Cell strain for stably expressing platelet activating factor receptor, construction method and application

Abstract

The invention discloses a cell strain capable of stably expressing a platelet activating factor receptor, a construction method and application. The CRISPR-Cas9 expression system and the cell strain comprise a recombinant expression vector for stably expressing a platelet activating factor receptor, the recombinant expression vector is obtained by stably knocking a target gene of the platelet activating factor receptor into a cell genome through a gene editing method, and the cell strain and cell beads can be stably subcultured. And the condition of losing is avoided. The cell strain can be used for drug development and function detection with a platelet activating factor receptor as a target spot.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to a cell line, construction method and application for stably expressing platelet activating factor receptor, and more specifically to a recombinant expression vector, CRISPR-Cas9 expression system, cell line, construction method and application for stably expressing platelet activating factor receptor. Background Art

[0002] Platelet activating factor (PAF) is an endogenous phospholipid transmitter with a wide range of biological activities. It is released by a variety of cells, such as IgE-sensitized basophils, premature macrophages, mast cells, platelets, endothelial cells, monocytes and neutrophils. In the central nervous system, neurons and glial cells can also synthesize low levels of PAF. Platelet activating factor receptor (PAFR) is a member of the GPCRs superfamily group A. PAFR is a seven-transmembrane G protein-coupled receptor that is mainly located in lipid rafts and / or cell membrane caveolae in the cell membrane. After binding to PAF, PAFR can activate a variety of signal transduction pathways, such as the phosphatidylinositol-calcium ion second messenger system.

[0003] PAFR is expressed in a variety of cells, including platelets, neutrophils, macrophages, monocytes, and eosinophils. In the central nervous system, PAFR is present in neurons, microglia, astrocytes, and oligodendrocyte progenitor cells. PAF can activate multiple signaling pathways, including Gq protein, by binding to PAFR, thereby triggering a series of physiological and pathological effects, and is an important target for drug development.

[0004] In the process of lead compound screening and verification of PAFR-related functions in drug development targeting PAFR, there are two main methods for testing at the cellular level: transient transfection and construction of stable expression cell lines. The transient transfection method has low efficiency, large batch differences, and high costs, which seriously affect the progress of the experiment. The existing PAFR-expressing cell lines are constructed using the traditional random integration method, and the expression is not stable enough. As the cells are transmitted, the target gene is easily lost, affecting the experiment. Moreover, since the receptor does not carry a suitable detection label, the use of the cell line is greatly restricted. Summary of the invention

[0005] In view of this, the purpose of the present application is to provide a cell line, construction method and application that stably expresses platelet activating factor receptor. The CRISPR-Cas9 expression system and cell line include a recombinant expression vector that stably expresses platelet activating factor receptor, and the recombinant expression vector is obtained by stably knocking the platelet activating factor receptor target gene into the cell genome through a gene editing method, and the cell bead can be stably subcultured. The cell line can be used for receptor protein preparation, receptor protein expression detection, receptor protein function detection, receptor protein labeling, receptor protein imaging and / or drug screening.

[0006] To achieve the above objectives, this application provides at least the following technical solutions:

[0007] In a first aspect, the present application provides a recombinant expression vector of platelet activating factor receptor, which is obtained by inserting a gene encoding human platelet activating factor receptor into a pAAVS1-Puro-DNR expression vector.

[0008] In a second aspect, the present application provides a CRISPR-Cas9 expression system for platelet activating factor receptor, which includes the aforementioned recombinant expression vector.

[0009] In a third aspect, the present application provides a cell line that stably expresses platelet-activating factor receptor, which includes the aforementioned recombinant expression vector or the aforementioned CRISPR-Cas9 expression system.

[0010] In a fourth aspect, the present application provides a method for constructing the aforementioned cell line, which comprises the step of transfecting the aforementioned recombinant expression vector or expression system into cells.

[0011] In a fifth aspect, the present application provides the use of the aforementioned cell lines in receptor protein preparation, receptor protein expression detection, receptor protein function detection, receptor protein labeling, receptor protein imaging and / or drug screening.

[0012] The technical solution provided in this application is mainly used for drug development and functional testing targeting PAFR. Compared with the existing technology, it has at least the following beneficial effects:

[0013] (1) The present application utilizes gene editing methods to stably knock the PAFR target gene into the cell genome, and the obtained cell line can stably replicate along with the cell replication and will not be lost during cell line generation.

[0014] (2) This cell line uses human HEK293 cells as host cells, which are more suitable for drug screening.

[0015] (3) The cell line carries a GPCR membrane signal peptide, which ensures that the receptor can be stably transported to the cell membrane after being expressed in the cell, thus ensuring the function of the receptor.

[0016] (4) This cell line carries an HA tag and can be used in various detection and labeling experiments, making it convenient to conduct various functional detection experiments on PAFR.

[0017] (5) After stable subculture and expression testing, the cell line was able to maintain stable expression of the target gene when it was subcultured to the 10th generation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a map of the recombinant expression vector provided in the examples of this application.

[0019] Figure 2 This is the mRNA expression detection result of the PAFR receptor gene provided in the examples of the present application.

[0020] Figure 3 These are the HA tag expression detection results of the cell lines provided in the examples of the present application, wherein (a) is the HA tag expression detection result of the cell line when it is passed to the 5th generation, (b) is the HA tag expression detection result of the cell line when it is passed to the 5th generation, and (c) is the HA tag expression detection result of the cell line when it is passed to the 5th generation.

[0021] Figure 4 This is the PAFR activated calcium flow function test result provided in the examples of the present application.

[0022] Figure 5 This is the detection result of the PAFR function being inhibited by the inhibitor provided in the embodiment of the present application. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0024] Those skilled in the art will appreciate that, unless otherwise stated, the words "said", "the", and "the aforementioned" used in the text of the present application may also include plural forms. It should be further understood that the wording "including" used in the specification of the present application means the presence of the features, steps, and operations, but does not exclude the presence or addition of one or more other features, integers, and steps.

[0025] Those skilled in the art will appreciate that, for any specific experimental steps or conditions not specified in the examples, the experiments may be carried out according to the conventional experimental steps or conditions described in the literature in the art; any raw materials or instruments used without specifying the manufacturer are conventional products that can be purchased commercially.

[0026] Those skilled in the art will understand that, unless otherwise specified in the present application, when a numerical range is given in an embodiment, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application are consistent with the prior art mastery of those skilled in the art and the records of this application, and any methods, equipment and materials of the prior art similar or equivalent to the methods, equipment and materials described in the embodiments of this application may also be used to implement this application.

[0027] pAAVS1-Puro-DNR is an expression vector for mammalian cells, particularly suitable for CRISPR / Cas9-mediated gene editing to achieve the integration of the target gene at the AAVS1 site in the human genome. In order to construct a cell line that stably expresses platelet-activating factor receptor, the present embodiment of the application inserts the platelet-activating factor receptor gene into the pAAVS1-Puro-DNR expression vector using the CRISPR-Cas9 gene editing method, thereby obtaining a recombinant expression vector that can stably express platelet-activating factor receptor, and then obtaining a CRISPR-Cas9 expression system and cell line including the recombinant expression vector, which is suitable for drug development and functional testing targeting PAFR.

[0028] Based on this, an embodiment of the present application provides a recombinant expression vector of platelet activating factor receptor, which is obtained by inserting a gene encoding human platelet activating factor receptor into the pAAVS1-Puro-DNR expression vector.

[0029] The pAAVS1-Puro-DNR expression vector provided in the embodiment of the present application can ensure that the gene encoding the human platelet-activating factor receptor is stably knocked into the host cell. The pAAVS1-Puro-DNR expression vector has an AAVS1 site. For the gene encoding the human platelet-activating factor receptor, inserting this site can ensure that the gene fragment is transcribed normally and stably to achieve the expected function. And compared with other commonly used expression vectors, inserting the gene fragment encoding the human platelet-activating factor receptor into the pAAVS1-Puro-DNR expression vector will not disrupt the function of endogenous genes and / or other regulatory factors, and has no side effects on cells.

[0030] In certain embodiments, the nucleotide sequence of the gene encoding human platelet activating factor receptor is shown as SEQ ID NO:1.

[0031] In certain embodiments, a signal peptide gene is further inserted into the pAAVS1-Puro-DNR expression vector, and the nucleotide sequence of the signal peptide gene is shown in SEQ ID NO: 2. By adding the signal peptide gene to the expression vector, the expressed signal peptide can ensure that the platelet activating factor receptor protein expressed by the aforementioned recombinant expression vector is efficiently transferred to the cell membrane.

[0032] In certain embodiments, a gene sequence encoding a protein tag is also inserted into the pAAVS1-Puro-DNR expression vector, and the protein tag is selected from at least one of an HA tag, a Flag tag, a Halo tag, a SNAP tag, a His6 tag, an eGFP tag, an eYFP tag, and an eCFP tag.

[0033] In certain preferred embodiments, the protein tag is an HA tag, and the nucleotide sequence of the gene encoding the HA tag is shown in SEQ ID NO: 3. By expressing the HA tag, it is convenient to detect the expression of platelet activating factor receptor and conduct other related studies that can be carried out using the HA tag.

[0034] In certain preferred embodiments, the recombinant expression vector is a pAAVS1-Puro-DNR-HA-PAFR recombinant plasmid having the following Figure 1 Structure of the spectrum shown.

[0035] Based on this, an embodiment of the present application provides a CRISPR-Cas9 expression system of platelet activating factor receptor, which includes the aforementioned recombinant expression vector.

[0036] In certain embodiments, the CRISPR-Cas9 expression system further comprises a pCas-Guide-AAVS1 donor plasmid.

[0037] Based on this, an embodiment of the present application also provides a cell line that stably expresses platelet-activating factor receptor, which includes the aforementioned recombinant expression vector or the aforementioned CRISPR-Cas9 expression system.

[0038] In certain embodiments, the cell line is a human cell.

[0039] In certain preferred embodiments, the human cells are HEK293 cells.

[0040] In certain preferred embodiments, the cell line is classified and named as: HEK293-HA-PAFR, a cell line stably expressing platelet activating factor receptor, which has been deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, China, the deposit date is: January 3, 2025, and the deposit number is: CCTCC NO: C202522.

[0041] Based on this, an embodiment of the present application provides a method for constructing the aforementioned cell line, which includes the step of transfecting the aforementioned recombinant expression vector or expression system into cells.

[0042] In certain embodiments, the cell is a human cell.

[0043] In certain preferred embodiments, the human cells are HEK293 cells.

[0044] In certain preferred embodiments, the transfection method is liposome transfection.

[0045] In certain preferred embodiments, the construction method further comprises the step of using puromycin to perform resistance screening on cells.

[0046] In certain preferred embodiments, the method comprises the following specific steps:

[0047] (1) transfecting the recombinant expression vector or the expression system into HEK293 cells using a liposome transfection method to obtain transfected cells;

[0048] (2) using 1 to 3 μg / ml of puromycin to screen the cells obtained in step (1) for resistance, thereby obtaining cells with puromycin resistance;

[0049] (3) suspending the puromycin-resistant cells in (2) and sorting them using a flow cytometer to obtain monoclonal cells;

[0050] (4) Screening and verifying the monoclonal cells obtained in (3) to obtain a positive monoclonal cell line.

[0051] Based on this, the embodiments of the present application provide the use of the aforementioned cell lines in receptor protein preparation, receptor protein expression detection, receptor protein function detection, receptor protein labeling, receptor protein imaging and / or drug screening.

[0052] In certain embodiments, the cell line can be used for calcium flux detection, and then for drug function detection during receptor drug development.

[0053] In certain embodiments, the drug is a drug for treating sickle cell disease (SCD), thrombosis, asthma, cerebral ischemia and encephalitis, liver, pancreas, stomach, intestinal damage and the like.

[0054] The technical solution of the present application and the technical effects achieved are described in detail below through more specific embodiments.

[0055] Target, host and expression plasmid construction

[0056] 1. Target

[0057] The target gene information is shown in Table 1.

[0058] Table 1

[0059] Gene ID (NCBI) Gene name Species origin 5724 PAFR people

[0060] 2. Host cells: human HEK293

[0061] 3. Resistance: Puromycin (Puro)

[0062] 4. Construction of expression plasmid

[0063] The platelet activating factor receptor target gene (PAFR, nucleotide sequence as shown in SEQ ID NO: 1), signal peptide gene (signal peptid, nucleotide sequence as shown in SEQ ID NO: 2), HA tag (nucleotide sequence as shown in SEQ ID NO: 3) were constructed into the pAAVS1-Puro-DNR expression vector by restriction endonuclease digestion and T4 DNA ligase ligation method to obtain the recombinant plasmid: pAAVS1-Puro-DNR-HA-PAFR (structure as shown in Figure 1 The pAAVS1-Puro-DNR-HA-PAFR recombinant plasmid is a specific gene knock-in vector. When co-transfected with the donor pCas-Guide-AAVS1 plasmid, the target gene can be stably knocked into the AAVS site of the cell genome. This site is a relatively safe gene knock-in site in the cell and has little effect on cell function. It is the most commonly used gene knock-in site.

[0064] Cell culture protocol

[0065] 1. Cell Recovery

[0066] Take out the cells stored in liquid nitrogen or -80℃ refrigerator, and quickly move the cells to a 37℃ water bath to thaw; prepare a 15ml centrifuge tube in advance and add 3ml of fresh culture medium; add 1ml of cell suspension in the cryopreservation tube to the prepared culture medium, and centrifuge it in a centrifuge at 1000rpm / min for 3 minutes; during this period, prepare a 100mm cell culture dish and add 10ml of fresh culture medium; after centrifugation, discard the supernatant, resuspend the cells with 1ml of fresh culture medium, evenly add the cell suspension to the prepared culture dish, and place it in a 37℃, 5% CO2 incubator for culture.

[0067] 2. Cell Passaging

[0068] (1) First, observe the cell status under a microscope to determine whether to perform cell subculture. After determining the subculture, first discard the old culture medium, then use PBS (5 mL PBS / 100 mm culture dish) to wash away dead cells and cell debris. The number of washes should be determined based on the cell status, generally 1 to 2 times is appropriate.

[0069] (2) Add trypsin at a ratio of 1 mL trypsin / 100 mm culture medium for digestion. After adding trypsin, place the culture dish in a 37°C incubator for digestion for 1 to 3 minutes. The degree of cell digestion can be observed under a microscope. When the cells become round and bright, digestion can be terminated.

[0070] (3) Add complete culture medium to the culture dish after digestion to terminate cell digestion; use a pipette to blow the cells several times to form a cell suspension, mix well, and transfer the cell suspension to a 15 mL centrifuge tube.

[0071] (4) Place a 15 ml centrifuge tube in a centrifuge and centrifuge at 1000 rpm / min for 3 min. During the centrifugation process, culture dishes or culture plates prepared in advance can be added with culture medium. After centrifugation, discard the cell supernatant. Resuspend the cells in complete culture medium and add them to a new cell culture dish or culture plate prepared in advance in a certain proportion.

[0072] (5) Finally, the cells were cultured in a 37°C, 5% CO2 cell culture incubator.

[0073] 3. Cell preservation

[0074] The steps of cell preservation are similar to those of cell passaging. First, wash the cells with PBS, then add 1 ml of trypsin for cell digestion. After digestion, add fresh culture medium to terminate cell digestion, use a pipette to blow to suspend all the cells in the culture medium, and transfer the cell suspension to a centrifuge tube for centrifugation. After centrifugation, suspend the cells with 1 ml of cell freezing solution, transfer them to a cell freezing tube, and note the cell name, preservation time, name of the preserver and other information. Store them in a -80°C refrigerator and transfer them to a liquid nitrogen tank the next day. The freezing solution is fetal bovine serum containing 10% DMSO.

[0075] Cell line construction

[0076] 1. Determination of Puromycin Screening Concentration

[0077] (1) HEK293 cells were seeded in 6-well plates.

[0078] (2) Designing a drug concentration gradient of puromycin, including 5 concentration gradients of 1 μg / ml, 2 μg / ml, 4 μg / ml, 6 μg / ml, and 8 μg / ml; adding puromycin drugs of different concentration gradients after the cell density in the 6-well plate grows to 80%;

[0079] (3) After that, fresh culture medium containing puromycin was replaced every 24 hours, and the survival and growth status of the cells were observed. The lowest puromycin concentration at which a large number of cells died after 2 days and all cells died after 4 days was selected as the optimal screening concentration. Finally, we selected 2 μg / ml as the puromycin screening concentration.

[0080] 2. Lipofectamine Transfection

[0081] (1) One day in advance, seed HEK293 cells into a 60 mm culture dish and observe the cell status on the second day. When the cell density is about 70% and the cells are in good condition, the lipid transfer experiment can be performed.

[0082] (2) Aspirate the culture medium, add 1 ml of OMEM, and place in a 37°C incubator.

[0083] (3) Prepare solution A: 200 μl OMEM, 4 μl Lipofectamine 2000, mix well and let stand at room temperature for 5 min.

[0084] (4) Prepare solution B: 200 μl OMEM, 1 μg pCas-Guide-AAVS1 plasmid, and 2 μg pAAVS1-Puro-DNR-HA-PAFR plasmid.

[0085] (5) Mix solution A and solution B thoroughly and let stand at room temperature for 20 min.

[0086] (6) Add the transfection solution dropwise to the cells, shake gently to mix, place in a 37°C incubator for 20 min, then add 3 ml of complete culture medium and transfect for 24 h.

[0087] 3. Drug Screening

[0088] 48 hours after transfection, cells were screened for puromycin at a drug concentration of 2 μg / ml. Fresh culture medium containing puromycin was replaced every 24 hours until all remaining cells were resistant to puromycin. The screening cycle was approximately 4 to 6 days.

[0089] 4. Flow Cytometry Isolation of Monoclonal Cell Lines

[0090] (1) Inoculate polyclonal cells in a 100 mm culture dish and check the cell growth after 48 hours. When the cells fill the dish and grow well, prepare to collect the cells.

[0091] (2) Discard the old culture medium, wash the cells twice with PBS, and then add 1 ml of trypsin to digest the cells. After about 2 to 3 minutes, add 3 ml of fresh culture medium to terminate the digestion. Use a pipette to repeatedly blow the cells to obtain a cell suspension and transfer it to a 15 ml centrifuge tube. Place it in a centrifuge at 1000 rpm / min and centrifuge for 3 minutes.

[0092] (3) Discard the upper culture medium and resuspend the cells in 1 ml of PBS (you can choose to add 1% serum depending on the specific situation).

[0093] (4) The cell suspension was briefly filtered and transferred to a flow cytometer analysis tube.

[0094] (5) Prepare five 96-well culture plates in advance and add 100 μl of complete culture medium to each well.

[0095] (6) Fix the flow tube containing the cell suspension on the sorter and start sorting, with one cell being sorted in each well.

[0096] 5. Establishment of Monoclonal Cell Lines

[0097] After the cells were fully grown in the 96-well plate, they were transferred to 24-well plates, 6-well plates, etc. for expansion and culture. Subsequently, multiple monoclonal cell lines were screened and verified to obtain positive monoclonal cell lines. Two monoclonal cell lines had good expression and function, and were named PAFR-11 and PAFR-27.

[0098] Target gene expression and functional verification

[0099] The following functional tests were performed on the cell lines PAFR-11 and PAFR-27 prepared above:

[0100] 1. The mRNA expression level of human platelet-activating factor receptor gene was detected by qPCR method. The qPCR primers are shown in Table 2.

[0101] Table 2

[0102] Primer name Primer sequences PAFR-F SEQ ID NO:4 PAFR-R SEQ ID NO:5 β-actin-F SEQ ID NO:6 β-actin-R SEQ ID NO:7

[0103] qPCR results Figure 2 As shown, it can be seen from the figure that compared with the mock group (normal HEK293 cells, not transfected with the recombinant expression vector expressing the PAFR gene), the mRNA of the PAFR gene in the two cell lines PAFR-11 and PAFR-27 is stably expressed.

[0104] 2. The expression of the HA tag at the N-terminus on the cell membrane surface was detected by ELISA. The results were as follows: Figure 3As shown in the figure, compared with the mock group (normal HEK293 cells, not transfected with the recombinant expression vector expressing the PAFR gene), the PAFR-11 and PAFR-27 cell lines can still express efficiently and stably when they are passed to the 5th generation (P5), the 7th generation (P7), and the 10th generation (P10). This further shows that the cell line still stably expresses PAFR when it is passed to the 10th generation.

[0105] 3. Use flipr to detect PAFR induced activation of downstream calcium signals under agonist stimulation; the results are as follows Figure 4 As shown in the figure, it can be seen that compared with the mock group (normal HEK293 cells, not transfected with the recombinant expression vector expressing the PAFR gene), the PAFR expressed by the two cell lines PAFR-11 and PAFR-27 can significantly activate calcium signals under agonist stimulation.

[0106] 4. Use flipr to detect the inhibition of downstream calcium signal activation by PAFR-specific inhibitor (ABT-491); the results are as follows Figure 5 As shown in the figure, it can be seen that compared with the mock group (normal HEK293 cells, not transfected with the recombinant expression vector expressing the PAFR gene), ABT-491 can effectively inhibit the activation of downstream calcium signals induced by PAFR expressed by the two cell lines PAFR-11 and PAFR-27 under agonist stimulation.

[0107] In summary, the present application successfully constructed an expression vector for stably expressing platelet activating factor receptor using gene editing and gene recombination methods, as well as an expression system and cell line including the expression vector, and an HA tag is attached to the N-terminus of the platelet activating factor receptor gene of the expression vector, which can be used for receptor expression detection, imaging, labeling and other experiments, receptor binding and activation and other functions, and can directly detect downstream calcium signals. The cell line can be used for the screening of platelet activating factor receptor drugs.

[0108] The present application is described in detail above. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the present application and its core ideas. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A recombinant expression vector of platelet activating factor receptor, characterized in that: It is obtained by inserting the gene encoding human platelet-activating factor receptor into the pAAVS1-Puro-DNR expression vector.

2. The recombinant expression vector according to claim 1, characterized in that The nucleotide sequence of the gene encoding human platelet activating factor receptor is shown in SEQ ID NO:

1.

3. The recombinant expression vector according to claim 1, characterized in that A signal peptide gene is also inserted into the pAAVS1-Puro-DNR expression vector, and the nucleotide sequence of the signal peptide gene is shown in SEQ ID NO:

2.

4. The recombinant expression vector according to claim 1, characterized in that A gene sequence encoding a protein tag is also inserted into the pAAVS1-Puro-DNR expression vector, and the protein tag is selected from at least one of an HA tag, a Flag tag, a Halo tag, a SNAP tag, a His6 tag, an eGFP tag, an eYFP tag, and an eCFP tag; When the protein tag is an HA tag, the nucleotide sequence of the gene encoding the HA tag is shown in SEQ ID NO:

3.

5. A CRISPR-Cas9 expression system for platelet activating factor receptor, characterized in that: A recombinant expression vector comprising any one of claims 1 to 4; Preferably, the CRISPR-Cas9 expression system further comprises a pCas-Guide-AAVS1 donor plasmid.

6. A cell line stably expressing platelet activating factor receptor, characterized in that: Comprising the recombinant expression vector according to any one of claims 1 to 4 or the CRISPR-Cas9 expression system according to claim 5; Preferably, the cell line is a human cell; more preferably, the human cell is a HEK293 cell.

7. The cell line according to claim 6, characterized in that The cell line is classified and named as: HEK293-HA-PAFR, a cell line stably expressing platelet activating factor receptor, and has been deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, China, the deposit date is: January 3, 2025, and the deposit number is: CCTCC NO: C202522.

8. The method for constructing the cell line according to claim 6, characterized in that: The method comprises the steps of transfecting the recombinant expression vector according to any one of claims 1 to 4 or the CRISPR-Cas9 expression system according to claim 5 into a cell; Preferably, the transfection method is liposome transfection; Preferably, the construction method further comprises the step of using puromycin to screen the cells for resistance.

9. The construction method according to claim 8, characterized in that: The following steps are involved: Transfecting the recombinant expression vector or the expression system into HEK293 cells using liposome transfection to obtain transfected cells; The transfected cells were screened for resistance using 1-3 μg / ml puromycin to obtain cells with puromycin resistance; The cells with puromycin resistance were suspended and sorted using a flow cytometer to obtain monoclonal cells; The monoclonal cells were screened and verified to obtain positive monoclonal cell lines.

10. Use of the cell line according to claim 6 in receptor protein preparation, receptor protein expression detection, receptor protein function detection, receptor protein labeling, receptor protein imaging and / or drug screening; Preferably, the cell line can be used for calcium flow detection, and then for drug function detection in the process of receptor drug development; Preferably, the drug is a drug for treating sickle cell disease (SCD), thrombosis, asthma, cerebral ischemia and encephalitis, liver, pancreas, stomach, intestinal damage and other diseases.

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