A cell for measuring the biological activity of GLP-2 analog drugs

Abstract

The present invention discloses a cell for determining the biological activity of a GLP-2 analog drug, and provides a method for determining the biological activity of a GLP-2 analog drug using the cell, the method comprising adding a GLP-2 analog to the cell provided by the present invention, incubating, and determining the biological activity of the GLP-2 analog drug according to the measured reporter gene signal value. The method is simple to operate, has a short test cycle, is sensitive to reaction, has small variability, strong specificity, and high accuracy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology. Specifically, the present invention relates to a cell for determining the biological activity of a GLP-2 analog drug. More specifically, the present invention relates to a D9 cell that stably expresses GLP-2R and its application in detecting the biological activity of a GLP-2 analog drug. Background Art

[0002] Glucagon-like peptide-2 (GLP-2) is a single-chain polypeptide hormone with a molecular weight of 3.9kDa secreted by intestinal L cells. It is a member of the glucagon-like peptide (GLP) family and plays an important role in the body. GLP-2 is a specific intestinal epithelial growth factor that promotes the repair of intestinal mucosal damage, inhibits cell apoptosis, improves intestinal absorption of nutrients, and enhances intestinal barrier function. It also plays an important role in maintaining the continuity and integrity of the intestinal mucosa.

[0003] GLP-2 analogs are analogs based on glucagon-like peptide-2 (GLP-2) with potential therapeutic applications. Among them, Teduglutide (also known as Teduglutide, Duglutide) is the first GLP-2 analog approved in China for the treatment of short bowel syndrome and is suitable for adults and children aged 1 year and above. Short bowel syndrome is a rare disease in which the effective absorption area of ​​the intestine of patients is significantly reduced, leading to nutrient absorption disorders and multiple metabolic disorders. Teduglutide can increase the height of the patient's residual intestinal villi and the depth of the crypts, promote the recovery of the digestive tract absorption function, and help patients reduce their dependence on parenteral nutrition. In addition, GLP-2 analogs also include Axiglutide, Apalutide, and Glepaglutide. Axiglutide can increase cell proliferation and reduce intestinal cell apoptosis, and has great application potential in the treatment of gastrointestinal mucositis, including chemotherapy-induced diarrhea; in a Phase II clinical trial for the treatment of patients with short bowel syndrome (SBS), apalutamide showed that it can significantly reduce patients' dependence on parenteral nutrition support. As a potential new therapy for rare gastrointestinal diseases, it has broad application prospects; Glepaglutide is mainly used in the research of inflammatory bowel disease (IBD) and Crohn's disease, and is also being developed as a potential treatment option for short bowel syndrome (SBS).

[0004] In the past, research on GLP-2 analogs has mainly focused on construction and medical use, and there are relatively few studies on the detection method of the biological activity of GLP-2 analog drugs. Based on this, the present invention constructs cells that stably express GLP-2 receptors and quantifies the biological activity of drugs by detecting changes in reporter gene expression after GLP-2 analog drug stimulation. The method has the advantages of simple operation, short test cycle, sensitive reaction, small variability, strong specificity and high accuracy, and can be used for product release inspection, which is of great significance for the quality control and clinical application of teduglutide. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a cell for determining the biological activity of a GLP-2 analog drug.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a method for constructing a cell line for determining the biological activity of a GLP-2 analog drug.

[0008] Further, the method comprises the following steps:

[0009] 1) The expression vector of GLP-2R gene and the expression vector of CRE-miniP-reporter gene are transfected into effector cells;

[0010] 2) Screening to obtain a monoclonal cell line expressing GLP-2R and CRE-miniP-reporter gene; the CRE-miniP is a complete genome formed by connecting the CRE gene and the miniP gene, the sequence of the CRE gene is shown in SEQ ID NO.7, the sequence of the miniP gene is shown in SEQ ID NO.8, and the sequence of the CRE-miniP gene is shown in SEQ ID NO.9.

[0011] Those skilled in the art can select different methods to synthesize the CRE-miniP gene according to the sequences of the CRE gene and the miniP gene. In a specific embodiment of the present invention, the CRE gene and the miniP gene are handed over to Suzhou Dongling Biotechnology Co., Ltd. for connection using gene total synthesis technology.

[0012] Furthermore, the reporter gene described in step 1) includes GUS gene, NPTII gene, CAT gene, NOS gene, LUC gene, and GFP gene.

[0013] Furthermore, the reporter gene is a LUC gene.

[0014] The term "reporter gene" is a gene that encodes a protein or enzyme that is easily detected in the background of endogenous proteins. The reporter gene is linked to the target gene or regulatory sequence, and directly or indirectly detects the signal of the reporter gene encoding product, such as reporter protein, mRNA, enzyme, etc., to directly reflect the transcriptional activity or expression level of the gene in the cell. The characteristics of general reporter genes are non-toxic, non-immunogenic, easy to detect, and have a certain specificity.

[0015] In a specific embodiment of the present invention, the reporter gene used is the LUC gene, i.e., the luciferase reporter gene. Luciferase comes from organisms that can emit light in nature. It is a general term for a class of enzymes that can catalyze the oxidation of luciferin or fatty aldehydes to produce light. Depending on the source organism, luciferase can be divided into firefly luciferase (FL) and bacterial luciferase (BL). At present, the luciferase gene from the North American firefly is the most widely used. This gene can encode a luciferase protein of 550 amino acids. The FL gene comes from fireflies and is found in Mg. 2+ , ATP, O2, catalyzes the oxidative decarboxylation of D-luciferin to produce activated oxyluciferin, and emits photons to produce 550-580nm fluorescence. Luciferase reporter gene detection is an important tool in the field of modern molecular biology research to analyze the interaction between potential cis elements (such as promoters, enhancers and silencers, etc.) and trans-acting factors in the flanking regions of structural genes. The luciferase reporter gene system is a reporter system that uses luciferin as a substrate to detect the activity of firefly luciferase. Luciferase can catalyze the oxidation of luciferin to oxyluciferin. During the oxidation process of luciferin, bioluminescence will be emitted, which is then measured by a chemiluminometer or liquid scintillation meter.

[0016] Furthermore, the expression vector described in step 1) includes a plasmid vector, a phage vector, and a viral vector.

[0017] Furthermore, the expression vector is a plasmid vector.

[0018] The term "plasmid" is a naked double-stranded circular DNA molecule independent of the DNA in the cell nucleus, which has the ability to replicate autonomously and can be stably inherited in the cell. Plasmids are mainly found in bacteria, but can also be found in eukaryotes. Specific DNA sequences are introduced into the plasmid, which encode a certain or certain specific proteins, thereby giving the host cell new traits or functions. For example, the plasmid can carry a target gene for gene therapy, or a coding sequence for protein production, or give the cell a new function through transfection. In the present invention, those skilled in the art can choose different plasmids to introduce the GLP-2R gene and the CRE-miniP-reporter gene into the cell.

[0019] In a specific embodiment of the present invention, the plasmid containing the GLP-2R gene is pLU.cmv.Zeo.GLP2R-Flag; the plasmid containing the CRE-miniP-luciferase reporter gene is pLU.CRE.miniP.Luc.Puro.

[0020] The term "transfection" refers to the process of introducing foreign DNA (such as plasmids) into eukaryotic cells, allowing the foreign genes to be expressed in the cells. Plasmid transfection is an important technology in genetic engineering and is widely used in gene function research, protein production and other fields.

[0021] Furthermore, the effector cells in step 1) are HEK293.

[0022] There are many cell types commonly used for transfection, including but not limited to the following:

[0023] 1) Primary cells and their derivatives. These cells are directly isolated from biological tissues without long-term in vitro culture and maintain high physiological activity and differentiation potential.

[0024] 2) Tumor cell lines: such as human breast cancer cells (MCF-7), human lung cancer cells (A549), human cervical cancer cells (HeLa), etc. These cell lines have high division rate and stability and are often used in gene function research and drug screening.

[0025] 3) Cell lines of specific tissue origin:

[0026] Human embryonic kidney cells (HEK-293): have high transfection efficiency and stability, and are often used in gene cloning and vaccine production.

[0027] Human embryonic lung cells (HEL): used as recipient cells in some experiments.

[0028] Human umbilical vein endothelial cells (HUVEC): used to study vascular biology and cardiovascular diseases, etc.

[0029] 4) Animal cell lines:

[0030] Baby Hamster Kidney Cells (BHK-21): Widely used in virology research and other biomedical research.

[0031] Mouse embryonic fibroblasts (NIH3T3): have low immunogenicity and high division rate, and are often used for gene knockout and drug screening.

[0032] Other special cell types:

[0033] Neuronal cells: used in neurobiology research.

[0034] Rat hepatocytes: used for drug metabolism and toxicity studies.

[0035] Rat glioma cells (C6): highly differentiated and stable, often used in drug screening and tumor research.

[0036] Those skilled in the art can select different effector cells according to the prior art. In a specific embodiment of the present invention, the effector cell is HEK293.

[0037] Furthermore, the transfection method described in step 1) includes physical mediation, chemical mediation, and biological mediation.

[0038] Preferably, the transfection method is biologically mediated.

[0039] More preferably, the transfection method is lentiviral infection.

[0040] The methods of cell transfection include physical mediation, chemical mediation, and biological mediation. The physical mediation includes electroporation, microinjection, and gene gun; the chemical mediation includes calcium phosphate coprecipitation, cationic polymer, and cationic liposome; the biological mediation is mainly viral mediation, such as lentivirus, adenovirus, etc. In a specific embodiment of the present invention, the transfection method is lentivirus transfection.

[0041] Furthermore, the screening described in step 2) includes the following steps: pressure screening, single clone isolation and culture, and flow cytometry analysis.

[0042] The term "pressure screening" refers to the screening and retention of successfully transfected cells through specific selection pressure. Generally, pressure screening methods include antibiotic screening and gene expression product screening.

[0043] In a specific embodiment of the present invention, the pressure screening is performed by adding antibiotics.

[0044] In cell screening, antibiotics are often used as a resistance screening tool to distinguish successfully transfected cells from untransfected cells. These antibiotics inhibit the growth of untransfected cells or kill them, thereby allowing cells that are successfully transfected and express antibiotic resistance genes to survive.

[0045] Furthermore, the antibiotics include but are not limited to: a penicillin-streptomycin mixture, G-418 (Geneticin), Blasticidin, Hygromycin B, Puromycin, 6-thioguanine, Ampicillin, and Zeocin.

[0046] Preferably, the antibiotics are bleomycin and puromycin.

[0047] More preferably, the concentrations of bleomycin and puromycin are 200 μg·mL -1 , 3μg·mL -1 .

[0048] A second aspect of the present invention provides a cell for determining the biological activity of a GLP-2 analog drug.

[0049] Further, the cell is named GLP2R-GRE-Luc-293, and is deposited at the General Microbiology Center of China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNO.46046, the classification name is Human HEK293 cells, and the deposit date is September 3, 2024.

[0050] Furthermore, the cell is constructed by the construction method described in the first aspect of the present invention.

[0051] In a specific embodiment of the present invention, the D9 cells have the highest positive rate of expressing GLP2R, reaching 97.4%, and at the same time have the strongest response to the GLP-2 analog drug teduglutide. Using this cell to determine the biological activity of GLP-2 analog drugs is more effective. Therefore, this cell is preserved and named GLP2R-GRE-Luc-293, and was deposited in the General Microbiology Center of China National Committee for the Collection of Microbiological Cultures on September 3, 2024.

[0052] The third aspect of the present invention provides a method for rapidly determining the biological activity of a GLP-2 analog drug.

[0053] Furthermore, the method comprises the following steps: adding a GLP-2 analog to the cells described in the second aspect of the present invention, incubating, and determining the biological activity of the GLP-2 analog drug based on the measured reporter gene signal value.

[0054] In the present invention, a four-parameter curve is fitted after measuring the reporter gene signal value to determine the biological activity of the GLP-2 analog drug. The term four-parameter curve fitting is a mathematical method used to describe a set of data by fitting a curve and obtain a functional equation that can describe the data relationship in order to predict future data. A four-parameter curve is usually defined by four parameters, which have practical significance in a biological context. For example, in detecting the biological activity of a drug, these four parameters may represent: the lower limit of absorbance (D), the upper limit of absorbance (A), that is, the minimum and maximum values ​​of absorbance; the half-response concentration (C), that is, the value when the drug concentration reaches half the maximum response; and the slope of the curve (B). Through these parameters, experimental data can be understood and analyzed more accurately, thereby providing deeper insights into biological research.

[0055] Furthermore, the effective concentration range of the GLP-2 analog drug is 10×10 -7 -10×10 -5 μg·mL -1 .

[0056] Furthermore, the GLP-2 analogs include teduglutide, whose amino acid sequence is H-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH.

[0057] Further, the GLP-2 analogue includes aciglutide, whose amino acid sequence is His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Ar g-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys.

[0058] Further, the GLP-2 analogs include apalutamide, whose amino acid sequence is His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Nle-{D-Phe}-Thr-Ile-Leu-Asp-Leu-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-NH2.

[0059] Furthermore, the GLP-2 analogues also include Glepaglutide, whose amino acid sequence is His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys-NH2.

[0060] The term "GLP-2 analogues" refers to a class of compounds that are similar in structure to glucagon-like peptide-2 (GLP-2) but may have undergone certain modifications to improve their biological activity or stability. GLP-2 analogues generally retain or enhance certain key biological functions of GLP-2, such as promoting intestinal absorption and improving intestinal health. Due to the important role of GLP-2 in intestinal health, GLP-2 analogues have broad application prospects in the treatment of diseases related to intestinal function.

[0061] Teduglutide described in the present invention is a glucagon-like peptide-2 (GLP-2) analog, which was approved by the China National Medical Products Administration (NMPA) on February 23, 2024 for the treatment of short bowel syndrome (SBS) in adults and children aged 1 year and above. It is the first GLP-2 analog in China to treat short bowel syndrome, which helps increase intestinal absorption area, reduce patients' dependence on parenteral nutrition, and improve their quality of life. Its molecular formula is C 170 H 259 N 47 O 56 S1, with an average molecular weight (MW) of 3889.2728 g / mol, contains 33 amino acids in its sequence, as shown in SEQ ID NO.1, wherein X is H and Z is OH.

[0062] The elsiglutide of the present invention is a GLP-2 analog and an orally active and selective GLP-2 receptor agonist, which can increase cell proliferation and reduce intestinal cell apoptosis. Its sequence contains 39 amino acids, as shown in SEQ ID NO.2, and it can improve the diarrhea symptoms of rats induced by Lapatinib (HY-50898). Its molecular formula is C 197 H 323 N 53 O 58 , the average molecular weight (MW) is 4362.0519 g / mol.

[0063] Apraglutide is a synthetic 33-amino acid peptide and a long-acting GLP-2 analogue that can enhance the adaptability and linear intestinal growth of newborn piglets with short bowel syndrome after total ileal resection. 172 H 263 N 43 O 52 The average molecular weight (MW) is 3765.2371 g / mol, and the sequence is shown in SEQ ID NO.3, wherein X is Nle, Z is NH2, and the F at the 11th position is D-Phe.

[0064] The Glepaglutide of the present invention is a long-acting GLP-2 analog and an effective GLP-2R agonist. It can reduce fecal excretion and increase intestinal absorption, and has the potential to reduce small intestinal inflammation. Glepaglutide is mainly used in the study of inflammatory bowel disease (IBD) and Crohn's disease, and is also developed as a potential treatment option for short bowel syndrome (SBS). Its sequence contains 39 amino acids, as shown in SEQ ID NO.4, where Z is NH2 and its molecular formula is C 198 H 326 N 54 O 56 , the average molecular weight (MW) is 4359.0946 g / mol.

[0065] In a specific embodiment of the present invention, the initial concentration of the GLP-2 analog teduglutide drug is 10 μg mL -1 , and then tested after 5-, 6-, and 7-fold serial dilutions.

[0066] Preferably, the optimal dilution multiple of teduglutide is 7 times.

[0067] Furthermore, the cell seeding density is 8×10 5 -1×10 6 cells·mL -1 .

[0068] Furthermore, the incubation time is 4-6 hours.

[0069] Preferably, the incubation time is 5 h.

[0070] The fourth aspect of the present invention provides a product for rapidly determining the biological activity of a GLP-2 analog drug.

[0071] Furthermore, the product comprises the cells described in the second aspect of the present invention.

[0072] Further, the product type includes a kit.

[0073] In the present invention, the components of the test kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the test kit usually include at least a vial, test tube, flask, PET bottle, syringe or other container, in which a component can be placed, and preferably, can be appropriately divided. When there is more than one component in the test kit, the test kit will usually also include a second, third or other additional container, in which additional components are placed separately. However, components of different combinations can be contained in a vial. The test kit of the present invention will usually also include a container for accommodating reactants, sealed for commercial sale. This container can include a plastic container of injection molding or blow molding, in which the required vial can be retained.

[0074] A fifth aspect of the present invention provides a system for evaluating the pharmaceutical activity of GLP-2 analogs.

[0075] Furthermore, the system comprises the following components: the cells described in the second aspect of the present invention, a GLP-2 analog drug sample and a reference product.

[0076] In the present invention, the term "system" refers to the drug quality control system, which refers to the control of various factors in the drug production process through scientific management methods to ensure that the drug quality meets the predetermined quality standards and requirements.

[0077] A sixth aspect of the present invention provides application of any of the following aspects.

[0078] Furthermore, the application includes:

[0079] 1) Use of the cell according to the second aspect of the present invention in the preparation of a product for detecting the biological activity of a GLP-2 analog drug;

[0080] 2) Application of the system described in the fifth aspect of the present invention in the quality control of GLP-2 analog drugs.

[0081] The term "sample" used herein refers to a sample to be identified and tested. In the present invention, the GLP-2 analog drug sample includes a drug sample produced in industrial production and to be tested for its biological activity.

[0082] In the present invention, unless otherwise specified, the terms "comprise", "include" and "contain" are open expressions, meaning that in addition to the listed elements, components and steps, other unspecified elements, components and steps may also be included.

[0083] Unless otherwise defined, all technical and scientific terms in the context of the present invention have the same meanings as understood by one of ordinary skill in the art.

[0084] The advantages and beneficial effects of the present invention are as follows: The present invention provides a cell for rapidly determining the biological activity of a GLP-2 analog drug, and provides a method for using the cell line to detect the biological activity of a GLP-2 analog drug. The method has the advantages of simple operation, short test cycle, sensitive response, small variability, strong specificity and high accuracy, and can be used for product release inspection, which is of great significance for the quality control and clinical application of teduglutide. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0086] Figure 1 Shown are graphs confirming that CRE-miniP-luc-GLP2R-HEK293 cells express GLP2R;

[0087] Figure 2 A graph showing the response of different CRE-miniP-luc-GLP2R-HEK293 monoclonal cell lines to tedugutide;

[0088] Figure 3 A graph showing the optimization results for different dilution factors;

[0089] Figure 4 Graph showing the results of optimization of different cell densities;

[0090] Figure 5 Graph showing the results of optimization of different incubation times;

[0091] Figure 6 A graph showing the response of D9 cell line to different GLP-2 analogs. DETAILED DESCRIPTION

[0092] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0093] The idea of ​​the present invention is to use the cAMP response element (CRE) to construct a luciferase reporter gene vector and a GLP-2 receptor expression vector, transfect the reporter gene and the GLP-2 receptor expression gene into HEK293 cells through a lentiviral packaging system, and obtain 5 monoclonal cell lines with stable expression by pressure screening, monoclonal isolation culture and flow analysis. After functional verification, all 5 cell lines responded to teduglutide to varying degrees. Among them, monoclonal D9 had the strongest response to teduglutide. After that, monoclonal cell D9 was selected to further optimize the drug dilution multiple, cell density and incubation time of the method, and a reporter gene method capable of quantitatively detecting the biological activity of teduglutide was established. The principle of this method is that after the GLP-2 receptor of the CRE-miniP-luc-GLP2R-HEK293 monoclonal cell line binds to teduglutide, it can activate the expression of the reporter gene luciferase through a series of signal transduction. The activity of teduglutide is quantified by detecting the changes in luciferase expression after teduglutide stimulation.

[0094] Example 1 Cell construction and positive cell screening

[0095] 1. Experimental methods

[0096] The GLP-2R gene was constructed into the pLU.cmv.Zeo vector through the BamHI and SalI cloning sites, and the CRE gene and the miniP gene were handed over to Suzhou Dongling Biotechnology Co., Ltd. for connection. Then, the CRE-miniP gene was constructed into the pLU.CMV.Luc.PGK.PURO vector through the ClaI and XbaⅠ cloning sites. The plasmid was then amplified and purified. The lentivirus was packaged with a virus packaging kit and the pLU-cmv-GLP-2R-Zeo plasmid and infected with HEK293T cells. Then, 200 μg·mL -1 zeocin (bleomycin) and 3 μg mL -1The cells were screened with puromycin to obtain stable transfected cells, and 5 monoclonal cell lines were obtained by limiting dilution. The cell line was named CRE-miniP-luc-GLP2RHEK293 and preserved by this laboratory. Finally, the expression of GLP-2R on the cell surface was analyzed by flow cytometry to determine the positive monoclonal cells.

[0097] To facilitate flow cytometry detection, a Flag tag was added to the C-terminus of the GLP-2R gene. The sequences of the GLP-2R gene and the Flag tag are shown in SEQ ID NOs. 5 and 6, respectively. The sequences of the CRE gene, miniP gene and CRE-miniP gene are shown in SEQ ID NOs. 7, 8 and 9, respectively. The pLU.cmv.Zeo vector and pLU.CMV.Luc.PGK.PURO vector used in this example were purchased from Suzhou Dongling Biotechnology.

[0098] 2. Experimental results

[0099] The flow cytometry analysis results are as follows Figure 1 As shown, the highest positive rate of GLP2R expression among the five monoclonal cell lines was D9, with a positive rate of 97.4%. The positive rates of the other four cell lines were: D5: 93.9%; E11: 92.4%; F2: 93.2%; G11: 93.9%.

[0100] The five cell lines were cultured at 3.0×10 4 The cells were plated at a density of 100 cells / well and the biological activity of the same sample of teduglutide was determined. The results were as follows: Figure 2 As shown in the figure, all five cell lines had a dose-effect response to teduglutide stimulation, and the level of luciferase expressed by the cells increased with the increase of drug concentration. The ratio of the upper and lower asymptotes of the S curve fitted by the four-parameter model of the five cell lines was greater than 20. Among them, monoclonal clone D9 had the strongest response to teduglutide, with a ratio of 35 between the upper and lower asymptotes of the curve, which was consistent with the highest expression level of GLP-2R in monoclonal clone D9 detected by flow cytometry. Therefore, monoclonal clone D9 cells were selected for subsequent experiments and named GLP2R-GRE-Luc-293, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCCNO.46046, a classification name of Human HEK293 cells, and a deposit date of September 3, 2024.

[0101] Example 2 Method Parameter Optimization

[0102] 1. Optimization of the best dilution ratio

[0103] Experimental method: Dilute teduglutide to a starting concentration of 10 μg mL -1 Then, 5-, 6- and 7-fold gradient dilutions were performed, with a total of 10 concentration points, to verify the response of D9 cell line to teduglutide.

[0104] Experimental results: Figure 3 As shown in the figure, when the dilution factor is 5 or 6 times, there is only one point on the platform under the curve, and no obvious lower platform can be formed. When the dilution factor is 7 times, there are 2 points on the upper and lower platforms of the curve, and 3 points in the linear part in the middle, and the points of the curve are evenly distributed. Therefore, the optimal dilution factor of the determination method is 7 times.

[0105] 2. Optimization of cell density

[0106] Experimental method: After determining the optimal dilution factor, the cell density was optimized to 10 μg mL -1 The teduglutide standard was used as the starting concentration for 7-fold serial dilutions, and the cell density was adjusted to 4×10 5 , 6×10 5 , 8×10 5 , 1×10 6 cells·mL -1 , 50 μl per well was added to a 96-well plate.

[0107] Experimental results: The results are as follows Figure 3 As shown in the figure, cells at four different densities can all obtain typical S-shaped proliferation curves. As the cell density increases, the cell response value also increases accordingly. 5 cells·mL -1 and 1×10 6 cells·mL -1 There was no significant difference in the highest cell response value when the cells were cultured. Therefore, the optimal cell density was determined to be 8×10 5 ~1×10 6 cells·mL -1 .

[0108] 3. Optimization of action time

[0109] Experimental method: The experiment was carried out with the optimal starting concentration, dilution gradient and cell density of teduglutide, and the cell response at action times of 4, 5 and 6 hours was detected.

[0110] Experimental results: The results are as follows Figure 5As shown in the figure, all three action times can produce S-shaped proliferation curves, and as the action time increases, the relative light unit (RLU) value is higher, and there is no significant difference in RLU between 5h and 6h. Considering the RLU value and time cost, the optimal action time of the method is determined to be 5h.

[0111] Example 3 Method Specificity Study

[0112] 1. Experimental methods

[0113] Four GLP-2 analogs, Teduglutide, Elsiglutide, Apraglutide and Glepaglutide, were diluted to the same starting molar concentration of 2.665uM, and 7-fold gradient dilutions were performed for a total of 10 concentration points to verify the response of D9 cell line to the four GLP-2 analogs.

[0114] 2. Experimental results

[0115] The results are as follows Figure 6 As shown in Figure 2, all four GLP-2 analogs can induce D9 cells to express luciferase and form a sigmoidal dose-response curve. EC values ​​of the dose-response curves of Teduglutide, Elsiglutide, Apraglutide and Glepaglutide 50 The values ​​were 1.078, 2.118, 1.428, and 0.592 uM, respectively, indicating that this method can distinguish the structural differences of the four GLP-2 analogs.

[0116] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A cell for determining the biological activity of a GLP-2 analog drug, characterized in that: The cell is named GLP2R-GRE-Luc-293, and is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNO.46046, the classification name is Human HEK293 cells, and the deposit date is September 3, 2024.

2. A method for rapidly determining the biological activity of a GLP-2 analog drug, characterized in that: The method comprises the following steps: adding a GLP-2 analog to the cells of claim 1, incubating the cells, and determining the biological activity of the GLP-2 analog drug based on the measured reporter gene signal value.

3. The method according to claim 2, characterized in that The effective concentration range of the GLP-2 analog drug is 10×10 -7 -10×10 -5 μg·mL -1 .

4. The method according to claim 2, characterized in that: The GLP-2 analogs include tedugutide, whose amino acid sequence is H-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH.

5. The method according to claim 2, characterized in that: The GLP-2 analogs include aciglutide, whose amino acid sequence is His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys.

6. The method according to claim 2, characterized in that The GLP-2 analogs include apalutamide, whose amino acid sequence is His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Nle-{D-Phe}-Thr-Ile-Leu-Asp-Leu-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-NH2.

7. The method according to claim 2, characterized in that The GLP-2 analogs include Glepaglutide, whose amino acid sequence is His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys-NH2.

8. The method according to claim 2, characterized in that: The cell seeding density was 8×10 5 -1×10 6 cells·mL -1 .

9. The method according to claim 2, characterized in that: The incubation time is 4-6 h.

10. The method according to claim 9, characterized in that The incubation time is selected as 5h.

11. A product for rapidly determining the biological activity of a GLP-2 analog drug, characterized in that: The product comprises the cells of claim 1.

12. A system for evaluating the drug activity of a GLP-2 analog, characterized in that: The system comprises the following components: the cells according to claim 1, a GLP-2 analog drug sample and a reference product.

13. The application of any one of the following aspects, characterized in that: The applications include: 1) Use of the cell according to claim 1 in the preparation of a product for detecting the biological activity of a GLP-2 analog drug; 2) Use of the system according to claim 12 in the quality control of GLP-2 analog drugs.

Images