Method for determining biological activity of FGF21 (fibroblast growth factor 21)

Abstract

The invention relates to a method for determining the biological activity of FGF21 (Fibroblast Growth Factor Receptor 21), which comprises the following steps: infecting and transfecting HEK293T cells by using an overexpression lentivirus containing FGFR1 and beta-Klotho genes and a reporter gene plasmid containing an SER reaction element so as to obtain a reporter gene-containing cell strain capable of stably expressing FGFR1, beta-Klotho and SRE; after FGF21 stimulation is added, the expression of a luciferase reporter gene at the downstream of the SER-containing promoter is activated; after co-incubation culture, adding a luciferase substrate, and fitting a four-parameter curve according to a measured reporter gene signal value, so as to determine the biological activity of the FGF21. According to the method provided by the invention, the test period is shortened to 24 hours, the detection sensitivity for FGF21 can reach a nanogram level and is improved by 1000 times compared with that of a traditional glucose oxidation method, and the result is consistent. The method provided by the invention can be used for evaluating the biological activity of FGF21 and analogues thereof as candidate drugs for treating metabolic diseases such as diabetes mellitus and NASH, and plays an important guiding role in quality control in research, development and production of FGF21 drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biological activity detection of biological drugs, and in particular to a method for determining the biological activity of FGF21. Background Art

[0002] Fibroblast growth factor 21 (FGF21) is a metabolic hormone produced in the liver, and its main biological functions are in glucose and lipid metabolism, improving insulin resistance, etc. At present, FGF21 has become a new candidate drug for the treatment of chronic diseases such as obesity, diabetes, hyperlipidemia and non-alcoholic fatty liver disease.

[0003] Early detection of the biological activity of FGF21 was to use radioactive isotope-labeled 2-deoxyglucose (2-DOG) to detect the radioactivity of [3H]-2-DOG inside differentiated and mature 3T3-L1 adipocytes, thereby reflecting the efficiency of glucose absorption by cells. However, due to the radioactivity of this method, it is difficult to apply to conventional laboratories. Subsequently, the biological activity detection method of FGF21 was improved, mainly the glucose oxidase method (GOD-POD). Although GOD-POD has been certified to be used to evaluate the biological activity of FGF21 in candidate drugs for the treatment of diabetes, it has limitations for other metabolic diseases (such as NASH). On the other hand, GOD-POD has the disadvantages of too high initial drug concentration (FGF21 reaches 1200μg / ml), long test period (4d~5d), and large variability in results during the determination. In addition, many companies or institutions have developed methods for detecting the biological activity of FGF21. For example, by utilizing the fact that FGF21 can promote cell proliferation and growth, the FGF21 to be detected is mixed with the cell culture medium, and the cell proliferation can be observed to preliminarily evaluate the biological activity of FGF21. K, Bartels T, Schwahn U, Kabiri M. Investigation of the Proliferative Potential of FGF21 orFGF19 in Liver-Specific FGFR4-Deficient Mice. Toxicol Pathol. 2023; 51(1-2): 27-38). FGF21 exerts its biological function by activating specific signaling pathways, such as activating the PI3K / Akt signaling pathway or the AMPK signaling pathway, and the biological activity of FGF2 1 is evaluated by detecting the activity of the signaling pathway (Arias-Calderón M, Casas M, Balanta-Melo J, et al. Fibroblast growth factor 21 is expressed and secreted from skeletal muscle following electrical stimulation via extracellular ATP activation of the PI3K / Akt / mTOR signaling pathway. Front Endocrinol (Lausanne). 2023; 14: 1059020). FGF21 can promote the oxidative metabolism of fatty acids, thereby regulating lipid metabolism. The effect of FGF21 is evaluated by measuring the intracellular fatty acid oxidation rate (Geller S, Arribat Y, Netzahualcoyotzi C, et al. Tanycytes Regulate Lipid Homeostasis by Sensing Free Fatty Acids and Signaling to Key Hypothalamic Neuronal Populations via FGF21Secretion. Cell Metab. 2019; 30(4): 833-844.e7). The current bioassay method has the defects of cumbersome operation, long test cycle, low accuracy and large error (Liu Lan et al., Chinese Journal of Biological Products, 2002; 15(3): 175-176).

[0004] Therefore, this field still needs to further develop efficient, stable, accurate, and widely applicable methods for determining the biological activity of FGF21.

[0005] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background technology. Summary of the invention

[0006] In view of the deficiencies of the prior art, the first aspect of the present invention provides a method for determining the biological activity of human fibroblast growth factor-21 (FGF21) based on a reporter gene method. The method is based on the activation of the MAPK / ERK signaling pathway after FGF21 forms a complex with its receptor and β-Klotho protein, and then activates the ELK-1 / SRF transcription factor. The ELK-1 / SRF transcription factor activates the transcriptional expression of the downstream luciferase reporter gene after binding to the SER response element. The method comprises the following steps: constructing a transgenic cell line with high responsiveness to FGF21; culturing the cell line, and detecting the biological activity of FGF21 in the sample to be tested by the luciferase reporter gene method.

[0007] According to a preferred embodiment, the method for determining the biological activity of FGF21 is obtained by a method comprising the following steps: obtaining a cell line that stably expresses FGFR1, β-Klotho and SRE; stimulating reporter gene expression with FGF21 and its analogs; and fitting a four-parameter curve based on the measured reporter gene signal value to determine the biological activity of FGF21 and its analogs.

[0008] According to a preferred embodiment, the cell line stably expressing FGFR1, β-Klotho and SRE is obtained by a method comprising the following steps:

[0009] (1) The target cells were infected with lentiviruses that overexpress FGFR1 and β-Klotho, and the MAPK / ERK signaling pathway was analyzed by antibiotic pressure screening and WB to obtain monoclonal cell lines that stably and highly expressed FGFR1 and β-Klotho proteins and were highly responsive to FGF21;

[0010] (2) Introducing a plasmid containing a reporter gene into the monoclonal cell described in (1), and using antibiotic pressure screening to obtain a monoclonal cell line containing a reporter gene that stably expresses SRE and has a high efficiency in responding to the reporter gene after FGF21 stimulation, and that stably expresses FGFR1, β-Klotho and SRE.

[0011] According to a preferred embodiment, the method for determining the biological activity of FGF21 comprises the following steps:

[0012] (1) Determine the biological activity of FGF21 using reporter gene-containing cell lines that stably express FGFR1, β-Klotho, and SRE;

[0013] (2) FGF21 and reference products were diluted to the pre-dilution concentration, and then added to the cells described in (1) under the incubation conditions of 37° C. and 5% CO2;

[0014] (3) Add luciferase substrate and fit a four-parameter curve according to the measured reporter gene signal value to determine the biological activity of FGF21.

[0015] According to a preferred embodiment, the cell line containing reporter genes that stably express FGFR1, β-Klotho and SRE is HEK293T cells.

[0016] According to a preferred embodiment, the FGF21-highly responsive FGFR1 / β-Klotho high-expressing monoclonal cell line is obtained by infecting HEK293T cells with FGFR1 and β-Klotho overexpressing lentivirus, and then undergoing antibiotic pressure screening and MAPK / ERK signaling pathway activation level evaluation, and is named FGFR1 + / β-Klotho + HEK293T.

[0017] According to a preferred embodiment, a monoclonal cell line with high reporter gene response is obtained by introducing a plasmid containing the reporter gene into a monoclonal cell with high FGFR1 / β-Klotho expression, and then screening with antibiotic pressure and evaluating the reporter gene expression level after FGF21 stimulation, and is named FGFR1 + / β-Klotho + HEK293T-Luc.

[0018] According to a preferred embodiment, the method for determining the biological activity of FGF21 is based on the change in the expression of luciferase gene in the interaction between FGF21 and transgenic cells. The specific steps of the detection method are as follows:

[0019] (1) The cell resuspension medium, activity detection reaction medium, and dilution medium for FGF21 and reference products were all high-glucose DMEM containing 10% FBS-1.0 μg / ml puromycin-0.25 mg / ml hygromycin B;

[0020] (2) The number of cells plated was 1.0×10 4 ~6.0×10 4 / hole, preferably 3.0×10 4 / hole;

[0021] (3) The pre-dilution concentration of FGF21 is 900 to 2000 ng / ml, preferably 1200 ng / mL,

[0022] (4) the dilution gradient of FGF21 is 3-5 times, preferably 3 times;

[0023] (5) After adding FGF21, the interaction time with cells is 6 to 12 hours, preferably 8 hours;

[0024] (6) Read the chemiluminescence value and determine the EC50 values ​​of FGF21 and the reference product by fitting a four-parameter curve, so as to reflect the biological activity of FGF21 by relative biological activity.

[0025] According to a preferred embodiment, the antibiotics include puromycin and hygromycin B, the screening concentration of puromycin is 1.0-10.0 μg / ml, preferably 2.0 μg / ml; the screening concentration of hygromycin B is 0.05-1.0 mg / ml, preferably 0.5 mg / ml.

[0026] According to a preferred embodiment, the FGFR1 stably expresses FGFR1, β-Klotho and SRE + / β-Klotho + -HEK293T-Luc cells, preferably the vector containing the SRE response element is the commercially available pGL4.33[luc2p / SRE / Hygro] plasmid; preferably, a double overexpression lentivirus of FGFR1 and β-Klotho genes.

[0027] According to a preferred embodiment, the reporter gene is a firefly luciferase reporter gene.

[0028] According to a preferred embodiment, high-glucose DMEM containing 10% FBS-1.0 μg / ml puromycin-0.25 mg / ml hygromycin B is used when resuspending the cells; the activity detection reaction solution is high-glucose DMEM containing 0.5-10% FBS-1.0 μg / ml puromycin-0.25 mg / ml hygromycin B, preferably 0.5% FBS-1.0 μg / ml puromycin-0.25 mg / ml hygromycin B high-glucose DMEM.

[0029] According to a preferred embodiment, the diluent for FGF21 and the reference substance is high-glucose DMEM containing 0.5% FBS-1.0 μg / ml puromycin-0.25 mg / ml hygromycin B.

[0030] According to a preferred embodiment, the changes in luciferase gene expression caused by the interaction of FGF21 with transgenic cells are detected.

[0031] Preferably, the chemiluminescence value read on a BioTek Synergy H1 microplate reader is used to determine the EC50 values ​​of FGF21 and the reference product by fitting a four-parameter curve, so that the relative biological activity reflects the biological activity of FGF21.

[0032] The second aspect of the present invention provides a cell line for determining the biological activity of FGF21, which is a monoclonal cell line containing reporter genes that stably expresses FGFR1, β-Klotho and SRE and is obtained by the method for determining the biological activity of FGF21 provided by the first aspect of the present invention.

[0033] The third aspect of the present invention provides a method for constructing a cell line for determining the biological activity of FGF21, the construction method comprising the following steps: successively infecting and transfecting target cells with an overexpression lentivirus containing FGFR1 (Genebank No: NM_001174063.2) and β-Klotho (Genebank No: NM_175737.4) genes and a reporter gene plasmid containing a SER response element, and using antibiotic pressure screening to obtain a monoclonal cell line containing a reporter gene that stably expresses FGFR1, β-Klotho and SRE, wherein the target cell is a HEK293T cell and the reporter gene is a firefly luciferase reporter gene.

[0034] The fourth aspect of the present invention provides a product for measuring the biological activity of FGF21, which comprises the cell line provided by the second aspect of the present invention or the third aspect of the present invention and a luciferase substrate.

[0035] In a fifth aspect, the present invention provides a recombinant lentiviral vector for co-overexpressing FGFR1 protein and β-klotho protein, the vector comprising FGFR1 gene and β-klotho gene, wherein the nucleotide sequence of the FGFR1 gene is shown in Seq ID NO.1; the nucleotide sequence of the β-klotho gene is shown in Seq ID NO.2. The present invention also provides a product comprising the recombinant lentiviral vector and a luciferase substrate.

[0036] According to a preferred embodiment, the FGFR1 gene is a human gene, and the β-klotho gene is a human gene.

[0037] According to a preferred embodiment, the human FGFR1 gene and the human β-klotho gene are inserted between the BamH I and Xho I sites of the lentiviral vector.

[0038] The sixth aspect of the present invention provides a recombinant lentivirus, which is a packaging body obtained by co-transfecting a host cell with the recombinant lentiviral vector provided by the fifth aspect of the present invention and a lentiviral auxiliary packaging plasmid.

[0039] The seventh aspect of the present invention provides a method for producing monoclonal antibodies, comprising the following steps: using the recombinant lentiviral vector provided by the sixth aspect of the present invention to transduce host cells; extracting the expressed human FGFR1 protein and human β-klotho protein from the transduced host cells; and using the extracted proteins to immunize experimental animals to produce antibodies that specifically bind to the proteins.

[0040] The eighth aspect of the present invention provides a monoclonal antibody produced by the method provided in the eighth aspect of the present invention, which can specifically bind to at least one epitope of human FGFR1 protein and / or human β-klotho protein.

[0041] The ninth aspect of the present invention also provides a method for determining the biological activity of FGF21, the method comprising:

[0042] Construction of a FGF21-high responsive cell line containing the FGFR1 gene and the β-klotho gene;

[0043] culturing the cell line;

[0044] The biological activity of FGF21 in the test samples was detected by luciferase reporter gene method.

[0045] The tenth aspect of the present invention provides the use of the methods provided in the first, seventh and ninth aspects of the present invention, the cell lines provided in the second or third aspects of the present invention, the products provided in the fourth aspect of the present invention, the recombinant lentiviral vectors and products provided in the fifth aspect of the present invention, the recombinant lentivirus provided in the sixth aspect of the present invention, and the monoclonal antibodies provided in the eighth aspect of the present invention in the quality control of new candidate drugs for treating chronic diseases such as diabetes, hyperlipidemia and NASH.

[0046] Technical effects of the present invention:

[0047] In the prior art, the method that can be used to evaluate the biological activity of FGF21 is the glucose oxidase method, which can be used to detect the biological activity of candidate drugs for the treatment of diabetes, but has certain limitations for other metabolic diseases (such as NASH). The reporter gene method is a biological activity detection method based on the drug signal transduction mechanism, which indirectly determines the biological activity of the drug by detecting the expression of the reporter gene. The signal transduction mechanism of FGF21 is to activate the downstream MAPK / ERK upstream signaling pathway after binding to the FGFR1 receptor and the auxiliary factor β-klotho to form a complex, so that ELK-1 and SRF in the cell nucleus are activated, and then recruited to the ETS and SRE binding sites in the GLUT1 promoter. SRF can specifically recognize SRE, thereby turning on the mechanism of GLUT1 transcription. Therefore, the constructed reporter gene cell line should contain three basic elements: cell receptor / auxiliary factor, transcription factor DNA response element and reporter gene.

[0048] HEK293T cells are human embryonic kidney cells. They have high transfection efficiency, express protein structures close to the conformation of endogenous proteins in the human body, and grow rapidly. They have been widely used in the research and development and production of recombinant proteins, virus packaging, and drugs. A small amount of FGFR1 receptors and auxiliary factor β-klotho are expressed on the surface of HEK293T cells. In addition, luciferase has the advantages of rapid response, short half-life, high sensitivity, and non-radioactivity, and is widely used in the activity determination of many biological products. Therefore, in order to improve its responsiveness to FGF21, the present invention successively transferred FGFR1, β-klotho, and SRE into HEK293T cells, and successfully constructed a cell line FGFR1 for RGA detection of the biological activity of FGF21. + / β-Klotho + HEK293T-Luc, and obtained a monoclonal cell line 3G10 with a good response. FGF21 binds to FGFR1 and β-klotho to stimulate the MAPK / ERK signaling pathway, thereby activating the expression of the luciferase gene downstream of SRE. The activity of FGF21 is determined based on the positive correlation between the intensity of luciferase gene expression and the content of FGF21 bound to the FGFR1 receptor on the target cell membrane.

[0049] The present invention utilizes the principle of reporter gene combined with the signal transduction mechanism of FGF21 to evaluate its biological activity, solves the problems of long experimental cycle, cumbersome operation and low result accuracy, and provides a powerful experimental means for studying the function of FGF21 and drug screening. Furthermore, in the process of establishing biological activity, the present invention optimizes aspects such as the density of plated cells, the selection of culture medium, the pre-dilution concentration of FGF21, the dilution gradient of FGF21, and the action time of FGF21, and performs methodological verification. The results show that by optimizing the aforementioned experimental conditions, the reporter gene method shows good repeatability and specificity in detecting the biological activity of FGF21, which can ensure that it shows good performance in subsequent research applications, and helps to improve the quality and reliability of related drugs.

[0050] The method for determining the biological activity of human fibroblast growth factor-21 (FGF21) based on the reporter gene method provided by the present invention shows obvious advantages in terms of accuracy of test results, experimental cycle, cost-effectiveness and ease of operation. The RGA method can provide data accuracy and repeatability comparable to or higher than the GOD-POD method, which is an important advantage. Since the method for detecting the biological activity of FGF21 provided by the present invention only requires a shorter experimental cycle, the demand for cell culture is reduced, which means that the RGA method is more environmentally friendly in terms of resource consumption and waste generation. Due to its rapidity, the RGA method of the present invention is more suitable for experimental applications that require rapid results, and its high sensitivity may make it suitable for a wider range of application fields, including drug screening and quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The results are from the measurement of FGFR1 levels on the surface of HEK293T and HL-7702 cells by cell immunofluorescence assay;

[0052] Figure 2 is the killing curve of puromycin on HEK293T cells;

[0053] Figure 3 It is the efficiency of FGFR1 / β-klotho co-overexpressing lentivirus infection in HEK293T cells and the functional verification results of the polyclonal cell line;

[0054] Figure 4 FGFR1 + / β-Klotho + -HEK293T monoclonal cell line screening and verification results;

[0055] Figure 5 is the killing curve of hygromycin B on 1B8 cells;

[0056] Figure 6It is the validation of the effect of pGL4.33-Luc plasmid transfection into 1B8 cells and the functional validation of polyclonal stable transfected cells;

[0057] Figure 7 FGFR1 + / β-Klotho + -Performance evaluation results of HEK293T-Luc monoclonal cell line;

[0058] Figure 8 It is the parameter optimization result of the reporter gene method for determining the biological activity of FGF21;

[0059] Fig. 9 is the linear regression equation of the logarithmic value of the theoretical value and the logarithmic value of the measured value of FGF21 potency;

[0060] Fig.10 This is the response curve of different cell generations of 3G10 cell line. DETAILED DESCRIPTION

[0061] The following is a detailed description with reference to the accompanying drawings.

[0062] The abbreviations used in the present invention are explained as follows.

[0063] RGAs: Reporter gene assays. In the examples, RGA is also used to represent reporter gene assays. FGFR1: Fibroblast Growth Factor Receptor 1. DMSO: Dimethyl sulfoxide. EDTA: Edetate disodium. FBS: Fetal bovine serum. PBS: Phosphate buffer saline. MTT: 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide. PVDF: polyvinylidene difluoride. TEMED: N,N,N′,N′-Tetramethylethylenedia. AP: ammonium persulfate. DAPI: 4′,6-diamidino-2-phenylindole. GLUT1: Glucose transporters1. SRE: Sterol response element. SRF: Serum response factor. WB: Western blot.

[0064] The reagents, materials and equipment used in the present invention are as follows.

[0065] Cells: HEK293T cells were purchased from Wuhan Pronocell Life Science Co., Ltd.; HL7702 cells were retained in the laboratory.

[0066] Reagents and materials: High-glucose DMEM, RPMI-1640 (without phenol red), trypsin, and fetal bovine serum (FBS) were purchased from GIBCO; FGFR1 and β-Kloth gene double overexpression lentivirus was constructed and packaged by Shanghai GeneCare Gene Medical Technology Co., Ltd.; pGL4.33[luc2p / SRE / Hygro] plasmid was purchased from Shanghai Core Biotechnology Co., Ltd.; Lipofectamine TM3000 reagent and fluorescence quencher were purchased from Thermo Company, USA; puromycin and hygromycin-B were purchased from Beijing Solebao Technology Co., Ltd.; donkey anti-sheep IgG-HRP was purchased from proteintech; Humanβ-Klotho Antibody was purchased from R&D Systems; Anti-FGFR1 was purchased from Abcam; HiScript IIQ RT SuperMix for qPCR (+gDNA wiper) was purchased from Nanjing Novozymes Biotech Co., Ltd.; white bottom transparent 96-well plates, luciferase detection kits and goat serum were purchased from Beyotime Biotechnology Co., Ltd.; human FGF21 and its reference products were prepared and retained by Zhejiang Key Laboratory of Biotechnology and Pharmaceutical Engineering.

[0067] Equipment and instruments: CO2 cell constant temperature incubator and agarose gel electrophoresis instrument were purchased from Thermo Company, USA; protein electrophoresis instrument, Gel Doc TM XR+ gel imaging system and fluorescence quantitative PCR instrument were purchased from Bio-rad, USA; MD microplate reader 190 was purchased from Molecular Devices Instrument Co., Ltd.; laser scanning confocal microscopy was purchased from Nikon, Japan; BioTekSynergy H1 multifunctional microplate reader was purchased from Agilent Technologies Co., Ltd.; cell counter was purchased from Shanghai Ruiyu Company; GraphPad Prism 8..0 was used for data statistical analysis.

[0068] The control group in the figures is represented by con or control.

[0069] The present invention selects HEK293T as a construction cell and provides a method for constructing a cell line for measuring FGF21 activity. The method is divided into two parts: ① using a lentivirus containing β-klotho (single-chain transmembrane protein) and FGFR1 to infect HEK293T cells to obtain a high-expression monoclonal cell line; and ② transfecting a reporter gene plasmid based on the above steps to finally obtain a reporter gene stably transfected cell line.

[0070] Example 1

[0071] Construction of a monoclonal HEK293T cell line co-overexpressing β-klotho and FGFR1.

[0072] This embodiment provides a method for constructing a stable cell line containing β-klotho / FGFR1, including the steps of reagent preparation, vector construction, β-klotho / FGFR1-containing lentivirus construction, cell recovery, cell passaging, cell freezing, cell counting and plating, determination of puromycin screening concentration, lentivirus infection of HEK293T cells, HEK293T-β-klotho / FGFR1 cell verification, and the like.

[0073] 1.1 Reagent preparation

[0074] (1) HEK293T cell complete culture medium: DMEM basal culture medium + 10% fetal bovine serum + 1% double antibody;

[0075] (2) HEK293T cell starvation medium: DMEM basal medium + 1% fetal bovine serum + 1% double antibody;

[0076] (3) DMEM screening medium 1: DMEM basal medium + 10% fetal bovine serum + 1% double antibody + 2.0 μg / ml puromycin;

[0077] (4) DMEM maintenance medium 1: DMEM basal medium + 10% fetal bovine serum + 1% double antibody + 1.0 μg / ml puromycin;

[0078] (5) DMEM starvation medium 1: DMEM basal medium + 1% fetal bovine serum + 1% double antibody + 1.0 μg / ml puromycin;

[0079] (6) Complete culture medium for HL-7702 cells: 1640 basal culture medium + 10% fetal bovine serum + 1% double antibody.

[0080] The dual antibody in this embodiment refers to penicillin-streptomycin dual antibody.

[0081] 1.2 Detection of FGFR1 levels on the surface of HEK293T cells

[0082] Methods: According to the literature, HL-7702 cells have low expression of FGFR1 on their surface. In this example, HL-7702 cells were used as a positive control, and the FGFR1 level on the surface of HEK293T cells was detected by cell immunofluorescence. HL-7702 cells and HEK293T cells were cultured at 6×10 5 The cells were inoculated at a density of 100 cells / well in a 6-well plate and cultured overnight for cell climbing, followed by fixation in 4% paraformaldehyde for 20 min, permeabilization with 0.5% Triton-×100 at room temperature for 10 min, blocking with 5% goat serum at room temperature for 30 min, incubation with anti-FGFR1 antibody (1:1000 dilution) at 4°C overnight, incubation with fluorescent secondary antibody at room temperature for 1 h and counter-staining with DAPI. Finally, the slides were sealed and the fluorescence intensity was observed under an inverted microscope to analyze the expression level of FGFR1.

[0083] The results are as follows Figure 1 As shown, the expression level of FGFR1 receptor on the surface of HEK293T cells is extremely low, so the level of FGFR1 receptor on its surface needs to be increased to enhance the responsiveness to FGF21.

[0084] 1.3 Puromycin killing curve determination of HEK293T cells

[0085] Methods: HEK293T cells were cultured at 1.0×10 4 Each well was inoculated with 100 cells / well in a 96-well plate, and the incubation conditions were: 37°C, 5% CO2 for 12-16 hours. Subsequently, the old culture medium was discarded, and fresh complete culture medium containing different concentrations of puromycin (1.0μg / ml, 0.9μg / ml, 0.8μg / ml, 0.7μg / ml, 0.6pg / ml, 0.5pg / ml, 0.4μg / ml, 0.3μg / ml, 0.2μg / ml, 0.1μg / ml, 0μg / ml) was added to continue the culture, and 3 replicates were made for each concentration. Fresh culture medium was replaced every 2 days, and cell survival was observed under a microscope every day. After 10 days of culture, the number of viable cells was determined by the MTT method, and the killing curve was established with the absorbance at 570nm as the ordinate and the puromycin concentration as the abscissa.

[0086] The results are as follows Figure 2 As shown in the figure, when the antibiotic concentration is 0 μg / ml, the cell survival rate is 100%; when the antibiotic concentration is between 0 and 1 μg / ml, the cell survival rate remains stable; when the antibiotic concentration is between 1.0 and 2.0 μg / ml, the cell survival rate decreases with the increase of puromycin concentration, and when the antibiotic concentration is 2.0 μg / ml, the cell survival rate is 0. Therefore, the minimum effective concentration of puromycin to effectively kill HEK293T cells is 2.0 μg / ml, and the maintenance concentration of cell culture can be 1.0 μg / ml.

[0087] 1.4 FGFR1 + / β-Klotho + - Preparation of HEK293T polyclonal stable cell lines

[0088] (1) Experimental methods

[0089] Lentivirus construction: The human FGFR1 gene (sequence number NM-001174063, 744-3026, Seq ID NO.1) and the 21-3155 sequence of human β-klotho (sequence number NM-175737, Seq ID NO.2) were retrieved from the NCBI database and inserted between the BamH I and Xho I restriction sites of the vector GV717 to design an overexpression plasmid vector. Subsequently, a gene company was commissioned to complete the construction, packaging, concentration and purification of the FGFR1 / β-klotho co-overexpression lentiviral vector.

[0090] Lentivirus infection: HEK293T cells were diluted to 2 × 10 5Cell suspension of 100 cells / ml was inoculated into 6-well plates, 2 ml per well. After culturing at 37°C, 5% CO2 for 24 hours, the culture medium was discarded, and FGFR1 / β-klotho co-overexpression lentivirus suspension (MO1 value = 10) diluted in complete culture medium was added, mixed, and cultured for 8 to 16 hours before replacing with fresh complete culture medium. After culturing at 37°C, 5% CO2 for 72 hours, half of the cells were taken for evaluation of lentivirus infection effect.

[0091] Evaluation of lentiviral infection efficiency: The above lentiviral infected cells were diluted to 2 × 10 5 / ml, inoculated in 6-well plates, 2ml per well, and made 3 replicates, and HEK293T cells that were not infected with lentivirus were used as negative controls. After culturing at 37°C, 5% CO2 for 18-24h, replace with starvation medium and continue culturing for 12-16h. Subsequently, replace the starvation medium containing 100ng / ml FGF21 and incubate for 6h. Discard the old medium, wash 3 times with PBS, and add 80μl of cell lysis buffer (RIPA lysis buffer: PMSF protease inhibitor: phosphatase inhibitor mixture = 100:1:1) to each well. After lysis on ice for 10min, collect the cells in a pre-cooled EP tube with a cell scraper, and then centrifuge at 4°C, 12000rpm for 20min to collect the supernatant sample. The protein concentration of the supernatant sample was determined using a BCA protein concentration assay kit (Biyuntian), and the sample was prepared according to a loading system of 20 μg / 10 μl, followed by SDS-PAGE electrophoresis and Western blotting to determine the expression levels of FGFR1 and β-klotho proteins, as well as the phosphorylation level of ERK.

[0092] Preparation and verification of polyclonal stable cell lines: The other half of the cells were collected and transferred to T25 culture flasks for antibiotic pressure screening. DMEM screening medium 1 was replaced every 2 days. The culture was continued for 8-10 days until there was no cell shedding and death, and then replaced with DMEM maintenance medium 1. After the cells that failed to integrate the exogenous gene were basically removed for 5-7 days, they were transferred to T75 culture flasks for continued amplification and culture to obtain a large amount of FGFR1 + / β-Klotho + -HEK293T polyclonal stable cell line. Finally, according to the operation of evaluating the efficiency of lentiviral infection, the polyclonal stable cell line was functionally verified, and the results are shown in Figure 3 .

[0093] (2) Experimental results

[0094] Figure 3The results show the efficiency of HEK293T cells infected with FGFR1 / β-klotho co-overexpressing lentivirus and the functional verification of polyclonal cell lines. WB detected the differences in (3A) the expression levels of FGFR1 and β-klotho and (3B) the activation levels of downstream signaling pathways under 100ng / ml FGF21 stimulation before and after FGFR1 / β-klotho co-overexpressing lentivirus infected HEK293T cells. WB detected the differences in (3C) the expression levels of FGFR1 and β-klotho (n=3) and (3D) the activation levels of downstream signaling pathways under 100ng / ml FGF21 stimulation (n=3) of polyclonal stable transfectants obtained by puromycin pressure screening after 20 passages. Compared with the normal group, ***P≤0.001, **P≤0.01, *P≤0.05.

[0095] The final lentiviral titer was 3 × 10 8 Tu / ml, which has a very high infection efficiency for HEK293T cells, and the β-klotho / FGFR1 gene can be integrated into the cell chromosome and activate the downstream signaling pathway (see Figure 3 A and 3B). Subsequently, the cells that failed to integrate the exogenous gene were removed by puromycin pressure selection for 15 days, and a polyclonal stable transfection cell line was obtained. In addition, after 20 passages, the polyclonal line could still stably express FGFR1 and β-Klotho genes ( Figure 3 C), and significantly increased the activation level of downstream signaling pathways after FGF21 stimulation ( Figure 3 D).

[0096] 1.5 FGFR1 + / β-Klotho + - Preparation and verification of HEK293T monoclonal cell line

[0097] (1) Experimental methods

[0098] Limiting dilution plating: dilute the polyclonal stable cell line with DMEM maintenance medium 1 to 1×10 4 Take 200μl of the cell suspension of 100 / ml and inoculate it in well A1 of a 96-well plate. Then make a 2-fold gradient dilution from well A1 to well H1 (vertical gradient dilution), and each well finally contains 100μl of cell suspension. Add 100μl of DMEM maintenance medium 1 to each well from A1 to H1 and mix well. Then, make a 2-fold gradient dilution from well A1 to well A12 (horizontal), and each well from A1 to H1 finally contains 100μl of cell suspension.

[0099] Monoclonal cell line marking and selection: After 6 hours of incubation at 37°C, 5% CO2, observe the wells with only one cell under a microscope; after 2-3 days of incubation, observe the clones of the marked wells under a microscope, and mark the wells with monoclonal cells and round and bright cell colonies again, while avoiding selecting clones growing at the edge of the wells. Replace the medium with fresh DMEM every 2 days.

[0100] Expansion culture of monoclonal cell lines: When the cell confluence reaches 100%, the marked selected monoclonal cell lines are transferred to 24-well culture. Subsequently, each monoclonal cell line is expanded and cultured in the manner of 24-well plate → 12-well plate → 6-well plate → T25 culture flask → T75 culture flask every time the cell confluence reaches 90%. Finally, the cells are collected in serum freezing solution (90% FBS + 10% DMSO), and divided into cell cryopreservation tubes for long-term storage in liquid nitrogen.

[0101] Monoclonal cell line performance evaluation: According to the experimental method for evaluating the lentiviral infection efficiency in 1.4 of Example 1, monoclonal stable cell lines in the logarithmic growth phase were selected for functional evaluation and verification, and the monoclonal cell lines with relatively optimal performance were selected. At the same time, RT-qRCR was used to detect and evaluate the mRNA expression levels of FGFR1 and β-Klotho in the monoclonal cell lines with the best performance, such as Figure 4 shown.

[0102] (2) Experimental results

[0103] Figure 4 FGFR1 + / β-Klotho + -HEK293T monoclonal cell line screening and validation results. (4A) WB detection of the differences in the expression levels of FGFR1 and β-klotho proteins in different monoclonal cell lines (n=3). (4B) WB detection of the differences in the activation levels of the downstream MAPK / ERK signaling pathway in different monoclonal cell lines after FGF21 stimulation (n=3). (4C) Differences in the mRNA expression levels of FGFR1 and β-Klotho between the 1B8 monoclonal cell line and normal HEK29T cells. Compared with the normal group, ***P≤0.001, **P≤0.01, *P≤0.05.

[0104] Finally, four monoclonal cells were obtained, which were named 1D6, 1B8, 1B9 and 2F4 according to the plate number of the labeled 96-well plate cells and the well position of the cells. Figure 4As shown in A, these four monoclonal cell lines all highly expressed β-klotho and FGFR1, but there was no statistical difference in the expression levels. At the same time, compared with HEK293T cells, after FGF21 stimulation, the activation level of the downstream MAPK / ERK signaling pathway of the four monoclonal cell lines was significantly enhanced, among which the 1B8 strain had the highest increase level, that is, the 1B8 strain had the best response to FGF21 ( Figure 4 B). In addition, compared with HEK293T cells, the mRNA expression levels of FGFR1 and β-Klotho in IB8 strain were also significantly increased ( Figure 4 C). Therefore, the 1B8 monoclonal cell line was selected for subsequent reporter gene cell construction.

[0105] The lentiviral system is a commonly used gene delivery tool that can stably and efficiently express exogenous genes into host cells to achieve long-term stable expression. This example used an overexpression lentivirus containing FGFR1 and β-klotho genes to infect HEK293T cells, and successfully constructed a monoclonal cell line with stable expression. The four monoclonal cells selected by antibiotic pressure all highly expressed FGFR1 and β-klotho genes ( Figure 4 A), and can activate the downstream ERK1 / 2 signaling pathway ( Figure 4 ).

[0106] Using overexpression lentivirus carrying the target gene to infect cells has stronger specificity and higher infection efficiency, greatly increasing the probability of obtaining target cells and greatly shortening the screening time of monoclonal cells.

[0107] The monoclonal cell line 293T-β-klotho-FGFR1 (1B8 cells) successfully constructed in this example can be used for the construction of a reporter gene cell line.

[0108] Example 2

[0109] FGFR1 with reporter gene + / β-Klotho + -Construction of HEK293T monoclonal cell line.

[0110] This example uses the FGFR1 successfully constructed in Example 1. + / β-Klotho + -HEK293T monoclonal cell line (1B8 cell line) was used as the basis for the construction of reporter gene cell line, and the repeated contents will not be repeated here.

[0111] 1.1 Solution preparation

[0112] (1) DMEM screening medium 2: DMEM basal medium + 10% fetal bovine serum + 1% double antibody + 2.0 μg / ml puromycin + 0.50 mg / ml hygromycin B;

[0113] (2) DMEM maintenance medium 2: DMEM basal medium + 10% fetal bovine serum + 1% double antibody + 1.0 μg / ml puromycin + 0.25 mg / ml hygromycin B;

[0114] (3) DMEM starvation medium 2: DMEM basal medium + 1% fetal bovine serum + 1% double antibody + 1.0 μg / ml puromycin + 0.25 mg / ml hygromycin B;

[0115] (4) Liquid LB medium (g / L): 5 g yeast powder + 5 g peptone + 10 g sodium chloride;

[0116] (5) Solid LB medium (g / L): 5 g yeast powder + 10 g peptone + 10 g sodium chloride + 11 g agar powder;

[0117] (6) 100 mg / ml ampicillin: Dissolve 1 g of ampicillin sodium salt in 10 ml of sterile water, filter with a 0.22 μM filter membrane, dispense into 1.5 ml sterile centrifuge tubes, and store at -20°C;

[0118] (7) 100 mg / ml Hygromycin B: Weigh 1 g of Hygromycin B and dissolve it in 10 ml of 1× PBS solution. After it is fully dissolved, filter it with a 0.22 μM filter membrane and store it in aliquots at -20°C.

[0119] (8) 50×TAE: In a 1-liter beaker, add 242 g of tris(hydroxymethyl)aminomethane (Tris) and ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-Na2), add 600 ml of deionized water to dissolve, add 57.1 ml of acetic acid solution and stir thoroughly, and then dilute to 1 L with deionized water.

[0120] 1.2 Determination of the killing curve of hygromycin B in 1B8 cell line

[0121] Methods: 1B8 cells were cultured at 1×10 4Each well was inoculated with 100 μl / well in a 96-well plate at 37°C and 5% CO2 for 12-16 hours. Subsequently, the old culture medium was discarded, and fresh DMEM containing different concentrations of hygromycin B (1.0 mg / ml, 0.75 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.10 mg / ml, 0.05 mg / ml) was added to maintain the culture medium 1 and continued to culture. Three replicates were made for each concentration. Fresh culture medium was replaced every 2 days, and cell survival was observed under a microscope every day. After 10-12 days of culture, the number of viable cells was determined by the MTT method, and a killing curve was established with the absorbance at 570 nm as the ordinate and the concentration of hygromycin B as the abscissa.

[0122] The results are as follows Figure 5 As shown, after about 10 to 12 days of culture, combined with naked eye observation and MTT test results, it can be seen that when the concentration of hygromycin B is between 0.05 and 0.50 mg / ml, the cells are in rapid growth, the culture medium turns yellow, but the cell survival rate decreases with the increase of puromycin concentration; when the concentration of hygromycin B is ≥ 0.50 mg / ml, the cells are basically dead, the cell survival rate is close to 0, and the culture medium has no obvious changes. Therefore, the minimum effective concentration of hygromycin B to effectively kill 1B8 cells is 0.50 mg / ml, and the maintenance concentration of cell culture can be 0.25 mg / ml.

[0123] 1.3 FGFR1 + / β-Klotho + -Preparation of HEK293T-Luc polyclonal stable cell lines

[0124] (1) Experimental methods

[0125] Reporter gene plasmid transfection: 1B8 cells were diluted to 2.5 × 10 5 100 μl / ml, 2 ml per well, inoculated in 6-well plates. Culture at 37°C, 5% CO2 for 24 h until the cell confluence reaches 50%, then replace with complete medium without dual antibodies, add 250 μl of plasmid complex (pGL4.33[1uc2p / SRE / Hygro] plasmid 11.45 μl + DMEM basal medium 500 μl + P3000 TM Reagent 8.70μl) and liposome complex (Lipofectamine TM 3000 reagent 15μl + DMEM basal medium 500μl) 1:1 mixed DNA-liposome complex, mix well. After culturing for 24 hours, replace with DMEM maintenance medium 1, continue culturing for 24 hours, and perform luciferase detection to evaluate the transfection effect.

[0126] Plasmid transfection assessment: 1B8 cells transfected with luciferase reporter gene were diluted with DMEM maintenance medium 1 and the concentration was 2 × 10 5 / ml, 100μl per well, inoculated in a 96-well plate with a white bottom and light-transmitting. After culturing at 37℃, 5% CO2 for 12-16h, replace with DMEM starvation medium 1, and continue starvation culture for 12-14h. Replace the starvation medium, pre-dilute FGF21 to 900ng / ml with the starvation medium, and add FGF21 for 24h in a 4-fold gradient dilution, a total of 7 gradients, 2 replicates per well, and 200μl / well. Discard the old culture medium, add 100μl of cell lysis to each well according to the operating instructions of the luciferase detection kit, lyse at room temperature for 10min, then add 100μl of luciferase substrate and shake thoroughly for 5min, detect the fluorescence signal value at a wavelength of 560nm, and fit the four-parameter curve.

[0127] Antibiotic pressure screening of polyclonal stable cell lines: Digest the above transfected cells and transfer them to a T25 culture flask, and use DMEM maintenance medium 1 to culture for 8 hours. After the cells reattach to the wall, replace them with DMEM screening medium 2 and perform antibiotic pressure screening. Wash twice with PBS every 2 days to wash away dead cells and shed cells, and replace with fresh medium once. On the 7th day of culture, observe the cells and find that some cells adhere to the wall and proliferate, and the number of shed cells decreases. Replace the screening medium with maintenance medium and continue to culture and expand the cells. When the cell confluence reaches 90%, transfer them to a T75 culture flask for continued expansion culture in the T75 culture flask to harvest a large number of FGFR1 + / β-Klotho + -HEK293T-Luc polyclonal stable transfection cell line, placed in serum freezing solution (90% FBS + 10% DMSO), and stored in liquid nitrogen for a long time. In addition, according to the operation of plasmid transfection evaluation, polyclonal stable transfection cell line after three consecutive passages was taken for functional verification, such as Figure 6 shown.

[0128] (2) Experimental results

[0129] Figure 6 The results of the validation of the effect of pGL4.33-Luc plasmid transfection in 1B8 cells and the functional validation of polyclonal stable transfected cells. (6A) Luciferase signal response curve of 1B8 cells transfected with pGL4.33-Luc plasmid 48 hours after stimulation with different concentrations of FGF21. (6B) FGFR1 + / β-Klotho + -Luciferase signal response curve of HEK293T-Luc polyclonal stable transfected cells stimulated by different concentrations of FGF21.

[0130] 24 hours after 1B8 cells were transfected with pGL4.33[1uc2p / SRE / Hygro] plasmid, the cells were in good condition and proliferated. After 48 hours, the cell confluence reached 80%. Figure 6 As shown in A, after FGF21 stimulation, there is a dose-response relationship curve between the intracellular luciferase and the concentration of FGF21, and its EC 50 The value is 99.29ng / ml, R 2 It is 0.980, which indicates that the plasmid was successfully transfected into 1B8 cells, making them respond well to FGF21, and the next step of stable strain screening can be carried out.

[0131] Subsequently, the FGFR1 + / β-Klotho + -HEK293T-Luc polyclonal stable cell line. Functional validation results showed that compared with before pressure screening, the responsiveness of this polyclonal stable cell line to FGF21 was significantly improved, and its EC 50 The value is 28.29ng / ml, R 2 It is 0.992, which indicates that cells that have not successfully integrated the exogenous gene have been effectively removed and can be used for the next step of monoclonal cell line screening.

[0132] 1.4 FGFR1 + / β-Klotho + - Preparation and verification of HEK293T-Luc monoclonal cell line

[0133] Experimental method: FGFR1 in the logarithmic growth phase was selected + / β-Klotho + -HEK293T-Luc polyclonal stable transfected cells, according to the method of limiting dilution method for screening monoclonal cells in 1.5 of Example 1, a large number of monoclonal cell lines were obtained. Among them, DMEM maintenance medium 2 was used for cell growth culture, and DMEM starvation medium 2 was used for starvation culture. In addition, according to the conditions of plasmid transfection evaluation in 1.3 of Example 2, the intensity of the fluorescence signal under the stimulation of 100ng / ml FGF21 was used to screen the monoclonal cell line with the best responsiveness to FGF21, and the four-parameter curve of the fluorescence signal response value under the stimulation of different concentrations of FGF21 was determined, as shown in FIG. Figure 7 shown.

[0134] Figure 7 FGFR1 + / β-Klotho +- Performance evaluation results of HEK293T-Luc monoclonal cell lines. (7A) Differences in luciferase expression levels of different monoclonal cell lines under 100ng / ml FGF21 stimulation. (7B) Luciferase signal response curve of 3G10 monoclonal cells under different concentrations of FGF21 stimulation. Compared with the normal group, ***P≤0.001, **P≤0.01, *P≤0.05.

[0135] Experimental results: By using the limiting dilution method, the stable polyclonal cell lines obtained by pressure screening were continuously diluted in gradients, and finally 10 monoclonal cell lines containing the reporter gene luciferase were screened out. They were named 1E9, 3G10, 5E8, 4G10, 2F8, 3D9, 3F8, 2G8, 5F10 and 3F10 according to the number of the culture plate and the location where the cells were obtained. Figure 7 As shown in A, after stimulation with FGF21, 7 monoclonal cell lines showed high expression of luciferase, among which the fluorescence signal response value of 3G10 was the highest. In addition, 3G10 also showed a good dose effect to FGF21 stimulation, as shown in Figure 7 As shown in B, the fluorescence signal values ​​under the stimulation of different concentrations of FGF21 showed a typical four-parameter curve, with an EC50 value of 0.61 ng / ml, SRN of 28.30, and R 2 The value of 3G10 monoclonal cell line was 0.988. This also shows that compared with the polyclonal stable cell line, the responsiveness of 3G10 monoclonal cell line to FGF21 is significantly improved. Therefore, 3G10 cell line was selected as the detection cell for the reporter gene method to measure FGF21 activity for subsequent detection method development and validation research.

[0136] Example 3

[0137] Establishment and methodological validation of the fluorescence reporter gene method.

[0138] In this example, the FGFR1 constructed in the previous example is + / β-Klotho + -Hek293T-Luc monoclonal cell line (3G10 cell line) was used as the basis for establishing a reporter gene method for determining the biological activity of FGF21, optimizing the conditions and verifying the methodology.

[0139] 1.1 Establishment and optimization of the reporter gene assay for FGF21

[0140] 1.1.1 Optimization of FGF21 pre-dilution concentration

[0141] Experimental method: 3G10 cells were diluted with DMEM maintenance medium 2 at a concentration of 1.5×10 4 / well, 100μl / well, inoculated in a 96-well plate with a white bottom and translucent, incubated at 37℃, 5% CO2 for 16-18h. Discard the old culture medium and replace it with DMEM starvation medium 2, and continue to culture for 14-18h. Replace the starvation medium, pre-dilute FGF21 to 10μg / ml with DMEM starvation medium 2, and add FGF21 solution to incubate for 24h according to the dosage method of 3-fold gradient dilution, 15 gradients in total, 200μl / well, and 2 replicate wells in each well. Discard the old culture medium, and according to the operating instructions of the luciferase detection kit, add 100μl of cell lysis to each well, lyse at room temperature for 10min, then add 100μl of luciferase substrate and shake thoroughly for 5min, detect the fluorescence signal value at a wavelength of 560nm, and fit the four-parameter curve, as shown Figure 8 shown.

[0142] Figure 8 Parameter optimization results of the reporter gene method for determining the biological activity of FGF21. (8A) Response curves of 3G10 cell lines at different pre-dilution concentrations. (8B) Optimization of 3G10 cell line activity detection reaction solution and FGF21 dilution solution. (8C) Response curves of 3G10 cell lines at different dilution multiples. (8D) Response curves of 3G10 cell lines at different action times. (8E) Response curves of 3G10 cell lines at different cell densities.

[0143] Experimental results: A qualified four-parameter curve should be S-shaped, and it should at least ensure that the upper and lower platforms of the curve have 2 test values. Figure 8 As shown in A, the detection values ​​of 15 FGF21 concentrations were fitted to form an S-shaped four-parameter curve, of which 4 detection values ​​were on the upper platform of the curve, and 6 detection values ​​were on the lower platform. The EC50 value was 11.62 ng / ml, the SRN was 27.03, and the R 2 Therefore, the pre-dilution concentration of FGF21 was set at 1200 ng / ml.

[0144] 1.1.2 Optimization of activity detection reaction solution and FGF21 dilution solution

[0145] Experimental method: According to the method and results of FGF21 pre-dilution concentration optimization in 1.1.1 of Example 3, the FGF21 pre-dilution concentration was 1200 ng / ml, and 10 concentrations were diluted in a 3-fold series. DMEM starvation culture 2 (containing 0.5% FBS) or DMEM maintenance medium 2 (containing 10% FBS) was used for starvation culture and preparation of FGF21 solutions of different concentrations. After 24 hours of action, the fluorescence signal value was detected and the four-parameter curve was fitted.

[0146] Experimental results: Figure 8As shown in B, after treatment with 0.5% FBS medium, the EC50 of the test results was 18.32 ng / ml, SNR was 16.92, and R 2 After treatment with 10% FBS medium, the EC50 of the test result was 12.57 ng / ml, SNR was 38.84, and R 2 The EC50 values ​​of the test results after two different treatments were similar, but the signal-to-noise ratio (SNR) of the test results treated with 0.5% FBS medium was higher. After comprehensive consideration, DMEM maintenance medium 2 containing 10% FBS was selected as the FGF21 diluent and activity detection reaction solution, and the operation of starvation culture for 14-18h could be removed.

[0147] 1.1.3 Optimization of FGF21 dilution gradient

[0148] Experimental method: According to the method and results of culture medium optimization in 1.1.2 of Example 3, FGF21 was pre-diluted to 1200 ng / ml using DMEM maintenance culture medium 2 (containing 10% FBS), and 10 concentrations were serially diluted in 3-fold, 4-fold or 5-fold ratios. After 24 hours of action, the fluorescence signal value was detected and a four-parameter curve was fitted.

[0149] Experimental results: Figure 8 As shown in C, when the pre-dilution concentration is 1200 ng / ml and a 3-fold gradient dilution is performed, the platform on the curve can have 2 detection values, but when a 4-fold or 5-fold gradient dilution is performed, the platform on the curve cannot have 2 detection values. In addition, when a 3-fold gradient dilution is performed, the EC50 is 38.07 ng / ml, the SNR is 53.11, and the R 2 When the dilution ratio was 4 times, the EC50 was 90.25 ng / ml, the SNR was 32.77, and the R 2 When the dilution ratio was 5 times, the EC50 was 134.90 ng / ml, the SNR was 310.13, and the R 2 The value was 0.999. After comprehensive consideration, 1200 ng / ml was selected as the pre-dilution concentration, and 10 gradients of 3-fold serial dilution were performed.

[0150] 1.1.4 Optimization of FGF21 action time

[0151] Experimental method: According to the method and results of FGF21 dilution gradient optimization in 1.1.3 of Example 3, FGF21 was pre-diluted to 1200 ng / ml using DMEM maintenance medium 2 (containing 10% FBS), and 10 concentrations were diluted in a 3-fold series. After 6h, 8h, 10h or 12h of action, the fluorescence signal value was detected and a four-parameter curve was fitted.

[0152] Experimental results: According to the results of transient transfection and stable transfection, FGF21 stimulation for 24 hours has an effect. The fluorescence signal was detected after 6h, 8h, 10h and 12h of stimulation and a four-parameter curve was drawn. Figure 8 As shown in D, when FGF21 was applied for 6 h, the EC50 was 13.00 ng / ml, the SNR was 24.99, and the R 2 was 0.997; after 8 hours of action, EC50 was 14.48 ng / ml, SNR was 55.56, R 2 is 0.998; after 10 hours of action, EC50 is 11.39ng / ml, SNR is 34.58, R 2 was 0.998; after 12 hours of action, EC50 was 14.27 ng / ml, SNR was 42.07, R 2 The SNR of the test results after different action time was 0.999. In addition, the EC50 of the test results after different action time was not much different, but the SNR of 8h was the largest. Therefore, 8h was selected as the action time of FGF21.

[0153] 1.1.5 Optimization of cell density

[0154] Experimental method: According to the method and results of optimizing the dilution gradient of FGF21 in 1.1.4 of Example 3, 3G10 cells were diluted with DMEM maintenance medium 2 to 1.0×10 4 , 2.0×10 4 , 3.0×10 4 4.0×10 4 5.0×10 4 or 6.0×10 4 Each well was inoculated in 96-well culture, FGF21 was pre-diluted to a concentration of 1200 ng / ml, and 10 concentrations were serially diluted 3-fold. After 8 hours of action, the fluorescence signal value was detected and a four-parameter curve was fitted.

[0155] Experimental results: Figure 8 As shown in E, when the cell density is 1.0×10 4 pcs / hole, EC 50 is 5.62ng / ml, SNR is -32.95, R 2 is 0.989; when the cell density is 2.0×10 4 pcs / hole, EC 50 was 6.23 ng / ml, SNR was -36.01, R 2 is 0.995; when the cell density is 3.0×10 4 pcs / hole, EC 50 is 7.47ng / ml, SNR is 116.14, R 2 is 0.999; when the cell density is 4.0×104 pcs / hole, EC 50 is 7.78ng / ml, SNR is 62.78, R 2 is 0.999; when the cell density is 5.0×10 4 pcs / hole, EC 50 is 9.09 ng / ml, SNR is 42.85, R 2 is 0.996; when the cell density is 6.0×10 4 pcs / hole, EC 50 is 10.19 ng / ml, SNR is 37.73, R 2 The EC value of the test results after inoculation at different cell densities was 0.999. 50 The difference was not significant, but when the cell density was 3.0×10 4 The SNR is the largest when the number of cells / well is greater than that of cells / well. Cell density, EC50 (half effective concentration), signal-to-noise ratio (SNR) and determination coefficient (R 2 ) is a key parameter in drug screening and cell culture experiments. When the cell density is high, cell-to-cell interactions and the accumulation of metabolites may increase, which may affect the distribution of the drug and the sensitivity of the cell to the drug, thereby affecting the determination of EC50. The signal-to-noise ratio (SNR) is the ratio of signal intensity to background noise. In cell experiments, higher cell density may increase the intensity of the signal, but it may also increase the background noise. Therefore, it is necessary to find an appropriate cell density to obtain the best SNR. In the drug dose-response curve, a higher R 2 The value indicates that the model fits the data well. The appropriate choice of cell density can affect the quality of experimental data and R 2 value.

[0156] Therefore, the number of plates was selected to be 3.0 × 10 4 The cell density for activity detection is 100 cells / well. Appropriate cell density can avoid nutrient competition and metabolic waste accumulation caused by overcrowding of cells, thereby maintaining high cell activity. Appropriate cell density can reduce experimental variation caused by uneven cell growth and improve experimental stability. At the same time, reliable experimental results can be obtained in a shorter time, improving detection efficiency.

[0157] According to the above results, the biological activity parameters of FGF21 protein detected by 3G10 cells, namely the optimized parameters of biological activity parameters of FGF21 protein detected by RGA are finally determined as follows:

[0158] The pre-dilution concentration was 1200 ng / ml; the dilution gradient was 3-fold; the FGF21 action time was 8 h; the FGF21 dilution solution and activity detection reaction solution were DMEM maintenance medium 2 containing 10% FBS; the cell density was 3.0×104 No starvation culture operation.

[0159] 1.2 Method Validation

[0160] 1.2.1 Experimental methods

[0161] According to the guidelines for validation of biological activity / potency assays for biological products in the 2020 edition of the Chinese Pharmacopoeia, the accuracy, precision, linearity and range of the reporter gene assay for FGF21 biological activity were validated, and the validation experiments for the four indicators were combined and designed. The specific operations are as follows:

[0162] a. Dilute 3G10 cells with DMEM maintenance medium 2 to 1.5×10 4 100 μl / well was inoculated into a 96-well plate with a white bottom and transparent surface, and cultured at 37°C and 5% CO2 for 16 to 18 hours.

[0163] b. Use DMEM maintenance medium 2 to pre-dilute the FGF21 active reference to 1200ng / ml; pre-dilute the FGF21 test samples to 1800, 1500, 1200, 900, and 600ng / ml, respectively, even if the relative potency levels of the five test samples are 50%, 75%, 100%, 125%, and 150%, respectively. Then, discard the old culture medium, add the FGF21 active reference and test sample solutions in a 3-fold gradient dilution, a total of 10 gradients, 200μl / well, and 2 replicates for each well, and react for 8h, detect the fluorescence signal value and fit the four-parameter curve.

[0164] c. The experiment was completed by two analysts at four time points. Cells of different generations were used at different time points. Each analyst independently performed two measurements at each time point and each titer level. The geometric mean of the results of the two analysts was reported each time.

[0165] 1.2.2 Experimental Results

[0166] The test results of the two analysts on the five samples with different relative potency levels are summarized in Table 1, and the accuracy, precision, linearity and range verification results are statistically analyzed based on them, as follows:

[0167] Table 1. Results of FGF21 biological activity assay (3G10 cells / luciferase reporter gene method)

[0168]

[0169] (1) Relative accuracy

[0170] Relative accuracy is generally expressed as relative bias (RB, %). The relative bias calculation formula is:

[0171]

[0172] Acceptable standard: The RB of each potency level relative to the potency measurement value should be within the range of ±12%. The horizontal axis is the logarithm of the theoretical potency value, and the vertical axis is the logarithm of its corresponding potency measurement value. The linear regression equation is obtained, and the slope of the regression equation is in the range of 0.80 to 1.25.

[0173] Verification results: The relative bias and confidence interval of the relative potency measurement value of each potency level were calculated according to the formula (see Table 2). The average relative bias of the relative potency measurement values ​​of the five potency levels were 2.66%, -4.52%, -6.85%, 4.59%, and -2.71%, respectively, all within the range of ±12%; a linear regression was made with the logarithm of the theoretical potency value corresponding to the logarithm of the corresponding potency measurement value, and the regression equation was y = 1.0278x-0.0313, with a slope of 1.0278 within the range of 0.80 to 1.25 (see Fig. 9 ). This shows that the accuracy of this method is good.

[0174] Table 2. Relative bias and confidence interval of relative potency values ​​at different potency levels

[0175]

[0176] Note: The base of logarithmic conversion in the table is e.

[0177] (2) Intermediate precision

[0178] Intermediate precision is expressed by geometric coefficient of variation (GCV, %). The geometric coefficient of variation of the relative potency measured at each potency level is calculated as follows:

[0179] GSD=antilog(SD)

[0180] GCV=(GSD-1)×100%

[0181] Acceptable criteria: The GCV of each potency level relative to the potency measurement value should be no greater than 20%;

[0182] Verification results: The geometric standard deviation, geometric coefficient of variation and confidence interval of the relative potency measurement value of each potency level were calculated by formula (see Table 3). By calculating the GCV of the relative potency measurement values ​​of the five potency levels, they were 12.4%, 17.3%, 9.5%, 17.4% and 14.1%, respectively, all less than 20%, indicating that the method has good precision.

[0183] Table 3. Geometric standard deviation, geometric coefficient of variation and upper confidence limit of relative potency values ​​of FGF21 at different potency levels

[0184]

[0185] Note: The base of the antilogarithmic transformation in the table is e. When calculating the 95% confidence limit

[0186] (3) Linear

[0187] Acceptable standard: The horizontal axis is the logarithm of the theoretical value of the potency, and the vertical axis is the logarithm of the corresponding measured potency value for linear regression. The correlation coefficient of the linear regression equation should not be less than 0.98.

[0188] Verification results: The fitted linear regression equation (see Fig. 9 ). The fitted linear regression equation is y=1.0278x-0.0313, R 2 =0.9923, which is greater than 0.98, indicating that the linearity of the determination method is good.

[0189] (4) Scope

[0190] Acceptable criteria: The reported relative accuracy, intermediate precision and linearity are within the required potency level range, and the quality standard range of relative potency in this range (80% to 150%) is at least covered.

[0191] Verification results: The relative accuracy, intermediate precision and linearity of this experimental method all meet the required potency level range of 50% to 150%, covering its quality standard range (80% to 150%) and meeting the requirements.

[0192] 1.3 Method stability study

[0193] Methods: The 3G10 cell line was serially subcultured and cryopreserved. After the 45th generation, the 5th, 25th, and 45th generation cells were taken and resuscitated at the same time. After three subcultures, the biological activity of FGF21 was detected according to the optimized detection conditions of 1.1 in Example 3, so as to evaluate the influence of different generations of cells, i.e., cell status, on the stability of the activity detection method.

[0194] Results: Fig.10 As shown, the EC50 value of the fifth generation cells was 21.66 ng / ml, the SNR was -39.18, and the R 2 The EC50 value of the 25th generation cells was 23.44 ng / ml, the SNR was -30.98, and the R 2 The EC50 value of the 45th generation cells was 8.656 ng / ml, the SNR was -17.05, and the R 2The value of 0.979 indicates that a good response curve can still be obtained when the 3G10 cell line is continuously passaged to the 45th generation for activity testing. This also reflects that the 3G10 cell line has good stability in activity testing, that is, the cell generation has no significant effect on the biological activity test results of the FGF21 protein.

[0195] 1.4 Method consistency study

[0196] In the prior art, the method that can be used to evaluate the biological activity of FGF21 is the glucose oxidase method (GOD-POD), the principle of which is that glucose oxidase oxidizes glucose into gluconic acid and hydrogen peroxide under the conditions of oxygen and water, and in the presence of a chromogenic oxygen acceptor, a Trinder reaction occurs, that is, catalase oxidizes hydrogen peroxide into water and oxygen, and dehydrogenates and condenses the chromogenic oxygen acceptor 4-aminoantipyrine-coupled phenol into red quinone compounds. The amount of red quinone compounds generated is proportional to the glucose content in the culture medium. FGF21 promotes the absorption of glucose by cells, so the part with a high concentration of FGF21 generates less red quinone substances, while the part with a low concentration generates more red quinone substances, which can be used to detect the biological activity of FGF21.

[0197] In this example, the biological activity of the same FGF21 sample was determined using the glucose oxidase method and the established reporter gene method, and the differences in the detection results of the two were compared.

[0198] (1) Experimental methods

[0199] Glucose oxidase method to measure the biological activity of FGF21: HL7702 cells were diluted with complete medium at a concentration of 1.6-1.8×10 4 100 μl / well, inoculated in a 96-well plate, cultured at 37°C, 5% CO2 for 18-24h. Replace with starvation medium (1640 basal medium + 10% fetal bovine serum + 1% double antibody), and continue starvation culture for 18-24h. Discard the old starvation medium, add FGF21 active reference (pre-dilution concentration 80AU / ml) and the sample to be tested (1200μg / ml), and dilute 6 gradients in a 4-fold series, with 2 replicates each. After continuing to culture for 46-50h, add 200μl of the mixed working solution of the glucose assay kit R1 and R2 to each well in a new 96-well cell culture plate (volume ratio, R1: R2 = 10: 1); then, aspirate 4μl of the supernatant culture solution from each well of the HL-7702 cell plate and add it to the corresponding well of the 96-well plate with the detection working solution. Finally, after incubation at 37°C for 15 min, the absorbance value was measured at a wavelength of 510 nm, and a four-parameter curve was fitted.

[0200] The biological activity of FGF21 was measured by reporter gene method: The biological activity of the same FGF21 was measured according to the optimized detection conditions of 1.1 in Example 3.

[0201] (2) Experimental results

[0202] The present invention uses the reporter gene method and the glucose oxidase method to measure the biological activity of the same FGF21 sample, respectively, and repeats 6 times. The unpaired / independent sample (unpaired) t test is used to perform statistical analysis on the test results. The results show that there is no statistical difference in the test results of the two methods, and they have good consistency (t test, p=0.0563) (see Table 4). In Table 4, the relative potency mean of the RGA method is 96.30%, the standard deviation (SD) is 13.65, and the coefficient of variation (CV) is 14.17%, indicating that in repeated experiments, the results of the RGA method for detecting the biological activity of FGF21 have low variability and high sensitivity, that is, the test results have strong stability and reliability. In addition, as shown in Tables 4 and 5, compared with the glucose oxidase method, the reporter gene method established by the present invention has the characteristics of rapid detection and small method variability. In Table 5, the experimental cycle required for the detection method of the present invention is only 1 day, the cell growth cycle is only 2 days, cell starvation culture is not required, and the detection range is 0.06-1200ng / ml; while the experimental cycle required for the glucose oxidase method is 5 days, the cell growth cycle is 4 days, cell starvation culture is required, and the detection range is 0.3-1200ng / ml. The method for determining the biological activity of FGF21 provided by the present invention greatly shortens the test time, significantly reflects the time advantage of the RGA method for determining the biological activity of FGF21, avoids the long-term cell culture and the generation of uncertain factors to interfere with the experimental results, and reduces the complexity and potential errors in the experimental process. In the RGA method, the test steps are reduced (the test process is simplified), which has advantages in terms of time cost or consumables cost; in addition, the detection range of the RGA method is 0.06-1200 ng / ml, which is more sensitive than the 0.3-1200 μg / ml of the GOD-POD method. This result means that the RGA method can detect samples with lower concentrations and has advantages for micro or trace analysis. Therefore, the detection method provided in this embodiment can reduce the test cost while maintaining high detection quality.

[0203] Table 4. Comparison of RGA and GOD-POD in detecting biological activity of FGF21

[0204]

[0205] Table 5 Comparison of RGA and GOD-POD methods for FGF21 bioactivity determination

[0206]

[0207] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the present invention specification and its drawings are illustrative and do not constitute a limitation of the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The present invention specification contains multiple inventive concepts, such as "preferably" and "according to a preferred embodiment", both of which indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the features guided by "preferably" are only an optional method and should not be understood as being required. Therefore, the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. A method for determining the biological activity of human fibroblast growth factor-21 (FGF21) based on a reporter gene method, characterized in that: The method is based on the activation of MAPK / ERK signaling pathway after FGF21 forms a complex with its receptor and β-Klotho protein, thereby activating ELK-1 / SRF transcription factor, and ELK-1 / SRF transcription factor activates the transcriptional expression of downstream luciferase reporter gene after binding to SER response element. Wherein, the method comprises the following steps: Construction of transgenic cell lines with high FGF21 responsiveness; A plasmid that binds to the SER response element ELK-1 / SRF transcription factor is inserted into the cell; The cell line is cultured, and the biological activity of FGF21 in the sample to be tested is detected by luciferase reporter gene method.

2. The method for determining the biological activity of FGF21 according to claim 1, characterized in that: The method for determining the biological activity of FGF21 is obtained by a method comprising the following steps: Obtain cell lines that stably express FGFR1, β-Klotho, and SRE; Stimulate reporter gene expression with FGF21 and its analogs; The biological activities of FGF21 and its analogs were determined by fitting a four-parameter curve based on the measured reporter gene signal values.

3. The method for determining the biological activity of FGF21 according to claim 1 or 2, characterized in that: The cell line stably expressing FGFR1, β-Klotho and SRE is obtained by a method comprising the following steps: (1) The target cells were infected with lentiviruses that overexpress FGFR1 and β-Klotho, and the MAPK / ERK signaling pathway was analyzed by antibiotic pressure screening and WB to obtain monoclonal cell lines that stably and highly expressed FGFR1 and β-Klotho proteins and were highly responsive to FGF21; (2) Introducing a plasmid containing a reporter gene into the monoclonal cell described in (1), and using antibiotic pressure screening to obtain a monoclonal cell line containing a reporter gene that stably expresses SRE and has a high efficiency in responding to the reporter gene after FGF21 stimulation, and that stably expresses FGFR1, β-Klotho and SRE.

4. The method for measuring the biological activity of FGF21 according to any one of claims 1 to 3, characterized in that: The steps include: (1) Determine the biological activity of FGF21 using reporter gene-containing cell lines that stably express FGFR1, β-Klotho, and SRE; (2) Diluting FGF21 and reference products to the pre-dilution concentration, performing multiple dilutions, and adding them to the cells described in (1) under the incubation conditions of 37° C. and 5% CO2; (3) Add luciferase substrate and fit a four-parameter curve according to the measured reporter gene signal value to determine the biological activity of FGF21.

5. The method for determining the biological activity of FGF21 according to claim 1, characterized in that: HEK293T cells were selected as the cell line containing reporter genes that stably expressed FGFR1, β-Klotho and SRE.

6. The method for determining the biological activity of FGF21 according to claim 1, characterized in that: The reporter gene is the firefly luciferase reporter gene.

7. The method for determining the biological activity of FGF21 according to claim 4, wherein the method detects the change in luciferase gene expression caused by the interaction of FGF21 with transgenic cells, characterized in that: In step (2), the specific steps of the detection method are as follows: (1) The cell resuspension medium, activity detection reaction medium, and dilution medium for FGF21 and reference products were all high-glucose DMEM containing 10% FBS-1.0 μg / ml puromycin-0.25 mg / ml hygromycin B; (2) The number of cells plated was 1.0×10 4 ~6.0×10 4 / hole, preferably 3.0×10 4 / hole; (3) The pre-dilution concentration of FGF21 is 900 to 2000 ng / ml, preferably 1200 ng / mL, (4) the dilution gradient of FGF21 is 3-5 times, preferably 3 times; (5) After adding FGF21, the interaction time with cells is 6 to 12 hours, preferably 8 hours; (6) Read the chemiluminescence value and determine the EC of FGF21 and the reference product by fitting the four-parameter curve 50 The relative biological activity reflects the biological activity of FGF21.

8. A recombinant lentiviral vector, characterized in that: The vector is used for co-overexpressing FGFR1 protein and β-klotho protein, and the vector comprises FGFR1 gene and β-klotho gene, wherein the nucleotide sequence of FGFR1 gene is shown in Seq ID NO.1; the nucleotide sequence of β-klotho gene is shown in Seq ID NO.2, Preferably, the FGFR1 gene and the β-klotho gene are inserted between the BamH I and Xho I sites of the recombinant lentiviral vector. Preferably, the recombinant lentiviral vector can be co-transfected with a lentiviral auxiliary packaging plasmid into a host cell to obtain a packaging body. Preferably, the recombinant lentiviral vector is used to transduce host cells; the expressed FGFR1 protein and β-klotho protein are extracted from the transduced host cells; and the extracted proteins are used to immunize experimental animals, thereby producing monoclonal antibodies that specifically bind to the proteins.

9. A product for measuring the biological activity of FGF21, characterized in that: The product comprises a luciferase substrate and the recombinant lentiviral vector as claimed in claim 8.

10. A method for determining the biological activity of human fibroblast growth factor-21 (FGF21), characterized in that: The method comprises: Construction of a FGF21-high responsive cell line containing the FGFR1 gene and the β-klotho gene; culturing the cell line; The biological activity of FGF21 in the test samples was detected by luciferase reporter gene method.

11. A monoclonal antibody, characterized in that By transducing host cells with the recombinant lentiviral vector as described in claim 8; by extracting the expressed FGFR1 protein and β-klotho protein from the transduced host cells; and using the extracted proteins to immunize experimental animals, thereby producing monoclonal antibodies that specifically bind to the proteins, wherein the monoclonal antibodies can specifically bind to at least one epitope of the FGFR1 protein and / or the β-klotho protein.

12. Use of the methods of claims 1 to 7 and 10, the recombinant lentiviral vector and packaging body of claim 8, the monoclonal antibody of claim 11, and the product of claim 9 in the quality control of new candidate drugs for treating chronic diseases such as diabetes, hyperlipidemia and NASH.

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