CN-120685920-B - Method for detecting in-vitro relative biological activity of bovine growth hormone

CN120685920BCN 120685920 BCN120685920 BCN 120685920BCN-120685920-B

Abstract

The invention discloses a method for detecting the in-vitro relative biological activity of bovine growth hormone, belonging to the technical field of biological detection. The method comprises the following steps of pbGHR construction, HEK293-bGHR-Luc cell construction and bovine growth hormone in-vitro relative biological activity detection. 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 bovine growth hormone is not detected at the cell level, and has higher application value in research and development and quality control of bovine growth hormone medicaments.

Inventors

LIU HAICHANG
HU WENLONG
HU YIFENG
HU DONG
LIU DONG
Zhu Fupei
YIN JINRU
LIANG PING
WEN QING
LI JIAXIN

Assignees

浙江毓昌生物技术有限公司

Dates

Publication Date: 20260512
Application Date: 20250620

Claims (10)

1. The method for detecting the in-vitro relative biological activity of bovine growth hormone is characterized by comprising the following steps of: S1, pbGHR, obtaining a bovine growth hormone receptor DNA sequence shown as SEQ ID NO.1 through NCBI database, connecting a Kozak sequence to the 5' end of the bovine growth hormone receptor DNA sequence, inserting a gene sequence between a BamHI enzyme cutting site and an XbaI enzyme cutting site of pcDNA3.1 (+) plasmid, transferring the recombinant plasmid into competent cells, screening antibiotics, shake-flask culturing, and extracting the plasmid to obtain a plasmid pbGHR capable of expressing the bovine growth hormone receptor; S2, constructing HEK293-bGHR-Luc cells, namely culturing HEK293 cells, preparing transfection mixture by using pbGHR and luciferase reporter plasmid pGL4.32[ Luc2P/NF-kB-RE/Hygro ], then carrying out cell transfection, and screening out target cells to obtain HEK293-bGHR-Luc cells; S3, detecting the in-vitro relative biological activity of bovine growth hormone, namely culturing HEK293-bGHR-Luc cells in a cell culture plate, pre-diluting bovine growth hormone test sample solution and bovine growth hormone reference substance solution, respectively carrying out gradient dilution treatment, adding the diluted bovine growth hormone test sample solution and bovine growth hormone reference substance solution into the cell culture plate for culturing HEK293-bGHR-Luc cells, adding a fluorogenic enzyme substrate, detecting a fluorescence value, fitting a four-parameter curve, and calculating the relative biological activity of bovine growth hormone.
2. The method according to claim 1, wherein in step S1, the competent cells are DH5 a competent cells and the antibiotic is ampicillin.
3. The method for detecting the in vitro relative biological activity of bovine growth hormone according to claim 1, wherein in the step S2, the culture solution for culturing HEK293 cells is DMEM culture solution, and the culture condition is 5% CO 2 and the culture is carried out for 18-24 hours at 37 ℃; The preparation method of the transfection mixture comprises the steps of diluting luciferase reporter plasmid and pbGHR into DMEM culture solution, lightly mixing and culturing at room temperature to obtain solution 1, diluting transfection reagent into DMEM culture solution, lightly mixing and culturing at room temperature to obtain solution 2, mixing the solution 1 and the solution 2 at room temperature to obtain transfection mixture; The cell transfection step comprises pouring out culture solution after HEK293 cell culture is completed, adding transfection mixture for transfection, pouring out transfection mixture, adding DMEM culture solution containing 10% fetal calf serum, incubating at 5% CO 2 and 37 ℃, pouring out DMEM culture solution containing 10% fetal calf serum, adding DMEM screening culture solution, and performing amplification culture.
4. The method for detecting the in vitro relative biological activity of bovine growth hormone according to claim 1, wherein in the step S2, the step of screening cells is to digest cells with trypsin, stop digestion when the cells are in a quicksand shape, add DMEM culture solution containing 10% fetal bovine serum, centrifuge to remove supernatant, add DMEM complete culture solution for washing, centrifuge, dilute the cells to a cell culture plate with the DMEM complete culture solution after being resuspended with the DMEM complete culture solution, enlarge the culture to obtain monoclonal cell strains, complete the primary screening, select a plurality of monoclonal cell strains, and perform the secondary screening according to the primary screening step.
5. The method for detecting the in vitro relative biological activity of bovine growth hormone according to claim 1, wherein in the step S3, the specific steps of detecting are as follows: (1) HEK293-bGHR-Luc cells were cultured with DMEM complete medium to a cell confluence of 90%; (2) Discarding the DMEM complete culture solution, washing, digesting by trypsin, collecting cells, preparing cell suspension by using RPMI1640 culture solution, and inoculating to a cell culture plate for culture; (3) Respectively diluting a bovine growth hormone test sample solution and a bovine growth hormone reference substance solution to the same concentration by using an RPMI1640 culture solution, then carrying out gradient dilution by using the RPMI1640 culture solution according to the concentration ratio of 3 times and the concentration of 8, adding the bovine growth hormone test sample solution and the bovine growth hormone reference substance solution subjected to gradient dilution into the cell culture plate in the step (2), and placing the cell culture plate in an incubator for culture; (4) And (3) standing the cell culture plate at room temperature, adding a fluorogenic enzyme substrate, uniformly mixing, incubating at room temperature in a dark place, detecting fluorescence values, fitting a four-parameter curve, and calculating the relative biological activity of bovine growth hormone.
6. The method for detecting the in vitro relative biological activity of bovine growth hormone according to claim 3, wherein the mass ratio of the luciferase reporter plasmid to pbGHR is 1:1, the volume ratio of the transfection reagent to the DMEM culture solution is 1:50, the transfection reagent is Sinofection, and the volume ratio of the solution 1 to the solution 2 is 1:1; The DMEM screening culture solution contains 10% of fetal bovine serum, 1% of double-antibody liquid and 89% of DMEM culture solution, wherein the double-antibody liquid is a mixed liquid of G418 and hygromycin B, the concentration of G418 is 1000 mug/mL, and the concentration of hygromycin B is 200 mug/mL.
7. The method for detecting the in vitro relative biological activity of bovine growth hormone according to claim 4 or 5, wherein the DMEM complete culture solution comprises 10% fetal bovine serum, 1% dual-antibody solution and 89% DMEM culture solution, wherein the dual-antibody solution is a mixed solution of G418 and hygromycin B, 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 in vitro relative biological activity of bovine growth hormone according to claim 5, wherein in the step (2), the washing reagent is PBS buffer solution, the cell concentration of the cell suspension is 5.71×10 5 cells/mL, and the culture condition is 5% CO 2 and 36.5+ -0.5 ℃ for 18-24 hours.
9. The method according to claim 5, wherein in step (3), the culture conditions are 5% CO 2 and the culture is carried out in an incubator at 37 ℃ for 5 hours, and the same concentration is 30 μg/mL.
10. The method for detecting the in-vitro relative biological activity of bovine growth hormone according to claim 1 or 5, wherein the method for preparing the bovine growth hormone test solution is characterized in that bovine growth hormone to be detected is prepared into bovine growth hormone test solution with the concentration of 1mg/mL by taking 1% PBS solution as a solvent, and the method for preparing the bovine growth hormone control solution is prepared into bovine growth hormone control solution with the concentration of 1mg/mL by taking recombinant bovine growth hormone and taking 1% PBS solution as a solvent.

Description

Method for detecting in-vitro relative biological activity of bovine growth hormone Technical Field The invention relates to the technical field of biological detection, in particular to a method for detecting the in-vitro relative biological activity of bovine growth hormone. Background Bovine growth hormone is highly used, but detection difficulty is high, and operation is complex and continuous attention is paid. Currently, there are Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), animal immunization. Radioimmunoassay (RIA), which uses radioactive substances to compete with bGH in a sample for binding to antibodies, involves a safety risk, is complicated to operate, and takes a long time. Enzyme-linked immunosorbent assay (ELISA) detects the antigenicity of bovine growth hormone, i.e. the structure of the protein, rather than its biological activity. Animal immunization, the activity of bovine growth hormone is assessed by observing its biological effect on experimental animals (e.g., rats). For example, the weight gain and bone growth of rats are measured, but the growth index changes such as weight bones are observed in a period of weeks or even months, the responses of different experimental animals to bovine growth hormone are different, a large number of experimental animals are needed, and the cost is high in design of animal feeding management and the like. In some cases, bovine growth hormone may be denatured, modified or folded, and although antigenicity is still present, biological activity is lost, and the immunoassay method cannot accurately reflect the actual activity state. Disclosure of Invention The invention aims to overcome the defects of the prior art, and provides the method for detecting the in-vitro relative bioactivity of the bovine growth hormone, which is quick, convenient and small in variation, fills up the blank of the cell-free bioactivity detection method of the bovine growth hormone, and has higher application value in research and development and quality control of bovine growth hormone medicaments. In order to achieve the above purpose, the present invention adopts the following technical scheme: the method for detecting the in-vitro relative biological activity of bovine growth hormone comprises the following steps: the construction of pbGHR, namely obtaining a bovine growth hormone receptor DNA sequence shown as SEQ ID NO.1 through NCBI database, connecting a Kozak sequence to the 5' end of the bovine growth hormone receptor DNA sequence, inserting the gene sequence between a BamHI enzyme cutting site and an XbaI enzyme cutting site of pcDNA3.1 (+) plasmid, transferring the recombinant plasmid into competent cells, screening antibiotics, shake flask culturing, and extracting the plasmid to obtain plasmid pbGHR capable of expressing the bovine growth hormone receptor; S2, constructing HEK293-bGHR-Luc cells, namely culturing HEK293 cells, preparing transfection mixture by using pbGHR and luciferase reporter plasmid, then carrying out cell transfection, and screening out target cells to obtain HEK293-bGHR-Luc cells; S3, detecting the in-vitro relative biological activity of bovine growth hormone, namely culturing HEK293-bGHR-Luc cells in a cell culture plate, pre-diluting bovine growth hormone test sample solution and bovine growth hormone reference substance solution, respectively carrying out gradient dilution treatment, adding the diluted bovine growth hormone test sample solution and bovine growth hormone reference substance solution into the cell culture plate for culturing HEK293-bGHR-Luc cells, adding a fluorogenic enzyme substrate, detecting a fluorescence value, fitting a four-parameter curve, and calculating the relative biological activity of bovine growth hormone. Further, the vector plasmid in the step S1 is pcDNA3.1 (+) plasmid, the competent cells are DH5 alpha competent cells, and the antibiotic is ampicillin. Further, in the step S2, the culture solution for culturing HEK293 cells is DMEM culture solution, and the culture condition is 5% CO 2 and the culture is carried out for 18-24 hours at 37 ℃; The preparation method of the transfection mixture comprises the steps of diluting luciferase reporter plasmid and pbGHR into DMEM culture solution, lightly mixing and culturing at room temperature to obtain solution 1, diluting transfection reagent into DMEM culture solution, lightly mixing and culturing at room temperature to obtain solution 2, mixing the solution 1 and the solution 2 at room temperature to obtain transfection mixture; The cell transfection step comprises pouring out culture solution after HEK293 cell culture is completed, adding transfection mixture for transfection, pouring out transfection mixture, adding DMEM culture solution containing 10% fetal calf serum, incubating at 5% CO 2 and 37 ℃, pouring out DMEM culture solution containing 10% fetal calf serum, adding DMEM screening culture solution, and performing amplificatio