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CN-122012670-A - EST protease activity detection method

CN122012670ACN 122012670 ACN122012670 ACN 122012670ACN-122012670-A

Abstract

The invention discloses an EST protease activity detection method which comprises the following steps of a, setting an EST experiment group, a negative control group and a blank control group, b, incubating the EST experiment group, the negative control group and the blank control group to obtain respective reaction mixtures, c, detecting the influence of the reaction mixtures on proliferation of breast cancer cells to obtain proliferation rates of the groups, d, calculating enzyme activity of EST proteins. The method is simple and easy to implement, has good repeatability and can effectively detect the activity of EST protease.

Inventors

  • LI YUXIN
  • LIU XIAOLING

Assignees

  • 烟台大学

Dates

Publication Date
20260512
Application Date
20260211

Claims (10)

  1. 1. A method for detecting the activity of EST protease, comprising the steps of: a. EST experimental group, negative control group and blank control group were set up: adding a buffer system with the same volume into each group, then respectively adding EST proteins to be detected and E2 into an EST experimental group, adding E2 into a negative control group, and adding solvents with the same concentration and final volume as those of the first two groups into a blank control group; wherein the buffer system comprises Tris-HCl buffer solution, mgCl 2 and DTT, BSA, PAPS; b. incubating the EST experimental group, the negative control group and the blank control group to obtain respective reaction mixtures; c. detecting the influence of the reaction mixture on the proliferation of breast cancer cells to obtain proliferation rates of each group; d. The enzyme activity of the EST protein was calculated according to the following formula: enzyme Activity of EST protein = { [ (proliferation rate of negative control group-proliferation rate of Experimental group)/(proliferation rate of negative control group-proliferation rate of blank control group) ] Estradiol content }/incubation time.
  2. 2. The method according to claim 1, wherein in step a, the buffer system has a Tris-HCl buffer pH of 7.4 and a concentration of 50 mM, mgCl 2 of 1 mM, DTT of 1 mM, BSA of 625. Mu.g/mL, and PAPS of 10. Mu.M.
  3. 3. The method according to claim 1, wherein in step a, the concentrations of EST protein and E2 to be tested in the EST test group are 0.3. Mu.g/50. Mu.l and 1. Mu.M, respectively, and the concentration of E2 in the negative control group is 1. Mu.M.
  4. 4. The method according to claim 3, wherein in step a, E2 is dissolved in DMSO, and in the blank group, the solvent is DMS0 and water.
  5. 5. The method according to any one of claims 1 to 4, wherein in step b, the incubation is performed by incubating each group at 37 ℃ and then ending the reaction with an ice bath.
  6. 6. The method according to claim 5, wherein the incubation time is 10 minutes and the ice bath time is 30 minutes.
  7. 7. The method according to claim 1, wherein in step c, MCF-7 cells are cultured for 6-8 days by using phenol red free DMEM containing 10% antiestrogen serum, the cell density is adjusted to 2X 10 4 cells/ml, the cells are inoculated into a 96-well plate, placed in an incubator for culturing for 24 hours and then cultured for 24 hours, and the proliferation rate of the cells is detected.
  8. 8. The method according to claim 7, wherein the method of dosing is: After 50. Mu.l of the reaction mixture from step b was mixed with 950. Mu.l of the medium, 100. Mu.l of each well of the 96-well plate was added.
  9. 9. The method for detecting according to claim 7 or 8, wherein, The culture medium is 89% of DEME culture medium without phenol red, 10% of antiestrogen serum and 1% of penicillin-streptomycin double antibody.
  10. 10. The method according to claim 7, wherein the cell proliferation rate is measured by CCK-8 method.

Description

EST protease activity detection method Technical Field The invention belongs to the technical field of biological medicine detection, and particularly relates to an EST protease activity detection method. Background EST (Estrogen Sulfotransferase) gene, also called SULT1E1, encodes an estrogen sulfotransferase, belonging to the family of sulfotransferase genes (SULTs), the family of gene products being key enzymes for the sulfation metabolism of the body, which catalyze the transfer of sulfate (SO 3-) groups from the universal sulfate donor PAPS (3 '-phosphoadenosine-5' -phosphosulfate ) to hydroxyl or amino residues of other acceptor substrates, a process known as sulfonation. Among the SULT1 subfamily, SULT1E1 mainly acts on steroid sex hormones, and SULT1E1 protein has affinity for steroid sex hormones such as estrone (E1), estradiol (E2) and the like and plays the greatest role when the above several substances are sulfated, so SULT1E1 is also called EST (estrogen sulfotransferase). At present, EST protease activity research is focused on a human species, and the human EST protein can inactivate the sulfation of estrogen and plays an important role in regulating the content of the estrogen and the gonadal development and maturation. However, the research on the activity of human EST protease mainly depends on an isotope labeling method, so that the method has the advantages of high cost, complex operation, high technical threshold and health hazard to human body, and more influencing factors, such as selection of sampling time points, tracer dose screening, purity and chemical morphology of a marker and the like, can influence the accuracy of enzyme activity measurement. In addition, some companies also employ methods for detecting the sulfate transfer activity of the SULT family to which they belong, which methods, although indirectly reflecting the activity of enzymes related to the SULT family, lack specific recognition ability for EST enzymes themselves. Therefore, development of an EST-specific enzyme activity detection method which is simple to operate, lower in cost, safe and reliable has become an urgent need for current research. Disclosure of Invention In order to solve the above problems, the present invention aims to provide a simple, easy-to-carry, specific method for detecting the activity of EST protease. In order to achieve the above object, the present invention provides the following technical solutions: A method for detecting the activity of EST protease, comprising the steps of: a. EST experimental group, negative control group and blank control group were set up: Adding a buffer system with the same volume into each group, then respectively adding EST protein to be detected and E2 (17-beta estradiol) into an EST experimental group, adding E2 into a negative control group, and adding solvents with the same concentration and final volume as those of the first two groups into a blank control group; wherein the buffer system comprises Tris-HCl buffer solution, mgCl 2 and DTT, BSA, PAPS; b. incubating the EST experimental group, the negative control group and the blank control group to obtain respective reaction mixtures; c. detecting the influence of the reaction mixture on the proliferation of breast cancer cells to obtain proliferation rates of each group; d. The enzyme activity of the EST protein was calculated according to the following formula: enzyme Activity of EST protein = { [ (proliferation rate of negative control group-proliferation rate of Experimental group)/(proliferation rate of negative control group-proliferation rate of blank control group) ] Estradiol content }/incubation time. Wherein in the step a, the pH of the Tris-HCl buffer solution is 7.4, the concentration is 50mM, the concentration of MgCl 2 is 1mM, the concentration of DTT is 1mM, the concentration of BSA is 625 mug/mL, and the concentration of PAPS is 10 mu M. In the step a, the concentrations of the EST protein to be detected and the E2 in the EST test group are respectively 0.3 mug/50 mu l and 1 mu M, and the concentration of the E2 in the negative control group is 1 mu M. In the step a, E2 is dissolved in DMSO, and in a blank control group, the solvent is DMS0 and water. Wherein in step b, the incubation reaction is to incubate each group at 37 ℃ and then the ice bath ends the reaction. Further, the incubation time is 10 minutes, and the ice bath time is 30 minutes. In the step c, MCF-7 cells are taken, cultured for 6-8 days by using phenol red-free DMEM containing 10% of antiestrogen serum, the cell density is adjusted to be 2X 10 4 cells/ml, inoculated into a 96-well plate, placed in an incubator for culturing for 24 hours, added with medicine, and then cultured for 24 hours continuously, and the cell proliferation rate is detected. Wherein, the method for adding the medicine comprises the following steps: After 50. Mu.l of the reaction mixture from step b was mixed with 950. Mu.l of the medium, 100.