A method for detecting the in vitro relative bioactivity of bovine growth hormone

Abstract

The application discloses a bovine growth hormone in vitro relative biological activity detection method and belongs to the technical field of biological detection. The method comprises the following steps: constructing pbGHR, constructing HEK293-bGHR-Luc cells, and detecting bovine growth hormone in vitro relative biological activity. The method has the characteristics of low cost, strong stability, strong specificity, high accuracy and precision, simple subsequent operation, short detection time and the like, solves the problem that bovine growth hormone relative biological activity cannot be detected at the cell level at present, and has high application value in bovine growth hormone drug research and development and quality control.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and more specifically, to a method for detecting the relative bioactivity of bovine growth hormone in vitro. Background Technology

[0002] Bovine growth hormone (BGH) is widely used, but its detection is difficult and complex, leading to ongoing concerns. Currently, there are three main methods: radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and animal immunoassay. RIA utilizes radiolabeled GH to competitively bind to antibodies against bGH in the sample. This method uses radioactive materials, posing safety risks, and is complex and time-consuming. ELISA detects the antigenicity of BGH, i.e., the protein structure, rather than its biological activity. Animal immunoassay assesses its activity by observing the biological effects of BGH on experimental animals (such as rats). For example, it measures weight gain and bone growth in rats. However, experiments require weeks or even months to observe changes in growth indicators such as weight and bone density. Individual animal responses to BGH vary, and a large number of animals are needed, along with the design of animal husbandry and management, resulting in high costs. In some cases, BGH may have undergone denaturation, modification, or misfolding. Although its antigenicity remains, its biological activity has been lost, and immunoassay cannot accurately reflect its true activity state. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide an in vitro relative bioactivity detection method for bovine growth hormone. This method is fast, convenient, and has low variability, filling the gap in cell-free bioactivity detection methods for bovine growth hormone and has high application value in the research and development and quality control of bovine growth hormone drugs.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] A method for detecting the relative bioactivity of bovine growth hormone in vitro, comprising the following steps:

[0006] S1: Construction of pbGHR: The bovine growth hormone receptor DNA sequence shown in SEQ ID NO.1 was obtained from the NCBI database. The Kozak sequence was ligated to the 5' end of the bovine growth hormone receptor DNA sequence, and the gene sequence was inserted between the BamHI restriction site and the XbaHI restriction site of the pcDNA3.1(+) plasmid. The recombinant plasmid was then transformed into competent cells. After antibiotic selection and shake-flask culture, the plasmid was extracted to obtain the plasmid pbGHR that expresses the bovine growth hormone receptor.

[0007] S2: Construction of HEK293-bGHR-Luc cells: HEK293 cells were cultured, and a transfection mixture was prepared using pbGHR and luciferase reporter plasmid. Cell transfection was then performed, and target cells were screened to obtain HEK293-bGHR-Luc cells.

[0008] S3: Detection of the relative biological activity of bovine growth hormone in vitro: HEK293-bGHR-Luc cells were cultured in cell culture plates. The bovine growth hormone test solution and bovine growth hormone control solution were pre-diluted, then serially diluted and added to the cell culture plates containing HEK293-bGHR-Luc cells. Fluorescent enzyme substrate was added, fluorescence values ​​were detected, and a four-parameter curve was fitted to calculate the relative biological activity of bovine growth hormone.

[0009] Furthermore, in step S1, the vector plasmid is pcDNA3.1(+) plasmid; the competent cells are DH5α competent cells; and the antibiotic is ampicillin.

[0010] Furthermore, in step S2, the culture medium for culturing HEK293 cells is DMEM culture medium, and the culture conditions are 5% CO2, cultured at 37°C for 18-24 hours;

[0011] The transfection mixture was prepared as follows: the luciferase reporter plasmid and pbGHR were diluted in DMEM culture medium, gently mixed, and cultured at room temperature to obtain solution 1; the transfection reagent was diluted in DMEM culture medium, gently mixed, and cultured at room temperature to obtain solution 2; solution 1 and solution 2 were mixed at room temperature to obtain the transfection mixture.

[0012] The cell transfection steps are as follows: After HEK293 cells are cultured, pour out the culture medium, add the transfection mixture for transfection, pour out the transfection mixture, add DMEM culture medium containing 10% fetal bovine serum, incubate at 5% CO2 and 37°C, pour out the DMEM culture medium containing 10% fetal bovine serum, add DMEM selection culture medium, and expand the culture.

[0013] Furthermore, in step S2, the cell screening steps are as follows: digest the cells with trypsin, and when the cells are in a quicksand-like state, add DMEM culture medium containing 10% fetal bovine serum to stop the digestion, centrifuge to remove the supernatant, add DMEM complete culture medium to wash, centrifuge, resuspend in DMEM complete culture medium, dilute the cells with DMEM complete culture medium to cell culture plates, expand the culture to obtain monoclonal cell lines, and complete the first screening; select multiple monoclonal cell lines and perform a second screening according to the steps of the first screening.

[0014] Furthermore, in step S3, the specific detection steps are as follows:

[0015] (1) HEK293-bGHR-Luc cells were cultured in DMEM complete medium until the cell confluence reached 90%;

[0016] (2) Discard the DMEM complete culture medium, wash, digest with trypsin, collect the cells, prepare a cell suspension with RPMI 1640 culture medium, and seed them into cell culture plates for culture;

[0017] (3) Take bovine growth hormone test solution and bovine growth hormone control solution, and dilute them to the same concentration with RPMI 1640 culture medium. Then, use RPMI 1640 culture medium to perform serial dilution at 3-fold ratio and 8 concentrations. Add the serially diluted bovine growth hormone test solution and bovine growth hormone control solution to the cell culture plate in step (2) and place them in an incubator for culture.

[0018] (4) Place the cell culture plate at room temperature, add the luciferase substrate, mix well, incubate at room temperature in the dark, detect the fluorescence value, fit the four-parameter curve, and calculate the relative biological activity of bovine growth hormone.

[0019] Furthermore, when preparing the transfection mixture, the mass ratio of luciferase reporter plasmid to pbGHR was 1:1, the volume ratio of transfection reagent to DMEM culture medium was 1:50, the transfection reagent was Sinofection, and the volume ratio of solution 1 to solution 2 was 1:1.

[0020] The DMEM screening culture medium contains 10% fetal bovine serum, 1% double antibiotic solution, and 89% DMEM culture medium; the double antibiotic solution is a G418-hygromycin B mixture, wherein the concentration of G418 is 1000 μg / mL and the concentration of hygromycin B is 200 μg / mL.

[0021] Furthermore, the DMEM complete culture medium contains 10% fetal bovine serum, 1% double antibiotic solution, and 89% DMEM culture medium; the double antibiotic solution is a G418-hygromycin B mixture, wherein the concentration of G418 is 500 μg / mL and the concentration of hygromycin B is 200 μg / mL.

[0022] Furthermore, in step (2), the washing reagent is PBS buffer; the cell concentration of the cell suspension is 5.71 × 10⁻⁶. 5 Cells / mL; culture conditions: 5% CO2, 36.5±0.5℃ for 18~24h.

[0023] Furthermore, in step (3), the culture conditions are 5% CO2, cultured in an incubator at 37°C for 5 hours, and the same concentration is 30 μg / mL.

[0024] Furthermore, the method for preparing the bovine growth hormone test solution is as follows: take the bovine growth hormone to be tested and prepare a bovine growth hormone test solution with a concentration of 1 mg / mL using 1% PBS solution as a solvent; the method for preparing the bovine growth hormone reference solution is as follows: take recombinant bovine growth hormone and prepare a bovine growth hormone reference solution with a concentration of 1 mg / mL using 1% PBS solution as a solvent.

[0025] In summary, the present invention has the following beneficial effects:

[0026] This invention constructs a plasmid expressing the bovine growth hormone receptor (BGH) receptor by inserting the bovine growth hormone receptor DNA sequence into the pcDNA3.1(+) plasmid. A luciferase reporter plasmid and a pbGHR plasmid are simultaneously transfected into HEK293 cells. After antibiotic selection, the cells are diluted to obtain monoclonal cell lines. Clones are isolated to obtain target cells highly responsive to bovine growth hormone. Then, bovine growth hormone test solution, bovine growth hormone control solution, and luciferase substrate are added to the cells. Fluorescence values ​​are detected, and a four-parameter curve is fitted to calculate the relative activity of bovine growth hormone. This invention features low cost, high stability, high specificity, high accuracy and precision, simple subsequent operations, and short detection time. It solves the current problem of lacking cellular-level detection of the relative biological activity of bovine growth hormone and has high application value in the research and quality control of bovine growth hormone drugs. Attached Figure Description

[0027] Figure 1 The image shows the pbGHR map of the bovine growth hormone receptor expression plasmid in Example 1.

[0028] Figure 2 This is a reaction curve diagram for the preliminary verification of the target cell activity in Example 1;

[0029] Figure 3 This is a reaction curve diagram used in Example 1 to verify the activity of the target cells;

[0030] Figure 4 This is a linear result graph showing the verification of the precision of the detection method in Example 2. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] Step 1: Building pbGHR

[0034] The bovine growth hormone receptor (BGH) DNA sequence was obtained from the NCBI database, as shown in SEQ ID NO.1. A CRO was commissioned to ligate the Kozak sequence to the 5' end of the BGH receptor DNA sequence. This gene sequence was then inserted between the BamHI and XbaHI restriction sites of the pcDNA3.1(+) plasmid (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) to obtain a recombinant plasmid. The recombinant plasmid was transformed into *E. coli* DH5α competent cells using a heat shock method. Screening was performed using 100 μg / mL ampicillin (purchased from China National Pharmaceutical Group Co., Ltd.). The cells were cultured overnight at 37°C and 250 rpm. Plasmids expressing the BGH receptor were extracted to obtain a plasmid (named pbGHR, e.g., ...). Figure 1 (As shown).

[0035] SEQ ID NO.1:

[0036]

[0037] Step 2: Construction of HEK293-bGHR-Luc cells

[0038] 1. HEK293 cells (purchased from ATCC) were cultured at a concentration of 0.3 × 10⁻⁶. 6 Cells were seeded at a concentration of cells / mL in 96-well cell culture plates (100 μL per well in DMEM medium (purchased from Gibco)) and cultured at 37°C for 20 h in 5% CO2 (the culture time can be adjusted between 18 and 24 h depending on cell adhesion).

[0039] 2. Preparation of the transfection mixture:

[0040] (1) Dilute 0.2 μg of luciferase reporter plasmid pGL4.32 [luc2P / NF-kB-RE / Hygro] (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) and 0.2 μg of pbGHR into DMEM culture medium (total 25 μL) and mix gently. Incubate at room temperature for 5 min to obtain solution 1;

[0041] (2) Dilute 0.5 μL of Sinofection transfection reagent (purchased from Beijing Sinofection Technology Co., Ltd.) into DMEM culture medium (total 25 μL), mix gently, and incubate at room temperature for 5 min to obtain solution 2;

[0042] (3) Mix solution 1 and solution 2 at room temperature for 20 minutes to obtain the transfection mixture.

[0043] 3. Cell transfection:

[0044] After cell adhesion, pour out the DMEM culture medium from the 96-well cell culture plate, leaving the adherent HEK293 cells. Add 50 μL of transfection mixture to each well and transfect for 6 hours (the transfection time can be adjusted between 4-6 hours depending on the actual situation). Pour out the transfection mixture and add DMEM culture medium containing fetal bovine serum (purchased from Gibco) (10% fetal bovine serum and 90% DMEM culture medium). Incubate at 37°C with 5% CO2 for 24 hours, then pour out the DMEM culture medium containing fetal bovine serum. Cells were cultured in DMEM selection medium [containing 10% fetal bovine serum, 1% penicillin-antibody solution (a mixture of G418 and hygromycin B, with G418 at 1000 μg / mL and hygromycin B at 200 μg / mL), and 89% DMEM medium]. The DMEM selection medium was changed every 4 days for a total of 4 times. Cells were collected by centrifugation and cultured in DMEM selection medium in 24-well and 6-well cell culture plates to a scale-up of 25 cm⁻¹. 2 Cell culture flasks are used to achieve 80% cell confluence.

[0045] 4. Preliminary verification of the activity of the target cells:

[0046] Partially expanded cultured cells were digested with trypsin until they became fibrous. Digestion was stopped by adding DMEM medium containing 10% fetal bovine serum. After centrifugation at 1000 rpm for 5 min, the supernatant was removed, and the cells were reconstituted with 2 mL of RPMI 1640 medium (purchased from Gibco). Cells were counted using a cell counter and then diluted with RPMI 1640 medium to a cell concentration of 5 × 10⁻⁶ cells / mL. 5 cells / mL, according to 4×10 4 Cells at a concentration of [number] cells / well were added to 96-well cell culture plates and cultured at 5% CO2 and 37°C for 20 h (adjusted between 18 and 22 h depending on cell culture progress). Bovine growth hormone was added at serially diluted levels (starting concentration 30 μg / mL, 3-fold serial dilutions, 8 concentration gradients). 100 μL of GMOne-Step 2.0 luciferase substrate (purchased from Jiman Biotechnology (Shanghai) Co., Ltd.) was added. Fluorescence values ​​were detected using a microplate reader, and a four-parameter curve was fitted. The results are shown below. Figure 2 As shown in the figure, there is a positive correlation between bovine growth hormone concentration and luciferase expression level.

[0047] 5. Target cell screening:

[0048] The remaining cells after expansion culture were digested with trypsin until they became slurry-like. Digestion was stopped by adding DMEM medium containing fetal bovine serum (10% fetal bovine serum and 90% DMEM medium). The cells were centrifuged at 1000 rpm for 5 min, the supernatant was removed, and the cells were washed with DMEM complete medium (containing 10% fetal bovine serum, 1% antibiotic solution (G418-hygromycin B mixture, where G418 concentration is 1000 μg / mL and hygromycin B concentration is 200 μg / mL), and 89% DMEM medium). The cells were centrifuged at 1000 rpm for 5 min, resuspended in DMEM complete medium, and then diluted to 0.5 cells per well in 96-well cell culture plates to ensure monoclonal colonies. Cells were then serially expanded to 25 cm⁻¹ in 24-well and 6-well cell culture plates. 2 In cell culture flasks, cell confluence was increased to 80%, completing the first screening. Ten monoclonal cell lines were selected and subjected to a second screening following the same steps. Stimulation was performed with serially diluted bovine growth hormone (initial concentration 30 μg / mL, 3-fold serial dilution, 8 concentration gradients). 100 μL of GMOne-Step 2.0 luciferase substrate was added, and fluorescence values ​​were detected using a microplate reader. A four-parameter curve was fitted to obtain the target cells, namely HEK293-bGHR-Luc cells. In this step, four target cell lines were selected (four-parameter curves are shown below). Figure 3 (As shown).

[0049] Step 4: Detection of in vitro bioactivity of bovine growth hormone

[0050] (1) Select one target cell (i.e., one HEK293-bGHR-Luc cell) obtained in step 5 and culture it in DMEM complete culture medium in a 75cm2 cell culture flask until the cell confluence reaches 90%.

[0051] (2) Discard the DMEM complete culture medium, wash once with PBS buffer, then digest with trypsin, collect the cells, and prepare a culture medium containing 5.71 × 10⁻⁶ cells per mL using RPMI 1640 medium. 5 Cell suspensions of 1,000 cells were seeded into cell culture plates at 70 µL per well, for a total of 32 wells. The cells were then cultured at 5% CO2 and 36.5 ± 0.5 °C for 20 h (the culture time can be adjusted between 18 and 24 h depending on the culture conditions).

[0052] (3) Take the bovine growth hormone to be tested and prepare a bovine growth hormone test solution with a concentration of 1 mg / mL using 1% PBS solution as solvent; take recombinant bovine growth hormone and prepare a bovine growth hormone reference solution with a concentration of 1 mg / mL using 1% PBS solution as solvent (the bovine growth hormone used as the reference can be purchased or synthesized according to existing methods; in this example, recombinant bovine growth hormone with a biological activity of 3 IU / mg purchased from Hangzhou Jiedi Biotechnology Co., Ltd. is used as the reference); dilute with RPMI 1640 culture medium to prepare a 30 µg / mL bovine growth hormone test solution and a 30 µg / mL bovine growth hormone reference solution;

[0053] (4) Place the 30µg / mL bovine growth hormone test solution and the 30µg / mL bovine growth hormone control solution in a cell culture plate, and dilute them 3-fold and 8 times with RPMI 1640 culture medium, with 2 wells for each concentration gradient, for a total of 32 wells;

[0054] (5) Add the bovine growth hormone test solution and bovine growth hormone control solution, which are serially diluted, to the cell culture plate of the cells cultured in step (2), 70 µL per well, and incubate in a 5% CO2, 37℃ incubator for 5 h;

[0055] (6) Remove the cell culture plate and let it stand at room temperature for 20 min. Add 100 μL of GMOne-Step2.0 luciferase substrate to each well, mix thoroughly, and incubate in the dark at room temperature for 5 min. Detect the fluorescence value with a microplate reader, fit a four-parameter curve, and calculate the relative biological activity of bovine growth hormone according to the following formula:

[0056]

[0057] In the formula: Pr represents the biological activity of the reference standard, IU / mg.

[0058] In this embodiment, the reference solution Ln (EC50) was 2.596, the test solution Ln (EC50) was 2.569, and Pr was 3 IU / mg. The relative biological activity of the bovine growth hormone test sample was calculated to be 3.03 IU / mg.

[0059] Example 2: Methodological Validation of Bovine Growth Hormone Bioactivity Assay

[0060] 1. Specificity verification

[0061] This method is for detecting the biological activity of bovine growth hormone, and its specificity needs to be verified. It can effectively detect the biological activity of bovine growth hormone and has a clear ability to detect degraded bovine growth hormone. Four samples of bovine growth hormone were taken and treated in a 60℃ water bath for 0, 3, 6, and 9 days, respectively, and then tested according to step 4 of Example 1. The results are shown in Table 1. With the increase of the 60℃ water bath time, the biological activity of bovine growth hormone showed a gradual decreasing trend. This method can effectively detect the actual biological activity of bovine growth hormone.

[0062] Table 1

[0063]

[0064] In Table 1, the relative potency is calculated as: bovine growth hormone control solution Ln(EC50) / sample Ln(EC50).

[0065] 2. Joint verification of accuracy, linearity, range, and precision.

[0066] Two researchers tested bovine growth hormone diluted to relative potencies of 50%, 71%, 100%, 141%, and 200% on different dates, following the method in step 4 of Example 1. The test results are shown in Table 2.

[0067] Table 2

[0068]

[0069] The accuracy of the data was analyzed with reference to the current version of the Pharmacopoeia, and the results are shown in Tables 3 and 4.

[0070] Table 3

[0071]

[0072] Table 4

[0073]

[0074] The results showed that the relative bias of the five relative potency levels was less than ±5%, which meets the requirement of less than ±12% in the current version of the pharmacopoeia. A linear regression was performed on the logarithm of the theoretical potency value (x-axis) and the logarithm of the corresponding potency measurement value (y-axis). The slope of the regression equation was 0.9397, which is in the range of 0.80 to 1.25, indicating that the accuracy meets the standards of the current version of the pharmacopoeia.

[0075] Precision, linearity, and range analyses were performed on the data with reference to the current edition of the Pharmacopoeia. The results are shown in Table 5.

[0076] Table 5

[0077]

[0078] The geometrical coefficient of variation (CIGCV, %) of the actual measured values ​​of the theoretical relative potency of bovine growth hormone were 5.535%, 4.529%, 6.520%, 5.874%, and 5.151%, respectively, all less than 20%, indicating that they precisely meet the standards of the current version of the pharmacopoeia.

[0079] Plotting the theoretical relative potency of bovine growth hormone on the x-axis and the corresponding measured relative potency on the y-axis, the linear results are as follows: Figure 4 As shown, the regression equation is Y = 0.9397X + 0.0366, R0 2 It is 0.9819.

[0080] Using the logarithm of the theoretical relative potency of bovine growth hormone as the x-axis and the corresponding logarithm of the measured relative potency as the y-axis, a linear regression was performed using the least squares method. The correlation coefficient of the regression equation was 0.9835 > 0.98, indicating that the linearity conforms to the current version of the pharmacopoeia.

[0081] Range validation results: The accuracy, intermediate precision, and linear relative valence ranged from 50% to 200% in 8 experiments.

[0082] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting the relative bioactivity of bovine growth hormone in vitro, characterized in that, Includes the following steps: S1: Construction of pbGHR: The bovine growth hormone receptor DNA sequence shown in SEQ ID NO.1 was obtained from the NCBI database. The Kozak sequence was ligated to the 5' end of the bovine growth hormone receptor DNA sequence, and the gene sequence was inserted between the BamHI restriction site and the XbaHI restriction site of the pcDNA3.1(+) plasmid. The recombinant plasmid was then transformed into competent cells. After antibiotic screening and shake-flask culture, the plasmid was extracted to obtain the plasmid pbGHR that expresses the bovine growth hormone receptor. S2: Construction of HEK293-bGHR-Luc cells: HEK293 cells were cultured, and a transfection mixture was prepared using pbGHR and luciferase reporter plasmid pGL4.32 [luc2P / NF-kB-RE / Hygro]. Cell transfection was then performed, and target cells were screened to obtain HEK293-bGHR-Luc cells. S3: Detection of the relative biological activity of bovine growth hormone in vitro: HEK293-bGHR-Luc cells were cultured in cell culture plates. The bovine growth hormone test solution and bovine growth hormone control solution were pre-diluted, then serially diluted and added to the cell culture plates containing HEK293-bGHR-Luc cells. Fluorescent enzyme substrate was added, fluorescence values ​​were detected, and a four-parameter curve was fitted to calculate the relative biological activity of bovine growth hormone.

2. The method for detecting the relative bioactivity of bovine growth hormone in vitro according to claim 1, characterized in that, In step S1, the competent cells are DH5α competent cells, and the antibiotic is ampicillin.

3. The method for detecting the relative bioactivity of bovine growth hormone in vitro according to claim 1, characterized in that, In step S2, the culture medium for culturing HEK293 cells is DMEM culture medium, and the culture conditions are 5% CO2, 37°C, for 18-24 hours. The transfection mixture was prepared as follows: the luciferase reporter plasmid and pbGHR were diluted in DMEM culture medium, gently mixed, and cultured at room temperature to obtain solution 1; the transfection reagent was diluted in DMEM culture medium, gently mixed, and cultured at room temperature to obtain solution 2; solution 1 and solution 2 were mixed at room temperature to obtain the transfection mixture. The cell transfection steps are as follows: After HEK293 cells are cultured, pour out the culture medium, add the transfection mixture for transfection, pour out the transfection mixture, add DMEM culture medium containing 10% fetal bovine serum, incubate at 5% CO2 and 37°C, pour out the DMEM culture medium containing 10% fetal bovine serum, add DMEM selection culture medium, and expand the culture.

4. The method for detecting the relative bioactivity of bovine growth hormone in vitro according to claim 1, characterized in that, In step S2, the cell screening steps are as follows: digest the cells with trypsin, and when the cells are in a quicksand-like state, add DMEM culture medium containing 10% fetal bovine serum to stop the digestion, centrifuge to remove the supernatant, add DMEM complete culture medium to wash, centrifuge, resuspend in DMEM complete culture medium, dilute the cells with DMEM complete culture medium to cell culture plates, expand the culture to obtain monoclonal cell lines, and complete the first screening; select multiple monoclonal cell lines and perform a second screening according to the steps of the first screening.

5. The method for detecting the relative bioactivity of bovine growth hormone in vitro according to claim 1, characterized in that, In step S3, the specific detection steps are as follows: (1) HEK293-bGHR-Luc cells were cultured in DMEM complete medium until the cell confluence reached 90%; (2) Discard the DMEM complete culture medium, wash, digest with trypsin, collect the cells, prepare a cell suspension with RPMI 1640 culture medium, and seed them into cell culture plates for culture; (3) Take bovine growth hormone test solution and bovine growth hormone control solution, and dilute them to the same concentration with RPMI 1640 culture medium. Then, use RPMI 1640 culture medium to perform serial dilution at 3-fold ratio and 8 concentrations. Add the serially diluted bovine growth hormone test solution and bovine growth hormone control solution to the cell culture plate in step (2) and place them in an incubator for culture. (4) Place the cell culture plate at room temperature, add the luciferase substrate, mix well, incubate at room temperature in the dark, detect the fluorescence value, fit the four-parameter curve, and calculate the relative biological activity of bovine growth hormone.

6. The method for detecting the relative bioactivity of bovine growth hormone in vitro according to claim 3, characterized in that, When preparing the transfection mixture, the mass ratio of luciferase reporter plasmid to pbGHR was 1:1, the volume ratio of transfection reagent to DMEM culture medium was 1:50, the transfection reagent was Sinofection, and the volume ratio of solution 1 to solution 2 was 1:

1. The DMEM screening culture medium contains 10% fetal bovine serum, 1% double antibiotic solution, and 89% DMEM culture medium; the double antibiotic solution is a G418-hygromycin B mixture, wherein the concentration of G418 is 1000 μg / mL and the concentration of hygromycin B is 200 μg / mL.

7. A method for detecting the relative bioactivity of bovine growth hormone in vitro according to claim 4 or 5, characterized in that, The DMEM complete culture medium contains 10% fetal bovine serum, 1% double antibiotic solution and 89% DMEM culture medium; the double antibiotic solution is a G418-hygromycin B mixture, wherein the concentration of G418 is 500 μg / mL and the concentration of hygromycin B is 200 μg / mL.

8. The method for detecting the relative bioactivity of bovine growth hormone in vitro according to claim 5, characterized in that, In step (2), the washing reagent is PBS buffer; the cell concentration of the cell suspension is 5.71 × 10⁻⁶. 5 Cells / mL; culture conditions: 5% CO2, 36.5±0.5℃ for 18~24h.

9. The method for detecting the relative bioactivity of bovine growth hormone in vitro according to claim 5, characterized in that, In step (3), the culture conditions are 5% CO2, cultured in an incubator at 37°C for 5 hours, and the same concentration is 30 μg / mL.

10. A method for detecting the relative bioactivity of bovine growth hormone in vitro according to claim 1 or 5, characterized in that, The method for preparing the bovine growth hormone test solution is as follows: take the bovine growth hormone to be tested and prepare a bovine growth hormone test solution with a concentration of 1 mg / mL using 1% PBS solution as a solvent; The method for preparing bovine growth hormone reference solution is as follows: Take recombinant bovine growth hormone and use 1% PBS solution as solvent to prepare a bovine growth hormone reference solution with a concentration of 1 mg / mL.

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