In vitro biological activity determination method of follicle-stimulating hormone based on cAMP

Abstract

The present disclosure relates to a method for determining the biological activity of follicle-stimulating hormone in vitro based on cAMP, the method comprising: (1) constructing effector cells that stably express FSHR-fluorescent protein reporter genes; (2) culturing effector cells, then adding FSH protein samples and continuing to culture; (3) after cell lysis, fitting is performed based on the cAMP content in the measured cell lysate to determine the biological activity of FSH. The present disclosure can be effectively applied to the detection of the biological activity of recombinant pFSH-Fc fusion protein in vitro by constructing effector cells that stably express FSHR-fluorescent protein reporter genes and based on the cAMP content in the measured cell lysate.

Description

Technical Field

[0001] The present disclosure mainly relates to the field of biological detection, and in particular to an in vitro determination method of FSH biological activity based on the cAMP content in cells. Background Art

[0002] Follicle-stimulating hormone (FSH), also known as follicle-stimulating hormone, is a glycoprotein hormone synthesized and secreted by the pituitary gland. The FSH molecule is a heterodimer composed of two subunits, the α subunit and the β subunit, which are non-covalently bound. In the same species, the amino acid sequence of the α subunit of FSH is the same as that of other glycoprotein hormones such as luteinizing hormone (LH) and thyroid stimulating hormone (TSH), which consists of 92-96 amino acids (different mammals have different compositions). The FSH β subunit gene consists of 109-115 amino acids, which determines the biological specificity of FSH. Neither the α nor the β subunit alone has biological activity, and can only play a biological role when the two are combined.

[0003] In women, the function of FSH is to promote the development and maturation of follicles, and to cooperate with luteinizing hormone (LH) to promote the secretion of estrogen and ovulation of mature follicles, and participate in the formation of normal menstruation. FSH can promote the development and maturation of follicles. If the FSH level is lower than normal, you may suffer from diseases such as polycystic ovary syndrome. In animal husbandry, FSH is often used in animal superovulation, in vitro fertilization, etc., and is closely related to animal littering.

[0004] Bioactivity assay is an important indicator to characterize the correctness of product molecular structure and function, and the effectiveness of product. As of 2021, the rat ovarian weight gain method is still the only FSH in vivo activity assay method approved and recognized by domestic and foreign regulatory agencies. However, animal experiments have the disadvantages of large individual differences, long experimental cycles, large variability, low sensitivity to activity changes, and high operating requirements, which restrict its use in the research and development process. With the international attention to animal welfare, it has become a consensus to vigorously develop alternative methods for animal experiments in the field of activity evaluation in drug research and development. Cell-based in vitro bioactivity analysis has been widely used in the evaluation and release inspection of monoclonal antibodies and recombinant cytokine products. In recent years, the reporter gene method has emerged in the field of in vitro cell activity assay of biotechnology drugs in my country, and has gradually become a platform technology. The reporter gene method for detecting the biological activity of type I interferon was included in the 2015 edition of the "Pharmacopoeia of the People's Republic of China". In addition, a variety of biological products have successfully developed the reporter gene method into a biological activity release method.

[0005] It is common to construct human reporter gene cell lines using CHO cells, but there is uncertainty in the expression of porcine genes. It is uncertain whether porcine FSHR can be expressed in CHO-K1 cell lines, and there is a problem of low expression level, which leads to difficulties in screening. Therefore, there is an urgent need to develop a high-precision in vitro method for detecting the biological activity of porcine FSH without the use of experimental animals. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present disclosure provides a method for determining the biological activity of FSH, which utilizes p2A to connect pFSHR and a reporter gene (such as eGFP) and express them in cells (such as CHO-K1 cell line), which is the first porcine FSHR reporter gene cell line. After 2A peptide cleavage, the fluorescence in the cells can be well expressed, which is conducive to screening and convenient for application in the detection of the biological activity of recombinant pFSH fusion protein in vitro.

[0007] The present disclosure provides a method for determining the biological activity of FSH, the method comprising:

[0008] (1) Constructing effector cells that stably express the FSHR-fluorescent protein reporter gene;

[0009] (2) Cultivate the effector cells, then add the FSH protein sample and continue culturing;

[0010] (3) After the cells are lysed, the FSH biological activity is determined by fitting based on the cAMP content in the measured cell lysate;

[0011] Preferably, the fitting is a four-parameter fitting.

[0012] 2A peptides are short peptides (18-25 amino acids) derived from viruses. They are often called "self-cleaving" peptides, which can produce multiple proteins from one transcript. 2A peptides are not completely "self-cleaving", but work by causing the ribosome to skip the synthesis of the glycine and proline peptide bonds at the C-terminus of the 2A element, ultimately resulting in the separation of the end of the 2A sequence and the downstream product. The C-terminus of the upstream protein will have some additional 2A residues added, while the N-terminus of the downstream protein will have additional prolines. There are currently four commonly used 2A peptides, P2A, T2A, E2A and F2A, which are derived from four different viruses. Among the four commonly used 2A peptides (P2A, T2A, E2A and F2A), P2A usually has the highest cleavage efficiency (close to 100% in some cases), followed by T2A, and then E2A and F2A. The cleavage efficiency of F2A is only about 50%, and it is usually recommended to use P2A or T2A in polycistronic. Inserting a signal peptide gene sequence upstream or downstream of the 2A peptide can localize the target gene to the nucleus, chloroplasts, mitochondria, membranes or cytoplasmic microtubules after translation. Self-cleaving 2A peptides have important applications in the field of bioimaging. The target gene is connected to the fluorescent protein through the 2A peptide, and the expression of the target gene can be preliminarily judged based on whether the fluorescent protein is expressed.

[0013] FSHR (FSH receptor) belongs to a structurally unique glycoprotein hormone receptor subfamily of G protein-coupled receptors (GPCRs) with an unusually long extracellular domain (ECD), seven transmembrane domains, three short intracellular loops, three extracellular loops, and an intracellular tail (Jiang et al., 2012). According to the hand-clasp model, dimeric FSH binds to the ectodomain of FSHR, triggering conformational changes in FSHR, followed by activation of G proteins and adenylate cyclase, leading to increased production of cyclic adenosine monophosphate (cAMP).

[0014] The present invention inserts the P2A sequence into the pig FSHR and eGFP genes. During the translation process, P2A undergoes "self-cleavage" to produce two proteins. The pig FSHR is located on the cell membrane, while the eGFP protein is in the cytoplasm. Green fluorescence is observed under a fluorescence microscope, indicating that the pig FSHR is also expressed at the same time. In order to evaluate the biological activity of porcine FSH in vitro, the present invention constructs CHO cells of porcine FSH receptor (FSHR), stimulates with different FSH samples, and determines the cAMP level by competitive ELISA to obtain the cAMP content of different FSHs, and obtains the relative biological activity level by comparing the reference product with the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the specification and, together with the description, serve to explain the principles of the specification.

[0016] Figure 1 The gene sequence design of the FSHR-eGFP reporter gene is shown; Figure (A) shows the break site after the reporter gene is expressed, and Figure (B) shows the amino acid sequence of the P2A protein.

[0017] Figure 2 The VIA changes of CHO-K1 cell lines screened by G418 are shown. Among them, "w / o G418" is the normal cell control group; "Pig FSHR-GFP" is the transfection screening group; "With G418" is the negative control group.

[0018] Figure 3 The figure shows CHO-K1 cells into which the Pig-FSHR-eGFP fusion gene was transferred and CHO-K1 cells without the Pig-FSHR-eGFP fusion gene being transferred observed under a fluorescence microscope; the left figure shows CHO-K1-Pig-FSHR-eGFP cells, and the right figure shows CHO-K1 cells.

[0019] Figure 4 Fluorescent clone-positive CHO-K1-Pig-FSHR-eGFP cells are shown.

[0020] Figure 5 A cAMP standard curve is shown.

[0021] Figure 6 The in vitro biological activity curve of FSH-Fc fusion protein based on cAMP content determination is shown.

[0022] Figure 7 The regression curve and equation of the theoretical value of the relative biological activity of the test sample and its corresponding measured value are shown.

[0023] Figure 8 It was shown that the FSH-Fc fusion protein exhibited an obvious dose response under this method, while the blank control and the negative control had no obvious dose response.

[0024] Fig. 9 The relationship between the degree of photodenaturation and biological activity of FSH-Fc fusion protein under this method is shown. DETAILED DESCRIPTION

[0025] I. Definition

[0026] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0027] As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0028] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0029] As used herein, the term "reporter gene" refers to a gene whose expression confers a phenotype that can be easily identified and measured in a cell. For example, a reporter gene can include a fluorescent protein gene or a selection gene. The selection gene can be used to confer a specific phenotype on a host cell. When a host cell expresses a selection gene to survive in a selective medium, the gene is considered to be a positive selection gene. The selection gene can also be used to select for host cells containing a specific gene; the selection gene used in this way is referred to as a negative selection gene.

[0030] The term "2A peptide" refers to short peptides (18-25 amino acids) derived from viruses. They are often called "self-cleaving" peptides, which can produce multiple proteins from one transcript. 2A peptides are not completely "self-cleaving", but work by causing the ribosome to skip the synthesis of the glycine and proline peptide bonds at the C-terminus of the 2A element, ultimately resulting in the separation of the end of the 2A sequence and the downstream product. The C-terminus of the upstream protein will have some additional 2A residues added, while the N-terminus of the downstream protein will have additional prolines. There are currently four commonly used 2A peptides, namely P2A, T2A, E2A and F2A, which are derived from four different viruses. Among the four commonly used 2A peptides (P2A, T2A, E2A and F2A), P2A usually has the highest cleavage efficiency (close to 100% in some cases), followed by T2A, and then E2A and F2A. The cleavage efficiency of F2A is only about 50%, and it is usually recommended to use P2A or T2A in polycistronic. Inserting a signal peptide gene sequence upstream or downstream of the 2A peptide can localize the target gene to the nucleus, chloroplasts, mitochondria, membranes or cytoplasmic microtubules after translation. Self-cleaving 2A peptides have important applications in the field of bioimaging. The target gene is connected to the fluorescent protein through the 2A peptide, and the expression of the target gene can be preliminarily judged based on whether the fluorescent protein is expressed.

[0031] The term "FSHR (FSH receptor)" belongs to a structurally unique subfamily of glycoprotein hormone receptors of G protein-coupled receptors (GPCRs), which have an unusually long extracellular domain (ECD), seven transmembrane domains, three short intracellular loops, three extracellular loops, and an intracellular tail (Jiang et al., 2012). According to the hand-clasp model, dimeric FSH binds to the ectodomain of FSHR, triggering conformational changes in FSHR, followed by activation of G proteins and adenylate cyclase, leading to increased production of cyclic adenosine monophosphate (cAMP).

[0032] The term "Fc region" is used to define the C-terminus of an immunoglobulin heavy chain. The "Fc region" may be a native sequence of an Fc region or an Fc region variant. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as a sequence from amino acid residue Cys226 or from Pro230 to its carboxyl terminus. The Fc region of an immunoglobulin generally includes two constant regions, CH2 and CH3.

[0033] The term "fluorescent protein" refers to any protein that can fluoresce when excited by appropriate electromagnetic radiation. This includes fluorescent proteins whose amino acid sequences are natural or engineered. Many cnidarians use green fluorescent protein ("GFP") as an energy transfer acceptor in bioluminescence. For example, green fluorescent protein is a protein that emits green light, and blue fluorescent protein is a protein that emits blue light, etc. GFP has been isolated from the Pacific Northwest jellyfish Victoria multi-tube luminous jellyfish (Aequorea victoria), sea nelum (Renilla reniformis), and Philalidium gregarium. Proteins from these organisms have been cloned, sequenced, and engineered and are well known in the art, including their primary and tertiary sequences. For example, the relevant GFPs of various Aequorea species with useful excitation and emission spectra have been engineered by modifying the amino acid sequence of the naturally occurring GFP from Victoria multi-tube luminous jellyfish. In addition to multi-tube jellyfish, there is also a green crown protein ZsGreen in coral polyps, which is brighter than eGFP. Since the mutant GFP could not produce red fluorescence, DsRed protein and DsRed2 protein were isolated from the coral Discosoma striata. In addition, there are many other fluorescent proteins isolated from corals or Victoria Aequorea, such as mWasabi, EBFP, EBFP2, mtagBFP, ECFP, EYFP, venus, tdTomato or TagRFP.

[0034] II. Detailed description of specific implementation plan

[0035] In one aspect, the present disclosure provides a method for determining the biological activity of FSH, the method comprising:

[0036] (1) Constructing effector cells that stably express the FSHR-fluorescent protein reporter gene;

[0037] (2) Cultivate the effector cells, then add the FSH protein sample and continue culturing;

[0038] (3) After the cells are lysed, the biological activity of FSH is determined by fitting based on the measured cAMP content in the cell lysate.

[0039] In some preferred embodiments, the above fitting is a four-parameter fitting.

[0040] In some embodiments, the FSHR-fluorescent protein reporter gene is a reporter gene formed by inserting a 2A peptide sequence into the target gene FSHR and the fluorescent protein gene.

[0041] In some preferred embodiments, the fluorescent protein is selected from eGFP, GFP, mWasabi, AcGFP, EBFP, EBFP2, mtagBFP, ECFP, EYFP, venus, tdTomato or TagRFP.

[0042] In some preferred embodiments, the fluorescent protein is eGFP.

[0043] In some preferred embodiments, the nucleic acid sequence of the fluorescent protein is as shown in SEQ ID NO: 3 or any variant thereof.

[0044] In some preferred embodiments, the amino acid sequence of the fluorescent protein is as shown in SEQ ID NO: 7 or any variant thereof.

[0045] In some preferred embodiments, the nucleic acid sequence of the reporter gene is as shown in SEQ ID NO: 4 or any variant thereof.

[0046] In some preferred embodiments, the amino acid sequence of the reporter gene is as shown in SEQ ID NO: 8 or any variant thereof.

[0047] In some embodiments, the 2A peptide is selected from any one of P2A, T2A, E2A or F2A.

[0048] In some preferred embodiments, the 2A peptide is P2A.

[0049] In some preferred embodiments, the nucleic acid sequence of the 2A peptide is as shown in SEQ ID NO: 2 or any variant thereof.

[0050] In some preferred embodiments, the amino acid sequence of the 2A peptide is as shown in SEQ ID NO: 6 or any variant thereof.

[0051] In some embodiments, the FSHR gene is porcine FSHR.

[0052] In some preferred embodiments, the nucleic acid sequence of porcine FSHR is as shown in SEQ ID NO: 1 or any variant thereof.

[0053] In some preferred embodiments, the amino acid sequence of porcine FSHR is as shown in SEQ ID NO: 5 or any variant thereof.

[0054] In some embodiments, the effector cells stably expressing the FSHR-fluorescent protein reporter gene are selected from any one of Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, 293 cells, 3T3 cells, TF-1 cells, NSO, SP2 cells, HeLa cells, monkey kidney cells (COS), human hepatoma cells, and 549A cells.

[0055] In some preferred embodiments, the effector cells are Chinese hamster ovary (CHO) cells.

[0056] In some embodiments, step (1) comprises: transfecting effector cells with a plasmid containing the FSHR-fluorescent protein reporter gene.

[0057] In some preferred embodiments, the plasmid is a pcDNA3.1 plasmid.

[0058] In some embodiments, step (1) includes: performing G418 pressure screening, monoclonal screening and fluorescence intensity screening on the effector cells after plasmid transfection.

[0059] In some preferred embodiments, the concentration of G418 is 500 μg / mL.

[0060] In some embodiments, step (2) comprises: culturing effector cells.

[0061] In some preferred embodiments, the effector cells are diluted to 1E6 cells / mL with complete culture medium and cultured overnight at 100 μL / well.

[0062] In some preferred embodiments, step (2) further comprises: diluting the FSH protein sample to 1.28 ng / mL-100 μg / mL with an assay medium.

[0063] In some preferred embodiments, step (2) further comprises: continuing to culture the effector cells to which the diluted FSH protein sample has been added for 4 hours.

[0064] In some embodiments, step (3) comprises: detecting the cAMP content using a cAMP detection kit.

[0065] In some preferred embodiments, the cAMP detection kit from R&D systems is used.

[0066] The present disclosure provides a CHO-K1 cell line that utilizes p2A to connect pFSHR and eGFP genes to enable their expression. The reporter gene cell line is the first porcine FSHR reporter gene cell line. The FSH in vitro detection method implemented using the cell line meets the verification requirements of specificity, precision, linearity and range, accuracy, and photostability, and can be effectively applied to the detection of the in vitro biological activity of the recombinant pFSH-Fc fusion protein.

[0067] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0068] Example

[0069] Example 1: Construction and screening of cell lines stably expressing FSHR-eGFP

[0070] 1. CHO-K1-FSHR-eGFP cell construction

[0071] (1) Gene sequence design

[0072] Insert the P2A sequence (SEQ ID NO: 2) into the pig FSHR (SEQ ID NO: 1) and eGFP gene (SEQ ID NO: 3) to form the pig FSHR-P2A-eGFP gene sequence (SEQ ID NO: 4) encoding the FSHR-eGFP fusion protein. After the FSHR-eGFP fusion protein is expressed, it will spontaneously break through the P2A site to form the Pig-FSHR protein and the eGFP protein, respectively. Figure 1 shown.

[0073] (2) Sequence synthesis and expression vector

[0074] After the Pig-FSHR-eGFP fusion gene was synthesized, the Pig-FSHR-eGFP fusion gene was first amplified by PCR, and then the pCDNA3.1 vector was linearized with NheI and ApaI restriction endonucleases, and finally the fusion gene was connected to the NheI and ApaI restriction sites of the pCDNA3.1 vector by seamless cloning. The ligation product was transformed into Escherichia coli Top10 competent cells, and a single colony was picked for sequencing verification after plating. The single colony that was successfully transformed was expanded and cultured, and the endotoxin removal kit was used for plasmid extraction to finally obtain the FSHR-eGFP-pcDNA3.1 plasmid.

[0075] (3) Transfection pressure screening

[0076] Before transfection, the FSHR-eGFP-pcDNA3.1 plasmid was linearized with the pvuI restriction endonuclease to facilitate correct integration into the host genome, and then electroporated (electroporation conditions: voltage 1620V, time 10ms, pulse 3 pulses) into the CHO-K1 cell line. After electroporation, the CHO-K1-FSHR-eGFP cells were transferred to complete culture medium (Quacell CD02+6mM L-Glutamine) for culture. After 48 hours of culture, G418 pressure screening (500μg / mL) was performed. The pressure screening samples were divided into three groups: (1) Normal cell control group: CHO-K1 cells that were not transfected or added with G418, cultured normally, and passaged every 3 to 4 days; (2) Transfection screening group: CHO-K1 cells that were transfected and added with G418; (3) Negative control group: CHO-K1 cells that were not transfected and added with G418. The screening results are shown in the figure below. Figure 3 As shown, on the 11th day of screening, the cell viability began to rebound (as shown in Figure 2 ), and strong fluorescence can be seen under fluorescent conditions (such as Figure 3 as shown).

[0077] 2. Monoclonal screening of effector cell lines

[0078] After obtaining a stable CHO-K1-FSHR-eGFP effector cell line, monoclonal screening was performed, 0.3 cells / well, 0.2 mL / well were plated, and after 14 days of culture in a 96-well plate, cell clones were counted and monoclonal fluorescence was observed. Cells with good cell growth and strong fluorescence were selected for expansion (such as Figure 4 After establishing a cell line stably expressing Pig-FSHR-eGFP, the analysis method was developed.

[0079] Example 2: Determination of biological activity of FSH-Fc fusion protein

[0080] CHO-K1-pig-FSHR-eGFP cells were diluted to 1E6 cells / ml, 100 μL / well with complete medium (DMEM / F12 medium containing 0.25% G418 and 10% FBS), and incubated at 37°C overnight. FSH-Fc fusion protein (1 mg / mL) was diluted 5-fold to 1.28 ng / mL-100 μg / mL with assay medium (DMEM / F12 medium containing 0.5 mM IBMX), with a total of 8 concentration points. The complete medium in the cell well plate was discarded, and the sample dilutions were added to the corresponding wells in turn, and incubated at 37°C with 5% CO 2After incubation in the incubator for 4 hours, the cells were lysed with the cell lysis buffer in the cAMP detection kit (R&D systems), and the cAMP content in the cell lysate was determined according to the method in the kit. The specific steps are as follows:

[0081] (1) Prepare all reagents, working standards, and samples in advance.

[0082] (2) Add 50 μL of primary antibody solution to each well of the 96-well plate except the NSB (non-specific binding) wells and incubate at room temperature for 1 h at 500 rpm ± 50 rpm.

[0083] (3) Wash four times with 400 μL / well washing solution.

[0084] (4) Add 50 μL / well of cAMP conjugate solution to all wells.

[0085] (5) Add 100 μL of cAMP standard, samples (test sample (pFSH-Fc fusion protein sample to be tested) and reference sample (pFSH-Fc fusion protein standard)) to the corresponding wells within 15 min.

[0086] (6) Add 100 μL of diluent to the NSB (nonspecific binding) well and the standard 0 (B0) well and incubate at room temperature for 2 h at 500 rpm ± 50 rpm.

[0087] (7) Repeat step (3) to wash the plate.

[0088] (8) Add 200 μL of substrate solution to all wells and incubate at room temperature for 30 min.

[0089] (9) Add 100 μL of stop solution to terminate the reaction.

[0090] (10) Read the plate at 450 nm, using 540 nm or 570 nm as the reference wavelength.

[0091] (11) Four-parameter fitting was performed with the concentration of cAMP standard as the horizontal axis and 450 nm as the vertical axis. The four-parameter equation is Y = (AD) / (1+(X / C)B)+D, where the C value is the corresponding EC 50 value.

[0092] (12) The cAMP content of all sample wells was calculated using the standard curve, and a four-parameter fit was performed with the sample concentration as the horizontal axis and the cAMP content as the vertical axis.

[0093] (13) Relative biological activity of the test article (%) = EC of the reference article 50 Value / EC value of the test article 50 value*100%.

[0094] The obtained cAMP standard curve is as follows Figure 5 As shown, the biological activity of FSH-Fc fusion protein in vitro was fitted with a four-parameter curve, as shown in Figure 6 As shown, the curve has obvious upper and lower platforms. With the increase of sample concentration, the cAMP content also shows an upward trend. The curve has an obvious linear relationship, indicating that the biological activity method of FSH-Fc fusion protein has been successfully established.

[0095] Example 3: Methodological Validation of FSH-Fc Fusion Protein Biological Activity Assay

[0096] 1. Precision

[0097] Dilute FSH-Fc fusion protein with analytical medium to 64%, 80%, 100%, 125% and 156% of the initial working mass concentration as test samples of different concentrations. Perform 5-fold gradient dilution to obtain 8 concentration points of the standard curve. Two analysts will measure on different 4 days, and each person will prepare 1 test sample at 5 different concentrations every day. Each standard curve concentration point of each test sample will be measured in parallel with 2 replicates. The relative standard deviation (RSD) of the relative biological activity test results of each group of test samples should be less than 20.0%.

[0098] As shown in the data of Table 1, the relative standard deviations of the relative biological activity test results of five different concentrations of the test samples (64%, 80%, 100%, 125% and 156%) were 10.7%, 10.6%, 7.8%, 7.7% and 11.0%, respectively. The precision of the method proposed in the present disclosure is good.

[0099] Table 1. Precision - Summary of relative biological activity test data of FSH-Fc fusion protein samples at different concentrations

[0100]

[0101] 2. Accuracy

[0102] Dilute FSH-Fc fusion protein with analytical medium to 64%, 80%, 100%, 125% and 156% of the initial working mass concentration as test samples of different concentrations. Perform 5-fold gradient dilution to obtain 8 concentration points of the standard curve. Two analysts will measure on different 4 days, and each person will prepare 1 test sample at 5 different concentrations every day. Each standard curve concentration point of each test sample will be measured in parallel with 2 replicates. The average recovery rate of the relative biological activity test results of each group of test samples should be within the range of 80%~120%; the relative standard deviation of the recovery rate of each concentration of the test sample should be less than 20.0%; the measured average value should be within the range of ±20% of the theoretical value.

[0103] As shown in the data of Table 2, the average recoveries of the relative biological activity of the five different concentrations of the test samples (64%, 80%, 100%, 125% and 156%) were 99%, 96%, 98%, 93% and 103%, respectively, which were within the range of 80% to 120%. The relative standard deviations of the recoveries of the corresponding concentrations were 10.7%, 10.6%, 7.8%, 7.7% and 11.0%, respectively; as shown in the data of Table 3, the measured average values ​​of the five different concentrations of the test samples were within the range of ±20% of the theoretical value, and the accuracy of the method proposed in the present disclosure was good.

[0104] Table 2. Accuracy - Recovery data summary of FSH-Fc fusion protein samples at different concentrations

[0105]

[0106] Table 3. Accuracy - Summary of the difference between the measured and theoretical values ​​of FSH-Fc fusion protein samples at different concentrations

[0107]

[0108] 3. Linearity and range

[0109] The FSH-Fc fusion protein was diluted with analytical culture medium to 64%, 80%, 100%, 125% and 156% of the initial working mass concentration as test samples of different concentrations. A 5-fold gradient dilution was performed to obtain 8 concentration points of the standard curve. Two analysts were assigned to perform the measurements on 4 different days. Each person prepared 1 test sample at 5 different concentrations each day. Each standard curve concentration point of each test sample was measured in parallel with 2 replicate wells. Linear fitting was performed with the theoretical relative biological activity value as the horizontal axis and the measured relative biological activity value as the vertical axis. The slope of the straight line should be in the range of 0.8~1.2; the correlation coefficient R 2 It should not be less than 0.95; if the concentration range meets both the linearity and accuracy acceptance criteria, it is the detection range.

[0110] like Figure 7 As shown in the figure, within the concentration range of 64% to 156%, the theoretical value of the relative biological activity of the test sample and its corresponding measured value are linearly related, with a slope of 1.032 and a linear correlation coefficient R 2 It is 0.986. The method proposed in the present disclosure has a good linear relationship within this range.

[0111] Based on the results of accuracy and linearity, the detection range of this method is 64%~156%.

[0112] 4. Exclusivity

[0113] Use the assay medium solution as a blank control and recombinant protein X (gonadotropin, GTH) as a negative control. Dilute the sample with the assay medium to a starting working mass concentration of 100 μg / ml, and then perform a 5-fold gradient dilution to obtain 8 concentration points of the standard curve. The FSH-Fc fusion protein should show an obvious dose response, a typical S-shaped curve, a signal-to-noise ratio of not less than 2, and a fitted four-parameter curve R 2 It should be greater than 0.95; the blank control and negative control should have no obvious dose response.

[0114] like Figure 8 As shown, FSH-Fc fusion protein has an obvious dose response, showing a typical S-shaped curve; the signal-to-noise ratio is 3.0; the fitting four-parameter curve R 2 is 0.997; the negative control recombinant protein X (gonadotropin GTH) has no obvious dose response, and the method proposed in the present disclosure has good specificity.

[0115] 5. Photostability test

[0116] After irradiating the FSH-Fc fusion protein for 1d, 3d, 5d, 8d and 16d, samples with different denaturation degrees were obtained. The samples were analyzed by SEC-HPLC and the peak area integration showed that the proportion of monomers in the fusion protein decreased with the increase of irradiation time, which were 93.9%, 89.6%, 82.8%, 73.9% and 53.9% respectively. At the same time, the samples without light denaturation were used as references to determine the biological activity of the samples. The relative biological activities were 98%, 91%, 85%, 72% and 48% respectively. The results are as follows: Fig. 9 From these two sets of data, it can be seen that as the monomer content decreases, its biological activity also decreases accordingly. The two sets of data are linearly fitted, R 2 =0.9976, which is a good fit, proving that the biological activity test method can effectively detect the loss of fusion protein activity caused by light denaturation, and further proves the reliability of the test method.

Claims

1. A method for measuring the biological activity of FSH, the method comprising: (1) Constructing effector cells that stably express the FSHR-fluorescent protein reporter gene; (2) Cultivate the effector cells, then add the FSH protein sample and continue culturing; (3) After the cells are lysed, fitting is performed based on the measured cAMP content in the cell lysate to determine the biological activity of FSH; the fitting is a four-parameter fitting; The FSHR-fluorescent protein reporter gene is a reporter gene formed by inserting a 2A peptide sequence between the target gene FSHR and the fluorescent protein gene; The fluorescent protein is eGFP; The nucleic acid sequence of the fluorescent protein is shown in SEQ ID NO: 3; The nucleic acid sequence of the reporter gene is shown in SEQ ID NO: 4; The 2A peptide is P2A; The nucleic acid sequence of the 2A peptide is as shown in SEQ ID NO: 2; The FSHR is porcine FSHR; The nucleic acid sequence of the porcine FSHR is shown in SEQ ID NO: 1; The step (1) comprises: transfecting effector cells with a plasmid containing the FSHR-fluorescent protein reporter gene; The step (1) comprises: performing G418 pressure screening, monoclonal screening and fluorescence intensity screening on the effector cells after plasmid transfection.

2. The method of claim 1, wherein the effector cells stably expressing the FSHR-fluorescent protein reporter gene are selected from any one of Chinese hamster ovary CHO cells, baby hamster kidney BHK cells, 293 cells, 3T3 cells, TF-1 cells, NSO, SP2 cells, HeLa cells, monkey kidney cells COS, human hepatoma cells or A549 cells.

3. The method according to claim 2, wherein: The effector cells are Chinese hamster ovary CHO cells.

4. The method of claim 1, wherein the plasmid is a pcDNA3.1 plasmid.

5. The method of claim 1, wherein the concentration of G418 is 500 μg / mL.

6. The method of claim 1, wherein the step (2) comprises: The effector cells were cultured and diluted to 10 6 / mL, 100μL / well and culture overnight.

7. The method of claim 6, wherein the step (2) further comprises: The FSH protein sample was diluted to 1.28 ng / mL-100 μg / mL with assay medium.

8. The method of claim 7, wherein step (2) further comprises: The effector cells to which the diluted FSH protein sample was added were cultured for a further 4 h.

9. The method according to any one of claims 1 to 8, wherein the step (3) comprises: The cAMP content was detected using a cAMP detection kit.

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