Method for detecting in-vitro relative biological activity of cattle growth hormone

Abstract

The invention discloses a cattle growth hormone in-vitro relative biological activity detection method, and belongs to the technical field of biological detection. The method comprises the following steps: construction of pbGHR, construction of HEK293-bGHR-Luc cells, and detection of the in-vitro relative biological activity of the bovine growth hormone. The method disclosed by the invention has the characteristics of low cost, strong stability, strong specificity, high accuracy and precision, simple subsequent operation and short detection time, solves the problem that the relative biological activity of the current cattle growth hormone is not detected at a cellular level, and has a relatively high application value in research and development and quality control of cattle growth hormone drugs.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and more particularly to a method for detecting the relative biological activity of bovine growth hormone in vitro. Background Art

[0002] Bovine growth hormone (BGH) is widely used, but its detection is challenging and complex, drawing ongoing attention. Currently, methods include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and animal immunoassays. The RIA utilizes radiolabeled GH to compete with bGH in a sample for binding to antibodies. This method, which uses radioactive materials, poses safety risks, is complex, and time-consuming. The ELISA detects the antigenicity of BGH—that is, the protein's structure—rather than its biological activity. The animal immunoassay assesses its activity by observing the biological effects of BGH on experimental animals (such as rats). For example, weight gain and bone growth can be measured in rats. However, these experiments require weeks or even months to observe changes in these growth indicators. Individual experimental animals vary in their response to BGH, and the large number of experimental animals required, along with the cost of animal husbandry and management, is high. In some cases, BGH may undergo denaturation, modification, or misfolding. While its antigenicity remains, its biological activity has been lost. In these cases, immunoassays cannot accurately reflect its true activity. Summary of the Invention

[0003] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a method for detecting the relative bioactivity of bovine growth hormone in vitro. The method is fast, convenient, and has low variability, filling the gap in the cell-free bioactivity detection method of bovine growth hormone and having high application value in the research and development and quality control of bovine growth hormone drugs.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for detecting the relative biological activity of bovine growth hormone in vitro comprises the following steps:

[0006] S1: Construction of pbGHR: The bovine growth hormone receptor DNA sequence (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 and XbaⅠ restriction sites 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 bovine growth hormone receptor-expressing plasmid pbGHR.

[0007] S2: Construction of HEK293-bGHR-Luc cells: HEK293 cells were cultured, and a transfection mixture was prepared using pbGHR and luciferase reporter plasmids. Subsequently, cells were transfected and the target cells were screened to obtain HEK293-bGHR-Luc cells.

[0008] S3: Detection of relative bioactivity 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 reference solution were pre-diluted and then added to the cell culture plates in which HEK293-bGHR-Luc cells were cultured. Luminescence enzyme substrate was added, the fluorescence value was detected, and a four-parameter curve was fitted to calculate the relative bioactivity 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 and 37°C for 18-24 hours;

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

[0012] The cell transfection steps are as follows: after the HEK293 cell culture is completed, the culture medium is poured out, the transfection mixture is added for transfection, the transfection mixture is poured out, DMEM culture medium containing 10% fetal bovine serum is added, and the cells are incubated under 5% CO2 and 37°C conditions, the DMEM culture medium containing 10% fetal bovine serum is poured out, DMEM screening culture medium is added, and the culture is expanded.

[0013] Furthermore, in step S2, the step of screening cells is as follows: digesting the cells with trypsin, adding DMEM culture medium containing 10% fetal bovine serum to terminate the digestion when the cells become a quicksand, centrifuging to remove the supernatant, adding DMEM complete culture medium to wash, centrifuging, resuspending with DMEM complete culture medium, and then diluting the cells with DMEM complete culture medium to a cell culture plate, expanding the culture to obtain a monoclonal cell line, and completing the first screening; selecting multiple monoclonal cell lines, and performing a second screening according to the steps of the first screening.

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

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

[0016] (2) Discard the DMEM complete culture medium, wash, trypsinize, collect the cells, prepare a cell suspension with RPMI1640 culture medium, and inoculate into a cell culture plate for culture;

[0017] (3) Take the bovine growth hormone test solution and the bovine growth hormone reference solution, dilute the bovine growth hormone test solution and the bovine growth hormone reference solution to the same concentration with RPMI1640 culture medium, then use RPMI1640 culture medium and perform gradient dilution at a ratio of 3 times and 8 concentrations, add the gradient diluted bovine growth hormone test solution and bovine growth hormone reference 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 fluorescent enzyme 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 the luciferase reporter plasmid and pbGHR was 1:1, the volume ratio of the transfection reagent and DMEM culture medium was 1:50, and the transfection reagent was Sinofection; the volume ratio of solution 1 and solution 2 was 1:1;

[0020] The DMEM screening culture medium contains 10% fetal bovine serum, 1% double antibody solution and 89% DMEM culture medium; the double antibody 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 antibody solution and 89% DMEM culture medium; the double antibody 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 5 The culture conditions were 5% CO2, 36.5±0.5℃ for 18~24h.

[0023] Furthermore, in step (3), the culture conditions are 5% CO2, 37°C in an incubator 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: to take the bovine growth hormone to be tested, a bovine growth hormone test solution with a concentration of 1 mg / mL is prepared using 1% PBS solution as a solvent; the method for preparing the bovine growth hormone reference solution is as follows: to take recombinant bovine growth hormone, a bovine growth hormone reference solution with a concentration of 1 mg / mL is prepared using 1% PBS solution as a solvent.

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

[0026] The present invention constructs a plasmid capable of expressing bovine growth hormone receptor by inserting a bovine growth hormone receptor DNA sequence into a pcDNA3.1(+) plasmid, simultaneously transfecting a luciferase reporter plasmid and a pbGHR plasmid into HEK293 cells, adding antibiotics for screening, diluting, obtaining a monoclonal cell line, isolating and cloning, obtaining target cells highly responsive to bovine growth hormone, then adding a bovine growth hormone test solution, a bovine growth hormone reference solution, and a luciferase substrate to the cells, detecting fluorescence values, fitting a four-parameter curve, and calculating the relative activity of the bovine growth hormone. The present invention has the characteristics of low cost, strong stability, strong specificity, high accuracy and precision, simple subsequent operation, and short detection time, solves the problem that there is no cell-level detection of the relative biological activity of bovine growth hormone in the current situation, and has high application value in the research and development and quality control of bovine growth hormone drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the map of the bovine growth hormone receptor expression plasmid pbGHR in Example 1;

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

[0029] Figure 3 This is a response curve diagram for verifying the activity of target cells in Example 1;

[0030] Figure 4 This is a linear result diagram for verifying the precision of the detection method in Example 2. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Example 1

[0033] Step 1: Construction of pbGHR

[0034] The bovine growth hormone receptor DNA sequence was obtained from the NCBI database, as shown in SEQ ID NO. 1. A CRO company was commissioned to ligate the Kozak sequence to the 5' end of the bovine growth hormone receptor DNA sequence, and the gene sequence was inserted between the BamHI and XbaⅠ 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 Escherichia coli DH5α competent cells using the heat shock method, screened with 100 μg / mL ampicillin (purchased from China National Pharmaceutical Group Co., Ltd.), and cultured overnight at 37°C and 250 rpm. The plasmid was extracted to obtain a plasmid expressing bovine growth hormone receptor (named pbGHR, as shown in FIG). Figure 1 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 0.3×10 6 Cells were seeded at a concentration of 100 μL / mL in a 96-well cell culture plate (DMEM (purchased from Gibco) as the culture medium, 100 μL per well), and cultured at 37°C with 5% CO2 for 20 h (the culture time can be adjusted between 18 and 24 h depending on the cell adhesion).

[0039] 2. Preparation of 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 (a total of 25 μL) and gently mix. Incubate at room temperature for 5 min to obtain solution 1;

[0041] (2) Dilute 0.5 μL of Sinofection transfection reagent (purchased from Beijing Sino-Bio Technology Co., Ltd.) into DMEM culture medium (a total of 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 a transfection mixture.

[0043] 3. Cell transfection:

[0044] After the cells adhered, the DMEM culture medium in the 96-well cell culture plate was poured out, leaving the attached HEK293 cells. 50 μL of transfection mixture was added to each well and transfection was carried out for 6 hours (the transfection time can be adjusted between 4-6 hours according to actual conditions). The transfection mixture was poured out and DMEM culture medium containing fetal bovine serum (purchased from Gibco) (10% fetal bovine serum and 90% DMEM culture medium) was added. After incubation for 24 hours under 5% CO2 and 37°C, the DMEM culture medium containing fetal bovine serum was poured out. DMEM screening culture medium [containing 10% fetal bovine serum, 1% double antibody solution (G418-hygromycin B mixture, where the G418 concentration is 1000 μg / mL and the hygromycin B concentration is 200 μg / mL), and 89% DMEM culture medium] was added for culture. The DMEM screening culture medium was replaced every 4 days during the culture period and a total of 4 times. The cells were collected by centrifugation and cultured in 24-well cell culture plates and 6-well cell culture plates in a stepwise manner until the cells were 25 cm thick. 2 Cell culture flasks were plated to allow the cells to reach 80% confluence.

[0045] 4. Preliminary verification of target cell activity:

[0046] Part of the expanded cultured cells were digested with trypsin. When the cells became a quicksand, DMEM containing 10% fetal bovine serum was added to terminate the digestion. After centrifugation at 1000 rpm for 5 min, the supernatant was removed and the cells were re-dissolved with 2 mL of RPMI1640 culture medium (purchased from Gibco). After counting with a cell counter, the cells were diluted with RPMI1640 culture medium to a cell concentration of 5 × 10 5 / mL, according to 4×10 4 Cells were added to a 96-well cell culture plate at a concentration of 100 μg / well and cultured at 5% CO2 and 37°C for 20 h (adjusted between 18 and 22 h depending on the cell culture conditions). Doubly diluted bovine growth hormone was added for stimulation (initial concentration 30 μg / mL, 3-fold dilution, 8 concentration gradients), and 100 μL GMOne-Step2.0 luciferase substrate (purchased from Jiman Biotechnology (Shanghai) Co., Ltd.) was added. The fluorescence value was detected by a microplate reader and a four-parameter curve was fitted. The results are shown in the figure. Figure 2 As shown in the figure, the concentration of bovine growth hormone and the expression level of luciferase are positively correlated.

[0047] 5. Target cell screening:

[0048] The remaining expanded cells were digested with trypsin. When the cells became a quicksand, DMEM culture medium containing fetal bovine serum (10% fetal bovine serum and 90% DEME culture medium) was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was removed, and DMEM complete culture medium (containing 10% fetal bovine serum, 1% double antibody solution (G418-hygromycin B mixture, in which the G418 concentration was 1000 μg / mL and the hygromycin B concentration was 200 μg / mL), 89% DMEM culture medium) was added for washing. The cells were centrifuged at 1000 rpm for 5 min, and resuspended with DMEM complete culture medium. The cells were diluted with DMEM complete culture medium to 96-well cell culture plates, with 0.5 cells per well to ensure monoclonality. The cells were gradually expanded to 25 cm in 24-well cell culture plates and 6-well cell culture plates. 2 In the cell culture flask, the cell confluence reached 80%, completing the first screening. 10 monoclonal cell lines were selected and secondary screening was performed according to the steps of the first screening. Bovine growth hormone was added for stimulation (starting concentration 30μg / mL, 3-fold dilution, 8 concentration gradients), 100μL GMOne-Step2.0 luciferase substrate was added, and the fluorescence value was detected by microplate reader. The four-parameter curve was fitted to obtain the target cells, namely HEK293-bGHR-Luc cells. In this step, a total of 4 target cells were screened (the four-parameter curve is shown in Figure 2). Figure 3 shown).

[0049] Step 4: In vitro bioactivity testing of bovine growth hormone

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

[0051] (2) Discard the DMEM complete culture medium, wash once with PBS buffer, then digest with trypsin, collect the cells, and prepare RPMI1640 culture medium to contain 5.71×10 cells per 1 mL. 5 The cell suspension of 10 cells was inoculated into a cell culture plate, 70 μL per well, for a total of 32 wells, and cultured in 5% CO2, 36.5±0.5℃ for 20 hours (the time can be adjusted between 18 and 24 hours according to the culture conditions);

[0052] (3) Take the bovine growth hormone to be tested and use 1% PBS solution as solvent to prepare a bovine growth hormone test solution with a concentration of 1 mg / mL; 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 (the bovine growth hormone used as the reference substance can be purchased or synthesized according to existing methods; this embodiment uses recombinant bovine growth hormone with a biological activity of 3 IU / mg purchased from Hangzhou Jedi Biotechnology Co., Ltd. as the reference substance); dilute with RPMI1640 culture medium to prepare 30 μg / mL bovine growth hormone test solution and 30 μg / mL bovine growth hormone reference solution;

[0053] (4) Place 30µg / mL bovine growth hormone test solution and 30µg / mL bovine growth hormone reference solution in a cell culture plate, and dilute them 3-fold and 8 times with RPMI1640 culture medium, with 2 wells for each concentration gradient, for a total of 32 wells;

[0054] (5) Add the concentration gradient diluted bovine growth hormone test solution and bovine growth hormone reference solution to the cell culture plate where the cells have been cultured in step (2), 70 μL per well, and culture in a 5% CO2, 37°C incubator for 5 h;

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

[0056]

[0057] Where: Pr is the biological activity of the reference substance, IU / mg.

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

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

[0060] 1. Specificity verification

[0061] This method is a method for detecting the biological activity of bovine growth hormone, and its specificity needs to be verified. While it can effectively detect the biological activity of bovine growth hormone, it also has a clear detection capability for degraded bovine growth hormone. Take 4 portions of bovine growth hormone to be tested, treat them in a 60°C water bath for 0, 3, 6, and 9 days, respectively, and then test them according to the method in step 4 of Example 1. The results are shown in Table 1. With the increase of the 60°C water bath time, the biological activity of bovine growth hormone shows a trend of gradually decreasing. This method can effectively detect the actual biological activity of bovine growth hormone.

[0062] Table 1

[0063]

[0064] In Table 1, relative potency = bovine growth hormone reference solution Ln (EC50) / sample Ln (EC50).

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

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

[0067] Table 2

[0068]

[0069] The data were analyzed for accuracy with reference to the current edition 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 met the requirement of less than ±12% in the current edition of the Pharmacopoeia. A linear regression was performed on the logarithm of the theoretical potency value (horizontally) and the logarithm of the corresponding measured potency value (ordinately). The slope of the regression equation was 0.9397, which was within the range of 0.80~1.25, indicating that the accuracy met the standards of the current edition of the Pharmacopoeia.

[0075] The data were analyzed for precision, linearity, and range with reference to the current edition of the Pharmacopoeia. The results are shown in Table 5.

[0076] Table 5

[0077]

[0078] The geometric coefficient of variation (CIGCV, %) of the actual measured values ​​of each theoretical relative potency of bovine growth hormone was 5.535%, 4.529%, 6.520%, 5.874%, and 5.151%, all less than 20%, indicating that the precision met the current edition of the pharmacopoeia standards.

[0079] The theoretical relative potency of bovine growth hormone is taken as the horizontal axis, and the corresponding measured relative potency is taken as the vertical axis. The linear results are as follows: Figure 4 As shown, the regression equation is Y=0.9397X+0.0366, R 2 It is 0.9819.

[0080] The logarithm of the theoretical relative potency of bovine growth hormone was used as the horizontal axis, and the logarithm of the corresponding measured relative potency was used as the vertical axis. The least squares method was used for linear regression. The correlation coefficient of the regression equation was 0.9835>0.98, indicating that the linearity met the current pharmacopoeia standards.

[0081] Range validation results: The accuracy, intermediate precision and linearity of the relative potency 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 embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart 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 biological activity of bovine growth hormone in vitro, characterized in that: The following steps are involved: S1: Construction of pbGHR: The bovine growth hormone receptor DNA sequence (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 and XbaⅠ restriction sites 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 bovine growth hormone receptor-expressing plasmid pbGHR. S2: Construction of HEK293-bGHR-Luc cells: HEK293 cells were cultured, and a transfection mixture was prepared using pbGHR and luciferase reporter plasmids. Subsequently, cells were transfected and the target cells were screened to obtain HEK293-bGHR-Luc cells. S3: Detection of relative bioactivity 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 reference solution were pre-diluted and then added to the cell culture plates in which HEK293-bGHR-Luc cells were cultured. Luminescence enzyme substrate was added, the fluorescence value was detected, and a four-parameter curve was fitted to calculate the relative bioactivity of bovine growth hormone.

2. The method for detecting the relative biological activity of bovine growth hormone in vitro according to claim 1, characterized in that: In step S1, the vector plasmid is pcDNA3.1(+) plasmid; the competent cells are DH5α competent cells, and the antibiotic is ampicillin.

3. The method for detecting the relative biological activity of bovine growth hormone in vitro according to claim 1, wherein: In step S2, the HEK293 cells are cultured in DMEM medium at 5% CO2 and 37°C for 18-24 hours. The transfection mixture was prepared as follows: luciferase reporter plasmid and pbGHR were diluted into DMEM culture medium, gently mixed, and incubated at room temperature to obtain solution 1; transfection reagent was diluted into DMEM culture medium, gently mixed, and incubated at room temperature to obtain solution 2; solution 1 and solution 2 were mixed at room temperature to obtain a transfection mixture; The cell transfection steps are as follows: after the HEK293 cell culture is completed, the culture medium is poured out, the transfection mixture is added for transfection, the transfection mixture is poured out, DMEM culture medium containing 10% fetal bovine serum is added, and the cells are incubated under 5% CO2 and 37°C conditions, the DMEM culture medium containing 10% fetal bovine serum is poured out, DMEM screening culture medium is added, and the culture is expanded.

4. The method for detecting the relative biological activity of bovine growth hormone in vitro according to claim 1, wherein: In step S2, the steps of screening cells are as follows: digesting the cells with trypsin, adding DMEM culture medium containing 10% fetal bovine serum to terminate the digestion when the cells become a quicksand, removing the supernatant by centrifugation, adding DMEM complete culture medium for washing, centrifuging, resuspending the cells with DMEM complete culture medium, and then diluting the cells with DMEM complete culture medium onto a cell culture plate, expanding the culture to obtain a monoclonal cell line, and completing the first screening; selecting multiple monoclonal cell lines, and performing a second screening according to the steps of the first screening.

5. The method for detecting the relative biological activity of bovine growth hormone in vitro according to claim 1, characterized in that: In step S3, the specific steps of detection are: (1) HEK293-bGHR-Luc cells were cultured in DMEM complete medium until the cell confluence was 90%; (2) Discard the DMEM complete culture medium, wash, trypsinize, collect the cells, prepare a cell suspension with RPMI1640 culture medium, and inoculate into a cell culture plate for culture; (3) Take the bovine growth hormone test solution and the bovine growth hormone reference solution, dilute the bovine growth hormone test solution and the bovine growth hormone reference solution to the same concentration with RPMI1640 culture medium, then use RPMI1640 culture medium and perform gradient dilution at a ratio of 3 times and 8 concentrations, add the gradient diluted bovine growth hormone test solution and bovine growth hormone reference 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 fluorescent enzyme 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 biological activity 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 and pbGHR was 1:1, the volume ratio of transfection reagent and DMEM culture medium was 1:50, and the transfection reagent was Sinofection; the volume ratio of solution 1 and solution 2 was 1:1; The DMEM screening culture medium contains 10% fetal bovine serum, 1% double antibody solution and 89% DMEM culture medium; the double antibody 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 biological activity 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 antibody solution and 89% DMEM culture medium; the double antibody 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 biological activity 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 5 The culture conditions were 5% CO2, 36.5±0.5℃ for 18~24h.

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

10. A method for detecting the relative biological activity of bovine growth hormone in vitro according to claim 1 or 5, characterized in that: The bovine growth hormone test solution is prepared as follows: bovine growth hormone to be tested is taken and a 1% PBS solution is used as a solvent to prepare a bovine growth hormone test solution with a concentration of 1 mg / mL; The preparation method of 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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