CN117074703A - cAMP-based follicle stimulating hormone in vitro biological activity determination method - Google Patents

Abstract

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

Description

cAMP-based method for determining the in vitro biological activity of follicle-stimulating hormone

Technical field

The present disclosure mainly relates to the field of biological detection, and specifically relates to an in vitro determination method of FSH biological activity based on cAMP content in cells.

Background technique

Follicle-stimulating hormone (FSH), also known as follicle-stimulating hormone and follicle-stimulating hormone, is a glycoprotein, a hormone synthesized and secreted by the pituitary gland. The FSH molecule is a heterodimer composed of two subunits, α subunit and β subunit, which are non-covalently bonded. In the same species, FSH has the same amino acid sequence as the α subunit of other glycoprotein hormones such as luteinizing hormone (LH), thyrotropin (Thyroid Stimulating Hormone, TSH), etc., consisting of 92-96 amino acids. (Different mammals have different compositions). The FSH beta subunit gene consists of 109-115 amino acids, which determines the biological specificity of FSH. Neither the alpha nor beta subunits alone have biological activity, and can only exert biological effects when they are combined.

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 from mature follicles, and participate in the formation of normal menstruation. FSH can promote the development and maturation of follicles. If FSH levels are lower than normal, you may be suffering 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 birth.

Biological activity measurement is an important indicator to characterize the correctness of the product's molecular structure and function, and the effectiveness of the product. As of 2021, the rat ovary weight gain method is still the only FSH in vivo activity measurement method approved and recognized by domestic and foreign regulatory agencies. However, animal experiments have shortcomings such as large individual differences, long experimental cycles, large variability, low sensitivity to activity changes, and high operational requirements, which restrict their use in the research and development process. With the international emphasis on 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 biological activity analysis methods have been widely used in the evaluation of monoclonal antibodies and recombinant cytokine products. and release inspection. In the past few years, the reporter gene method has emerged in the field of in vitro cell activity determination of biotech drugs in my country and has gradually become a platform technology. The reporter gene method to detect 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, Reporter gene methods have been successfully developed as release methods for biological activity for a variety of biologics.

It is very common to use CHO cells to construct human reporter gene cell lines, but there is uncertainty in the expression of pig-derived genes. It is not sure whether the pig-derived FSHR can be expressed in the CHO-K1 cell line, and there is a problem of low expression level, resulting in its There are screening difficulties. Therefore, there is an urgent need to develop a high-precision in vitro biological activity detection method for porcine FSH that does not require the use of experimental animals.

Contents of the invention

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 uses p2A to connect pFSHR and a reporter gene (such as eGFP) and place it in cells (such as CHO-K1 cell line) Expression, this cell is the first porcine-derived FSHR reporter gene cell line. After 2A peptide cleavage, the fluorescence in the cells can be well expressed, which is convenient for screening and convenient for testing the in vitro biological activity of the recombinant pFSH fusion protein.

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

(1) Construct 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 lysing the cells, perform fitting based on the measured cAMP content in the cell lysate to determine the FSH biological activity;

Preferably, the fitting is a four-parameter fitting.

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. The 2A peptide is not completely "self-cleaving", but works by causing the ribosome to skip the synthesis of glycine and proline peptide bonds at the C-terminus of the 2A element, ultimately leading to the separation of the 2A sequence end and the downstream product. Some additional 2A residues will be added to the C-terminus of the upstream protein, while additional proline will be added to the N-terminus of the downstream protein. There are currently four commonly used 2A peptides, namely P2A, T2A, E2A and F2A, derived from four different viruses. Among the four commonly used 2A peptides (P2A, T2A, E2A, and F2A), P2A generally has the highest cleavage efficiency (nearly 100% in some cases), followed by T2A, followed by E2A and F2A. The cleavage efficiency of F2A is only about 50%, and it is generally recommended to use P2A or T2A in polycistrons. Inserting a signal peptide gene sequence upstream or downstream of the 2A peptide can enable the target gene to be localized to the nucleus, chloroplast, mitochondria, membrane or cytoplasmic microtubules after translation. Self-cleaving 2A peptide has important applications in the field of biological imaging. By connecting the target gene to the fluorescent protein through 2A peptide, the expression of the target gene can be preliminarily judged based on whether the fluorescent protein is expressed or not.

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, and three short intracellular loop, three extracellular loops and an intracellular tail (Jiang et al., 2012). According to the hand-buckle model, dimeric FSH binds to the ectodomain of FSHR, triggering conformational changes in FSHR and subsequently activating G proteins and adenylyl cyclase, resulting in increased production of cyclic adenosine monophosphate (cAMP).

This disclosure inserts the P2A sequence into the pig FSHR and eGFP genes. During the translation process, P2A "self-cleaves" to produce two proteins. The pig FSHR will be located on the cell membrane, while the eGFP protein will be in the cytoplasm, which can be observed with a fluorescence microscope. Green fluorescence was detected, indicating that pig FSHR was also expressed at the same time. In order to evaluate the biological activity of porcine FSH in vitro, the present disclosure constructs CHO cells with porcine FSH receptors (FSHR), stimulates them with different FSH samples, and determines cAMP levels by competitive ELISA to obtain the cAMP content of different FSHs. , and obtain the relative biological activity level by comparing the reference product with the sample.

Description of the drawings

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.

Figure 1 shows the gene sequence design of the FSHR-eGFP reporter gene; Figure (A) shows the cleavage site after expression of the reporter gene, and Figure (B) shows the amino acid sequence of the P2A protein.

Figure 2 shows the VIA changes of G418 pressurized screening of CHO-K1 cell line. 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.

Figure 3 shows the CHO-K1 cells transformed into Pig-FSHR-eGFP fusion gene and the CHO-K1 cells not transformed into Pig-FSHR-eGFP fusion gene observed under a fluorescence microscope; the left picture shows CHO-K1- Pig-FSHR-eGFP cells, the picture on the right shows CHO-K1 cells.

Figure 4 shows fluorescent clone-positive CHO-K1-Pig-FSHR-eGFP cells.

Figure 5 shows the cAMP standard curve.

Figure 6 shows the in vitro biological activity curve of FSH-Fc fusion protein based on cAMP content determination.

Figure 7 shows the regression curve and equation between the theoretical value of the relative biological activity of the test product and its corresponding measured value.

Figure 8 shows that the FSH-Fc fusion protein showed an obvious dose response under this method, while neither the blank control nor the negative control had an obvious dose response.

Figure 9 shows the relationship between the degree of photodenaturation and biological activity of FSH-Fc fusion protein under this method.

Detailed ways

I.Definition

In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used in this article are terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present disclosure, definitions and explanations of relevant terms are provided below.

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 rounding up or down to the nearest integer within plus or minus 10% of a given value or range.

As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," including their grammatical equivalents, are generally to be understood as open-ended and non-restrictive, e.g., not Exclude other elements or steps not listed.

As used herein, the term "reporter gene" refers to a gene whose expression confers a phenotype on a cell that can be readily identified and measured. For example, the reporter gene can include a fluorescent protein gene or a selection gene. Selective genes can be used to confer specific phenotypes on host cells. When a host cell expresses a selected gene in order to survive in a selective medium, the gene is considered to be a positively selected gene. Selective genes can also be used to select against host cells containing a specific gene; selectable genes used in this way are called negative selection genes.

The term "2A peptides" are short peptides (18-25 amino acids) derived from viruses. They are often called "self-cleaving" peptides and can produce multiple proteins from one transcript. The 2A peptide is not completely "self-cleaving", but works by causing the ribosome to skip the synthesis of glycine and proline peptide bonds at the C-terminus of the 2A element, ultimately leading to the separation of the 2A sequence end and the downstream product. Some additional 2A residues will be added to the C-terminus of the upstream protein, while additional proline will be added to the N-terminus of the downstream protein. There are currently four commonly used 2A peptides, namely P2A, T2A, E2A and F2A, derived from four different viruses. Among the four commonly used 2A peptides (P2A, T2A, E2A, and F2A), P2A generally has the highest cleavage efficiency (nearly 100% in some cases), followed by T2A, followed by E2A and F2A. The cleavage efficiency of F2A is only about 50%, and it is generally recommended to use P2A or T2A in polycistrons. Inserting a signal peptide gene sequence upstream or downstream of the 2A peptide can enable the target gene to be localized to the nucleus, chloroplast, mitochondria, membrane or cytoplasmic microtubules after translation. Self-cleaving 2A peptide has important applications in the field of biological imaging. By connecting the target gene to the fluorescent protein through 2A peptide, the expression of the target gene can be preliminarily judged based on whether the fluorescent protein is expressed or not.

The term "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-buckle model, dimeric FSH binds to the ectodomain of FSHR, triggering conformational changes in FSHR and subsequently activating G proteins and adenylyl cyclase, resulting in increased production of cyclic adenosine monophosphate (cAMP).

The term "Fc region" is used to define the C-terminal end of an immunoglobulin heavy chain. The "Fc region" may be a native Fc region sequence 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 the sequence from amino acid residues Cys226 or from Pro230 to its carboxy terminus. The Fc region of an immunoglobulin usually consists of two constant regions, CH2 and CH3.

The term "fluorescent protein" refers to any protein that fluoresces when excited by appropriate electromagnetic radiation. This includes fluorescent proteins whose amino acid sequence is natural or engineered. Many cnidarians use green fluorescent protein ("GFP") as an energy transfer receptor 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. GFP has been isolated from the Pacific Northwest jellyfish Aequorea victoria, Renilla reniformis, and Phialidium 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, various Aequorea-related GFPs with useful excitation and emission spectra have been engineered by modifying the amino acid sequence of the naturally occurring GFP from Aequorea victoria. In addition to Aequorea, there is also a green crown protein ZsGreen in coral polyps, which is brighter than eGFP. Since the mutant GFP cannot obtain red fluorescence, DsRed protein and DsRed2 protein were isolated from the Discosoma striata coral. In addition, there are many other fluorescent proteins isolated from corals or Aequorea victoria, such as mWasabi, EBFP, EBFP2, mtagBFP, ECFP, EYFP, venus, tdTomato or TagRFP.

II. Detailed description of specific implementation plans

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

(1) Construct 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 lysing the cells, perform fitting based on the measured cAMP content in the cell lysate to determine the FSH biological activity.

In some preferred embodiments, the above fit is a four-parameter fit.

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

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

In some preferred embodiments, the fluorescent protein is eGFP.

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

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

In some preferred embodiments, the nucleic acid sequence of the reporter gene is set forth in SEQ ID NO: 4 or any variant thereof.

In some preferred embodiments, the amino acid sequence of the reporter gene is set forth in SEQ ID NO: 8 or any variant thereof.

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

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

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

In some preferred embodiments, the 2A peptide has an amino acid sequence as set forth in SEQ ID NO: 6 or any variant thereof.

In some embodiments, the FSHR gene is porcine FSHR.

In some preferred embodiments, the nucleic acid sequence of porcine FSHR is set forth in SEQ ID NO: 1 or any variant thereof.

In some preferred embodiments, the amino acid sequence of porcine FSHR is set forth in SEQ ID NO: 5 or any variant thereof.

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

In some preferred embodiments, the effector cells are Chinese Hamster Ovary (CHO) cells.

In some embodiments, step (1) includes: transfecting effector cells with a plasmid comprising the FSHR-fluorescent protein reporter gene.

In some preferred embodiments, the plasmid is the pcDNA3.1 plasmid.

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

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

In some embodiments, step (2) includes: culturing effector cells.

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

In some preferred embodiments, step (2) further includes: diluting the FSH protein sample to 1.28ng/mL-100μg/mL with analysis medium.

In some preferred embodiments, step (2) further includes: continuing to culture the effector cells added with the diluted FSH protein sample for 4 hours.

In some embodiments, step (3) includes: detecting cAMP content using a cAMP detection kit.

In some preferred embodiments, a cAMP detection kit from R&D systems is used.

The present disclosure provides a CHO-K1 cell line that uses p2A to connect pFSHR and eGFP genes to express them. This reporter gene cell line is the first pig-derived FSHR reporter gene cell line. The FSH in vitro detection method implemented using this cell line meets the specificity , precision, linearity and range, accuracy and photostability and other verification requirements, and can be effectively applied to the detection of in vitro biological activity of recombinant pFSH-Fc fusion protein.

For purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the disclosure may include embodiments having combinations of all or some of the features described. Some embodiments.

Example

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

1.Construction of CHO-K1-FSHR-eGFP cells

(1) Gene sequence design

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

(2) Sequence synthesis and expression vector

After the Pig-FSHR-eGFP fusion gene is synthesized, the Pig-FSHR-eGFP fusion gene is first amplified by PCR, then the pCDNA3.1 vector is linearized with NheI and ApaI restriction endonucleases, and finally through seamless cloning. , connect the fusion gene to the NheI and ApaI restriction sites of pCDNA3.1 vector. The ligation product was transformed into E. coli Top10 competent cells. After plating, single colonies were picked for sequencing verification. The single colonies that were successfully transformed were expanded and cultured. The endotoxin kit was removed for plasmid extraction, and finally FSHR-eGFP-pcDNA3 was obtained. .1 Plasmid.

(3) Transfection pressure screening

Before transfection, the FSHR-eGFP-pcDNA3.1 plasmid was linearized with pvuI restriction endonuclease to facilitate correct integration into the host genome, and then electroporated (electroporation conditions: voltage 1620V, time 10ms, pulse: 3 pulses) were transferred into the CHO-K1 cell line. After electroporation, the CHO-K1-FSHR-eGFP cells were transferred into complete medium (Quacell CD02+6mM L-Glutamine) for culture. After 48 hours of culture, G418 pressure screening was performed ( 500μg/mL). Pressure screening samples were divided into three groups: (1) Normal cell control group: CHO-K1 cells without transfection or addition of G418, cultured normally, and passaged once every 3 to 4 days; (2) Transfection screening group: transfected CHO-K1 cells transfected with G418 added; (3) Negative control group: CHO-K1 cells not transfected with G418 added. The screening results are shown in Figure 3. On the 11th day of screening, the cell viability began to rebound (shown in Figure 2), and strong fluorescence could be seen under fluorescent conditions (shown in Figure 3).

2. Monoclonal screening of effector cell lines

After obtaining the stable CHO-K1-FSHR-eGFP effector cell line, perform monoclonal screening and plate 0.3 cells/well, 0.2mL/well. After culturing in a 96-well plate for 14 days, count cell clones and observe monoclonal fluorescence. Cells with good cell growth and strong fluorescence were selected for expansion (as shown in Figure 4), and after establishing a stable expression Pig-FSHR-eGFP cell line, analysis methods were developed.

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

CHO-K1-pig-FSHR-eGFP cells were diluted to 1E6 cells/ml, 100 μL/well with complete culture medium (DMEM/F12 medium containing 0.25% G418 and 10% FBS), and incubated at 37°C overnight. The FSH-Fc fusion protein (1mg/mL) was diluted 5-fold in the assay medium (DMEM/F12 medium containing 0.5mM IBMX) to 1.28ng/mL-100μg/mL, with a total of 8 concentration points. Discard the complete culture medium in the cell well plate, add the sample dilution solution to the corresponding wells in sequence, incubate for 4 hours in a 37°C 5% _CO2 incubator, and then use the cell lysis solution in the cAMP detection kit (R&D systems). Lyse and measure the cAMP content in the cell lysate according to the method in the kit. Specific steps are as follows:

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

(2) Except NSB (non-specific binding) wells, add 50 μL of primary antibody solution to the 96-well plate and incubate at room temperature at 500 rpm ± 50 rpm for 1 hour.

(3) Wash 4 times with 400 μL/well washing solution.

(4) Add 50 μL/well cAMP conjugate solution to all wells.

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

(6) Add 100 μL diluent to the NSB (non-specific binding) well and standard 0 (B0) well, and incubate at room temperature at 500 rpm ± 50 rpm for 2 hours.

(7) Repeat step (3) to wash the plate.

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

(9) Add 100 μL stop solution to terminate the reaction.

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

(11) Perform four-parameter fitting with cAMP standard concentration as the abscissa and 450nm as the ordinate. The four-parameter equation is Y=(AD)/(1+(X/C)B)+D, where the C value is the corresponding EC ₅₀ value.

(12) Use the standard curve to calculate the cAMP content of all sample wells, and perform four-parameter fitting with the sample concentration as the abscissa and the cAMP content as the ordinate.

(13) Relative biological activity of the test product (%) = EC ₅₀ value of the reference product / EC ₅₀ value of the test product * 100%.

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

Example 3: Methodological verification of biological activity determination of FSH-Fc fusion protein

1. Precision

The FSH-Fc fusion protein was diluted with the assay medium to 64%, 80%, 100%, 125% and 156% of the initial working mass concentration as test samples of different concentrations. Then perform 5-fold gradient dilution to obtain 8 concentration points of the standard curve. Two analysts conducted separate measurements on four different days. Each person prepared one test sample at five different concentrations every day, and each standard curve concentration point of each test sample was measured in parallel in two replicate wells. The relative standard deviation (RSD) of the relative biological activity test results of each group of test products should be less than 20.0%.

The accuracy is shown in the data in Table 1. The relative standard deviations of the relative biological activity detection results of five different concentrations of test products (64%, 80%, 100%, 125% and 156%) are 10.7% and 10.6 respectively. %, 7.8%, 7.7%, 11.0%, the precision of the method proposed in this disclosure is good.

Table 1 Precision - summary of relative biological activity determination data of different concentrations of FSH-Fc fusion protein test products

2. Accuracy

The FSH-Fc fusion protein was diluted with the assay medium to 64%, 80%, 100%, 125% and 156% of the initial working mass concentration as test samples of different concentrations. Then perform 5-fold gradient dilution to obtain 8 concentration points of the standard curve. Two analysts conducted separate measurements on four different days. Each person prepared one test sample at five different concentrations every day, and each standard curve concentration point of each test sample was measured in parallel in two replicate wells. The average recovery rate of the relative biological activity test results of each group of test products should be in the range of 80% to 120%; the relative standard deviation of the recovery rate of the test products at each concentration should be less than 20.0%; the actual measured average value should be within the theoretical value ± Within 20% range.

As shown in the data in Table 2, the average recovery rates of relative biological activity of five different concentrations of test products (64%, 80%, 100%, 125% and 156%) were 99%, 96%, 98%, 93%, 103%, in the range of 80% to 120%. The relative standard deviations of the recoveries at corresponding concentrations are 10.7%, 10.6%, 7.8%, 7.7% and 11.0% respectively; as shown in the data in Table 3, the measured average values of the above five different concentrations of test products are within the theoretical range. Within the range of ±20%, the accuracy of the method proposed in this disclosure is good.

Table 1 Accuracy - Summary of recovery data of different concentrations of FSH-Fc fusion protein test products

Table 2 Accuracy - Summary of data on differences between actual measured values and theoretical values of FSH-Fc fusion protein test products at different concentrations

3. Linearity and range

The FSH-Fc fusion protein was diluted with the assay medium to 64%, 80%, 100%, 125% and 156% of the initial working mass concentration as test samples of different concentrations. Then perform 5-fold gradient dilution to obtain 8 concentration points of the standard curve. Two analysts conducted separate measurements on four different days. Each person prepared one test sample at five different concentrations every day, and each standard curve concentration point of each test sample was measured in parallel in two replicate wells. Linear fitting was performed with the theoretical relative biological activity value as the abscissa and the measured relative biological activity value as the ordinate. The slope of the straight line should be in the range of 0.8 to 1.2; the correlation coefficient ^R2 should not be less than 0.95; if it meets the concentration range of both linearity and accuracy acceptance standards, it is the detection range.

As shown in Figure 7, in the concentration range of 64% to 156%, the theoretical relative biological activity value of the test product has a linear relationship with its corresponding measured value, the slope is 1.032, and the linear correlation coefficient ^R2 is 0.986. According to the disclosure The proposed method has good linearity in this range.

Based on the results of comprehensive accuracy and linearity, the detection range of this method is 64% to 156%.

4. Exclusiveness

The analysis medium solution was used as a blank control, and recombinant protein X (Gonadotropin Hormone, GTH) was used as a negative control. Dilute the sample with analytical medium to a starting working mass concentration of 100 μg/ml, and then perform 5-fold gradient dilution to obtain 8 concentration points of the standard curve. The FSH-Fc fusion protein should show an obvious dose response, showing a typical S-shaped curve, the signal-to-noise ratio should not be less than 2, and the ^R2 of the fitted four-parameter curve should be greater than 0.95; the blank control and negative control should have no obvious dose response.

As shown in Figure 8, the FSH-Fc fusion protein has an obvious dose response, showing a typical S-shaped curve; the signal-to-noise ratio is 3.0; the ^R2 of the fitted four-parameter curve is 0.997; the negative control recombinant protein X (Gonadotropin Hormone, GTH) has no obvious dose response, and the method proposed in this disclosure has good specificity.

5.Photostability test

After the FSH-Fc fusion protein was illuminated for 1d, 3d, 5d, 8d and 16d, samples with different degrees of denaturation were obtained. The samples were analyzed by SEC-HPLC and the peak area integration was performed. It was found that as the illumination time increased, the amount of denaturation in the fusion protein increased. The proportions of monomers decreased accordingly, to 93.9%, 89.6%, 82.8%, 73.9% and 53.9% respectively. At the same time, the samples that were not denatured by light were used as reference products to measure the biological activity of the samples. The relative biological activities were 98%, 91%, 85%, 72% and 48% respectively. The results are shown in Figure 9. It can be seen from these two sets of data that as the monomer content decreases, its biological activity also decreases accordingly. When these two sets of data are linearly fitted, R ² =0.9976, the fitting is good, proving that the biological The activity test method can effectively detect the loss of fusion protein activity due to light denaturation, further proving the reliability of the test method.

Claims (10)

1. A method for measuring the biological activity of FSH, the method comprising: (1) Construct effector cells that stably express the FSHR-reporter gene; preferably, the reporter gene is a fluorescent protein reporter gene; (2) Cultivate the effector cells, then add the FSH protein sample and continue culturing; (3) After lysing the cells, perform fitting based on the measured cAMP content in the cell lysate to determine the FSH biological activity; preferably, the fitting is a four-parameter fitting. 2. The method of claim 1, wherein 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; Preferably, the fluorescent protein is selected from eGFP, GFP, mWasabi, AcGFP, EBFP, EBFP2, mtagBFP, ECFP, EYFP, venus, tdTomato or TagRFP; More preferably, the fluorescent protein is eGFP; Preferably, the nucleic acid sequence of the fluorescent protein is as shown in SEQ ID NO: 3 or any variant thereof; Preferably, the amino acid sequence of the fluorescent protein is as shown in SEQ ID NO:7 or any variant thereof; More preferably, the nucleic acid sequence of the reporter gene is as shown in SEQ ID NO: 4 or any variant thereof; More preferably, the amino acid sequence of the reporter gene is as shown in SEQ ID NO: 8 or any variant thereof. 3. The method of claim 2, wherein the 2A peptide is selected from any one of P2A, T2A, E2A or F2A; More preferably, the 2A peptide is P2A; Preferably, the nucleic acid sequence of the 2A peptide is as shown in SEQ ID NO: 2 or any variant thereof; Preferably, the amino acid sequence of the 2A peptide is as shown in SEQ ID NO: 6 or any variant thereof. 4. The method according to any one of claims 1-3, wherein the FSHR gene is porcine FSHR; Preferably, the nucleic acid sequence of the porcine FSHR is as shown in SEQ ID NO: 1 or any variant thereof; Preferably, the amino acid sequence of the porcine FSHR is as shown in SEQ ID NO: 5 or any variant thereof. 5. The method of any one of claims 1-4, wherein the effector cells stably expressing the FSHR-fluorescent protein reporter gene are selected from Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, 293 cells, and 3T3 cells. , any one of TF-1 cells, NSO, SP2 cells, HeLa cells, monkey kidney cells (COS), human liver cancer cells, and 549A cells; Preferably, the effector cells are Chinese hamster ovary (CHO) cells. 6. The method of any one of claims 1-5, wherein said step (1) comprises: transfecting effector cells with a plasmid containing the FSHR-fluorescent protein reporter gene; Preferably, the plasmid is pcDNA3.1 plasmid. 7. The method according to any one of claims 1 to 6, wherein the step (1) includes: performing G418 pressure screening, monoclonal screening and fluorescence intensity screening on the effector cells after plasmid transfection; Preferably, the concentration of G418 is 500 μg/mL. 8. The method of any one of claims 1-7, wherein step (2) includes: culturing effector cells; Preferably, the effector cells are diluted to 1E6 cells/mL with complete culture medium and cultured at 100 μL/well overnight. 9. The method according to any one of claims 1 to 8, wherein the step (2) further comprises: diluting the FSH protein sample to 1.28ng/mL-100μg/mL with analysis medium; Preferably, the step (2) further includes: continuing to culture the effector cells added with the diluted FSH protein sample for 4 hours. 10. The method according to any one of claims 1 to 9, wherein the step (3) includes: using a cAMP detection kit to detect cAMP content; Preferably, the cAMP detection kit from R&D systems is used.