CN115141809A - Method and kit for detecting biological activity of GNRH (GNRH) binding protein - Google Patents
Method and kit for detecting biological activity of GNRH (GNRH) binding protein Download PDFInfo
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Abstract
The invention relates to a method and a kit for detecting the biological activity of GNRH binding protein, belonging to the field of medical detection. The invention aims to rapidly detect the biological activity of GNRH binding protein, and particularly relates to a method for rapidly detecting the biological activity of GNRH binding protein, which comprises the steps of constructing and screening Chinese hamster ovary cells CHOK1/GnRHR/NFAT co-expressing GNRHR and NFAT-luciferase reporter genes, then incubating a mixture containing the GNRH binding protein and effector cells, adding an enzyme reaction substrate after incubation, and fitting a four-parameter curve according to a measured signal value to determine the biological activity of the GNRH binding protein.
Description
Technical Field
The invention belongs to the field of biological activity detection of biological medicines, and particularly relates to a biological activity method for detecting GNRH binding protein.
Background
The Gonadotropin-releasing hormone (GNRH) is secreted from the hypothalamus, stimulates or inhibits the secretion of pituitary gonadotropins, plays an important role in the regulation of vertebrate reproduction, and has considerable activity potential, and GNRHR (Gonadotropin-releasing hormone Receptor) is a Receptor thereof.
The method for detecting biological activity mostly adopts a cell-based biological activity measuring method, and mainly comprises a cell proliferation inhibition method, a cytotoxicity method, an antibody-dependent cell-mediated cytotoxicity method, a complement-dependent cytotoxicity method, a cell competition ELISA method and a reporter gene method. The biological activity detection method of the reporter gene method is to construct a transgenic cell line expressing luciferase reporter genes and reflect the pharmaceutical activity of monoclonal antibodies by detecting luciferase chemiluminescence signals. Compared with a cell proliferation inhibition method, the reporter gene method has the characteristics of short experimental period and small variation, is suitable for evaluating biological activity of a plurality of samples in a short time, and is a rapid detection method developed in recent years.
Currently, there is still a need in the art for novel assay methods for the determination of the biological activity of GNRH binding proteins, and no report related to the use of reporter gene methods for detecting the biological activity of GNRH binding proteins is found.
Disclosure of Invention
The purpose of the invention is as follows: at present, aiming at the determination of the biological activity of the GNRH-binding protein, the invention provides a method and a kit for detecting the biological activity of a GNRH-binding protein drug.
The technical scheme is as follows:
in a first aspect, the invention provides a method for detecting the biological activity of a GNRH binding protein by constructing effector cells co-expressing GNRHR and a reporter gene with an immune-related signaling pathway response element, incubating a mixture comprising GNRH binding protein and effector cells, adding an enzyme reaction substrate after incubation, and determining the biological activity of GNRH binding protein by fitting a four parameter curve based on the measured signal values.
In some embodiments, the GNRH binding protein is a GNRH agonist or inhibitor.
In some embodiments, the reporter gene with an immune-related signaling pathway response element is CRE-luciferase, NFAT-luciferase, SRE-luciferase, NFAT-CRE-luciferase, and most preferably NFAT-luciferase.
In some embodiments, the effector cell is a CHO-K1 cell, an Expi293 cell, a 293T cell, and most preferably a CHO-K1 cell.
In some embodiments, the effector cell is a chinese hamster ovary cell CHOK1/GNRHR/NFAT co-expressing GNRHR and NFAT-luciferase reporter gene, which is deposited at the chinese type culture collection at 22 months 7/2022 at the collection address of university of wuhan, with the collection number of CCTCC NO: C2022163.
in some embodiments, the GNRH binding protein is a GNRH agonist and the method steps for detecting the biological activity of the GNRH agonist are as follows:
(1) Constructing and screening to obtain effector cells of the co-expression GNRHR and the reporter gene with the immune-related signal channel response element;
(2) Performing gradient dilution on the GNRH agonist, and respectively incubating the GNRH agonist after the gradient dilution with the effector cells in the step (1);
(3) Luciferase reaction substrate was added and the biological activity of GNRH agonists was determined by fitting a four parameter curve to the measured signal values.
In some embodiments, the GNRH agonist is GNRH.
In some embodiments, the GNRH binding protein is a GNRH inhibitor and the method steps for detecting the biological activity of the GNRH inhibitor are as follows:
(1) Constructing and screening to obtain effector cells of the co-expression GNRHR and the reporter gene with the immune-related signal channel response element;
(2) Performing gradient dilution on the GNRH inhibitor, and respectively incubating the GNRH inhibitor after the gradient dilution with the GNRH agonist and the effector cells in the step (1);
(3) Luciferase reaction substrate was added and the biological activity of the GNRH inhibitor was determined by fitting a four parameter curve to the measured signal values.
In some embodiments, the GNRH inhibitor is ganirelix acetate injection.
In some embodiments, the concentration of the GNRH agonist in the method for detecting a biological activity of a GNRH inhibitor is 0.005 to 0.05nm. In some embodiments, the concentration of the GNRH agonist in the method for detecting the biological activity of a GNRH inhibitor is preferably from 0.007 to 0.04nm. In some embodiments, the concentration of the GNRH agonist in the method of detecting a biological activity of a GNRH inhibitor is more preferably from 0.008 to 0.03nm, more preferably from 0.008nM, 0.009nM, 0.01nM, 0.02nM, 0.03nM, and most preferably 0.01nM. In some embodiments, the concentration of the GNRH agonist in the method of detecting a biological activity of a GNRH inhibitor is 0.01nM.
In some embodiments, the cell density of the effector cells used for incubation in detecting GNRH binding protein is 3 × 10 6 ~5×10 6 cell/mL, preferably from 3.3X 10 6 ~5×10 6 cell/mL, more preferably from 3.3X 10 6 ~4.7×10 6 cell/mL, more preferably from 3.5X 10 6 cell/mL、3.6×10 6 cell/mL、3.7×10 6 cell/mL、3.8×10 6 cell/mL、3.9×10 6 cell/mL、4×10 6 cell/mL、4.1×10 6 cell/mL、4.2×10 6 cell/mL、4.3×10 6 cell/mL、4.4×10 6 cell/mL、4.5×10 6 cell/mL、4.6×10 6 cell/mL、4.7×10 6 cell/mL, most preferably 4X 10 6 cell/mL. In some embodiments, the assay comprisesThe effector cell density for incubation was 4X 10 when measuring GNRH-binding proteins 6 cell/mL. In some embodiments, the effector cells used for incubation in detecting the GNRH-binding protein are CHOK1/GnRHR/NFAT Chinese hamster ovary cells co-expressing the GNRHR and NFAT-luciferase reporter gene, and the cell density of the cells is 4X 10 6 cell/mL. In some embodiments, the effector cells used for incubation in detecting GNRH binding protein are chinese hamster ovary cells CHOK1/GNRHR/NFAT co-expressing GNRHR and NFAT-luciferase reporter, deposited at the chinese type culture collection at 22/7/2022 at the deposition address of university of wuhan, china with the deposition number CCTCC NO: c2022163, CHOK1/GnRHR/NFAT, wherein the cell density of the deposited cells is 4 × 10 6 cell/mL。
In some embodiments, the incubation conditions for detecting GNRH binding protein are: 35 to 38 ℃ and 7 to 10% of CO 2 The incubation was performed under shaking conditions for 5 to 7 hours. In some embodiments, the incubation conditions for detecting GNRH binding protein are: preferably 36 to 37 ℃ and 7 to 9% by weight of CO 2 Incubating for 5.5 to 7 hours under the condition of (1). In some embodiments, the incubation conditions for detecting GNRH binding protein are: more preferably from 36.5 to 37 ℃ and from 7 to 9% by weight of CO 2 The incubation is performed under the condition of (1) for 5.5 to 6.5 hours with shaking. In some embodiments, the incubation conditions for detecting GNRH binding protein are: most preferably 36.5 ℃ and 8% CO 2 Conditions (2) were incubated with shaking for 6 hours.
In some embodiments, after incubation, the volume of the enzyme reaction substrate added to the incubation mixture is 1 to 1.5, preferably from 1 to 1.3, more preferably from 1 to 1.2, and most preferably from 1.
In a second aspect, the present invention provides a composition for determining the biological activity of a GNRH binding protein, comprising the following components: 1) Chinese hamster ovary cells CHOK1/GnRHR/NFAT co-expressing GNRHR and NFAT-luciferase reporter genes are preserved in China center for type culture Collection in 7 months and 22 days 2022 at the preservation address of university of Wuhan, china with the preservation number of CCTCC NO: c2022163; 2) Luciferase reaction substrate.
In a third aspect, the present invention provides a kit for determining the biological activity of a GNRH binding protein, said kit comprising a composition according to the second aspect.
In a fourth aspect, the invention provides a Chinese hamster ovary cell CHOK1/GnRHR/NFAT co-expressing a GNRHR and NFAT-luciferase reporter gene, which is preserved in the China center for type culture Collection in 2022, 7 months and 22 days, wherein the preservation address is Wuhan university in China, the preservation number is CCTCC NO: C2022163.
has the beneficial effects that:
(1) The biological activity of the GNRH agonist and the inhibitor is detected by adopting a reporter gene method for the first time;
(2) Successfully constructing and screening CHO-K1 cells co-expressing GNRH and NFAT-luciferase and sending the cells to the China center for type culture Collection for preservation;
(3) The method improves and optimizes the step conditions and the like of the detection method, and is more favorable for obtaining accurate experimental results.
Defining:
as used herein, the terms "NFAT", "CRE" and "SRE" are each response elements, i.e., short DNA sequences in the promoter region of a gene that bind to a specific transcription factor and regulate transcription of the gene.
The terms "CHO-K1 cells", "Expi293 cells" and "293T cells" as used herein refer to cell lines used in rapid, scalable suspension culture systems.
The term "Chinese hamster ovary cells CHOK1/GnRHR/NFAT" as used herein is the name of the deposited cells on the proof of deposit, and represents Chinese hamster ovary cells capable of stably co-expressing GNRHR and NFAT-luciferase.
Drawings
FIG. 1 is a bar graph of CHO-K1 cell density most useful for a method of detecting the biological activity of GNRH-binding proteins;
FIG. 2 is a four parameter plot of GNRH agonists stimulating CHO-K1 cells stably co-expressing GNRHR and NFAT-luciferase;
FIG. 3 is a four parameter plot of GNRH inhibitors inhibiting CHO-K1 cells stably co-expressing GNRHR and NFAT-luciferase;
FIG. 4 is a bar graph of signal to noise ratio of CHO-K1 monoclonal cells stably co-expressing GNRHR and NFAT-luciferase;
FIG. 5 is a four parameter plot of CHO-K1 monoclonal cells 4 stably co-expressing GNRHR and NFAT-luciferase following GNRH agonist stimulation screening;
FIG. 6 is a four parameter plot of CHO-K1 monoclonal cells 4 stably co-expressing GNRHR and NFAT-luciferase following GNRH inhibitor inhibition screening;
FIG. 7 is a four parameter plot obtained in a methodological validation.
Detailed Description
For the purpose of illustrating the objects, technical solutions and advantages of the present invention in detail, the present invention will be clearly illustrated by combining specific experiments.
Experimental materials
Untransfected CHO-K1 cells: from ATCC, cat No.: CCL-61; PURO: puromycin: from Biyun sky, with the product number ST551; and (3) NeoR: geneticin: from Biyun, with a cargo number ST081; culture medium 1: from Aopumai, cat # C602502; GNRH agonists: gnRH, from Genscript, cat.no.: c837YGJ110-1/PE8571; GNRH inhibitors: ganirelix acetate injection: from MERCK & co, inc, cat No.:0001297375; luciferase reaction substrate: from a luciferase reporter gene detection kit, wherein the kit is from Novozan company and has the cargo number DD1201; an enzyme-labeling instrument: from BioTek; carbon dioxide high-speed shaking incubator: derived from SEELEY; enzyme label plate: from Shanghai Jing' an, cat # J09602; deep hole plate: from Wuxi naisi, cat # 503062; a centrifuge: is from the middle-aged or the senior, and has a goods number of KDC-30; endotoxin-free plasmid extraction kit: from Sigma, cat No.: A33073.
example 1
1.1 construction of plasmids
Synthesizing NFAT gene by adopting a whole gene synthesis technology, adding SpeI enzyme cutting site at the 5 'end of the NFAT gene, adding BstBI enzyme cutting site at the 3' end of the NFAT gene, then constructing the synthesized NFAT gene to a carrier PGL-luc-NeoR (the carrier is obtained by reverse PCR) through the upstream SpeI enzyme cutting site and the downstream BstBI enzyme cutting site, and carrying out small extraction preparation of endotoxin-free plasmid by using an endotoxin-free plasmid extraction kit to obtain PGL-luc-NFAT-NeoR plasmid.
Synthesizing a Human GNRHR gene by adopting a whole gene synthesis technology, adding a HindIII single enzyme cutting site at the 5' end of the Human GNRHR gene, constructing the synthesized Human GNRHR gene to a vector PB-PurOR vector (containing a Puror screening marker) through an upstream HindIII single enzyme cutting site, and performing small extraction preparation of an endotoxin-free plasmid by using an endotoxin-free plasmid extraction kit to obtain a PCD004-PURO-Human GNRHR plasmid.
1.2 transfection of CHO-K1 cells
PCD004-PURO-Human GNRHR plasmids and PGL-luc-NFAT-NeoR plasmids are both introduced into CHO-K1 cells by an electrotransfer method. The transfection was carried out using 125mL shake flasks at a cell density of greater than 1X 10 6 cell/mL, volume of medium 1 was 10mL, and plasmid dose was 20. Mu.g. The voltage is 300V, and the pulse time is 20 ms/time. After 48 hours after the electrotransfer, the medium was changed to pressure culture, and the selection pressure was PURO 10. Mu.g/mL and NeoR 300. Mu.g/mL. And detecting after passage for 3-5 times.
1.3 CHO-K1 cell density screening
Untransfected CHO-K1 cells as a blank were counted in samples of CHOK1/GnRHR/NFAT Chinese hamster ovary cells stably co-expressing the GNRHR and NFAT-luciferase reporter, resuspended in 15.6mL assay medium (99% DMEM + 1% FBS) after centrifugation and diluted to the cell density as described in Table 1; GNRH agonist was diluted to 0.01nM using assay medium (99% DMEM + 1% FBS). Deep well plating was performed according to the system of table 1, and two types of CHO-K1 cells were mixed with GNRH agonist, respectively:
placing the deep-well plate containing the mixture in a carbon dioxide high-speed shaking incubator at 36.5 deg.C and 8% CO 2 After culturing for 6 hours under the conditions of (1), the deep-well plate was taken out and left at room temperature for 30 min. 100 μ L of the mixture was taken from each well of the deep well plate and addedIn the enzyme label plate, the mixture and luciferase reaction substrate are 1:1, adding a luciferase reaction substrate into the ELISA plate, namely adding 100 mu L of luciferase reaction substrate into each hole; standing at room temperature for at least 3 min, detecting with microplate reader for luminescence, and processing with Graphpad8.0 software to obtain histogram (FIG. 1). Final selection Chinese hamster ovary cells CHOK1/GnRHR/NFAT co-expressing the GNRHR and NFAT-luciferase reporter were set to a cell density of 4X 10 6 cell/mL, ready for use.
1.4 detection of GNRH agonists in CHO-K1 cells
Taking untransfected CHO-K1 cells as blank control, chinese hamster ovary cells CHOK1/GnRHR/NFAT stably co-expressing GNRHR and NFAT-luciferase reporter genes and untransfected CHO-K1 cells are respectively diluted to the density of 4 × 10 6 cells/mL, and each separately plated to a deep well plate at a volume of 390. Mu.L per well; GNRH agonists were diluted to 1nM, 0.1nM, 0.01nM, 1X 10, respectively -3 nM、1×10 -4 nM、1×10 -5 nM、1×10 -6 nM, 0nM, and gradient concentrations of GNRH agonist added to the deep well plate at a volume of 10 μ L per well, mixing the two CHO-K1 cells with GNRH agonist separately; placing the deep-well plate containing the mixture in a carbon dioxide high-speed shaking incubator at 36.5 deg.C and 8% CO 2 After 6 hours of incubation, 100. Mu.L of the mixture was taken from each well of the deep well plate and added to the microplate in a ratio of 1:1, adding a luciferase reaction substrate into the enzyme-linked immunosorbent assay plate, namely adding 100 mu L of the luciferase reaction substrate; standing at room temperature for at least 3 min, detecting with microplate reader for luminescence, and fitting with data processing using Graphpad8.0 to obtain an inverted S-shaped four-parameter curve (FIG. 2).
1.5 detection of GNRH inhibitor medicine by CHO-K1 cell
Taking untransfected CHO-K1 cells as blank control, the cell density of Chinese hamster ovary cells CHOK1/GnRHR/NFAT stably co-expressing GNRHR and NFAT-luciferase reporter genes and untransfected CHO-K1 cells is respectively diluted to 4 x 10 6 cells/mL, and each separately plated to a deep well plate at a volume of 370. Mu.L per well; dividing the concentration of GNRH inhibitor intoDiluting to 1 × 10 4 nM、1×10 3 nM, 100M, 10nM, 1nM, 0.1nM, 0nM, and each graded concentration of GNRH inhibitor was added separately to a deep well plate in a volume of 20. Mu.L per well; diluting the GNRH agonist to 0.01nM, adding to a deep well plate in a volume of 10. Mu.L per well, and mixing the two CHO-K1 cells, the GNRH inhibitor and the GNRH agonist at each gradient concentration; placing the deep-well plate containing the above mixture in a carbon dioxide high speed shaking incubator at 36.5 deg.C and 8% 2 After 6 hours of incubation, 100. Mu.L of the mixture was taken from each well of the deep-well plate and added to the microplate in a ratio of 1:1, adding a luciferase reaction substrate into the ELISA plate, namely adding 100 mu L of the luciferase reaction substrate; standing at room temperature for at least 3 min, detecting with microplate reader for luminescence, and fitting with data processing using Graphpad8.0 to obtain an inverted S-shaped four-parameter curve (FIG. 3).
1.6 Experimental results
The result shows that the constructed Chinese hamster ovary cells CHOK1/GnRHR/NFAT stably co-expressing the GNRHR and NFAT-luciferase reporter gene have better luciferase expression reactivity on the stimulation effect of a GNRH agonist and the inhibition effect of a GNRH inhibitor (shown in a figure 2-3), so the Chinese hamster ovary cells CHOK1/GnRHR/NFAT stably co-expressing the GNRHR and NFAT-luciferase reporter gene constructed by the method can be used as experimental cells.
Example 2
2.1 monoclonal screening of CHO-K1 cells
After the cell density and viability of the Chinese hamster ovary cells CHOK1/GnRHR/NFAT stably co-expressing the GNRHR and NFAT-luciferase reporter gene are recovered, the well plate is deeply paved by a limiting dilution method according to the density of 0.5 per well to screen the monoclone. During cell growth, which wells were monoclonals were observed and marked, and when the confluency of cells in the monoclonals reached more than 50%, cells were transferred and expanded for culture, with clone numbers 1-6 being marked. The density of each Chinese hamster ovary cell CHOK1/GnRHR/NFAT monoclonal cell stably co-expressing GNRHR and NFAT-luciferase reporter gene is diluted to 4 multiplied by 10 6 cell/mL。
Separately, hamster ovary cells CHOK1/GnRHR/NFAT stably overexpressing GNRHR and NFAT-luciferase in example 1 were used as a control, and monoclonal cells selected from 6 strains were plated in a deep well plate at a volume of 390. Mu.L/well; the GNRH agonist concentration was diluted to 0.01nM and added to the deep well plate in a volume of 10 μ L per well, and the two types of CHO-K1 cells were mixed with GNRH agonist, respectively; placing the deep-well plate containing the mixture in a carbon dioxide high-speed shaking incubator at 36.5 deg.C and 8% CO 2 After 6 hours of incubation, the deep well plate was removed and left at room temperature for 30 min. And (3) taking 100 mu L of mixture from each hole of the deep-hole plate, adding the mixture into an enzyme label plate, and mixing the mixture according to the ratio of the detection reagent to the luciferase reaction substrate of 1:1, adding a luciferase reaction substrate into the ELISA plate, namely adding 100 mu L of luciferase reaction substrate into each hole; standing at room temperature for at least 3 min, detecting with microplate reader for luminescence, and fitting with data processing using Graphpad8.0 to obtain an inverted S-shaped four-parameter curve (FIG. 4).
The signal to noise ratio of a hamster ovary cell CHOK1/GnRHR/NFAT monoclonal cell 4 stably co-expressing GNRHR and NFAT-luciferase reporter gene is the highest, the monoclonal cell 4 is selected for cell preservation, the cell is preserved in China type culture Collection in 2022 year 7-month-22, the preservation address is China. C2022163.
2.2 GNRH agonist assay of Chinese hamster ovary cells CHOK1/GnRHR/NFAT monoclonal cells 4
Taking untransfected CHO-K1 cells as blank control, the cell densities of Chinese hamster ovary cells CHOK1/GnRHR/NFAT monoclonal cells 4 and untransfected CHO-K1 cells were respectively diluted to 4 × 10 6 cells/mL, and each separately plated to a deep well plate at a volume of 390. Mu.L per well; GNRH agonists were diluted to 1nM, 0.1nM, 0.01nM, 1X 10, respectively - 3 nM、1×10 -4 nM、1×10 -5 nM、1×10 -6 nM, 0nM, adding the gradient GNRH agonist to each well of the deep well plate in a volume of 10. Mu.L per well, mixing the two CHO-K1 cells with the GNRH agonist, respectively; placing the deep hole plate filled with the mixture in a carbon dioxide high-speed shaking incubatorAt 36.5 deg.C, 8% CO 2 After 6 hours of incubation in the incubator, 100. Mu.L of the mixture was taken from each well of the deep-well plate and added to the microplate in a ratio of 1:1, adding a luciferase reaction substrate into the ELISA plate, namely adding 100 mu L of the luciferase reaction substrate; the mixture was allowed to stand at room temperature for at least 3 min, subjected to luminescence detection using a microplate reader, and subjected to data processing using Graphpad8.0 to fit an inverted S-shaped four-parameter curve (FIG. 5).
2.3 detection of GNRH inhibitors in Chinese hamster ovary cells CHOK1/GnRHR/NFAT monoclonal cells 4
Taking untransfected CHO-K1 cells as blank control, the cell density of Chinese hamster ovary cells CHOK1/GnRHR/NFAT monoclonal cells 4 and untransfected CHO-K1 cells were respectively diluted to 4 × 10 6 cells/mL, and each in 370. Mu.L per well volume separately to deep well plate; the GNRH inhibitor was diluted to 1X 10 concentrations 4 nM、1×10 3 nM, 100M, 10nM, 1nM, 0.1nM, 0nM, and 20. Mu.L per well of each gradient of GNRH inhibitor added to each well of the deep well plate; diluting the GNRH agonist to 0.01nM, spreading the diluted GNRH agonist to a deep well plate in a volume of 10. Mu.L per well, and mixing the two CHO-K1 cells, the GNRH inhibitor and the GNRH agonist at each gradient concentration; placing the deep-well plate containing the above mixture in a carbon dioxide high speed shaking incubator at 36.5 deg.C and 8% CO 2 After 6 hours of incubation in the incubator, 100. Mu.L of the mixture was taken from each well of the deep-well plate and added to the microplate in a ratio of 1:1, adding a luciferase reaction substrate into the ELISA plate, namely adding 100 mu L of the luciferase reaction substrate; the mixture was allowed to stand at room temperature for at least 3 min, subjected to luminescence detection using a microplate reader, and subjected to data processing using Graphpad8.0 to fit an inverted S-shaped four-parameter curve (FIG. 6).
2.4, results of the experiment
The result shows that the CHOK1/GnRHR/NFAT monoclonal cell 4 stably co-expressing the GNRHR and the NFAT-luciferase reporter gene obtained by screening has better luciferase expression reactivity to the stimulation effect of a GNRH agonist and the inhibition effect of a GNRH inhibitor (figures 5-6), so the CHOK1/GnRHR/NFAT monoclonal cell 4 stably co-expressing the GNRHR and the NFAT-luciferase reporter gene obtained by screening by the method can be used for detecting the biological activities of the GNRH agonist and the inhibitor.
Example 3 methodological validation
The samples and standards shown in table 2 were diluted with assay medium (99% DMEM + 1% FBS) and the final concentrations of the samples and standards were recorded in table 3:
a system as shown in table 4 was prepared:
the above system was spread on a deep well plate, and the above described Chinese hamster ovary cells CHOK1/GnRHR/NFAT monoclonal cells 4, GNRH inhibitor (standard or sample) and GNRH agonist were mixed at each gradient concentration.
Placing the deep-well plate containing the mixture in a high-speed shaking incubator with carbon dioxide at 36.5 deg.C and 8% CO 2 After culturing for 6 hours, the deep-well plate containing the mixture was taken out and left at room temperature for 30 min. From each well of the deep well plate, 100. Mu.L of the mixture was added to the microplate, and the ratio of the mixture to the luciferase reaction substrate per well was 1:1, adding a luciferase reaction substrate into the ELISA plate, namely adding 100 mu L of the luciferase reaction substrate; standing at room temperature for at least 3 min, detecting with enzyme labeling instrument, processing data with Graphpad8.0 software, fitting with reverse S-shaped four-parameter curve (FIG. 7), and obtaining IC50 and R of the sample and standard 2 See table 5. Thus, it was concluded that reporter gene assays, which can be used to detect the biological activity of GNRH-binding proteins, meet the acceptance criteriaThe biological activity of the GNRH binding protein was measured.
Example 4
A kit for determining the biological activity of a GNRH binding protein, the kit comprising a luciferase reaction substrate and the chinese hamster ovary cells CHOK1/GNRHR/NFAT co-expressing the GNRHR and NFAT-luciferase reporter gene deposited in example 2. The luciferase reaction substrate is not limited to be derived from a luciferase reporter gene detection product with the product number DD1201 of Novowed company; the preserved Chinese hamster ovary cells CHOK1/GnRHR/NFAT co-expressing the GNRHR and NFAT-luciferase reporter gene are preserved in the China center for type culture Collection at 7/22/2022 at the preservation address of China, wuhan university, with the preservation number of CCTCC NO: C2022163.
the method used in the kit was according to the experimental procedures 2.2 and 2.3 of example 2.
Claims (16)
1. A method for detecting the biological activity of GNRH-binding protein is characterized in that effector cells which co-express GNRHR and a reporter gene with an immune-related signal path response element are constructed, then a mixture containing the GNRH-binding protein and the effector cells is incubated, an enzyme reaction substrate is added after incubation, and the biological activity of the GNRH-binding protein is determined by fitting a four-parameter curve according to a measured signal value; the effector cells are Chinese hamster ovary cells CHOK1/GnRHR/NFAT which co-express GNRHR and NFAT-luciferase reporter genes, and are preserved in the China center for type culture Collection in 7 months and 22 days 2022, wherein the preservation address is China, wuhan university, the preservation number is CCTCC NO: C2022163.
2. the method of claim 1, wherein the GNRH-binding protein is a GNRH agonist or inhibitor.
3. The method of claim 1, wherein the GNRH binding protein is a GNRH agonist and the step of detecting the biological activity of the GNRH agonist is as follows: (1) Constructing and screening to obtain effector cells of the co-expression GNRHR and the reporter gene with the immune-related signal channel response element; (2) Performing gradient dilution on the GNRH agonist, and respectively incubating the GNRH agonist after the gradient dilution with the effector cells in the step (1); (3) Luciferase reaction substrate was added and the biological activity of GNRH agonists was determined by fitting a four parameter curve to the measured signal values.
4. The method of claim 1, wherein the GNRH binding protein is a GNRH inhibitor and the step of detecting the biological activity of the GNRH inhibitor is as follows: (1) Constructing and screening to obtain effector cells of the co-expression GNRHR and the reporter gene with the immune-related signal channel response element; (2) Performing gradient dilution on the GNRH inhibitor, and respectively incubating the GNRH inhibitor after the gradient dilution with the GNRH agonist and the effector cells in the step (1); (3) Luciferase reaction substrate was added and the biological activity of the GNRH inhibitor was determined by fitting a four parameter curve to the measured signal values.
5. The method of claim 3 or 4, wherein the effector cell density at the time of incubation is 3 x 10 6 ~5×10 6 cell/mL。
6. The method of claim 3 or 4, wherein the effector cell density is 3.3 x 10 upon incubation 6 ~5×10 6 cell/mL。
7. The method of claim 3, wherein the GNRH agonist is GNRH.
8. The method of claim 4, wherein the GNRH inhibitor is a ganirelix acetate injection.
9. The method of claim 4, wherein the GNRH agonist is present at a concentration of 0.005 to 0.05nM.
10. The method of claim 4, wherein the GNRH agonist is at a concentration of 0.007 to 0.04nM.
11. The method according to claim 1, wherein the GNRH-binding protein is detected by shake incubation for 5 to 7 hours under conditions of 35 to 38 ℃ and 7 to 10% CO2.
12. The method according to claim 1, wherein after the incubation, the volume of the enzyme reaction substrate added to the incubation mixture is 1 to 1.5.
13. The method according to claim 1, wherein after the incubation, the volume of the enzyme reaction substrate added to the incubation mixture is 1 to 1.3.
14. A composition for determining the biological activity of a GNRH binding protein comprising the following components: 1) Chinese hamster ovary cells CHOK1/GnRHR/NFAT co-expressing GNRHR and NFAT-luciferase reporter genes are preserved in China center for type culture Collection in 7 months and 22 days 2022 at the preservation address of university of Wuhan, china with the preservation number of CCTCC NO: c2022163; 2) Luciferase reaction substrate.
15. A kit for determining the biological activity of a GNRH binding protein, comprising the composition of claim 14.
16. Chinese hamster ovary cells CHOK1/GnRHR/NFAT co-expressing GNRHR and NFAT-luciferase reporter genes are preserved in China center for type culture Collection in 7 months and 22 days 2022 at the preservation address of university of Wuhan, china with the preservation number of CCTCC NO: C2022163.
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| CN104884457A (en) * | 2013-10-30 | 2015-09-02 | 上海恒瑞医药有限公司 | Pyrazolopyrimidone or pyrrolotriazone derivatives, method of preparing same, and pharmaceutical applications thereof |
| CN108949904A (en) * | 2018-07-26 | 2018-12-07 | 南京维立志博生物科技有限公司 | A method of measurement LAG3 protein binding molecule biological activity |
| CN110418799A (en) * | 2017-01-20 | 2019-11-05 | 免疫系统调节控股有限公司 | New compound (immunostimulatory peptides) |
| CN112961832A (en) * | 2021-03-05 | 2021-06-15 | 上海交通大学 | Cell strain and preparation method and application thereof |
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| CN104884457A (en) * | 2013-10-30 | 2015-09-02 | 上海恒瑞医药有限公司 | Pyrazolopyrimidone or pyrrolotriazone derivatives, method of preparing same, and pharmaceutical applications thereof |
| CN110418799A (en) * | 2017-01-20 | 2019-11-05 | 免疫系统调节控股有限公司 | New compound (immunostimulatory peptides) |
| CN108949904A (en) * | 2018-07-26 | 2018-12-07 | 南京维立志博生物科技有限公司 | A method of measurement LAG3 protein binding molecule biological activity |
| CN112961832A (en) * | 2021-03-05 | 2021-06-15 | 上海交通大学 | Cell strain and preparation method and application thereof |
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