat # Q711-02) for real-time fluorescent quantitative PCR experiments with GAPDH as internal reference and HEK293 as control sample.
As can be seen from FIG. 2, the TRPC5-478T >C mutant cell line is approximately 500-fold overexpressed as compared to the control.
Example 3
Whole-cell patch clamp screening and verification
The voltage stimulation protocol for whole-cell patch-clamp recording of TRPC5 currents was as follows:
the cell membrane after forming the whole cell seal is subjected to membrane rupture by clamping at-60 mV by using a voltage, the voltage is recorded and stepped from-60 mV to-100 mV for 1 ms, and then 300 ms Ramp stimulation is given to 100 mV. And repeatedly collecting data every 5 s, and observing the effect of the drug on the TRPC5 outward current peak value. Experimental data were collected by EPC 10 amplifiers (HEKA) and stored in PatchMaster (HEKA) software.
Drawing the capillary glass tube into a recording electrode by using a microelectrode drawing instrument, filling the electrode filled with intracellular fluid into an electrode tip, operating a microelectrode manipulator under an inverted microscope to immerse the electrode in extracellular fluid and recording the electrode resistance (Rpip). The electrode is contacted with the cell surface, negative pressure is applied to suction to form high-resistance sealing (G omega), at the moment, fast capacitance compensation is executed, negative pressure is applied continuously, cell membranes are broken through by suction, a whole cell recording mode is formed, slow capacitance compensation is carried out, experimental parameters such as membrane capacitance (Cm) and series resistance (Rs) are recorded, and electric leakage compensation is not applied.
Dosing was started after stabilization of TRPC5 current recorded in whole cells, and the next concentration was measured after each drug concentration had been applied for 5 min (or current to stabilization), with multiple concentrations measured for each test compound.
Placing the cover glass paved with cells in a recording bath under an inverted microscope, enabling blank control external liquid and working solution of a compound to be detected to sequentially flow through the recording bath from low concentration to high concentration by using a gravity perfusion method so as to act on the cells, and performing liquid exchange by using a peristaltic pump in recording. The current measured by each cell in the compound-free external fluid served as its own control, and at least three independent replicates of each concentration were used, all electrophysiological experiments were performed at room temperature.
The TRPC5 agonist used in this example was rosiglitazone (rosiglitazone) and the inhibitor was ML204. The extracellular fluid contains 145 mM NaCl,4 mM KC1,2 mM CaCl 2 ，2.1 mM MgCl 2 10 mM hepes,10 mM glucose, naoh adjusted pH =7.4; intracellular fluid contained 120 mM Cs-aspartic acid,20 mM CsCl 2 ，2 mM MgCl 2 ，8.8 mM CaCl 2 ，10 mM EGTA，10 mM HEPES and 2 mM Na 2 -ATP, csOH adjusted pH =7.2.
FIGS. 3-4 show the results of the measurement of TRPC5-WT cell line, wherein FIG. 3 shows ML204 inhibition curves, and FIG. 4 shows the current intensities at different concentrations of the inhibitor. By fitting, an IC50 of ML204 can be calculated to be 5.065 μ M.
FIGS. 5-6 show the results of testing TRPC5-478T > -C mutant cell lines, FIG. 5 shows the ML204 inhibition curve, and FIG. 6 shows the current intensities at different concentrations of inhibitor. By fitting, the IC50 of ML204 can be calculated to be 1.893 μ M, which meets the literature requirements.
Therefore, compared with the TRPC5-WT cell line, the TRPC5-478T >C mutant cell line effectively solves the problem of small detection current of the whole-cell patch clamp, and provides a foundation for high-throughput drug screening.
Example 4
FLIPR calcium current and membrane potential detection method
TRPC5-WT and TRPC5-478T one day in advance>The C mutant cell line was inoculated into a black 384-well plate at 10000/well and 5% CO at 37 ℃ 2 Culturing overnight;
on the day of the experiment, the culture medium is discarded, 20 mu L of HBSS buffer solution containing 20 mM HEPES and 1 Xdye loading Dye are added into each hole, the mixture is placed in an incubator at 37 ℃, the calcium flow Dye is incubated for 2 hours, and the membrane potential Dye is incubated for 30 min;
preparing a compound plate, diluting the agonist according to different concentrations, and adding the diluted agonist to a corresponding position of another 384-well plate;
and after the incubation is finished, the machine is operated, the fluorescence signal of the baseline when the agonist is not added is recorded for 20 times at an interval of 0.5 s every time, 10 mu L of the agonist is added, the recording is continued for 5 min, and the maximum value of the fluorescence signal under the agonists with different concentrations is selected to fit the EC50.
FIG. 7 shows the membrane potential detection result, and it can be known from the figure that TRPC5-478T >C mutant cell line can excite 1.8 times of signal window, and the EC50 of riluzole is 32.4 μ M; the TRPC5-WT cell line has the problems of weak signal and no agonism.
FIG. 8 shows the results of calcium flux assay, which shows that TRPC5-WT cell line can stimulate signal, but its EC50 is too high to be compatible with literature report, and it can not present "S" type curve, and the signal window is only 1.2 times; and the TRPC5-478T >.
Therefore, compared with the TRPC5-WT cell line, the TRPC5-478T >C mutant cell line has stronger signal intensity in FLIPR membrane potential and calcium current detection, and provides a basis for high-throughput drug screening.
In conclusion, the TRPC5-478T of the invention>C mutant cell line capable of increasing monovalent cation Na after opening TRPC5 channel by using agonist + And K + And divalent cation Ca 2+ The method can be used for improving the problem of small detection current of a whole-cell patch clamp and increasing the calcium current/membrane potential signal intensity in FLIPR detection, thereby perfecting the drug screening method of TRPC5 small-molecule inhibitors. Thus, based on TRPC5-478T>The establishment of the screening method of the C mutant cell strain high-flux TRPC5 small-molecule inhibitor lays a foundation for rapidly determining the screening of the small-molecule inhibitor candidate for the focal segmental glomerulosclerosis disease.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A TRPC5 mutant cell line, characterized in that the TRPC5 mutant cell line is a TRPC5-478T >.
2. The method for constructing the TRPC5 mutant cell line according to claim 1, comprising the steps of:
mutating the 478 th T of the base sequence of the TRPC5 coding region into C, constructing the mutated sequence into a vector, transferring the vector into cells by a liposome transfection method, and screening to obtain the TRPC5 mutant cell strain.
3. The method of claim 2, wherein the vector is pcDNA3.1/Zeo (+).
4. The construction method according to claim 2, characterized in that, in the transfection, the constructed TRPC5-478T > -C-pcDNA3.1/Zeo (+) plasmid is transferred to HEK293 cells by a liposome transfection method, and the cells are cultured to obtain polyclonal cells.
5. The method of construction according to claim 2, wherein the screening comprises antibiotic screening and monoclonal screening.
6. The construction method according to claim 5, wherein the antibiotic is selected by adding bleomycin with a final concentration of 80-120 μ g/mL into a culture medium of a polyclonal cell pool, and selecting the selected polyclonal cell strain for amplification culture for 4-6 days.
7. The method of construction according to claim 5, wherein the monoclonal screening comprises qRT-PCR screening and whole cell patch clamp screening.
8. The use of the TRPC5 mutant cell line according to claim 1, characterized in that the TRPC5 mutant cell line is used in drug screening.
9. The use according to claim 8, wherein in the drug screening, based on TRPC5 mutant cell lines, a whole-cell patch clamp method and a FLIPR calcium flux or membrane potential method are used to establish a high-throughput small molecule drug screening technology for TRPC5 target.
10. The use according to claim 9, wherein when the FLIPR calcium flux or membrane potential detection method is established, the FLIPR calcium flux or membrane potential method is established by verifying the channel agonist EC50 and the inhibitor IC50 by a whole-cell patch clamp method, and screening a monoclonal with an IC50 ≤ 3.
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| CN116930385A (en) * | 2023-08-08 | 2023-10-24 | 北京爱思益普生物科技股份有限公司 | Method for measuring concentration of compound in high-salt-concentration preparation |
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| CN112351781A (en) * | 2018-03-05 | 2021-02-09 | 金翅雀生物公司 | Imidazodiazepine diones and methods of use thereof |
| WO2021067946A1 (en) * | 2019-10-04 | 2021-04-08 | Goldfinch Bio, Inc. | Biomarker-based treatment of focal segmental glomerulosclerosis and diabetic kidney disease |
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| CN112351781A (en) * | 2018-03-05 | 2021-02-09 | 金翅雀生物公司 | Imidazodiazepine diones and methods of use thereof |
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