Method for screening functional nucleic acid aptamer targeting GPCR protein in combination with cell screening and function screening and use

Abstract

A method for screening a functional nucleic acid aptamer targeting a GPCR protein in combination with cell screening and function screening and the use. The natural configuration of a target protein is retained by means of performing cell screening for the target protein, and the obtained nucleic acid aptamer has better in-vivo specificity and targeting ability compared with a nucleic acid aptamer obtained from traditional protein SELEX. Cell-SELEX for model cells is constructed, which, compared with the traditional Cell-SELEX technique with an unclear target, has a higher targeting ability. According to the present invention, a functional aptamer that can affect a target protein signaling pathway and a cell phenotype can be directly obtained by means of performing only one round of function screening in cells after a series of target aptamers are screened out at a time, and has the advantages of high targeting ability, high affinity, low cost, low labor cost, broad-spectrum universality of the method, etc.

Description

A method and application for screening functional nucleic acid aptamers targeting GPCR proteins by combining cell screening and functional screening Technical Field

[0001] The present invention relates to the technical field of biotechnology, and in particular to a method for screening functional nucleic acid aptamers targeting GPCR proteins by combining cell screening and functional screening, and its application. Background Art

[0002] GPCRs are a large class of membrane protein receptors that play a central role in signal transduction and are responsible for important cellular physiological functions. Over half of the currently marketed drugs are developed for GPCRs. However, as membrane proteins, GPCRs are large and complex, making them challenging to synthesize and purify. In the absence of a lipid bilayer, they struggle to maintain their native conformation. Due to inherent technical barriers to GPCRs, current approaches to studying their potential as small molecule drugs are limited. Furthermore, aptamers specifically recognize targets with complementary shapes, making the selection and preparation of targets with native conformations crucial.

[0003] Aptamers are single-stranded oligonucleotides, typically derived through systematic evolution of ligands by exponential enrichment (SELEX), that possess unique tertiary conformations and interact with target topologies with relatively high shape complementarity. One of the most important features of SELEX or aptamers is the ability to generate aptamers with high affinity and specificity for a wide range of targets, from small molecules to proteins, bacteria, and cells, regardless of the target's complexity and molecular weight. Compared to antibodies, aptamers offer advantages such as simple synthesis and modification, low molecular weight, low immunogenicity, and good tissue penetration and recognition. Currently, various aptamers have emerged as therapeutic agents in the process of translating basic research into clinical trials, for example, pegaptanib, E10030, ARC1779, AS1411, NOXA12, and NOX-H94.

[0004] GPCRs, also known as G-coupled proteins, are cell membrane proteins. Currently, 40% of FDA-approved drug development targets GPCRs. As a second messenger, cAMP plays a crucial role in signal transduction. Cellular signaling via G protein-coupled receptors (GPCRs) can be assessed by measuring downstream effectors. Measuring the levels of cAMP, a second messenger produced by adenylate cyclase activation, is one of the most common methods for screening GPCR agonists and antagonists. Hormone → GPCR → G protein → adenylate cyclase → cAMP → cAMP-dependent protein kinase A → gene regulatory proteins → gene transcription. Therefore, using GPCRs as drug screening targets to identify specific receptor agonists or antagonists with high activity, long half-lives, and specificity is of great significance. However, due to the chemical properties of GPCRs and the scarcity of native samples, the development of novel compounds targeting GPCRs faces inherent technical barriers in effectively expressing and purifying proteins in their native conformation. Due to the inherent technical barriers of GPCR proteins, methods for studying small molecule inhibitor targets are limited. There are few reports on G protein-coupled receptor (GPCR)-targeted aptamers, and all reported small molecules targeting TSHR have only entered the preclinical stage. Because the extracellular domain of GPCRs is limited as an epitope and it is impossible to maintain its native conformation in the absence of a lipid bilayer, it is challenging to generate molecules that recognize the native GPCR conformation. Some previous GPCR studies have used recombinant GPCR proteins, such as NTSR1, β2-adrenergic receptor, and CCR5, whose overexpression, purification, and reconstitution are both laborious and time-consuming.

[0005] Summary of the Invention

[0006] In order to address the technical defects of the existing technology, the present invention provides a method and application for screening functional nucleic acid aptamers targeting GPCR proteins by combining cell screening and functional screening. The SELEX technology of nucleic acid aptamers targeting GPCR proteins by combining cell screening and functional screening can target specific proteins and screen out specific nucleic acid aptamers while maintaining the natural activity of the protein. Combined with further functional screening, functional nucleic acid aptamers that affect GPCR signaling pathways can be screened.

[0007] The technical solution adopted by the present invention is: a method for screening functional nucleic acid aptamers targeting GPCR proteins by combining cell screening and functional screening, comprising the following steps:

[0008] (1) Construct and validate stable model cells for cell screening;

[0009] (2) Construction of screening library;

[0010] (3) enriching the screening library by cell screening to obtain an enriched library;

[0011] (4) Sequencing of enriched libraries after cell screening;

[0012] (5) Family analysis and proportion analysis of candidate aptamers;

[0013] (6) Analysis of affinity and specificity of candidate aptamers: including flow cytometry screening to determine the target affinity of the aptamer; pull-down experiments to verify the target protein of the aptamer; target protein knockdown experiments to verify the target protein of the aptamer; tissue section and antibody localization to verify the target protein of the aptamer;

[0014] (7) Screening of candidate aptamers for their function against the second messenger cAMP;

[0015] (8) Screening of functional aptamers for functional detection of cell phenotypes.

[0016] The steps of constructing the model cell in step (1) are as follows:

[0017] (a) Construction of protein expression plasmid and empty plasmid: PCR primers were designed according to the gene database sequence and primer design principles to amplify the full-length CDS sequence of the target gene. The PCR product was cloned into a double-enzyme-digested plasmid to construct a target protein expression plasmid and an empty plasmid without target gene expression; wherein the 5' and 3' ends of the PCR product contained sequences homologous to the plasmid;

[0018] (b) Lentivirus packaging: The two plasmids and the helper plasmid were co-transfected into HEK293T cells using the calcium phosphate DNA precipitation method to produce high-titer lentivirus for viral packaging.

[0019] (c) Virus concentration: Collect the HEK293T cell supernatant, centrifuge to remove debris, and filter. Filter the culture medium into the collection bottle below. Discard most of the culture medium in the collection bottle. Then invert the filter and centrifuge to centrifuge the virus into the culture medium to concentrate the virus.

[0020] (d) Construction of target protein stably expressing cell lines: Two concentrated lentiviruses were inoculated into HEK293T cells. After 48 h of infection, cells were screened with antibiotic-containing medium, and the cells were harvested and amplified to obtain target protein stably expressing cell lines and non-target protein expressing cell lines.

[0021] (e) Verification of target protein expression: Flow cytometry, immunofluorescence, and Western Blot experiments were performed to verify the expression of target proteins in cell lines that stably expressed target proteins and cell lines that did not express target proteins.

[0022] The steps of cell screening and enrichment to obtain an enriched library in step (3) are as follows:

[0023] (1) After denaturing the original DNA library at 95°C and then renaturing it at 4°C, HEK-293T cells with high expression of the target protein were used as positive screening cells to obtain positive screening supernatant and positive screening cell fluid;

[0024] (2) After the positive screening cell fluid is denatured and renatured, a cell line without target protein expression is used as a counter-screening cell to obtain the counter-screening supernatant and the counter-screening cell fluid;

[0025] (3) performing positive screening on the reverse screening supernatant and the positive screening cells to obtain positive screening supernatant and positive screening cell fluid;

[0026] (4) performing PCR detection on the supernatant of the reverse screening and the cell fluid of the positive screening, and obtaining the product obtained in this round of screening based on the PCR results;

[0027] (5) After PCR amplification, the product is recovered after denaturation and desalting for the next round of screening. The progress of the screening library is detected by flow cytometry, and the enriched library of cell screening is obtained based on the flow cytometry results. The functional screening in step (7) includes:

[0028] (I) Several candidate aptamers were incubated with positive screening cells and a cyclic nucleotide phosphodiesterase inhibitor (IBMX) was added, and the cAMP content in the cells or cell supernatant was measured by ELISA;

[0029] (II) Screened aptamers that affect cAMP production in the GPCR protein signaling pathway are used to investigate their effects on cell phenotypes through cell phenotypic experiments;

[0030] (III) Explore its effects on signaling pathways through Western Blot.

[0031] A use of a functional nucleic acid aptamer targeting a GPCR protein obtained by the method in preparing a reagent or a kit or an imaging agent for specifically recognizing and quantifying a GPCR protein.

[0032] A method for preparing a GPCR protein molecule agonist or inhibitor is provided.

[0033] A use of a functional nucleic acid aptamer targeting a GPCR protein obtained by the method in preparing a drug for preventing and / or treating and / or reversing a GPCR-related disease.

[0034] The beneficial effects of the present invention are as follows: the present invention provides a method and application for screening functional nucleic acid aptamers targeting GPCR proteins by combining cell screening with functional screening. By screening cells for the target protein, the natural configuration of the target protein is maintained, and the obtained nucleic acid aptamers have better in vivo specificity and targeting than the nucleic acid aptamers obtained by traditional protein SELEX. Cell-SELEX for constructing model cells has stronger targeting than traditional Cell-SELEX technology with unclear targets, and a series of aptamers targeting the target can be obtained through a single screening, which can be used as a library for functional screening of cells. Compared with traditional screening methods that require screening and then investigating whether the aptamers have functions in cells, the present invention can screen out a series of target aptamers at one time, and only perform a round of functional screening in cells to directly obtain functional aptamers that can affect the target protein signaling pathway and affect the cell phenotype. The method has the advantages of strong targeting, high affinity, economy, low labor cost, and broad versatility. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a schematic diagram of the model cell construction.

[0036] Figure 2 shows the construction of model cells verified by flow cytometry.

[0037] FIG3 is the construction of the model cells verified by immunofluorescence.

[0038] Figure 4 shows the construction of model cells verified by Western Blot.

[0039] FIG5 shows the affinity of nucleic acid aptamer libraries with different rounds to positive cells and negative cells.

[0040] FIG6 shows the affinity of a series of candidate aptamers to positive cells and negative cells.

[0041] FIG7 shows the effect of candidate aptamers on cAMP content in TSHR-293T cells as determined by ELISA.

[0042] FIG8 shows the effect of nucleic acid aptamer YC3 on cell verification factors IL-6 and IL-8 as determined by ELISA. DETAILED DESCRIPTION

[0043] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0044] Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0045] Cell sources: The primary fibroblasts (GOFs) used in this experiment were extracted from adipose connective tissue isolated from orbital decompression surgery in patients with thyroid-related diseases; human embryonic kidney cell line (293T) was purchased from Wuhan Punosai Life Science Technology Co., Ltd., China.

[0046] Taking TSHR protein in GPCR as an example

[0047] Example 1

[0048] Stable model cells for cell screening were constructed and verified, namely positive screening cells stably transfected with high-expression TSHR plasmid by lentivirus and negative screening cells stably transfected with empty-loaded plasmid by lentivirus.

[0049] Construction of protein expression plasmid and empty plasmid

[0050] PCR primers were designed using Primer 5 software according to the NCBI gene database sequence and primer design principles (primer information is shown in Table 1) to amplify the full-length sequence of the target gene hTSHR CDS. The PCR reaction system was prepared as follows: template, primers, dNTPs, enzyme, reaction buffer, and ddH2O were added in appropriate proportions, slightly centrifuged, and placed in a PCR instrument (C1000, Thermal Cycle, BIO-RAD) to amplify the target fragment. The PCR product was cloned into a double-enzyme-digested plasmid using seamless cloning technology to construct a TSHR expression plasmid and an empty plasmid without target gene expression; wherein, the 5' and 3' ends of the PCR product contained sequences homologous to the plasmid;

[0051] Table 1. PCR primers and their sequences

[0052] Lentiviral packaging

[0053] The above two plasmids were co-transfected with helper plasmids into HEK293T cells using the calcium phosphate DNA precipitation method (clontech kit) to produce high-titer lentivirus and complete virus packaging;

[0054] HEK293T cells were passaged 24 hours before transfection and ready for transfection when the cell density reached 80% confluency. Three hours before transfection, the medium was exchanged with fresh medium and 15 μl of 50 mM Choloroquine was added. Vortex the necessary reagents for transfection. Prepare two centrifuge tubes for each 10 cm dish transfected. Add the following reagents in the following order:

[0055] While gently vortexing Solution A, gradually and gently add Solution B. When complete, the solution should become opaque due to the precipitate. Incubate at room temperature for 15 minutes. Vortex the transfection complex again before adding it to the cells. Gently add the transfection complex dropwise to the culture plate at multiple locations. Once complete, rock the plate back and forth to ensure even adsorption of the precipitate to the cell surface. Change the solution 8 hours after transfection.

[0056] Concentrated virus

[0057] The HEK293T cell supernatant was collected, centrifuged to remove debris, and then filtered through a 0.45 μm filter. Viruses were not filtered out and remained above the filter, while the culture medium was filtered into a collection flask below. Most of the culture medium in the collection flask was discarded, and the filter was inverted and centrifuged to concentrate the virus into the culture medium.

[0058] Construction of target protein stable expression cell line

[0059] HEK293T cells were inoculated with two concentrated lentiviruses and 4ug / mL polybrene. After 48h of infection, they were screened with a culture medium containing puromycin antibiotics for 3 days. The cells were collected and amplified to obtain cell lines stably expressing the target protein and cell lines without target protein expression.

[0060] Verify the expression of target protein

[0061] The expression of target protein was verified by flow cytometry, immunofluorescence and Western Blot experiments in cell lines stably expressing target protein and cell lines without target protein expression.

[0062] Flow cytometry

[0063] Both model cells were cultured for at least 48 hours to a cell density of 90%. Adherent cells were then dissociated from the culture dishes using 0.2% EDTA. Harvested cells were incubated with FITC-conjugated IgG2a (Bioss, China) for 30 minutes at room temperature, followed by incubation with an anti-TSHR antibody (ab27974, Abcam) for 1 hour at room temperature. After washing three times, cells were incubated with a FITC-conjugated secondary antibody for 30 minutes at room temperature, washed three times, and analyzed by flow cytometry.

[0064] Immunofluorescence experiments

[0065] Cells were seeded into the bottom of an optical culture dish and cultured for 24 hours. For aptamers, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature after rinsing with D-PBS. After washing with D-PBS, cells were blocked in 5% BSA / DPBS buffer for 1 hour and then incubated with anti-TSHR antibody (1:500, Abcam, ab27974) at 4°C overnight. After the wash step, cells were stained with secondary antibody at room temperature for 1 hour, followed by DAPI nuclei staining for 10 minutes. Images were acquired using a Cell Observer SD (Leica, Bannockburn, IL, USA).

[0066] Western Blot

[0067] Cultured cells were rinsed with cold D-PBS and lysed in RIPA lysis buffer with a protease inhibitor cocktail (EpiZyme). Protein concentration was determined using a protein detection reagent (EpiZyme). Protein samples (40 μg / well) were electrophoresed on a 10% SDS-PAGE gel electrophoresis solution with protein loading buffer. Proteins were then transferred to a 0.22-μm NC membrane using a Western blot transfer system (Bio-Rad) at 300 mA for 90 minutes. The NC membrane was washed with TBST (Tris-buffered saline containing 0.5% Tween 20) and blocked with 5% nonfat milk for 1 hour at room temperature. The membrane was then washed three times with TBST and incubated overnight at 4°C with anti-TSHR antibody (1:1000, Abcam, ab27974) and anti-β-actin antibody (1:1000, Proteintech, 66009-1-Ig). After washing three times with TBST, the cells were incubated with goat anti-mouse IgG (heavy and light chains) antibody (1:50,000, LI-COR, IRDye680CW) at room temperature for 1 hour and then detected with Odyssey LI-COR.

[0068] Example 2: Screening of nucleic acid aptamers for target proteins

[0069] Design of nucleic acid library and primers used (nucleic acid chains were synthesized by Shanghai Bioengineering):

[0070] Random single-stranded DNA library:

[0071] 5'-ACCGACCGTGCTGGACTCA(N)42ACTATGAGCGAGCCTGGCG-3' (N represents any of the four bases A, T, G, and C)

[0072] Upstream primer: 5'-FITC-ACCGACCGTGCTGGACTCA-3

[0073] Downstream primer: 5'-biotin-CGCCAGGCTCGCTCATAGT-3

[0074] Screening Process:

[0075] The present invention uses TSHR-293T cells that highly express TSHR as a positive screening target and MOCK cells that do not express TSHR as a counter-screening target.

[0076] Positive screening:

[0077] a. Incubation: Dissolve the random DNA library in binding buffer, denature at 95°C for 5 minutes, anneal on ice for 10 minutes, and then incubate with pretreated TSHR-293T cells that have been cultured for at least 48 hours and have reached approximately 90% confluency at 4°C for 1 hour.

[0078] b. Separation: Remove the supernatant after incubation and rinse the cells several times with wash buffer. Then, scrape the washed cells with sterile water and place them in a centrifuge tube. Denature at 95°C for 10 minutes, renature on ice for 10 minutes, and centrifuge at 5500 rpm for 3 minutes. Aspirate the supernatant to obtain the first-round screening nucleic acid library of TSHR-293T cells.

[0079] c. PCR amplification of the library: Using the library obtained in step b as a template and the above primers as primers, amplify the library using the following conditions: 95°C for 30 seconds, 55.9°C for 30 seconds, 72°C for 30 seconds for 8 cycles, and 72°C for 5 minutes. A preliminary amplification product is obtained, and then amplification is performed using the amplified product as a template for an appropriate number of cycles.

[0080] d. Preparation of single-stranded DNA: Use streptavidin-modified agarose beads to separate the biotin-labeled antisense strand of the PCR amplification product in step c, then denature the double-stranded DNA with 0.2 M NaOH, and collect the positive single-stranded DNA library labeled with fluorescein isothiocyanate by desalting.

[0081] Counter-screening: The single-stranded DNA library obtained in step d is incubated with counter-screening cells MOCK cells, and the supernatant is collected to exclude non-specifically bound nucleic acid molecules. The collected supernatant can be further incubated with positive screening cells for the next screening step.

[0082] Screening cycle: Repeat steps 2.2.1 and 2.2.2 until a library of aptamers with strong binding to the target TSHR-293T cells is identified. This process may need to be repeated for several to a dozen rounds. High-throughput sequencing: The final round of screening is combined with the largest library for high-throughput sequencing. Flow cytometry is used to analyze the resulting sequences for their ability to bind to TSHR-293T cells, thereby identifying the aptamers. Fluorescence detection is performed by flow cytometry, with the initial random DNA library serving as a control.

[0083] Example 3: Screening of functional aptamer chains

[0084] The cAMP content in the cell supernatant was measured by ELISA to screen aptamers that affect protein signaling pathways.

[0085] Cells were seeded in 24-well plates and starved overnight with 1% fetal bovine serum. The next day, cells were incubated in serum-free and antibiotic-free medium containing 0.5 mM cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthie (IBMX) (MedChemExpress, USA) for 30 minutes to prevent cAMP degradation, followed by the addition of TSH (100 ng / mL), M22 (100 ng / mL), or candidate aptamers for 6 hours. Cell supernatants were collected and stored at −80°C until use in a cAMP assay (KGE002B; R&D Systems, Minneapolis, Minnesota). A polyclonal antibody competitively binds to cAMP standards or sample supernatants. All cAMP release measurements were performed in duplicate as described previously.

[0086] Effects of aptamers on cell phenotypes

[0087] GOFs were seeded in 24-well plates and incubated overnight with 1% FBS. The next day, cells were cultured in DMEM supplemented with 1% FBS with or without TSH (100 ng / mL), M22 (100 ng / mL), the aptamer YC3, or Teprotumumab. After 48 hours, the cell culture supernatant was centrifuged at 5000 g for 10 minutes and stored at -80°C until further use. IL-6 and IL-8 levels were measured using commercial ELISA kits (FineTest, Wuhan, China) according to the manufacturer's instructions. Samples were diluted 1:10 before analysis and analyzed in triplicate.

[0088] Detection of the effects of aptamers on cellular pathways by Western Blot

[0089] Cultured cells were rinsed with cold D-PBS and lysed in RIPA lysis buffer supplemented with a protease inhibitor cocktail (EpiZyme) and phosphatase inhibitors (EpiZyme). Protein concentration was determined using a protein detection reagent (EpiZyme). Protein samples (40 μg / well) were added to protein loading buffer, boiled at 95°C for 5 minutes, and electrophoresed on a 10% SDS-PAGE. Proteins were then transferred to a 0.22-μm NC membrane using a Western blot transfer system (Bio-Rad) at 300 mA for 90 minutes. The NC membrane was washed with TBST (Tris-buffered saline containing 0.5% Tween 20) and blocked with 5% nonfat milk for 1 hour at room temperature. The membrane was then washed three times with TBST and incubated with the primary antibody and anti-actin antibody (1:1000, Proteintech, 66009-1-Ig) overnight at 4°C. After washing three times with TBST, the cells were incubated with secondary antibody (1:50,000, LI-COR) at room temperature for 1 hour and then detected with Odyssey LI-COR.

[0090] Technical personnel should note: Although the present invention has been described according to the above specific implementation methods, the inventive concept of the present invention is not limited to this invention. Any modification using the inventive concept will be included in the scope of protection of this patent.

[0091] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for screening functional nucleic acid aptamers targeting GPCR protein by combining cell screening and functional screening, characterized in that: The following steps are involved: (1) Construct and validate stable model cells for cell screening; (2) constructing a screening library; (3) enriching the screening library by cell screening to obtain an enriched library; (4) Sequencing of enriched libraries after cell screening; (5) Family analysis and ratio analysis of candidate aptamers; (6) Analysis of affinity and specificity of candidate aptamers: including flow cytometry screening to determine the target affinity of the aptamer; Pull-down experiments verify the target protein of the aptamer; target protein knockdown experiments verify the target protein of the aptamer; tissue sections and antibody localization verify the target protein of the aptamer; (7) Screening the candidate aptamers for their functions against the second messenger cAMP; (8) Screening of functional aptamers for functional detection of cell phenotypes.

2. The method according to claim 1, characterized in that The steps of constructing the model cell in step (1) are as follows: (a) Construction of protein expression plasmid and empty plasmid: According to the gene database sequence and primer design principles, PCR primers are designed to amplify the full-length CDS sequence of the target gene, and the PCR product is cloned into a double-enzyme-digested plasmid to construct a target protein expression plasmid and an empty plasmid without target gene expression; wherein the 5' and 3' ends of the PCR product carry sequences homologous to the plasmid; (b) Lentivirus packaging: The above two plasmids and the helper plasmid were co-transfected into HEK293T cells using the calcium phosphate DNA precipitation method to produce high-titer lentivirus and complete virus packaging; (c) Virus concentration: Collect the supernatant of HEK293T cells, centrifuge to remove debris, and then filter. Filter the culture medium into the collection bottle below, discard most of the culture medium in the collection bottle, and then invert the filter and centrifuge to centrifuge the virus into the culture medium, so that the virus is concentrated; (d) Construction of a cell line stably expressing the target protein: Two concentrated lentiviruses were inoculated into HEK293T cells. After 48 h of infection, cells were screened with a medium containing antibiotics, and the cells were collected and amplified to obtain a cell line stably expressing the target protein and a cell line without target protein expression; (e) Verification of target protein expression: Flow cytometry, immunofluorescence, and Western Blot experiments were performed to verify the expression of target proteins in cell lines that stably expressed target proteins and cell lines that did not express target proteins.

3. The method according to claim 1, characterized in that The step of screening and enriching cells to obtain an enriched library in step (3) is as follows: (i) After the original DNA library was denatured at 95°C and then renatured at 4°C, HEK-293T cells with high expression of the target protein were used as positive screening cells to obtain positive screening supernatant and positive screening cell fluid; (ii) After the positive screening cell fluid is denatured and renatured, a cell line without target protein expression is used as a counter-screening cell to obtain a counter-screening supernatant and a counter-screening cell fluid; (iii) performing positive screening on the reverse screening supernatant and the positive screening cells to obtain positive screening supernatant and positive screening cell fluid; (iv) performing PCR detection on the reverse screening supernatant and the forward screening cell fluid, and obtaining the product obtained in this round of screening based on the PCR results; (V) The products after PCR amplification are denatured, desalted, recovered and then subjected to the next round of screening. The progress of the screening library is detected by flow cytometry, and the enriched library of cell screening is obtained based on the flow cytometry results.

4. The method according to claim 1, characterized in that: The function screening in step (7) includes: (I) several candidate aptamers were incubated with positive screening cells and a cyclic nucleotide phosphodiesterase inhibitor (IBMX) was added, and then the cAMP content in the cells or in the cell supernatant was measured by ELISA; (II) Screened aptamers that affect cAMP production in the GPCR protein signaling pathway are used to explore their effects on cell phenotypes through cell phenotypic experiments; (III) Explore its effects on signaling pathways through Western Blot.

5. Use of a functional nucleic acid aptamer targeting a GPCR protein obtained by the method according to any one of claims 1 to 4 in the preparation of a reagent or a kit or an imaging agent for specific recognition and quantification of a GPCR protein.

6. Use of a functional nucleic acid aptamer targeting a GPCR protein obtained by the method according to any one of claims 1 to 4 in the preparation of a GPCR protein molecule agonist or inhibitor.

7. Use of a functional nucleic acid aptamer targeting a GPCR protein obtained by the method according to any one of claims 1 to 4 in the preparation of a drug for preventing and / or treating and / or reversing a GPCR-related disease.

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