A2aR-His6-G alpha16-HEK cell strain capable of stably expressing human adenosine A2aR and application of A2aR-His6-G alpha16-HEK cell strain

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an A2aR-His6-G alpha 16-HEK cell strain capable of stably expressing a human adenosine A2a receptor (A2aR). According to the invention, the Galpha16 protein and the A2aR which are determined by screening are sequentially expressed in HEK-FlpIn cells, and the A2aR-His6-Galpha16-HEK cell strain capable of stably expressing the human A2aR is obtained by screening; a cell evaluation model suitable for calcium signal detection is constructed, and the cell evaluation model can be used for screening and detecting A2aR-targeting agonists and antagonists; the kit has the characteristics of more reliable detection result, higher sensitivity, larger screening flux, short experimental period, simplicity and convenience in operation and the like, can greatly improve the screening and evaluation efficiency of A2aR-targeting agonists and antagonists, and meanwhile, has a significant meaning for A2aR-related diseases. Candidate drug molecules are provided for treatment of diseases such as motion control, neurodegenerative diseases, tumor immunoregulation, anti-depression effect, heart diseases, nerve regeneration and immunoregulation, the problem that at present, A2aR target-based drug screening does not have a high-throughput detection method based on calcium signal detection is solved, and the method has obvious practical application value.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to an A2aR-His6-Gα16-HEK cell line that stably expresses human adenosine A2aR and its applications. Background Technology

[0002] Adenosine receptors (ARs) are G protein-coupled receptors (GPCRs) containing a seven-transmembrane structure. ARs mainly include four receptor subtypes: A1, A2a, A2b, and A3. The A2a receptor (A2aR) consists of 410 amino acids, and its amino acid sequence shows high homology in mammals. A2aR plays an important role in various physiological and pathological processes, such as motor control, neurodegenerative diseases, tumor immunomodulation, antidepressant effects, heart disease, nerve regeneration, and immune regulation. After activation by endogenous ligands or exogenous agonists, A2aR further activates G proteins on the inner side of the plasma membrane. A2aR couples with the excitatory G protein Gs, which further activates adenylate cyclase and increases intracellular cAMP levels (Linden J., Annu. Rev. Pharmacol. Toxicol., 41: 775-87, 2001).

[0003] Currently, A2aR-based compound screening and detection methods mainly include cAMP detection, radioligand receptor binding detection, and FRET-based detection methods. However, these methods have the following drawbacks: long experimental cycles, cumbersome operation processes, low screening efficiency, and high costs.

[0004] Currently, there is a lack of stable cell models for screening A2aR-targeted drugs suitable for calcium signal analysis methods. Stable cell models for A2aR expression will make the screening and pharmacodynamic evaluation of A2aR-targeted compounds faster and more sensitive, greatly shortening the experimental cycle of A2aR drug discovery, simplifying the operation process, and improving screening efficiency. Summary of the Invention

[0005] The purpose of this invention is to address the lack of stable expression cell models for A2aR-targeting drug screening based on the FLIPR platform and applicable to calcium signal analysis methods. This invention provides a cell line that stably expresses A2aR and its construction method. A sequential expression strategy is used to construct a cell line that stably expresses human A2aR and Gα16, and a real-time fluorescence detection method is used to achieve rapid screening and evaluation of A2aR-targeting compounds.

[0006] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a method for screening compounds targeting A2aR, specifically comprising the following steps: cultured A2aR-His6-Gα16-HEK cell lines, adding the target compound to be detected; if intracellular calcium signal enhancement occurs with statistical significance, the target compound to be detected is an agonist targeting A2aR; if intracellular activated calcium signal is blocked with statistical significance, the target compound to be detected is an antagonist targeting A2aR. The method for preparing the A2aR-His6-Gα16-HEK cell line includes the following steps: (1) Preparation of human A2aR-His6-pcDNA5 / FRT receptor expression plasmid: Plasmid A2aR was ligated with the enzyme-digested expression vector pcDNA5 / FRT to construct recombinant expression plasmid A2aR-His6-pcDNA5 / FRT; (2) Preparation of Gα16 protein expression plasmid: The expression vector pIRES-puro3 after Gα16 enzyme digestion was ligated to construct the recombinant expression plasmid Gα16-pIRES-puro3; (3) Transfect the Gα16 protein expression plasmid into HEK-FlpIn cells and select cell lines that stably express Gα16 protein: Transfect the Gα16 protein expression plasmid into HEK-FlpIn cells and add puromycin to select positive cell lines; stimulate cells with A2aR agonist and detect changes in intracellular calcium signal by real-time fluorescence detection of FLIPR, and screen clones Gα16-HEK-FlpIn that meet the requirements of Gα16 protein expression signal to obtain Gα16-HEK-FlpIn cells; (4) Based on the established stable Gα16 protein cell line, the A2aR-His6-pcDNA5 / FRT and POG44 plasmid genes were co-transfected into Gα16-HEK-FlpIn cells, and cell lines stably expressing human A2aR were obtained through screening: human A2aR expression plasmid and FLP recombinase plasmid POG44 were co-transfected into Gα16-HEK-FlpIn cells, and puromycin and hygromycin were added to screen for positive cell lines; cells were stimulated with A2aR agonists, and changes in intracellular calcium signals were detected by FLIPR real-time fluorescence, and clones with human A2aR expression signals meeting the requirements were screened; after 5 to 10 passages, the pharmacological characteristics were identified using the A2aR agonist NECA, and the cell line A2aR-His6-Gα16-HEK stably expressing human A2aR and Gα16 proteins was obtained.

[0007] The expression vector connecting the human A2aR gene in step (1) is pcDNA5 / FRT. This plasmid contains the recombinase recognition site OFRT, which can be co-transfected with the FLP recombinase plasmid pOG44 for directional recombination. The screening marker is hygromycin.

[0008] Preferably, the A2aR agonist is NECA and the A2aR antagonist is ZM241385.

[0009] Preferably, in step (1), plasmid A2aR is used as a template, and hA2aR-His6-Nhe IF and A2aR-His6-Not IR are used as specific primers to perform PCR amplification to obtain the target gene, which is tagged with His at the C-terminus. The PCR amplification product is subjected to electrophoresis on an agarose gel to separate the target band and recover the target DNA. The sequence of the specific primer hA2aR-cHis6-Nhe IF is shown in SEQ ID NO.5, and the sequence of hA2aR-cHis6-Not IR is shown in SEQ ID NO.6.

[0010] Preferably, in step (1), the expression vector pcDNA5 / FRT is double-digested with Nhe I / Not I, and then ligated with the target DNA to construct the recombinant expression plasmid A2aR-His6-pcDNA5 / FRT.

[0011] Preferably, in step (2), plasmid Gα16 is used as a template, and Gα16-Age IF and Gα16-Not IR are used as specific primers to perform PCR amplification to obtain the target gene; the PCR amplification product is subjected to agarose gel electrophoresis to separate the target band and recover the target DNA; the sequence of the specific primer GNA15-Age IF is shown in SEQ ID NO.7, and the sequence of GNA15-Not IR is shown in SEQ ID NO.8.

[0012] Preferably, in step (2), the expression vector pIRES-puro3 is double-digested with Age I / Not I, and then ligated with the target DNA to construct the recombinant expression plasmid Gα16-pIRES-puro3.

[0013] Preferably, the method for identifying the pharmacological characteristics described in step (4) is as follows: an A2aR specific agonist or inhibitor is added to the constructed cell line, the change in fluorescence signal caused by calcium signal is measured, and the functional activity of the cell line is evaluated based on the result of the change in fluorescence signal.

[0014] Preferably, the method for screening compounds targeting A2aR is as follows: the cell line described in the first aspect is seeded into a cell plate; after overnight culture, the culture medium is removed, 40 μL of calcium-sensitive dye is added to each well, and the plate is incubated at 37°C in the dark for 120 min; freshly prepared compound stock solution is added to a new well plate to serve as the compound plate; the cell plate is then loaded into a fluorescence imaging plate reader (FLIPR). TETRA (MD Company, USA) Fluorescence readings were performed according to the parameter settings: excitation wavelength 470-495 nm, emission wavelength 515-575 nm, LED intensity 60-80%, gain 6.5-8, and exposure time 0.045-0.05 s. First, the detection program read for 10 seconds as a baseline. Then, using an automated liquid handling system, the compound solution in the compound plate was transferred to the corresponding wells in the cell plate at 10 μL / well. Data was collected once per second for 120 seconds. OriginLab 2019 software was used for data fitting and analysis to calculate EC50. 50 The value is used to quantify the agonistic effect of the compound on A2aR.

[0015] In a second aspect, the present invention provides an A2aR-His6-Gα16-HEK cell line that stably expresses human A2aR, which is prepared by the method described in the first aspect; the cell line is mammalian HEK-FlpIn cells sequentially transfected with human Gα16 gene and A2aR gene. The A2aR gene sequence is shown in SEQ ID NO.2, and has a His tag at the C-terminus; the gene sequence of the Gα16 protein (GNA15) is shown in SEQ ID NO.4; The mammalian cell is a HEK-FlpIn cell, which contains a stably integrated recombinase recognition sequence FRT site in the transcriptionally active region, enabling the expression vector to be site-specifically integrated at the same chromosomal site in each cell while ensuring high expression levels.

[0016] Preferably, the amino acid sequence of A2aR is as shown in SEQ ID NO.1, with a 6-His tag at the C-terminus; the amino acid sequence of the Gα16 protein is as shown in SEQ ID NO.3.

[0017] Thirdly, the present invention provides a method for preparing the cell line described in the first aspect above, the method comprising: preparing a human A2aR expression plasmid and a Gα16 protein expression plasmid; sequentially transfecting the human Gα16 protein expression plasmid and the A2aR expression plasmid into HEK-FlpIn cells; and obtaining a stable A2aR-His6-Gα16-HEK cell line expressing human A2aR through antibiotic selection and functional testing. Figure 1 ).

[0018] The present invention aims to obtain a stable A2aR-His6-Gα16-HEK cell line expressing human A2aR by sequentially co-expressing Gq homologous protein (screened and determined Gα16 protein) and A2aR in HEK-FlpIn cells; and in the cell line, when A2aR is activated, it induces a coupled Gα16 protein response, leading to intracellular Ca2+. 2+ Increase, using Ca 2+ Sensitive fluorescent dyes can rapidly and efficiently capture this change. Therefore, using real-time quantitative fluorescence detection (FLIPR) technology and a calcium signal change-based detection principle, a cell model and high-throughput detection method for screening A2aR-targeting drugs were established. This method offers more reliable detection results, higher sensitivity, larger screening throughput, shorter experimental cycle, and simpler operation, significantly improving the efficiency of screening and evaluating A2aR-targeting compounds. It also provides candidate drug molecules for the treatment of A2aR-related diseases, such as motor control, neurodegenerative diseases, tumor immunomodulation, antidepressant effects, heart disease, and nerve regeneration and immune regulation. This addresses the current lack of high-throughput detection methods based on calcium signals in A2aR-targeted drug screening, demonstrating significant practical application value. The beneficial effects of this invention are:

[0019] 1. This invention innovatively utilizes a sequential expression strategy. Gα16 protein expression plasmids are sequentially transfected into HEK-FlpIn cells. After screening for positive clones stably expressing the target protein, human A2aR expression plasmids and FLP recombinase plasmid POG44 are then transfected into Gα16-HEK-FlpIn cells, successfully constructing a cell line stably expressing human A2aR and Gα16 proteins. Functional assays of this cell line were also performed, highly reproducing the in vivo signal transduction mechanism of A2aR, a feat not previously reported. This cell line exhibits a sensitive response to compounds targeting A2aR, laying the foundation for further research on the biological characteristics of A2aR and enriching related research. The Gα16 protein converts the Gs-coupled signal of A2aR into calcium flux, which can be directly detected using calcium-sensitive dyes, avoiding interference from indirect pathways. The EC50 is at the nanomolar level, demonstrating high sensitivity. Furthermore, by employing a sequential transfection strategy, a monoclonal cell line that can be passaged for a long period of time can be obtained. This cell line retains its function after multiple passages, reducing batch-to-batch variation and making it suitable for large-scale screening.

[0020] 2. This invention provides a cell line that stably expresses human A2aR, and utilizes this cell line to establish a method for screening A2aR compound activity based on real-time fluorescence detection of calcium signals. Compared with existing detection methods that require transient plasmid transfection, this invention saves time and costs, while exhibiting good stability and uniformity. Compared with cAMP experiments, the detection results are more reliable, more sensitive, have higher screening throughput, shorter cycle time, and are simpler to operate. It can significantly improve the rapid detection and pharmacodynamic evaluation of A2aR-targeting compounds. Furthermore, it provides guidance for drug screening in diseases closely related to A2aR, such as motor control, neurodegenerative diseases, tumor immunomodulation, antidepressant effects, heart diseases, and nerve regeneration and immune regulation. It has broad value for basic and applied research.

[0021] 3. This invention is based on the FLIPR platform and directly captures the calcium flow signal after A2aR activation. The detection time is only tens to hundreds of seconds, supporting high-throughput screening of multi-well plates. The efficiency is several times higher than other screening pathways that rely on gene expression, making it suitable for the initial screening of large-scale compound libraries. Attached Figure Description

[0022] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0023] Figure 1 Flowchart for constructing the A2aR-His6-Gα16-HEK cell line Figure 2 Figure 1 shows the results of enzyme digestion and agarose gel electrophoresis for the A2aR gene construction. In the figure: a: lane1 is the target band, lane2 is the marker band; b: lane1-7 are 7 bacterial clones, lane8 is plasmid DNA (A2aR-His6-pcDNA5 / FRT7 positive control).

[0024] Figure 3 Map of the A2aR-His6-pcDNA5 / FRT vector

[0025] Figure 4 Figure 1 shows the results of enzyme digestion and agarose gel electrophoresis for the construction of the Gα16 gene. In the figure: a: lane1 is the undigested image, lane2 is the kpnl digested image, lane3 is the marker band image; b: 1kb marker band image. Figure 5 Map of the Gα16-pIRES-puro3 vector Figure 6 Figure showing the screening results of Gα16 positive clonal cell lines. In the figure: a: the horizontal axis represents the detection time, and the vertical axis represents the fluorescence intensity; b: the horizontal axis represents the clone number, and the vertical axis represents the fluorescence intensity.

[0026] Figure 7 To identify His tag expression in the A2aR-His6-Ga16-HEK cell line using immunofluorescence. In the image: the left image shows His-tagged immunofluorescence of clone 1, and the right image shows DAPI staining of clone 1.

[0027] Figure 8 Figure showing the screening results of A2aR / Gα16 positive clonal cell lines. In the figure: a: the horizontal axis represents the detection time, and the vertical axis represents the fluorescence intensity; b: the horizontal axis represents the clone number, and the vertical axis represents the fluorescence intensity.

[0028] Figure 9 A diagram illustrating the identification of A2aR protein expression in the A2aR-His6-Gα16-HEK stable cell line. In the figure: a: bright field image of cells; b: A2aR protein staining image; c: DAPI staining image; d: overlay image of A2aR protein staining and DAPI staining.

[0029] Figure 10 This diagram illustrates the dose-activation effect of the agonist NECA on the stable A2aR-His6-Gα16-HEK cell line. In the figure: a: signal map of A2aR activation by the agonist NECA; b: ECG fit of the agonist. 50 Value curve.

[0030] Figure 11 Figure showing the dose-antagonistic effect of antagonist ZM241385 on the stable A2aR-His6-Gα16-HEK cell line. In the figure: a: antagonistic signal of the antagonist ZM241385 on the activation of A2aR; b: fitted IC50 of the antagonist. 50 Value curve. Detailed Implementation

[0031] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Working principles not described in detail in this invention are prior art and common knowledge in the field, and should be known by those skilled in the art. The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0032] The reagents and materials used in this invention are as follows: HEK-FlpIn cells, POG44 plasmid, antibiotics Puromycin and Hygromycin, and transfection reagent Lipofectin 2000 were all purchased from Invitrogen. DH5α competent cells, PCR product recovery kits, and gel product recovery kits were all purchased from Tiangen Biotech Co., Ltd. Fetal bovine serum was purchased from Gibco, USA. The human A2aR gene was constructed into the pcDNA5 / FRT expression vector; the Gα16 gene was constructed into the pIRES-puro3 vector; the FLIPR calcium signal 6 detection kit was purchased from Meigu Molecular Instruments Co., Ltd.; the anti-His antibody was purchased from Zhongshan Jinqiao Co., Ltd.; the A2aR rabbit primary antibody and FITC-labeled goat anti-rabbit secondary antibody were purchased from Genetex, USA; DAPI was purchased from Solarbio, USA; the tool compound agonist NECA (N6-hydroxybenzyl adenosine) and antagonist ZM241385 were purchased from ABCAM; 384-well black-bottom permeable cell culture plates and 384-well compound plates were purchased from Greiner, Germany. The company; the 384-well FLIPR sample tip was purchased from MD Company, USA.

[0033] The instruments used in this invention are as follows: FLIPR Penta Fluorescence detection and analysis system, Molecullar Devices, USA; 18AIC cell culture incubator, SANYO, Japan; 2780 PCR instrument, Application Systems, USA; ChemiScope 6100Touch gel imaging system, Qinxiang, China; YT-CJ-1D clean bench, Asia Pacific Kolon, China; DW-86L388J benchtop low-temperature centrifuge, Haier, China; Cytation 1 cell imaging microplate detection system, BioTek, China; B3111 CO2 cell culture incubator, Thermo Fisher Scientific, USA; 838 dispensing workstation, Thermo Fisher Scientific, USA; Ti2-U inverted fluorescence microscope, Nikon, Japan; Stellaris 5 laser confocal microscope, Leica, Germany; Milli-QIQ-7000 pure water system, Millipore, USA. Example 1 1: Amplification of the A2aR gene

[0034] (1) PCR amplification reaction: Based on the human A2aR gene sequence (gene name A2aR, NM_001278497) in NCBI, specific primer pairs for the A2aR gene were designed using CE Design primer design software: hA2aR-cHis6-Nhe IF and hA2aR-cHis6-Not IR, respectively. ， and 3 ， The restriction enzyme sites Nhe I and Not I were introduced. The sequence of the specific primer hA2aR-cHis6-Nhe IF is shown in SEQ ID NO.5, and the sequence of hA2aR-cHis6-Not IR is shown in SEQ ID NO.6. The A2aR gene was amplified by PCR reaction using plasmid A2aR as a template.

[0035] The PCR reaction system consisted of: 10×PCR Buffer, 5 μL; 2 mM dNTPs, 5 μL; primer hA2aR-cHis6-NheI-F (10 μM), 1.0 μL; primer hA2aR-cHis6-Not IR (10 μM), 1.0 μL; template plasmid A2aR, 1.0 μL; DNA polymerase, 1.0 μL; dd H2O, 36 μL; and a total volume of 50 μL.

[0036] PCR reaction procedure: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 60℃ annealing for 20 sec, 72℃ extension for 120 sec, for a total of 30 cycles, and a final extension at 72℃ for 5 min. After PCR, 20 μL of the amplification product was electrophoresed and separated on an agarose gel, and the DNA was recovered and purified using a gel extraction kit.

[0037] (2) DNA agarose gel electrophoresis: The target band was obtained by agarose gel electrophoresis: 1% agarose gel was prepared, heated in a microwave oven to completely dissolve the agarose particles, cooled to 60°C, and then GV-II was added and mixed well and poured into a dry gel casting plate; the gel was poured, and a comb was inserted to avoid air bubbles; after the gel solidified, the comb was carefully removed; the gel was placed in the electrophoresis tank and electrophoresis buffer was added; 20 μL of PCR product was loaded and electrophoresed at 100V for 40 min.

[0038] (3) PCR product gel recovery: DNA was recovered according to the instructions of the agarose gel DNA recovery kit of Tiangen Biotech (Beijing) Co., Ltd. 2: Amplification of the Gα16 gene

[0039] (1) PCR amplification reaction: Based on the human Gq homolog Gα16 gene sequence (gene name GNA15, NM_002068) preserved in the laboratory, specific primer pairs for the Gα16 gene were designed using CE Design primer design software: GNA15-Age IF and GNA15-Not IR, respectively. ， and 3 ， The restriction enzyme sites Age I and Not I were introduced. The sequence of the specific primer GNA15-Age IF is shown in SEQ ID NO.7, and the sequence of GNA15-Not IR is shown in SEQ ID NO.8. The Gα16 gene was amplified by PCR using plasmid Gα16 as a template.

[0040] The PCR reaction system consisted of: 10×PCR Buffer, 5 μL; 2 mM dNTPs, 5 μL; primer GNA15-Age IF (10 μM), 1.0 μL; primer GNA15-Not IR (10 μM), 1.0 μL; template plasmid Gα16, 1.0 μL; DNA polymerase, 1.0 μL; dd H2O, 36 μL; total volume, 50 μL.

[0041] PCR reaction procedure: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 sec, 60℃ annealing for 20 sec, 72℃ extension for 120 sec, for a total of 30 cycles, and a final extension at 72℃ for 5 min. After PCR, 20 μL of the amplification product was electrophoresed and separated on an agarose gel, and the DNA was recovered and purified using a gel extraction kit.

[0042] (2) DNA agarose gel electrophoresis: The target band was obtained by agarose gel electrophoresis: 1% agarose gel was prepared, heated in a microwave oven to completely dissolve the agarose particles, cooled to 60°C, and then GV-II was added and mixed well and poured into a dry gel casting plate; the gel was poured, and a comb was inserted to avoid air bubbles; after the gel solidified, the comb was carefully removed; the gel was placed in the electrophoresis tank and electrophoresis buffer was added; 20 μL of PCR product was loaded and electrophoresed at 100V for 40 min.

[0043] (3) PCR product gel recovery: DNA was recovered according to the instructions of the agarose gel DNA recovery kit of Tiangen Biotech (Beijing) Co., Ltd. 3: Construct the recombinant expression plasmid A2aR-His6-pcDNA5 / FRT

[0044] (1) Vector digestion The blank vector pcDNA5 / FRT was digested with Nhe I and Not I. The digestion reaction system was as follows: Nhe I, 1.5 μL; Not I, 1.5 μL; plasmid pcDNA5 / FRT, 1 μL (1 μg / μL); 10×Buffer, 2 μL; ddH2O, 14 μL; total system, 20 μL.

[0045] The enzyme digestion system was reacted in a water bath at 37°C for 2 h. The enzyme digestion products were subjected to agarose gel electrophoresis, and the correct target bands were recovered by gel electrophoresis.

[0046] (2) Ligation of the target gene and the vector The A2aR gene recovered from the gel in Example 1 was ligated with the enzyme-digested vector pcDNA5 / FRT using T4 DNA ligase. The ligation system was reacted at 16°C for 24 h to construct the recombinant expression plasmid A2aR-His6-pcDNA5 / FRT.

[0047] The ligation system was as follows: T4 DNA ligase, 1 μL; 10×Buffer, 2 μL; linearized vector pcDNA5 / FRT, 1 μL (1 μg / μL); inserted A2aR gene, 5 μL; ddH2O, 11 μL; total volume, 20 μL.

[0048] (3) Plasmid transformation of competent Escherichia coli DH5α E. coli DH 5α competent cells were slowly thawed on ice for 10 min. 20 μL of competent cells were transferred to a sterile EP tube, and 5 μL of the constructed recombinant expression plasmid A2aR-His6-pcDNA5 / FRT was added. The mixture was incubated on ice for 30 min. The cells were then heat-shocked in a 42℃ water bath for 45 s, quickly transferred to ice, and incubated for 2 min. 500 μL of preheated (37℃) antibiotic-free LB medium was added, and the cells were incubated at 37℃ with shaking at 300 rpm for 1 h. 100 μL of competent cells were plated onto LB agar plates containing Ampicillin (100 μg / mL) and incubated at 37℃. After 12 h, colonies were checked in the culture dishes.

[0049] Select single colonies from agar plates and inoculate them into 5 mL of LB medium containing Ampicillin (100 μg / mL). Incubate at 37°C and 300 rpm for 12 h on a shaker.

[0050] (4) Plasmid extraction and identification of recombinant strains Plasmid extraction was performed according to the instructions of the Qiagen (Germany) plasmid extraction kit. The A2aR-his-pcDNA5 / FRT recombinant expression plasmid was obtained. OD was measured using NanoDrop. 260 and OD 280 The purity and concentration of the plasmid were calculated. Preliminary identification was performed by Nhe I / Not I digestion and agarose gel electrophoresis, and the results are as follows: Figure 2 As shown, further identification was performed by sequencing. The sequencing results showed that the bases were completely identical to the target sequence (see A2aR-his-pcDNA5 / FRT plasmid vector diagram). Figure 3 ). 4: Construct the recombinant expression plasmid Gα16-pIRES-puro3

[0051] (1) Vector digestion The blank vector pIRES-puro3 was digested with Age I / Not I. The digestion reaction system was as follows: Age I, 1.5 μL; Not I, 1.5 μL; plasmid pIRES-puro3, 1 μL (1 μg / μL); 10×Buffer, 2 μL; ddH2O, 14 μL; total system, 20 μL.

[0052] The enzyme digestion system was reacted in a water bath at 37°C for 2 h. The enzyme digestion products were subjected to agarose gel electrophoresis, and the correct target bands were recovered by gel electrophoresis.

[0053] (2) Ligation of the target gene and the vector The Gα16 gene recovered from the gel in Example 1 was ligated with the enzyme-digested vector pIRES-puro3 using T4 DNA ligase. The ligation system was reacted at 16°C for 24 h to construct the recombinant expression plasmid Gα16-pIRES-puro3.

[0054] The ligation system was as follows: T4 DNA ligase, 1 μL; 10×Buffer, 2 μL; linearized vector pIRES-puro3, 1 μL (1 μg / μL); inserted Gα16 gene, 5 μL; ddH2O, 11 μL; total volume, 20 μL.

[0055] (3) Plasmid transformation of competent Escherichia coli DH5α E. coli DH 5α competent cells were slowly thawed on ice for 10 min. 20 μL of competent cells were transferred to a sterile EP tube, and 5 μL of the constructed recombinant expression plasmid Gα16-pIRES-puro3 was added. The mixture was incubated on ice for 30 min. The cells were then heat-shocked in a 42℃ water bath for 45 s, quickly transferred to ice, and incubated for 2 min. 500 μL of preheated (37℃) antibiotic-free LB medium was added, and the cells were incubated at 37℃ with shaking at 300 rpm for 1 h. 100 μL of competent cells were plated onto LB agar plates containing Ampicillin (100 μg / mL) and incubated at 37℃. After 12 h, colonies were checked in the culture dishes.

[0056] Select single colonies from agar plates and inoculate them into 5 mL of LB medium containing Ampicillin (100 μg / mL). Incubate at 37°C and 300 rpm for 12 h on a shaker.

[0057] (4) Plasmid extraction and identification of recombinant strains Plasmid extraction was performed according to the instructions of the Qiagen (Germany) plasmid extraction kit. The recombinant expression plasmid Gα16-pIRES-puro3 was obtained. OD was measured using NanoDrop. 260 and OD 280 The purity and concentration of the plasmid were calculated. Preliminary identification was performed by AgeI / NotI digestion and agarose gel electrophoresis, and the results are as follows: Figure 4 As shown, further identification was performed by sequencing. The sequencing results showed that the bases were completely identical to the target sequence (see the image of the Gα16-pIRES-puro3 plasmid vector). Figure 5 ). 5: Construction of a stable Gα16-HEK cell line

[0058] The Gα16-pIRESpuro3 plasmid was transiently transfected into HEK-FlpIn cells. The next day, the cells were seeded at a low density, and 0.2 μg / mL of puromycin was added for screening of positive clones. Fresh culture medium and antibiotics were added every 3–4 days, gradually increasing the antibiotic concentration to 1.0 μg / mL each time. After approximately 2–3 weeks, resistant monoclonal cell clusters grew. Forty-eight monoclonal cells were picked and transferred to 24-well plates for further expansion and functional verification. Healthy clones typically take about 2–3 weeks to grow.

[0059] Eighteen clones grew rapidly, reaching nearly 70-80% confluence in 24-well plates. Cells were transiently transfected with A2aR cDNA and seeded in 384-well plates. High-expression positive clones were screened using calcium flux analysis. After FLIPR calcium flux screening, Gα16 clones #13 and #44 showed relatively high signal intensity under the action of the adenosine receptor agonist NECA 10 μM. Figure 6 Clone #44 was used for the next step of constructing a stable A2aR cell line. 6: Construction of a stable A2aR-His6-Gα16-HEK cell line

[0060] Based on the established stable Gα16 cell line, the A2aR-His6-pcDNA5 / FRT+POG44 plasmid gene was co-transfected into Gα16-HEK-FlpIn cells. The next day, the cells were seeded at a low density, and 25 μg / mL of Hygromycin and 1 μg / mL of Puromycin were added for screening of positive clones. The culture medium and antibiotics were changed every 3-4 days. After about two to three weeks, resistant monoclonal cell clusters grew, but there were relatively few healthy cell clones. About 9 monoclonal clones were picked and transferred to 24-well plates for further expansion culture and expression and functional verification. Healthy clones took about 2-3 weeks to grow.

[0061] For expression identification, since the C-terminus of the A2aR gene carries a His tag, immunofluorescence staining was used to identify A2aR-His6 expression. Cell clones #1, 2, 3, 5, 6, 7, and 8 showed positive immunofluorescence signals. Taking clone #1 as a representative, the immunofluorescence signal is as follows... Figure 7 As shown.

[0062] When clones #1, 2, 3, 5, 6, 7, and 8 reached a suitable growth stage for FLIPR experiments, the cells were digested, seeded in 384-well plates, and the FLIPR calcium flux signal was measured using NECA as an agonist. The results are as follows. Figure 8 As shown, clones #1 and #5 have the strongest signals. 7: Identification of A2aR protein expression in A2aR-His6-Gα16-HEK stable cell line

[0063] Clone #1 was passaged and cultured using double antibody selection. Cells with a growth density of approximately 80% were inoculated into 35 mm glass-bottom confocal dishes and allowed to grow until a large number of single cells adhered. The culture medium was discarded, and the cells were washed three times with PBS for 5 min each time. The cells were fixed by soaking in 4% paraformaldehyde for 20 min, followed by washing three times with PBS for 5 min each time. Immunofluorescence blocking solution was added to the confocal dish and the cells were blocked at room temperature for 30 min. The blocking solution was discarded, and the cells were washed three times with TPBS for 5 min each time. Sufficient A2aR rabbit primary antibody (1:200 dilution) was added to the confocal dish, and the dish was placed in a humidified chamber and incubated overnight at 4 °C. The dish was incubated at room temperature for 40 min, and then washed three times with PBST containing 0.05% Tween-20 for 5 min each time. FITC-labeled goat anti-rabbit secondary antibody (1:1000 dilution) was added to the slide, and the dish was placed in a humidified chamber and incubated at room temperature in the dark for 2 h. The confocal dish was washed three times with PBST for 5 minutes each time, followed by three more washes with PBS for 5 minutes each time. A mounting solution containing DAPI as an anti-fluorescence quencher was added, and the dish was stained for 10 minutes. The confocal dish was then washed four times with PBST for 5 minutes each time. Images were then observed and acquired under a confocal microscope. Results are as follows: Figure 9 As shown, A2aR is a membrane-expressed receptor. Fluorescent FITC labeling shows green color, and DAPI staining of the nucleus shows blue color. It was determined that A2aR-His6-Gα16-HEK cells stably express A2aR, which can be used for high-throughput screening of drugs targeting A2aR. Example 2 Calcium flow method for detecting the dose-dependent agonistic effect of NECA on A2aR-His6-Gα16-HEK stable cell lines

[0064] The activation effect of NECA on clone #1 was detected using the calcium flow method, and the dose-response curve of the NECA agonist was obtained. The specific steps were as follows: A2aR-His6-Gα16-HEK cells were seeded in 384-well plates and cultured overnight. Then, freshly prepared A2aR agonist NECA solution (maximum concentration 3.33 μM, serially diluted 3-fold) was used to stimulate the cells. Real-time sampling and recording were used to detect changes in calcium signal induced by different concentrations of the agonist. The results are shown below. Figure 10 As shown, the changes in calcium fluorescence signal values ​​induced by different concentrations of NECA exhibited a dose-dependent relationship. OriginLab 2019 software was used for data fitting analysis, and the changes in calcium fluorescence signal values ​​showed an S-shaped growth curve. The EC50 of the NECA agonist effect was calculated. 50The fluorescence intensity value is 8.18 nM, which greatly improves the detection sensitivity. Meanwhile, the cell seeding plate is a 384-well plate, capable of simultaneously detecting 384 compounds with changing fluorescence signal values, significantly increasing the detection throughput. The detection reaction time is only 120 s, simplifying the operation and thus greatly improving detection efficiency. Example 3 Calcium flow assay for the dose-dependent antagonistic effect of antagonist ZM241385 on A2aR-His6-Gα16-HEK stable cell lines

[0065] The antagonistic effect of ZM241385 on clone #1 was detected using the calcium flow method, and the dose-response curve of the antagonist ZM241385 was obtained. The specific steps were as follows: A2aR-His6-Gα16-HEK cells were seeded in 384-well plates and cultured overnight. Freshly prepared antagonist ZM241385 (maximum concentration 10 μM, serially diluted 3-fold) was added to the 384-well plates. After incubation for 15 min, a single concentration of the agonist NECA (according to EC50) was added. 80 The value was determined (1 μM in this embodiment), and the changes in calcium signal caused by different concentrations of antagonist were detected by real-time sampling and recording. The results are as follows: Figure 11 As shown, the changes in calcium fluorescence signal values ​​induced by different concentrations of ZM241385 exhibit an inverted S-shaped curve. OriginLab 2019 software was used to fit and analyze the data, and the IC50 was calculated. 50 The value is 0.053 μM.

[0066] Different concentrations of NECA and ZM241385 induced calcium signaling changes in A2aR-His6-Gα16-HEK cells in a dose-dependent manner, indicating that a stable A2aR-His6-Gα16-HEK cell line expressing A2aR has been successfully constructed. Furthermore, calcium signaling methods can be used to detect A2aR-targeting compounds, including agonists and antagonists. Drug testing using existing tools showed that this cell line provides more reliable detection results for agonist and antagonist compounds.

Claims

1. A method for screening compounds targeting A2aR, characterized in that, Specifically, the steps include: culturing A2aR-His6-Gα16-HEK cell lines, adding the target compound to be tested; if intracellular calcium signaling is enhanced and statistically significant, the target compound is an agonist targeting A2aR; if intracellular activated calcium signaling is blocked and statistically significant, the target compound is an antagonist targeting A2aR. The method for preparing the A2aR-His6-Gα16-HEK cell line includes the following steps: (1) Preparation of human A2aR-His6-pcDNA5 / FRT receptor expression plasmid: Plasmid A2aR was ligated with the enzyme-digested expression vector pcDNA5 / FRT to construct recombinant expression plasmid A2aR-His6-pcDNA5 / FRT; (2) Preparation of Gα16 protein expression plasmid: The expression vector pIRES-puro3 after Gα16 enzyme digestion was ligated to construct the recombinant expression plasmid Gα16-pIRES-puro3; (3) Transfect the Gα16 protein expression plasmid into HEK-FlpIn cells and select cell lines that stably express Gα16 protein: Transfect the Gα16 protein expression plasmid into HEK-FlpIn cells and add puromycin to select positive cell lines; stimulate cells with A2aR agonist and detect changes in intracellular calcium signal by real-time fluorescence detection of FLIPR, and screen clones Gα16-HEK-FlpIn that meet the requirements of Gα16 protein expression signal to obtain Gα16-HEK-FlpIn cells; (4) Based on the established stable Gα16 protein cell line, the A2aR-His6-pcDNA5 / FRT and POG44 plasmid genes were co-transfected into Gα16-HEK-FlpIn cells, and cell lines stably expressing human A2aR were obtained through screening: human A2aR expression plasmid and FLP recombinase plasmid POG44 were co-transfected into Gα16-HEK-FlpIn cells, and puromycin and hygromycin were added to screen for positive cell lines; cells were stimulated with A2aR agonists, and changes in intracellular calcium signals were detected by FLIPR real-time fluorescence, and clones with human A2aR expression signals meeting the requirements were screened; after 5 to 10 passages, the pharmacological characteristics were identified using the A2aR agonist NECA, and the cell line A2aR-His6-Gα16-HEK stably expressing human A2aR and Gα16 proteins was obtained.

2. The method for screening compounds targeting A2aR according to claim 1, characterized in that, The A2aR agonist is NECA, and the A2aR antagonist is ZM241385.

3. The method for screening compounds targeting A2aR according to claim 1, characterized in that, In step (1), plasmid A2aR is used as a template, and hA2aR-His6-Nhe IF and A2aR-His6-Not IR are used as specific primers to perform PCR amplification to obtain the target gene, which is tagged with His at the C-terminus. The PCR amplification product is subjected to electrophoresis on an agarose gel to separate the target band and recover the target DNA. The sequence of the specific primer hA2aR-cHis6-Nhe IF is shown in SEQ ID NO.5, and the sequence of hA2aR-cHis6-Not IR is shown in SEQ ID NO.

6.

4. The method for screening compounds targeting A2aR according to claim 1, characterized in that, In step (1), the expression vector pcDNA5 / FRT is double-digested with Nhe I / Not I, and then ligated with the target DNA to construct the recombinant expression plasmid A2aR-His6-pcDNA5 / FRT.

5. The method for screening compounds targeting A2aR according to claim 1, characterized in that, In step (2), plasmid Gα16 is used as a template, and Gα16-Age IF and Gα16-Not IR are used as specific primers to perform PCR amplification reaction to obtain the target gene; The PCR amplification products were subjected to agarose gel electrophoresis to separate the target band and recover the target DNA. The sequence of the specific primer GNA15-Age IF is shown in SEQ ID NO.7, and the sequence of GNA15-Not IR is shown in SEQ ID NO.

8.

6. The method for screening compounds targeting A2aR according to claim 1, characterized in that, In step (2), the expression vector pIRES-puro3 is double-digested with Age I / Not I, and then ligated with the target DNA to construct the recombinant expression plasmid Gα16-pIRES-puro3.

7. The method for screening compounds targeting A2aR according to claim 1, characterized in that, The method for identifying the pharmacological characteristics described in step (4) is as follows: A2aR specific agonist or inhibitor is added to the constructed cell line, the change in fluorescence signal caused by calcium signal is measured, and the functional activity of the cell line is evaluated based on the result of the change in fluorescence signal.

8. The method for screening compounds targeting A2aR according to claim 1, characterized in that, The method for screening compounds targeting A2aR is as follows: The cell line described in the first aspect is seeded into a cell plate; after overnight culture, the culture medium is removed, and 40 μL of calcium-sensitive dye is added to each well, incubated at 37°C in the dark for 120 min; freshly prepared compound stock solution is added to new wells to form the compound plate; the cell plate is then loaded into a fluorescence imaging plate reader (FLIPR). TETRA (MD Company, USA) Fluorescence readings were performed according to the parameter settings: excitation wavelength 470-495 nm, emission wavelength 515-575 nm, LED intensity 60-80%, gain 6.5-8, and exposure time 0.045-0.05 s. First, the detection program read for 10 s as a baseline. Then, using an automated liquid handling system, the compound solution in the compound plate was transferred to the corresponding wells in the cell plate at 10 μL / well. Data was collected once per second for 120 s. OriginLab 2019 software was used for data fitting analysis to calculate EC50. 50 The value is used to quantify the agonistic effect of the compound on A2aR.

9. A stable A2aR-His6-Gα16-HEK cell line expressing human A2aR, characterized in that, The cell line was prepared by the method described in any one of claims 1 to 8; the cell line was mammalian HEK-FlpIn cells sequentially transfected with human Gα16 gene and A2aR gene. The A2aR gene sequence is shown in SEQ ID NO.2, and has a His tag at the C-terminus; the gene sequence of the Gα16 protein (GNA15) is shown in SEQ ID NO.4; The amino acid sequence of A2aR is shown in SEQ ID NO.1, with a 6-His tag at the C-terminus; the amino acid sequence of the Gα16 protein is shown in SEQ ID NO.

3.

10. A method for preparing the cell line according to claim 9, the method comprising: preparing a human A2aR expression plasmid and a Gα16 protein expression plasmid; sequentially transfecting the human Gα16 protein expression plasmid and the A2aR expression plasmid into HEK-FlpIn cells; and obtaining an A2aR-His6-Gα16-HEK cell line stably expressing human A2aR through resistance selection and functional testing.

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