Application of expression cassette, vector or recombinant cell in screening MR inhibitor or detecting MR inhibitory activity

Abstract

The invention discloses an application of an expression cassette, a vector or a recombinant cell in screening of an MR inhibitor or detection of MR inhibitory activity, and relates to the technical field of targeted inhibition drugs of a mineralocorticoid receptor. The screening method of the mineralocorticoid receptor inhibitor or the method for detecting the inhibitory activity of the mineralocorticoid receptor has the advantages of good specificity, strong specificity and high accuracy; the precision is high, and the repeatability and the fluorescence presentation degree of a reference product and a test product are good. Therefore, by adopting the method provided by the invention, the biological activity of the MRAs can be simply, accurately and efficiently evaluated, which is of great significance to screening, research and development, quality control and clinical application of the MRAs.

Description

Technical Field

[0001] The present invention relates to the technical field of mineralocorticoid receptor targeted inhibitory drugs, and in particular to an application of an expression cassette, a vector or a recombinant cell in screening MR inhibitors or in detecting MR inhibitory activity. Background Art

[0002] Aldosterone (Aldo) is a mineralocorticoid hormone secreted by the adrenal cortex that plays an important role in regulating the balance of sodium, potassium, and water in the body. It affects the function of the kidneys, heart, blood vessels, and other organs by binding to mineralocorticoid receptors (MR). Aldo not only plays an important role under physiological conditions, but also plays a key role in the occurrence and development of a variety of chronic heart and kidney diseases. It leads to the progression of the disease by promoting myocardial and vascular fibrosis, inducing oxidative stress, aggravating inflammatory responses, and triggering tubular and glomerular dysfunction. Given its key role in cardiorenal diseases, drugs that target the inhibition of Aldo or its receptors, such as mineralocorticoid receptor antagonists (such as spironolactone and eplerenone), have become an important strategy for the treatment of these chronic diseases.

[0003] Recently, the third-generation Aldo inhibitor Finerenone developed by Bayer is currently in multiple clinical trial stages and has achieved positive results in multiple important trials. Finerenone is marketed under the trade name Kerendia and is mainly used to treat patients with chronic kidney disease (CKD) with type 2 diabetes. As of 2024, finerenone has been recognized in several key areas, especially in the treatment of patients with type 2 diabetes and chronic kidney disease. In the two large clinical trials of FIDELIO-DKD and FIGARO-DKD, finerenone significantly reduced the risk of renal and cardiovascular events, and has therefore been recommended for use in such patients by multiple international treatment guidelines. In addition, Bayer is also expanding the scope of indications for finerenone, including the field of heart failure. In the FINEARTS-HF trial, finerenone showed a significant effect in reducing cardiovascular mortality and hospitalization in patients with heart failure, a result that laid the foundation for its further development and regulatory approval. Therefore, MR inhibitors (MRAs) have the potential to become an effective treatment for chronic kidney disease and heart disease. Developing MRAs with higher selectivity and specificity to reduce non-target effects and reduce the risk of side effects is an important direction in current drug development. These highly selective MRAs are expected to provide advantages in patient tolerance and therapeutic effects. There are many large small molecule compound libraries in China, but how to efficiently screen out MRAs is a challenge faced in drug development.

[0004] At present, the conventional in vitro screening system and characteristics of MR targeted inhibitory drugs can be summarized as follows:

[0005] High-throughput screening (HTS): High-throughput screening using compound libraries to discover potential MR inhibitors. This method usually relies on changes in MR binding to ligands, or changes in MR-mediated gene expression to detect inhibitory activity. HTS requires high-end automated equipment, expensive reagents, and a large number of compound libraries, all of which will significantly increase screening costs. Although HTS has a high degree of automation, technicians are still required to perform system maintenance, data analysis, and subsequent verification experiments, which also increases labor costs. At the same time, HTS needs to be carried out under strictly controlled conditions, but even so, the repeatability and consistency of the experiment may be affected by various factors (such as temperature, reagent stability, cell status, etc.), resulting in data variation. There may be differences between different batches of compounds or reagents, affecting the repeatability of the screening results.

[0006] Cell reporter gene assay: A cell line is constructed using a reporter gene (such as luciferase), and potential MR antagonists are screened by detecting changes in reporter gene expression. Although cell reporter gene assays are relatively common, the experimental operation is relatively complex, including steps such as cell culture, transfection, and drug treatment, which increases the cost and time of the experiment. Cell reporter gene assays usually require specific reagents, such as luciferase substrates or detection buffers. The quality and cost of these reagents will also affect the experimental results and economy. Different cell lines may respond differently to the same compound, so the screening results may be limited to the selected cell line and difficult to generalize to other cell types or tissues.

[0007] Competition binding experiments: Evaluate the ability of candidate drugs to compete for the binding of MR to its natural ligand (such as Aldo). Competition binding experiments involve multiple steps, including preparation of targets and ligands, optimization of reaction conditions, data analysis, etc., which may be time-consuming and complicated to operate, increasing the cost of the experiment. If radiolabeled ligands are used, special equipment and processing procedures are required, which increases the cost and safety requirements of the experiment. Competition binding experiments rely on the reaction to reach equilibrium to accurately reflect the affinity of the compound. If the experimental conditions fail to reach equilibrium, it may lead to incorrect estimates of binding affinity. Competition binding experiments involve multiple steps, including preparation of targets and ligands, optimization of reaction conditions, data analysis, etc., which may be time-consuming and complicated to operate, increasing the cost of the experiment. If radiolabeled ligands are used, special equipment and processing procedures are required, which increases the cost and safety requirements of the experiment.

[0008] However, to date, there are no relevant systems and patents for screening MR inhibitor systems, especially for systems that can stably and accurately detect changes in MR activation states in living cells, which greatly limits the development of MRAs.

[0009] In view of this, the present invention is proposed. Summary of the invention

[0010] The object of the present invention is to provide an expression cassette, a vector or a recombinant cell for use in screening MR inhibitors or in detecting MR inhibitory activity to solve the above technical problems.

[0011] The present invention is achieved in that:

[0012] In a first aspect, the present invention provides an expression cassette, comprising: a fluorescent protein encoding gene and at least one MR protein targeting sequence; and the fluorescent protein encoding gene is located downstream of the at least one MR protein targeting sequence;

[0013] The MR protein targeting sequence is shown in SEQ ID NO: 1, and the MR protein targeting sequence can bind to the MR protein.

[0014] In a second aspect, the present invention provides a vector comprising the above-mentioned expression cassette.

[0015] In a third aspect, the present invention provides a virus particle comprising the above-mentioned vector and capsid.

[0016] In a fourth aspect, the present invention provides a recombinant cell, which comprises the above-mentioned expression cassette, the above-mentioned vector or the above-mentioned virus particle.

[0017] In a fifth aspect, the present invention provides the use of an expression cassette, a vector, the above-mentioned virus particle or the above-mentioned recombinant cell in screening mineralocorticoid receptor inhibitors, in quality control of mineralocorticoid receptor inhibitors or in detecting mineralocorticoid receptor inhibitory activity.

[0018] In a sixth aspect, the present invention provides a method for screening a mineralocorticoid receptor inhibitor, a method for quality control of a mineralocorticoid receptor inhibitor, or a method for detecting the inhibitory activity of a mineralocorticoid receptor, which comprises the following steps: obtaining the above-mentioned recombinant cells, mixing the recombinant cells with aldosterone, a mineralocorticoid receptor inhibitor, a test substance and aldosterone, and a blank control, respectively, and recording them as a model group, a positive drug group, an experimental group, and a control group, respectively; after culturing, taking a culture sample to detect at least one of the following indicators: detecting the number of cells positive for fluorescent protein, detecting the expression level of fluorescent protein, and detecting the fluorescence intensity.

[0019] The present invention has the following beneficial effects:

[0020] The method for screening mineralocorticoid receptor inhibitors (MRAs) or detecting the inhibitory activity of mineralocorticoid receptors used in the present invention has good specificity, strong specificity, high accuracy, high precision, repeatability, and good fluorescence presentation of reference and test samples. Therefore, the method provided by the present invention can be used to simply, accurately, and efficiently evaluate the biological activity of MRAs, which is of great significance for the screening, research and development, quality control, and even clinical application of MRAs.

[0021] By screening MR protein targeting sequences, it was found that the MR protein transferred to the nucleus after Aldo activation can specifically bind to a MR protein targeting sequence in the nucleus to form a complex, which in turn regulates the expression of downstream genes.

[0022] After adding exogenous Aldo, Aldo can bind to MR in the cytoplasm, thereby activating the activity of MR to regulate transcription, allowing the MR protein to enter the cell nucleus. Since the MR protein targeting sequence can specifically bind to the MR protein, it activates and starts the transcription of the fluorescent protein. After the fluorescent protein is translated in the cytoplasm and excited by the excitation light, it can produce fluorescence of a specific wavelength. Therefore, the intensity of fluorescence reflects the activation of MR. If an MR inhibitor is added to inhibit the binding of Aldo to MR in the cytoplasm, the MR cannot be activated, and then cannot enter the cell nucleus, and cannot start the expression of the fluorescent protein. Even if it is excited, it cannot produce fluorescence of a specific wavelength, reflecting the efficiency of the drug's inhibitory effect on MR.

[0023] Compared with the prior art, the present invention has the following significant advantages and effects:

[0024] 1. High-throughput MR activation detection: The recombinant cells provided by the present invention have real-time fluorescence detection function, which can detect the MR activation state in living cells in real time in situ, greatly improving the experimental efficiency and facilitating high-throughput screening of MRAs.

[0025] 2. High specificity and high sensitivity: The recombinant cells provided by the present invention are highly specific and can effectively and specifically present the MR activation state in real time.

[0026] 3. Rapid detection: The recombinant cells of the present invention can be directly intervened in experiments and subjected to fluorescence observation, thus facilitating in-depth research on MRAs.

[0027] 4. Renewability and stability: The recombinant cells of the present invention can not only be recycled, but also be stably expressed for qualitative analysis. In addition, the recombinant cells have stable properties and structures and can be stored and used for a long time without affecting their efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 is the constructed 4×HRE element promoter sequence;

[0030] Figure 2 A detailed process diagram for the construction of the 4×HRE element promoter sequence;

[0031] Figure 3 This is the Pμro08-4×HRE-EGFP plasmid map;

[0032] Figure 4 The fluorescence microscopy image of NKR-52E cells transfected with plasmids to activate MR by Aldo;

[0033] Figure 5 Map of the modified pCDH-Pμro-CMV-R plasmid vector;

[0034] Figure 6 Lentivirus pCDH-Pμro-CMVR-4×HRE-EGFP plasmid vector map;

[0035] Figure 7 This is a statistical result diagram of the expression level of EGFP fluorescent protein in different groups (NKR-52E cells and cells inserted with 4×HRE-EGFP elements) detected by Western-blot;

[0036] Figure 8 The fluorescence intensity graph of cells in different groups (model group and control group) observed by light microscope;

[0037] Fig. 9 Western-blot detection of EGFP fluorescent protein expression levels in different groups (model group and control group);

[0038] Fig.10 The graph is a statistical result of the intensity of green fluorescence after MR was stimulated by different concentrations of Aldo;

[0039] Fig.11 Western-blot was used to verify the expression level of collagen-Ⅲ (Col-Ⅲ) protein in different groups;

[0040] Fig.12The number of EGFP-positive cells in different groups was observed under a fluorescence microscope, and the statistical results of the EGFP-positive area in each group were analyzed using Image J;

[0041] Fig.13 This is a statistical result diagram of the fluorescence intensity of each group of cells detected by flow cytometry;

[0042] Fig.14 This is a statistical result diagram of the fluorescence OD value of each group of cells detected by the Lμminescence module of the multifunctional microplate reader;

[0043] Fig.15 MR fluorescence microscopy images of Aldo activation in NRK52E cell lines transfected with 1×, 2×, and 4× HRE-EGFP plasmids, respectively. DETAILED DESCRIPTION

[0044] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0045] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0046] In the present invention, the term "Aldo" is synonymous with aldosterone. The term "MR inhibitor" is synonymous with "MRAs" and "mineralocorticoid receptor inhibitors".

[0047] In a first aspect, the present invention provides an expression cassette, comprising: a fluorescent protein encoding gene and at least one MR protein targeting sequence; and the fluorescent protein encoding gene is located downstream of the at least one MR protein targeting sequence;

[0048] The MR protein targeting sequence is shown in SEQ ID NO: 1, and the MR protein targeting sequence can bind to the MR protein.

[0049] The sequence shown in SEQ ID NO: 1 is derived from rats. In other embodiments, the sequence shown in SEQ ID NO: 1 may also be derived from animals of other species, including but not limited to mice, rabbits, non-human primates, etc. By screening the MR protein targeting sequence, it was found that the MR protein transferred to the nucleus after Aldo activation can specifically bind to a MR protein targeting sequence in the nucleus to form a complex, and the formed complex further regulates the expression of downstream genes.

[0050] After adding exogenous Aldo, Aldo can bind to MR in the cytoplasm, thereby activating MR regulatory factors and allowing MR regulatory factors to enter the cell nucleus. Since the MR protein targeting sequence can specifically bind to the MR protein, it activates and starts the transcription of the fluorescent protein. After the fluorescent protein is translated in the cytoplasm and excited by the excitation light, it can produce fluorescence of a specific wavelength. Therefore, the intensity of fluorescence reflects the activation of MR. If an MR inhibitor is added to inhibit the binding of Aldo to MR in the cytoplasm, MR cannot be activated, and then cannot enter the cell nucleus, and cannot start the expression of the fluorescent protein, even if it is excited, it cannot produce fluorescence of a specific wavelength.

[0051] Based on the above mechanism, the present invention provides an expression cassette for co-expressing a fluorescent protein encoding gene and at least one MR protein targeting sequence to achieve detection or monitoring of the MR activation state.

[0052] In a preferred embodiment of the present invention, the expression cassette further includes a regulatory sequence and a long terminal repeat sequence (LTR), the regulatory sequence includes: a promoter; and the promoter is located between the fluorescent protein encoding gene and the MR protein targeting sequence; the long terminal repeat sequence is located between two adjacent MR protein targeting sequences.

[0053] In a preferred embodiment of the present invention, the long terminal repeat sequence is selected from the LTR sequence of MMTV, and its nucleotide sequence is shown in SEQ ID NO: 3. HRE can be separated by intervals to facilitate cloning and sequencing and improve transcription efficiency.

[0054] SEQ ID NO:3:

[0055] ATGTGAGACAAGTGGTTTCCTGACTTGGTTTGGTATCA.

[0056] In a preferred embodiment of the present invention, the promoter is selected from a constitutive promoter, an inducible promoter, a universal promoter, a tissue-specific promoter, a cell type-specific promoter or a developmental stage-specific promoter. In a preferred embodiment of the present invention, the promoter is a type II promoter or a type III promoter.

[0057] In a preferred embodiment of the present invention, the promoter is selected from EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter or UBC promoter;

[0058] In a preferred embodiment of the present invention, the CMV promoter is a minimal CMV promoter, and its nucleotide sequence is shown in SEQ ID NO: 2. Specifically, it is: GGTAGGCGTGTACGGTGGGAGGTTTATATAAGCAGAGCT.

[0059] In a preferred embodiment of the present invention, the expression cassette includes at least two MR protein targeting sequences, and the multiple MR protein targeting sequences are arranged in series, and the long terminal repeat sequence is located at the 5' end of the first MR protein targeting sequence or between two adjacent MR protein targeting sequences;

[0060] In a preferred embodiment of the present invention, the expression cassette includes 2-10 MR protein targeting sequences;

[0061] In a preferred embodiment of the present invention, the expression cassette includes four MR protein targeting sequences, and the long terminal repeat sequence is located between the second MR protein targeting sequence and the third MR protein targeting sequence from the 5' end.

[0062] In a preferred embodiment of the present invention, the fluorescent protein is selected from one or more of BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP, eCFP, eYFP, eBFP, DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP and tagBFP;

[0063] In a preferred embodiment of the present invention, the expression cassette further comprises a terminator, which is selected from SV40polyA, bGHpolyA, BETAPolyA or 6XT.

[0064] In a second aspect, the present invention provides a vector comprising the above-mentioned expression cassette.

[0065] Based on the above expression cassette, the present invention further provides a vector for convenient cell transfection, thereby constructing a stable cell line for screening MR inhibitors with biological activity or detecting MR inhibitory activity.

[0066] In a preferred embodiment of the present invention, the expression cassette also includes a selectable marker gene expression cassette; the term "selectable marker" refers to a gene that will help select cells that actively express the nucleic acid sequence. Examples of suitable selectable markers include enzymes encoding resistance to antibiotics (i.e., antibiotic resistance genes), such as kanamycin, neomycin, puromycin, hygromycin, blasticidin or bleomycin.

[0067] In addition, the selectable marker gene also comprises an amplifiable selectable marker gene. Mentioning " amplifiable selectable marker gene " as used herein refers to the gene that allows to increase this gene under suitable growth conditions. The amplifiable selectable marker gene can respond to the shortage of inhibitor or essential metabolite by amplification to increase the expression product (that is, by the expression of the protein encoded by the amplifiable selectable marker gene). In one embodiment, the amplifiable selectable marker gene can be characterized as being able to complement with an auxotrophic host.

[0068] In a preferred embodiment of the present invention, the vector is a recombinant adeno-associated virus vector, a lentivirus, an adenovirus or a poxvirus.

[0069] The AAV nucleic acid sequence of the recombinant adeno-associated virus is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or a combination thereof.

[0070] In a third aspect, the present invention provides a virus particle, which comprises the above-mentioned vector and capsid. The capsid (CAP) is composed of VP1, VP2, and VP3 encoded by the cap gene as capsid proteins.

[0071] In a fourth aspect, the present invention provides a recombinant cell, which comprises the above-mentioned expression cassette, the above-mentioned vector or the above-mentioned virus particle.

[0072] Based on the above vector transfection cells, a stable cell line was constructed. Exogenous Aldo was given to bind to MR, thereby mediating the synthesis and secretion of fluorescent protein, generating fluorescence at a specific wavelength (the excitation wavelength of fluorescent protein). The intensity of the fluorescence reflects the activation of MR. The detection of the activation state of cell MR is achieved by observing the fluorescence. When screening MRAs, Aldo and compounds can be given at the same time, and the fluorescence brightness when Aldo and compound intervention is compared with that when Aldo alone is treated, and MR inhibitors with biological activity can be preliminarily screened.

[0073] In a preferred embodiment of the present invention, the recombinant cells include but are not limited to: bacteria, fungi or 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6 cells, 293 series cells, Per6 cells and CHO cells.

[0074] In a preferred embodiment of the present invention, the recombinant cell is a cell that highly expresses MR protein; the cell that highly expresses MR protein can promote the expression of MR protein after adding Aldo. After adding MR inhibitor, the activation of MR protein by Aldo is inhibited, so that MR protein cannot be activated normally, and then the transcription of fluorescent protein cannot be made, so that a large fluorescence difference is reflected at the cellular level.

[0075] In a preferred embodiment of the present invention, the recombinant cells are renal tubular epithelial cells.

[0076] In a fifth aspect, the present invention provides the use of an expression cassette, a vector, the above-mentioned virus particle or the above-mentioned recombinant cell in screening a mineralocorticoid receptor inhibitor, in quality control of a mineralocorticoid receptor inhibitor or in detecting a mineralocorticoid receptor inhibitory activity;

[0077] In a preferred embodiment of the present invention, the application of screening mineralocorticoid receptor inhibitors or detecting mineralocorticoid receptor inhibitory activity includes the following application methods:

[0078] Recombinant cells were obtained, and the recombinant cells were mixed with aldosterone, a mineralocorticoid receptor inhibitor, a test substance and aldosterone, and a blank control, respectively, and recorded as a model group, a positive drug group, an experimental group, and a control group, respectively; after culture, a culture sample was taken to detect at least one of the following indicators: the number of cells positive for fluorescent protein, the expression level of fluorescent protein, and the fluorescence intensity.

[0079] Preferably, the final concentration of aldosterone added is 100-400 μM. This final concentration helps to fully activate MR and reduce the false positive rate of screening MRAs. For example, the final concentration is 100 μM, 150 μM, 160 μM, 180 μM, 200 μM, 220 μM, 250 μM, 300 μM, 350 μM or 400 μM. Preferably, it is 200 μM.

[0080] The application method of quality control mineralocorticoid receptor inhibitors includes: obtaining recombinant cells, mixing the recombinant cells with aldosterone, mineralocorticoid receptor inhibitor standards, test substances (quality control substances for mineralocorticoid receptor inhibitors) and aldosterone, and blank controls, respectively, and recording them as model group, standard substance group, experimental group and control group; after cultivation, taking culture samples to detect at least one of the following indicators: the number of cells positive for fluorescent protein, the expression level of fluorescent protein and the fluorescence intensity. According to the difference between the number of cells positive for fluorescent protein, the expression level of fluorescent protein and the fluorescence intensity and the standard, it is evaluated whether the mineralocorticoid receptor inhibitor meets the standard.

[0081] In a preferred embodiment of the present invention, if at least one of the number of fluorescent protein-positive cells, the expression level of fluorescent protein and the fluorescence intensity of the experimental group is decreased compared with the model group, the measured substance is judged to be a candidate drug for a mineralocorticoid receptor inhibitor or has mineralocorticoid receptor inhibitory activity;

[0082] In a preferred embodiment of the present invention, the method for detecting the number of fluorescent protein-positive cells is selected from at least one of the following: detection under a fluorescence microscope and a flow cytometer;

[0083] The method for detecting the fluorescence intensity is selected from at least one of the following: detection under a fluorescence microscope, an ELISA reader and a flow cytometer; the method for detecting the expression level of the fluorescent protein is selected from at least one of the following: an ELISA reader, Western-blot, ELISA and ELISpot;

[0084] In a preferred embodiment of the present invention, the mineralocorticoid receptor inhibitor is phenonidone;

[0085] In a preferred embodiment of the present invention, the culture conditions are: 37°C ± 0.5°C, 5% ± 0.05% CO2 in a cell culture incubator for 48h ± 2h;

[0086] In a preferred embodiment of the present invention, when a fluorescence microscope is used to detect the number of fluorescent protein-positive cells, the fluorescent protein-positive area (RLM) of multiple fields of view is obtained using image analysis software, wherein the control group is RLΜ1, the model group is RLΜ2, and the experimental group is RLΜ3; the MR protein inhibition rate or MR protein activation rate of the experimental group is calculated according to the RLΜ signal value, and the calculation method is: MR inhibition rate (%) = [1-(RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)]×100%; MR activation rate (%) = (RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)×100%;

[0087] The higher the MR protein inhibition rate or the lower the MR protein activation rate, the higher the probability that the substance being tested is a candidate drug for a mineralocorticoid receptor inhibitor or has a higher mineralocorticoid receptor inhibitory activity;

[0088] In a preferred embodiment of the present invention, when the flow cytometer reads the fluorescence intensity, the control group is recorded as RLΜ1, the model group is recorded as RLΜ2, and the experimental group is recorded as RLΜ3; the MR activation rate or MR protein activation rate of the experimental group is calculated according to the RLΜ signal value, and the calculation method is: MR inhibition rate (%) = [1-(RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)]×100%; MR activation rate (%) = (RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)×100%;

[0089] In a preferred embodiment of the present invention, when the fluorescence intensity is read using a microplate reader, the fluorescence OD value is detected; the control group is recorded as RLΜ1, the model group is recorded as RLΜ2, and the experimental group is recorded as RLΜ3; the MR activation rate or MR protein activation rate of the experimental group is calculated according to the RLΜ signal value, and the calculation method is: MR inhibition rate (%) = [1-(RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)]×100%; MR activation rate (%) = (RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)×100%.

[0090] In a sixth aspect, the present invention provides a method for screening a mineralocorticoid receptor inhibitor, a method for quality control of a mineralocorticoid receptor inhibitor, or a method for detecting the inhibitory activity of a mineralocorticoid receptor, which comprises the following steps: obtaining the above-mentioned recombinant cells, mixing the recombinant cells with aldosterone, a mineralocorticoid receptor inhibitor, a test substance and aldosterone, and a blank control, respectively, and recording them as a model group, a positive drug group, an experimental group, and a control group, respectively; after culturing, taking a culture sample to detect at least one of the following indicators: detecting the number of cells positive for fluorescent protein, detecting the expression level of fluorescent protein, and detecting the fluorescence intensity.

[0091] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0092] Materials and Reagents:

[0093] Materials: pCDH-Pμro-CMVR-4×HRE-EGFP recombinant plasmid and lentivirus (General Biotechnology (Anhui) Co., Ltd.); sodium dodecyl sulfate (Sodiμm dodecyl sμlfate, SDS), tetramethylethylenediamine (Tetramethylethylenediamine, TEMED), glycine, agar powder, 1M Tris-HCl buffer (PH = 6.8), 1M Tris-HCl buffer (PH = 8.8), 1M Tris-HCl buffer (PH = 7.5), sterile enzyme-free water, Tris (hydro×ymethyl) aminomethane (TRIS), persulfamide (Ammoniμm Persμlfate, APS), bovine serum albumin, Tween-20 (Beijing Solebold Technology Co., Ltd.); methanol, anhydrous ethanol (Chengdu Jinshan Chemical Reagent Co., Ltd.); 30% acrylamide (Pumike Biotechnology Co., Ltd.); low-glucose DMEM medium (Gibco); skim milk powder (Inner Mongolia Yili Industrial Group Co., Ltd.); sodium chloride (Tianjin Komio Chemical Reagent Co., Ltd.); fetal bovine serum, penicillin-streptomycin solution, 0.25% trypsin (Viva Cell); yeast extract, tryptone (Beyotime); tissue cell fixative (4% PFA) (LEAGENE); prestained protein marker (Thermo Scientific); primary antibody diluent (Beyotime); ECL luminescence kit (Changzhou Tiandirenhe Biotechnology Co., Ltd.); Lipofectamine 2000 reagent (Invitrogen); RIPA strong lysis buffer (Suzhou Biyuntian Biotechnology Co., Ltd.); serum-free cell freezing solution (Suzhou Xinsaimei Biotechnology Co., Ltd.); disposable PE gloves (Nanchang Quanxing Medical Equipment Co., Ltd.); disposable multi-purpose protective gloves (Koroway); medical surgical masks (Nanchang Fuda Medical Equipment Co., Ltd.); glass-bottomed culture dishes, 1.5mL microcentrifuge tubes, bromophenol blue, 5mL pipette tips, 1000μL pipette tips, 200μL pipette tips, 10μL pipette tips (Biosharp); 6-well plates, 96-well plates, subculture bottles, cryotubes, culture dishes (NEST); β-actin (β-actin) (Pume Biotechnology Co., Ltd.); rabbit anti-type III collagen (Col-Ⅲ) (Abcam); EGFP tag protein (Zhengneng Biotechnology); puromycin (Pμromycin) (Solarbio); horseradish peroxidase (HRP) labeled goat anti-rabbit monoclonal antibody IgG secondary antibody (Pume Biotechnology Co., Ltd.) Reagents: staining solution CCK-8 (purchased from Dojindo), RPMI1640 culture medium powder (purchased from Gibco), streptomycin / penicillin double antibody (purchased from GIBCO), trypsin (purchased from Gibco), FBS (purchased from Gibco). .

[0094] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0095] Example 1

[0096] This example provides a method for constructing a Pμro08-4×HRE-EGFP plasmid vector.

[0097] 1. Using the rat MR binding gene sequence (i.e., MR protein targeting sequence, HRE) as a template, design a 1×HRE element sequence: 5-GGTACATTTTGTTCTAGAACAAAATGTACC-3' (SEQ ID NO: 1) and then synthesize a 4×HRE element (the MR protein targeting sequence is also a promoter sequence). HRE is the binding site of endogenous NR2C3. In order to separate the two 2XHREs and facilitate cloning and sequencing, MMTV-LTR is inserted in the middle, followed by a minimal CMV promoter (which provides background transcription levels, is the core part of the promoter, contains the RNA polymerase binding site and the transcription start site. If this part is lost, transcription will not be possible), forming a promoter that can induce transcription, refer to Figure 1 As shown. The HRE element can be further doubled according to the corresponding demand for the fluorescent protein EGFP (such as 5×HRE), and the number of HRE response elements is positively correlated with the EGFP fluorescence intensity. The specific process of constructing the 4×HRE element promoter sequence is referred to Figure 2 shown.

[0098] 2. Insert the 4×HRE promoter fragment into the vector pCMVPμro08, replace the original CMV promoter (cloning site BglII-HindIII), and obtain the Pμro08-4×HRE-EGFP plasmid. The plasmid vector map is referenced Figure 3 shown.

[0099] 3. Transfect NK2-52E cells with Pμro08-4×HRE-EGFP plasmid. Prepare 4μg / mL plasmid and 10μl Lip2000 in 1mL serum-free DMEM and let stand for 5min, then mix the plasmid and Lip2000 at room temperature for 15min, add them to 30%-50% confluency NKR-52E cells, transfect for 24h, and then change to normal culture medium for culture. At the same time, add 200μm Aldo intervention for 48h according to the experimental grouping, and observe the green fluorescence under a fluorescence microscope.

[0100] like Figure 4 As shown, under the stimulation of Aldo, the fluorescence intensity of cells in the plasmid transfection group increased, indicating that this detection system can detect the activation state of MR in real time.

[0101] Example 2

[0102] Based on this, the present embodiment transforms the lentiviral pCDH-Pμro vector: the CMV promoter in the vector is flipped (the purpose of flipping the CMV promoter is to provide a suitable restriction site for convenient connection to 4XHRE-EGFP), and the pCDH-Pμro-CMV-R plasmid (cloning site SpeI-BamHI) can be obtained; then the 4×HRE-EGFP fragment is cut out from the p4×HRE-Pμro01 plasmid (restriction site BglII-EcoRI), and the fragment is connected to the pCDH-Pμro-CMV-R vector (the vector is cut with BamHI-EcoRI) to replace the CMV promoter; using the characteristics of BglII and BamHI being homotypic enzymes (the restriction site disappears after connection), the target lentiviral vector plasmid pCDH-Pμro-CMVR-4×HRE-EGFP is obtained for virus coating.

[0103] Specifically, the construction method of pCDH-Pμro-CMVR-4×HRE-EGFP lentiviral vector is as follows:

[0104] A. Transformation of lentiviral pCDH-Pμro vector: Flip the CMV promoter in the vector to obtain pCDH-Pμro-CMV-R plasmid (cloning site SpeI-BamHI), see Figure 5 ;

[0105] B. Cut the 4×HRE-EGFP fragment from the p4×HRE-Pμro08 plasmid (restriction sites BglII-EcoRI);

[0106] C. Connect the fragment in step B to the pCDH-Pμro-CMV-R vector (cut with BamHI-EcoRI), replace the CMV promoter; take advantage of the fact that BglII and BamHI are homozygous enzymes (the restriction site disappears after connection), and obtain the target lentiviral vector plasmid, pCDH-Pμro-CMVR-4×HRE-EGFP, see Figure 6 As shown, it is used to package viruses.

[0107] Example 3

[0108] Construction of NRK-52E-4×HRE-EGFP stable cell line.

[0109] Through the pre-infection experiment, the cell seeding density, the optimal MOI (mμltiplicity of infection) and other conditions are determined, and the required amount of virus is calculated. The NRK-52E cells in good condition are inoculated in a 96-well plate, and the cells are cultured in a normal sugar DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and placed in a 37°C, 5% CO2, 95% air incubator. After 1 to 2 days, they are observed under an inverted microscope. When the cell fusion reaches 30% to 40%, they can be infected. Before infection, the cells are replaced with serum-free medium, and then the diluted virus solution is evenly added dropwise, and 5-8μg / mL of polybrene is added at the same time. Shake gently, culture in a 37°C incubator for 24h, remove the medium containing the virus solution, replace it with fresh complete medium, and continue to culture at 37°C. After 48-72h of culture, 2μg / mL of puromycin (Pμromycin) is added to select monoclonal cells, and the cell line is relatively stable after 10 days for screening.

[0110] NRK52E-4×HRE-EGFP stable transfected cells and normal NRK-52E cells with a confluence of 80%-90% and good growth status were taken, digested and counted, and 3*10 4 2mL cell suspension of each cell was placed in a cell culture incubator at 37°C and 5% CO2 for 1 day; Aldo was diluted to 100nM with DMEM complete cell culture medium, 4μL Aldo (final concentration was 200μm) was added to the cell wells, and 3 replicate wells were set up for each cell type; the plate was placed in a cell culture incubator at 37°C and 5% CO2 for 48h; the 6-well plate was taken out of the incubator and equilibrated to room temperature for about 5min; the culture medium in the culture wells was aspirated and rinsed with PBS 3 times; equal volumes of RIPA reagent were added respectively, and the cells were scraped and lysed on ice; Western-blot was subsequently used to verify the expression levels of EGFP fluorescent protein in different groups.

[0111] like Figure 7As shown in the figure, NRK52E-4×HRE-EGFP stably transfected cells expressed fluorescent protein under the intervention of Aldo, but normal NRK-52E cells did not express fluorescent protein. The experiment was repeated with cells of different generations and the results were consistent, indicating that the NRK52E-4×HRE-EGFP stably transfected cell line was successfully constructed.

[0112] Example 4

[0113] The response of stably transfected cell lines to MR activation was tested.

[0114] The NRK52E-4×HRE-EGFP stably transfected cells with a confluence of 80%-90% and good growth status were selected, digested, counted, and 3×10 4 2mL cell suspension of each cell was placed in a cell culture incubator at 37°C and 5% CO2 for 1 day; Aldo was diluted to 100nM with DMEM complete cell culture medium, 4μL Aldo (final concentration was 200μm) was added to the cell wells as the model group, and a control group was set up (the only difference from the experimental group was that Aldo was not added), with 3 replicate wells for each experimental group and control group; the plate was placed in a cell culture incubator at 37°C and 5% CO2 for 48h; the 6-well plate was taken out of the incubator and equilibrated to room temperature for about 20min; the culture medium in the culture wells was aspirated, 1mL DMEM complete culture medium at room temperature was added, and the fluorescence intensity of cells in different groups was observed under a fluorescence microscope; the culture medium in the culture wells was aspirated, and PBS was rinsed 3 times; equal volumes of RIPA reagent were added respectively, and the cells were scraped and lysed on ice; Western-blot was used to verify the expression level of EGFP fluorescent protein in different groups; Image J was used to analyze the grayscale values ​​of the bands, and the relative expression of EGFP in the corresponding cells was obtained by using the grayscale value of the EGFP protein / the grayscale value of the housekeeping gene β-actin. The GraphpadPrism scientific drawing tool was used to draw graphs with mean values ​​and standard deviation error bars.

[0115] like Figure 8 As shown, under the stimulation of Aldo, the cell fluorescence intensity of the stably transfected cell line is greatly enhanced, which shows that the stably transfected cell line can reflect the MR activation in real time, the effect is intuitive, and the fluorescence is stable. The cells in the above different groups were subjected to Western-blot to verify the expression of EGFP fluorescent protein.

[0116] like Fig. 9As shown, compared with the normal stably transfected cell line, the expression of EGFP fluorescent protein in the stably transfected cell line increased under the stimulation of Aldo, and this result was consistent with the intensity of green fluorescence, which showed that the stably transfected cell line could reflect the MR activation in real time, with intuitive effect and stable fluorescence, and could provide a rapid analysis method for MR activation effect.

[0117] Example 5

[0118] The sensitivity of stably transfected cell lines to MR activation stimulated by different concentrations of Aldo.

[0119] The NRK52E-4×HRE-EGFP stably transfected cells with a confluence of 80%-90% and good growth status were selected, digested, counted, and 3*10 4 2mL cell suspension of 10 cells was added, and the plate was placed in a cell culture incubator at 37°C and 5% CO2 for 1 day; Aldo was diluted to 100nM with DMEM complete cell culture medium, and 0, 0.5, 1, 2, 4, and 8μL Aldo (final concentration of 0, 25, 50, 100, 200, and 400μM) were added to the cell wells to set up the experimental group, and a control group was set up (the only difference from the experimental group was that Aldo was not added), with 3 replicate wells for each experimental group and control group; the plate was placed in a cell culture incubator at 37°C and 5% CO2 for 48h; the 6-well plate was taken out of the incubator and equilibrated to room temperature and placed for about 20min; the culture medium in the culture wells was aspirated, the cells were digested with phenol red-free trypsin for 1min, complete culture medium was added to terminate the digestion, and then the cells were collected by centrifugation, and the fluorescence intensity was read using a flow cytometer.

[0120] Fig.10 As shown in the figure, with the increase of Aldo concentration, the green fluorescence gradually increased and reached a peak at 100 μM. In addition, the concentration range with strong green fluorescence intensity excitation was smaller than the conventional Aldo 150 μM intervention concentration, indicating that the stable cell line has good sensitivity. In order to fully activate MR and reduce the false positive rate of screening MRAs, the subsequent screening experiments of the samples to be tested used an Aldo 200 μM intervention concentration.

[0121] Example 6

[0122] The positive drug Finerenone was tested to inhibit the activity of MR.

[0123] The NRK52E-4×HRE-EGFP stably transfected cells with a confluence of 80%-90% and good growth status were selected, digested, counted, and 3×10 42mL cell suspension of each cell was placed in a cell culture incubator at 37°C and 5% CO2 for 1 day; Aldo was added to the cell wells (final concentration was 200μM) as the model group, and a control group (the only difference from the model group was that Aldo was not added) and a positive drug group Finerenone (10μm) (the only difference from the model group was that Finerenone was added) were set up, with 3 replicate wells for each treatment group; the 6-well plate was placed in a cell culture incubator at 37°C and 5% CO2 for 48h; the 6-well plate was taken out of the incubator, the culture medium in the culture wells was aspirated, and the plates were rinsed with PBS 3 times; equal volumes of RIPA reagent were added to scrape and lyse the cells on ice; Western-blot was subsequently used to verify the expression levels of collagen-Ⅲ (Col-Ⅲ) protein in different groups; Image J was used to analyze the grayscale value of the bands, and the relative expression of Col-Ⅲ in the corresponding cells was obtained by using the grayscale value of Col-Ⅲ protein / grayscale value of the housekeeping gene β-actin, and the Prism scientific research drawing tool was used to draw graphs with mean and standard deviation error bars.

[0124] like Fig.11 As shown, after the stably transfected cell line of the present invention is stimulated by Aldo, MR activation leads to aggravation of the fibrosis process, while MR positive inhibitors can improve this course of disease, inhibit MR activation, and thus improve the course of fibrosis.

[0125] Example 7

[0126] Real-time fluorescence microscopy, flow cytometry and microplate reader were used to detect the inhibitory activity of unknown compounds on MR.

[0127] The NRK52E-4×HRE-EGFP stably transfected cells with a confluence of 80%-90% and good growth status were selected, digested, counted, and 3×10 42mL cell suspension of 10 cells was added and the plate was placed in a cell culture incubator at 37°C and 5% CO2 for 1 day; Aldo was added to the cell wells (final concentration was 200μM) to set as the model group, and a control group (the only difference from the model group was that Aldo was not added) and a positive drug group Finerenone (10μM) (the only difference from the model group was that Finerenone was also added) were set up, with 3 replicate wells for each model group and control group; the plate was placed in a cell culture incubator at 37°C and 5% CO2 for 48h; the 6-well plate was taken out of the incubator and equilibrated to room temperature and placed for about 20min; the culture medium in the culture wells was aspirated, and 1mL DMEM complete culture medium at room temperature was added, and the number of EGFP-positive cells in different groups was observed under a fluorescence microscope, and the number of EGFP-positive cells in different groups was observed under an image microscope. JAnalyze the EGFP-positive area of ​​8 random fields in each group, and record the EGFP-positive value as the EGFP-positive area RLΜ, where the control group was recorded as RLΜ1, the model group was recorded as RLΜ2, and the positive drug group was recorded as RLΜ3; calculate the MR activation rate of the experimental group according to the RLΜ signal value, and the calculation method is: MR activation rate (%) = (RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)×100%.

[0128] like Fig.12 As shown, the MR activation of the stable cell line of the present invention after being stimulated by Aldo leads to enhanced green fluorescence, while the MR positive inhibitor can reduce this fluorescence intensity. The statistical table of MR activation rate of each group of results is as follows:

[0129] Table 1

[0130]

[0131] Example 8

[0132] Flow cytometry was used to detect the inhibitory activity of unknown compounds on MR.

[0133] The NRK52E-4×HRE-EGFP stably transfected cells with a confluence of 80%-90% and good growth status were selected, digested, counted, and 3*10 42mL cell suspension of each cell was placed in a cell culture incubator at 37°C and 5% CO2 for 1 day; Aldo was added to the cell wells (final concentration was 200μm) as the model group, and a control group (the only difference from the model group was that Aldo was not added) and a positive drug Finerenone (10μm) (the only difference from the model group was that Finerenone was also added), with 3 replicate wells for each control group; the culture was continued in a cell culture incubator at 37°C and 5% CO2 for 48h; the culture medium in the culture well was aspirated, the cells were digested with phenol red-free trypsin for 1min, complete culture medium was added to terminate digestion, and then the cells were collected by blowing and centrifugation. The fluorescence intensity was then read using a flow cytometer; the control group was recorded as RLΜ1, the model group was recorded as RLΜ2, and the positive drug group was recorded as RLΜ3; the MR activation rate of the experimental group was calculated based on the RLΜ signal value, and the calculation method was: MR activation rate (%) = (RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)×100%.

[0134] like Fig.13 As shown, the MR activation of the stable cell line of the present invention after being stimulated by Aldo leads to enhanced green fluorescence, while the MR positive inhibitor can reduce the fluorescence intensity. The statistical table of MR activation rate of each group of results is shown in Table 2 below:

[0135] Table 2

[0136]

[0137]

[0138] Example 9

[0139] The activity of unknown compounds in inhibiting MR was detected by an enzyme-labeled instrument.

[0140] NRK52E-4×HRE-EGFP stably transfected cell lines with a confluence of 80%-90% and good growth status were taken respectively, the cells were digested and counted, and 1 mL of cell suspension containing 1500 cells was added to each well of a 96-well plate, and the plate was placed in a cell culture incubator at 37°C and 5% CO2 for culture for 1 day; Aldo was added to the cell wells (final concentration was 200 μm) to set as the model group, and a control group (the only difference from the model group was that Aldo was not added) and a positive drug group Finerenone (10 μm) (the only difference from the model group was that Finerenone was not added) were set up, with 3 replicate wells for each model group and control group; the plates were placed in a cell culture incubator at 37°C and 5% CO2 for 48 hours; the 6-well plates were taken out of the incubator and equilibrated to room temperature, and left for about 20 minutes; the culture medium in the culture wells was aspirated, and 1 mL of room temperature was added DMEM complete culture medium was used, and the Lμminescence module of a multifunctional microplate reader was used to detect the fluorescence OD value; the control group was recorded as RLΜ1, the model group was recorded as RLΜ2, and the positive drug group was recorded as RLΜ3; the MR activation rate of the experimental group was calculated according to the RLΜ signal value, and the calculation method was: MR activation rate (%) = (RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)×100%.

[0141] like Fig.14 As shown, the stable cell line of the present invention is activated by MR after being stimulated by Aldo, resulting in enhanced green fluorescence, while the MR positive inhibitor can reduce this fluorescence intensity, and compound M32 can also reduce the green fluorescence enhanced by MR activation by Aldo. The statistical table of MR activation rate of each group of results is shown in Table 3 below:

[0142] Table 3

[0143]

[0144] Embodiment 11

[0145] The response of NRK52E cell lines transfected with 1×, 2×, and 4× HRE-EGFP plasmids to Aldo was tested.

[0146] Pμro08-1×HRE-EGFP, Pμro08-2×HRE-EGFP, and Pμro08-4×HRE-EGFP plasmids were transfected into NK2-52E cells. 4μg / mL plasmids and 10μl Lip2000 were prepared in 1mL serum-free DMEM and allowed to stand for 5min, then the plasmids and Lip2000 were mixed and incubated at room temperature for 15min, added to 30%-50% confluency NKR-52E cells, and transfected for 24h before changing to normal culture medium. At the same time, 200μmAldo was added according to the experimental grouping for 48h, and the green fluorescence was observed under a fluorescence microscope.

[0147] like Fig.15 As shown, under the stimulation of Aldo, the fluorescence intensity of cells in the plasmid transfection group increased with the increase in the number of HREs contained in the transfected HRE-EGFP plasmid, suggesting that increasing HRE within a certain range in this detection system can enhance the responsiveness of cells to Aldo.

[0148] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An expression cassette, characterized in that It includes: A fluorescent protein encoding gene and at least one MR protein targeting sequence; and the fluorescent protein encoding gene is located downstream of the at least one MR protein targeting sequence; The MR protein targeting sequence is shown in SEQ ID NO: 1, and the MR protein targeting sequence can bind to the MR protein.

2. The expression cassette according to claim 1, characterized in that The expression cassette further comprises a regulatory sequence and a long terminal repeat sequence (LTR), wherein the regulatory sequence comprises: a promoter; and the promoter is located between the fluorescent protein encoding gene and the MR protein targeting sequence; the long terminal repeat sequence is located between two adjacent MR protein targeting sequences; Preferably, the long terminal repeat sequence is selected from the LTR sequence of MMTV, and its nucleotide sequence is shown in SEQ ID NO: 3; The promoter is selected from EF1A promoter, EFS promoter, CMV promoter, CAGGS promoter, SV40 promoter, COPIA promoter, ACT5C promoter, TRE promoter, CBh promoter, PGK promoter or UBC promoter; Preferably, the CMV promoter is a minimal CMV promoter, and its nucleotide sequence is shown in SEQ ID NO:

2.

3. The expression cassette according to claim 2, characterized in that The expression cassette comprises at least two MR protein targeting sequences, and the multiple MR protein targeting sequences are arranged in series, and the long terminal repeat sequence is located at the 5' end of the first MR protein targeting sequence or between two adjacent MR protein targeting sequences; Preferably, the expression cassette comprises 2-10 MR protein targeting sequences; Preferably, the expression cassette comprises four MR protein targeting sequences, and the long terminal repeat sequence is located between the second MR protein targeting sequence and the third MR protein targeting sequence from the 5' end.

4. The expression cassette according to claim 1, characterized in that The fluorescent protein is selected from one or more of BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP, eCFP, eYFP, eBFP, DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP and tagBFP; Preferably, the expression cassette further comprises a terminator.

5. A carrier, characterized in that It comprises the expression cassette according to any one of claims 1 to 4.

6. The carrier according to claim 5, characterized in that The expression cassette also includes a selectable marker gene expression cassette; Preferably, the vector is a recombinant adeno-associated virus vector, a lentivirus, an adenovirus or a poxvirus.

7. A virus particle, characterized in that It comprises the vector and capsid described in any one of claims 5-6.

8. A recombinant cell, characterized in that It comprises the expression cassette of any one of claims 1 to 4, the vector of any one of claims 5 to 6, or the virus particle of claim 7; Preferably, the recombinant cell is a cell that highly expresses MR protein; Preferably, the recombinant cell is a renal tubular epithelial cell.

9. Use of the expression cassette according to any one of claims 1 to 4, the vector according to any one of claims 5 to 6, the viral particle according to claim 7 or the recombinant cell according to claim 8 in screening mineralocorticoid receptor inhibitors, in quality control of mineralocorticoid receptor inhibitors or in detecting mineralocorticoid receptor inhibitory activity; Preferably, the application of screening mineralocorticoid receptor inhibitors or detecting mineralocorticoid receptor inhibitory activity includes the following application methods: Obtaining recombinant cells, mixing the recombinant cells with aldosterone, a mineralocorticoid receptor inhibitor, a test substance and aldosterone, and a blank control, respectively, and recording them as a model group, a positive drug group, an experimental group, and a control group, respectively; after culturing, taking a culture sample to detect at least one of the following indicators: detecting the number of cells positive for fluorescent protein, detecting the expression level of fluorescent protein, and detecting the fluorescence intensity; Preferably, aldosterone is added to a final concentration of 100-400 μM; Preferably, if at least one of the number of fluorescent protein-positive cells, the expression level of fluorescent protein and the fluorescence intensity of the experimental group is decreased compared with the model group, the measured substance is judged to be a candidate drug for a mineralocorticoid receptor inhibitor or has mineralocorticoid receptor inhibitory activity; Preferably, the method for detecting the number of fluorescent protein-positive cells is selected from at least one of the following: detection under a fluorescence microscope and a flow cytometer; The method for detecting the fluorescence intensity is selected from at least one of the following: detection under a fluorescence microscope, an ELISA reader and a flow cytometer; the method for detecting the expression level of the fluorescent protein is selected from at least one of the following: an ELISA reader, Western-blot, ELISA and ELISpot; Preferably, the mineralocorticoid receptor inhibitor is phenonidone; Preferably, the culture conditions are: 37°C ± 0.5°C, 5% ± 0.05% CO2 in a cell culture incubator for 48h ± 2h; Preferably, when a fluorescence microscope is used to detect the number of fluorescent protein-positive cells, the fluorescent protein-positive area (RLM) of multiple fields of view is obtained using image analysis software, wherein the control group is RLΜ1, the model group is RLΜ2, and the experimental group is RLΜ3; the MR protein inhibition rate or MR protein activation rate of the experimental group is calculated according to the RLΜ signal value, and the calculation method is: MR inhibition rate (%) = [1-(RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)]×100%; MR activation rate (%) = (RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)×100%; The higher the MR protein inhibition rate or the lower the MR protein activation rate, the higher the probability that the substance being tested is a candidate drug for a mineralocorticoid receptor inhibitor or has a higher mineralocorticoid receptor inhibitory activity; Preferably, when the flow cytometer reads the fluorescence intensity, the control group is recorded as RLΜ1, the model group is recorded as RLΜ2, and the experimental group is recorded as RLΜ3; the MR activation rate or MR protein activation rate of the experimental group is calculated according to the RLΜ signal value, and the calculation method is: MR inhibition rate (%) = [1-(RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)]×100%; MR activation rate (%) = (RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)×100%; Preferably, when the fluorescence intensity is read using a microplate reader, the fluorescence OD value is detected; the control group is recorded as RLΜ1, the model group is recorded as RLΜ2, and the experimental group is recorded as RLΜ3; the MR activation rate or MR protein activation rate of the experimental group is calculated according to the RLΜ signal value, and the calculation method is: MR inhibition rate (%) = [1-(RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)]×100%; MR activation rate (%) = (RLΜ3-RLΜ1) / (RLΜ2-RLΜ1)×100%.

10. A method for screening a mineralocorticoid receptor inhibitor, a method for quality control of a mineralocorticoid receptor inhibitor, or a method for detecting mineralocorticoid receptor inhibitory activity, characterized in that: The method comprises the following steps: obtaining the recombinant cells according to claim 8, mixing the recombinant cells with aldosterone, a mineralocorticoid receptor inhibitor, a test substance and aldosterone, and a blank control, respectively, and recording them as a model group, a positive drug group, an experimental group and a control group, respectively; after culturing, taking a culture sample to detect at least one of the following indicators: detecting the number of cells positive for fluorescent protein, detecting the expression level of fluorescent protein and detecting the fluorescence intensity.

Images