Use of a pa28 gamma-t23 site phosphorylation antibody

Abstract

The application provides an application of a PA28gamma-T23 site phosphorylation antibody, and belongs to the field of biomedicine. The application finds a new phosphorylation site of PA28gamma, which is related to tumors, and on this basis, a PA28gamma-T23 site phosphorylation modification detection antibody is prepared, so as to provide more possibilities for analyzing the specific mechanism of the cancer-promoting function of PA28gamma and for finding new tumor treatment targets and prognostic indicators.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedicine, and particularly relates to preparation and application of a PA28 gamma-T23 site phosphorylation antibody. BACKGROUND

[0002] Tumorous diseases are a major category of diseases that threaten human health, and the treatment methods thereof mainly include surgical treatment, radiotherapy and chemotherapy, immunotherapy and molecular targeted therapy. The molecular targeted therapy is closely concerned due to advantages such as good curative effect and small toxic and side effects. Therefore, exploring the action mechanism of a new molecular marker of tumor occurrence and development has important significance for screening a new treatment target and a prognosis index, improving the survival rate of tumor patients and saving social resources.

[0003] Proteasome activator 28 subunit gamma (PA28 gamma) is an 11S-20S-11S proteasome activator, which can mediate peptide and protein degradation without dependence on ubiquitin and adenosine triphosphate (ATP), and currently, a variety of substrate molecules dependent on the PA28 gamma proteasome system have been found. The gene name of PA28 gamma is PSME3.

[0004] A plurality of studies have shown that the expression of PA28 gamma is closely related to tumorous diseases, and high expression thereof plays an important role in the occurrence and development of head and neck squamous cell carcinoma, lung cancer, breast cancer, prostate cancer, thyroid cancer, gastric cancer, colorectal cancer, liver cancer and pancreatic cancer, and is related to the poor prognosis of patients, but the detailed regulation mechanism is still unclear.

[0005] Post-translational modification of proteins has a huge impact on protein structure and function, but there are few reports on post-translational modification of PA28 gamma, especially the most widely studied phosphorylation modification method. So far, only S247 site phosphorylation modification related to apoptosis in human osteosarcoma cells has been reported for PA28 gamma.

[0006] In order to analyze the specific mechanism of the carcinogenic function of PA28 gamma and to provide more possibilities for finding new tumor treatment targets and prognosis indexes, more specific recognition of phosphorylation sites of PA28 gamma protein is needed. SUMMARY

[0007] The purpose of the present application is to provide preparation and application of a PA28 gamma-T23 site phosphorylation antibody.

[0008] The present application provides a new PA28 gamma protein, which is a PA28 gamma protein phosphorylated at the T23 site.

[0009] The application also provides a use of a reagent for inhibiting phosphorylation level of PA28gamma-T23 site in the preparation of a medicine for preventing and / or treating a tumor.

[0010] Further, the tumor is oral squamous cell carcinoma, lung cancer, breast cancer, prostate cancer, thyroid cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, uterine cancer and / or cervical cancer; preferably, the tumor is oral squamous cell carcinoma, lung adenocarcinoma and / or cervical cancer.

[0011] The application also provides a polypeptide with phosphorylation of PA28gamma-T23 site, characterized in that the amino acid sequence is shown in SEQ ID NO. 1.

[0012] The application also provides an antibody for detecting the phosphorylation level of PA28gamma-T23 site, characterized in that it is an antibody prepared by using the polypeptide shown in SEQ ID NO. 1 as an immunogen.

[0013] The antibody is used in the preparation of a reagent for detecting the phosphorylation level of PA28gamma-T23 site.

[0014] Further, the reagent for detecting the phosphorylation level of PA28gamma-T23 site is an ELISA detection reagent, a dot blot detection reagent, an immunoblot detection reagent, an immunohistochemical detection reagent, and an immunocytochemical detection reagent.

[0015] The application also provides a use of a reagent for detecting the expression level of phosphorylated PA28gamma-T23 site in the preparation of a tumor screening reagent.

[0016] Further, the tumor is oral squamous cell carcinoma, lung cancer, breast cancer, prostate cancer, thyroid cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, uterine cancer and / or cervical cancer; preferably, the tumor is oral squamous cell carcinoma, lung adenocarcinoma and / or cervical cancer.

[0017] The application also provides a tumor screening kit, characterized in that it comprises an optional reagent for detecting the expression level of phosphorylated PA28gamma-T23 site.

[0018] PA28gamma-T23 site refers to the 23rd amino acid site of PA28gamma protein, and the 23rd amino acid is threonine (T).

[0019] Amino acid sequence of human PA28γ protein (SEQ ID NO.3):MASLLKVDQEVKLKVDSFRERITSEAEDLVANFFPKKLLELDSFLKEPILNIHDLTQIHSDMNLPVPDPILLTNSHDGLDGPTYKKRRLDECEEAFQGTKVFVMPNGMLKSNQQLVDIIEKVKP EIRLLIEKCNTVKMWVQLLIPRIEDGNNFGVSIQEETVAELRTVESEAASYLDQISRYYITRAKLVSKIAKYPHVEDYRRTVTEIDEKEYISLRLIISELRNQYVTLHDMILKNIEKIKRPRSSNAETLY

[0020] To date, no antibodies have been reported that specifically recognize the phosphorylated T23 site of the PA28γ protein. The invention prepares antibodies to detect the phosphorylated T23 site of PA28γ, which will provide more opportunities for understanding the specific mechanisms of PA28γ's cancer-promoting function and for identifying new tumor treatment targets and prognostic indicators.

[0021] Experimental results show that the present invention provides an application of a PA28γ-T23 site phosphorylation antibody, and experimental verification shows that PA28γT23 site phosphorylation modification promotes the proliferation of oral squamous cell carcinoma. The present invention proves that by detecting the phosphorylation level of the PA28γT23 site, the growth rate of oral squamous cell carcinoma tumors with high T23 site phosphorylation levels is faster than the growth rate of tumors with low T23 site phosphorylation levels. By detecting the phosphorylation level of the PA28γT23 site, the risk of suffering from tumors containing the PSME3 gene, such as oral squamous cell carcinoma, lung adenocarcinoma, or cervical cancer, can be detected. The application of the PA28γ-T23 site phosphorylation antibody provided by the present invention can provide an effective basis for patients to take relevant treatment measures or make decisions, and has good prospects for clinical application.

[0022] The present invention also demonstrates, through simulated dephosphorylation and phosphorylation of the PA28γT23 site, that phosphorylation of the PA28γT23 site promotes cancer cell proliferation, and that inhibiting PA28γT23 phosphorylation can inhibit cancer cell proliferation. Therefore, agents that inhibit PA28γT23 phosphorylation can be used to prepare drugs for treating tumors, showing promising application prospects.

[0023] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0024] The above content of the present application will be further explained in detail by way of specific embodiments in the form of examples. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application falls within the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Preparation of antigen polypeptide for anti-PA28γ-T23 site phosphorylation modification antibody design and sequence accuracy detection.

[0026] Figure 2 Spot hybridization detection results of positive and negative modification polypeptides of PA28γ-T23 site.

[0027] Figure 3 Immunoblotting detection of PA28γ-T23 site phosphorylation modification in oral squamous cell carcinoma cell lines.

[0028] Figure 4 Immunohistochemical detection of PA28γ-T23 site phosphorylation modification in human lung adenocarcinoma tissues.

[0029] Figure 5 Immunocytochemical detection of PA28γ-T23 site phosphorylation modification in HeLa cells.

[0030] Figure 6 Effect of PA28γ T23 site phosphorylation modification on the growth of OSCC cells A. CCK8 experiment was performed on HSC-3 and UM1 cells stably knocked down endogenous PA28γ and overexpressed exogenous PA28γ T23 site different phosphorylation modification states or control EV. (**P<0.01, ***P<0.001, 3 independent repeats were represented by mean ± standard deviation).

[0031] Figure 7 Figure 1: Nude mouse subcutaneous xenograft model verifies the effect of PA28γ T23 site phosphorylation on the growth of OSCC tumors A. HSC-3 cells stably knocked down endogenous PA28γ and overexpressed exogenous PA28γ T23 site different phosphorylation states or control EV were used to construct nude mouse subcutaneous xenograft models, and the tumors of each group growing to the 25th day were photographed (n=6 for each group); B. The volume changes of the OSCC tumors in each group in Figure A were statistically analyzed (measured every 3 days); **P<0.01, 6 mice in each group were represented by mean ± standard deviation; C. The weights of the OSCC tumors in each group in Figure A were statistically analyzed on the 25th day. **P<0.01, 6 mice in each group were represented by mean ± standard deviation. DETAILED DESCRIPTION

[0032] The raw materials and equipment used in the present application are known products, which can be obtained by purchasing commercially available products.

[0033] KLH: Keyhole Limpet Hemocyanin (KLH) is a highly immunogenic protein macromolecule used as a carrier protein in the preparation of immunogens, cross-linking with haptens and other antigens to enhance their immunogenicity.

[0034] OSCC: Oral squamous cell carcinoma

[0035] Example 1: Detection of the T23 phosphorylation modification site of the PA28γ protein of the present invention

[0036] 1. Experimental Methods

[0037] (I) Preparation of PA28γ protein T23 site antigen and antibody

[0038] 1. PA28γ protein T23 phosphorylation site positive modified peptides and negative peptides

[0039] The present invention first discovered the existence of phosphorylation modification at T23 site of PA28γ protein through phosphorylation modification mass spectrometry. Targeting this modification site, the present invention designed T23 positive and negative peptides to develop specific antibodies for phosphorylation modification at this site ( Figure 1 A), antigen mass spectrometry detected the accuracy of positive and negative peptides ( Figure 1 B).

[0040] Positive polypeptide sequence SEQ ID NO. 1: T23-phosphorylated (p-T23): FRERI-pT-SEAEDC.

[0041] Negative polypeptide sequence SEQ ID NO. 2: T23-unphosphorylated (unphos): DSFRERITSEAEDC.

[0042] 2. Preparation of antibodies against the T23 phospho-site of PA28γ protein (rabbit immunization experiment)

[0043] 1) Peptide coupling

[0044] Peptide coupling: The positive polypeptides shown in SEQ ID NO. 1 were coupled with KLH respectively and used as immunogens for immunization of rabbits (healthy New Zealand rabbits).

[0045] 2) Rabbit immunization

[0046] (1) Preparation of immunization material: dilute the immunogen with physiological saline, then mix with Freund's adjuvant at 1:1. Mix the antigen and Freund's adjuvant completely to form a stable emulsion, draw the antigen mixture with a syringe, and inject the antigen at two points on the rabbit's double shoulder skin and two points on the double hind leg muscles. About 1 / 4 volume of immunogen is injected in each area. In this way, the immunogen can persist and thus improve the immune response.

[0047] (2) Immunization: each rabbit is immunized a total of 4 times, on day 1, day 21, day 28, and day 35;

[0048] (3) Blood collection: first blood collection: on day 45, collect 30 mL of whole blood and centrifuge, then collect the supernatant and send to the laboratory for serum screening tests, including ELISA or Dot blot; second / third / fourth blood collection: on day 50, day 65, and day 70, collect 20 mL of whole blood each time, centrifuge, collect the supernatant, and send to the laboratory for serum screening tests, including ELISA or Dot blot; collect the serum with positive test results for purification.

[0049] 3) Antibody purification

[0050] (1) Preparation of protein A affinity column: usually select 5 mL or 10 mL of protein A packing, mix equal volumes of packing and PBS buffer solution, stir, and draw air to remove air bubbles in the packing. Slowly add the protein A packing to a glass column and fill the chromatography column. Avoid column drying during this process. After filling, equilibrate the column with 10 volumes of pre-cooled PBS buffer solution.

[0051] (2) Protein A affinity chromatography: filter the serum and load it onto the equilibrated protein A chromatography column. To detect the binding efficiency of the antiserum and the packing, retain the flow-through. Wash the column with PBS buffer solution, then elute with 150 mM glycine buffer solution. Collect the eluate and add neutralization buffer solution to adjust the pH to 7.

[0052] (3) Enrichment of target antibodies: 13 / 17 load the crude IgG obtained after protein A purification onto the equilibrated antigen polypeptide affinity chromatography column to specifically enrich the target antibodies.

[0053] (4) Removal of non-specific antibodies: load the target antibodies obtained in the previous step onto the unmodified affinity chromatography column and directly collect the flow-through to remove non-specific antibody components.

[0054] (5) Antibody preservation: determine the protein content. Add 10% glycerol to preserve the antibodies, and store the purified antibodies at -20°C after aliquoting.

[0055] (II) Verification of the detection of PA28 gamma protein T23 phosphorylation modification sites by the aforementioned antibody 1. ELISA detection:

[0056] 1) Antigen coating: Dilute the antigen with coating solution, and add it to the enzyme-labeled plate at a dose of 50 ug / well, and incubate in the refrigerator at 4°C overnight or in a 37°C oven for 2 hours.

[0057] 2) Plate washing: Take out the enzyme-labeled plate coated the day before, and wash it with 1xTBST for 3 times.

[0058] 3) Blocking: After washing the enzyme-labeled plate, add 1% BSA blocking solution, and incubate at 37°C for 1 hour, and then wash 1-3 times.

[0059] 4) Primary antibody incubation: Dilute the antibody by 3 times gradient starting from 1:1K (1:1000). Adjust the dilution volume according to the actual situation. Add it to the enzyme-labeled plate in turn, and incubate at 37°C for 1.5 hours, and then wash 1-3 times.

[0060] 5) Secondary antibody incubation: Dilute the secondary antibody with 1% BSA blocking solution to 1:10K, and incubate at room temperature or 37°C for 45 minutes, and then wash 1-3 times.

[0061] 6) Color development: Add TMB color developing solution for 5-10 minutes, and then terminate the reaction with 1M sulfuric acid, and read the data with an enzyme-labeled instrument.

[0062] 7) Antibody ELISA detection results (ELISA titer (OD450>1.0), the recognition ability of the antibody for the positive modified polypeptide is not less than 1:54K dilution, and it does not recognize the non-modified negative polypeptide.):

[0063]

[0064] The antibody of the present application can effectively bind to the antigen. When the dilution multiple is 1:54K (1:54000), the ELISA titer OD450 is 1.319>1.0, indicating that the antibody still has recognition ability for the positive modified polypeptide, and the antibody does not have recognition ability for the negative modified polypeptide.

[0065] 2. Dot blotting detection:

[0066] 1) Dotting: Dot the uncrosslinked antigen polypeptide on the PVDF membrane according to the gradient of 1 ng, 4 ng, 16 ng, and 64 ng.

[0067] 2) Blocking: After the membrane surface is dried, add blocking solution, and block at room temperature for 60 minutes.

[0068] 3) Washing: Wash with 1xTBST for 10 minutes.

[0069] 4) Primary antibody incubation: dilute the antibody with 2.5-5% skim milk powder, incubate at room temperature for 2h, and then wash with 1xTBST for 3 times, each for 5-10min.

[0070] 5) Secondary antibody incubation: according to the property of the primary antibody, select the corresponding mouse or rabbit antibody; dilute the secondary antibody at a ratio of 1:10K, incubate at room temperature for 45min-1h, and then wash with 1xTBST for 3 times, each for 5-10min.

[0071] 6) After washing the membrane, expose it to the color developing substrate.

[0072] 7) Dot blot detection results (dot blot positive detection limit reaches 4ng, and there is no obvious cross signal with negative unmodified polypeptide):

[0073] Negative polypeptide: T23-unphosphorylated (unphos): DSFRERITSEAEDC

[0074] Positive polypeptide: T23-phosphorylated (p-T23): FRERI-pT-SEAEDC.

[0075] The foregoing ELISA detection results and dot blot detection results prove that the antibody of the application can effectively detect the positive polypeptide, and can accurately detect whether the T23 site of the PA28γ protein is phosphorylated.

[0076] (Three) using the foregoing antibody to detect the T23 phosphorylation modification site of PA28γ protein in the tissue to be detected

[0077] 1, Western blot detection:

[0078] 1) Cell lysis: select cancer cell lines according to experimental requirements, select different lysis methods to obtain sample total protein according to different samples, and determine the protein concentration by BCA method.

[0079] 2) Protein electrophoresis and membrane transfer: protein electrophoresis: according to the molecular weight of the target protein, select the corresponding concentration of separation gel. Add 10% ammonium persulfate and TEMED at the end of gel preparation. Each plate of separation gel is 4.7ml. Load 20-40ug sample per well and electrophorese; the electrophoresis condition is: concentrated gel 80v, separation gel 120v. Wet transfer membrane: the transfer solution is cooled before transfer, and the gel, membrane and filter paper are arranged in a sandwich structure in the transfer solution to avoid air bubbles. The transfer voltage is 80v-120v.

[0080] 3) Blocking: add blocking solution to the transferred membrane, and block at room temperature for 60min. After incubation, wash with 1xTBST for 10min.

[0081] 4) Primary antibody incubation: Dilute antibody with 2.5-5% non-fat dry milk, incubate at room temperature for 2 hours, wash with 1x TBST for 3 times, 5-10 minutes each time.

[0082] 5) Secondary antibody incubation: Choose the corresponding mouse anti or rabbit anti according to the primary antibody attribute; add secondary antibody with 1:10K dilution ratio, incubate at room temperature for 45 minutes to 1 hour, wash with 1x TBST for 3 times, 5-10 minutes each time.

[0083] 6) After washing the membrane, expose it to the color developing substrate.

[0084] Immunoblotting detection results: Immunoblotting detection of PA28γ-T23 site phosphorylation modification in oral squamous cell carcinoma cell lines, it can be seen that the antibodies all detect Figure 3 PA28γ-T23 site phosphorylation modification in cells.

[0085] 2. Immunohistochemical detection:

[0086] 1) De-waxing / hydration

[0087] ① Heat in an oven at 63-65 degrees Celsius for about 1 hour.

[0088] ② Use the following procedure for de-waxing / hydration: soak in xylene twice (5-10 minutes each time), soak in anhydrous ethanol twice (5 minutes each time), soak in 95% ethanol twice (5 minutes each time), soak in 85% ethanol (3 minutes), soak in 75% ethanol (3 minutes), and finally wash with distilled water three times.

[0089] 2) Antigen retrieval: Recommend using heat-induced antigen retrieval with a pressure cooker.

[0090] ① Add 500 milliliters of distilled water to the pressure cooker.

[0091] ② Soak the slides in the staining dish containing the antigen retrieval solution, then place the staining dish in the pressure cooker.

[0092] ③ Run the pressure cooker with the program of running at 125°C for 30 seconds or at 110°C for 15-30 minutes.

[0093] ④ After the pressure is over, take out the staining dish and cool it to room temperature naturally (10-30 minutes).

[0094] ⑤ Proceed to the staining step.

[0095] 3) Staining

[0096] ① Rinse the slides with distilled water three times (if a shaker is used, 3 minutes each time).

[0097] ② Soak the tissue in 3% hydrogen peroxide for 5-10 minutes to inactivate endogenous peroxidase.

[0098] ③ Wash the slides three times with TBST (if using a shaker, wash for 3 minutes each time).

[0099] ④ Block the sections with goat serum for 30-60 minutes.

[0100] ⑤ Dilute the primary antibody with primary antibody diluent according to the instructions.

[0101] ⑥ Place the slices in a humidified box and add primary antibody, then place the humidified box in a 4-degree refrigerator and incubate overnight.

[0102] ⑦ Wash the slides three times with TBST (if using a shaker, wash for 3 minutes each time).

[0103] ⑧Add secondary antibody and incubate at room temperature for 30 minutes.

[0104] ⑨Wash the slides three times with TBST (5 minutes each time).

[0105] ⑩ Add freshly prepared DAB substrate to the slices and incubate until brown staining occurs (10-60s,

[0106] Follow the instructions).

[0107] Rinse sections with distilled water.

[0108] Counterstain with hematoxylin (1-2 minutes, according to the instructions).

[0109] Rinse the slices with water.

[0110] Rinse with 75% ethanol (3 minutes), 95% ethanol (3 minutes), and absolute ethanol twice (3 minutes each), and then rinse twice with xylene or tissue clearing agent (5 minutes each).

[0111] Dry the slides with a hair dryer and fix the coverslips onto the slides using mounting medium.

[0112] Immunohistochemistry test results showed that the antibody detected phosphorylation modification of PA28γ-T23 site in human lung adenocarcinoma tissue, which was nuclear staining.

[0113] 3. Immunocytochemistry detection:

[0114] 1) Fixed

[0115] ① Adherent cells

[0116] a) Cells are grown in 12-well plates, allowing the cells to attach to the coverslips at the bottom of the 12-well plates. When the cells reach approximately 80% confluency, the plates are removed for immunofluorescence experiments.

[0117] b) The media is removed from the 12-well plates and the cells are washed once with 500 μl of lx PBS.

[0118] c) The cells are fixed with 200 μl of 4% paraformaldehyde for 20 minutes at room temperature or 200 μl of 100% ice cold methanol for 5 minutes.

[0119] d) The cells are washed 3 times with lx PBS (500 μl for 5 minutes each at room temperature).

[0120] e) The PBS is discarded and at least 500 μl of cell storage solution is added. The plates can be stored at 2-8°C

[0121] for 2 months.

[0122] ii) Suspension Cells

[0123] a) The cell density is adjusted to 1 x 106 cells per ml. One ml of the cell suspension is removed and centrifuged at 1000 rpm for 3 minutes in a 1.5 ml eppendorf tube.

[0124] b) The supernatant is removed and the cells are washed 3 times with 1 ml of lx PBS and centrifuged at 1000 rpm for 3 minutes.

[0125] c) The supernatant is removed and the cells are fixed with 1 ml of 4% paraformaldehyde for 20 minutes at room temperature or 1 ml of 100% ice cold methanol for 5 minutes.

[0126] d) The cells are centrifuged at 1000 rpm for 3 minutes and the supernatant is discarded. The cells are washed 2 times with 1 ml of lx PBS and centrifuged at 1000 rpm for 3 minutes.

[0127] e) The supernatant is discarded and the cells are resuspended in 1 ml of cell storage solution. The cell suspension can be stored at 2-8°C

[0128] for 1 month.

[0129] f) A hydrophobic pen is used to draw a circle on a glass slide. 10-15 μl of the cell suspension is added to each circle and the cell suspension is spread evenly across the circle using the tip of a gun.

[0130] g) The slides are baked at 37°C for 20 minutes and are ready to use when the slides are completely dry.

[0131] 2) Cell Permeabilization

[0132] ① Discard the cell storage solution, and wash the cells once with 1 x PBST (500 μl, 5 min each, room temperature).

[0133] ② Permeabilize the cells with 200 μl of 0.1% Triton X-100 for 5 min at room temperature.

[0134] ③ Wash the cells twice with 1 x PBST.

[0135] 3) Blocking: Block the cells with 200 μl (50-100 μl of cell suspension) of blocking solution for 1 h at room temperature.

[0136] 4) Antibody staining

[0137] ① Dilute the primary antibody according to the manufacturer's instructions.

[0138] ② Add 200 μl of the diluted primary antibody to the cells, and incubate overnight at 2-8 °C.

[0139] ③ Wash the cells three times with wash buffer (500 μl, 5 min each, room temperature).

[0140] ④ Add 200 μl of fluorescently labeled secondary antibody (1:1000 dilution) and DAPI (1:100 dilution) to the cells, and incubate for 45 min at room temperature in the dark.

[0141] ⑤ Wash the cells three times with wash buffer (500 μl, 5 min each, room temperature).

[0142] ⑥ Mount the cells with a fluorescent mounting medium, and store in the dark.

[0143] 5) Results of immunocytochemical detection

[0144] The antibody detects the phosphorylation modification of PA28γ-T23 in HeLa cells (cervical cancer cells), mainly in the nucleus.

[0145] The experimental results show that PA28γ-T23 phosphorylation can be detected by ELISA detection, dot blot detection, immunoblotting detection, immunohistochemical detection, and immunocytochemical detection to screen for oral squamous cell carcinoma, lung adenocarcinoma, cervical cancer, and other tumors.

[0146] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0147] Experimental Example 1, PA28γ T23 phosphorylation modification promotes the growth of OSCC cells

[0148] (I) CCK8 assay

[0149] 1. The cells to be tested are plated in a 96-well plate, 3000-5000 cells per well, 5-6 replicates. Set up a blank control group with only culture medium, add ddH2O to the periphery of the 96-well plate for moisture retention, prepare 4 identical plates, and place the 96-well plate in a cell culture incubator (37°C, 5% CO2) overnight.

[0150] 2. Perform CCK8 experiments at 24h, 48h, 72h, and 96h after plating, respectively.

[0151] 3. Add 10μL of CCK8 solution to each well (without generating bubbles), and incubate in a cell culture incubator (37°C, 5% CO2) for 1-4h.

[0152] 4. Place the 96-well plate in a multifunctional microplate reader to measure the absorbance at 450nm.

[0153] (B) Construction of OSCC xenograft tumor model in nude mice

[0154] 1. Prepare the cell strain HSC-3 oral squamous cell carcinoma cells needed for inoculation.

[0155] 2. Collect the cells in the logarithmic growth phase after trypsin digestion into a clean BD tube, resuspend and wash with PBS for 2 times, add serum-free and antibiotic-free culture medium, resuspend, count the cells, and adjust the cell concentration.

[0156] 3. Use BALB / c-nu nude mice for the experiment, 6 mice per group, disinfect the skin on the side of the back where the injection is needed with 75% alcohol, then inoculate 100μL of cell suspension subcutaneously with an insulin needle (mix the cell suspension gently before inoculation).

[0157] 4. Measure the volume of the transplanted tumor with a vernier caliper 7 days after inoculation, measure every 3 days, collect the transplanted tumor on the 25th day, measure the volume and weight, take photos, and perform statistical analysis.

[0158] To further explore whether the phosphorylation of PA28γ T23 site affects the proliferation of OSCC cells, CCK8 and plate colony formation experiments were performed.

[0159] T23A is a mutation of the 23rd amino acid of PA28γ to A (alanine) (pseudo-dephosphorylation), T23D is a mutation of the 23rd amino acid of PA28γ to D (aspartic acid) (pseudo-phosphorylation), and EV is an empty control set by transfection plasmid.

[0160] Firstly, CCK8 was performed on HSC-3 (human oral squamous cell carcinoma cells) and UM1 cells (human oral squamous cell carcinoma cells) stably knocked down endogenous PA28γ and overexpressed exogenous PA28γ WT, T23A, T23D or control EV, and the results showed that the cell growth rate of the T23A, WT, T23D groups increased in turn, indicating that PA28γ T23 site phosphorylation promoted the proliferation of OSCC cells.

[0161] The experimental results show that the cell growth rate of the T23A, WT, T23D groups increases in turn, indicating that PA28γ T23 site phosphorylation promotes the proliferation of OSCC (HSC-3 and UM1) cells, and inhibiting PA28γ T23 site phosphorylation can inhibit the proliferation of OSCC (HSC-3 and UM1) cells.

[0162] (Three) PA28γ T23 site phosphorylation modification can promote the growth of OSCC subcutaneous xenograft

[0163] In order to detect the effect of PA28γ T23 site phosphorylation modification on the growth of OSCC in vivo, a nude mouse subcutaneous xenograft model was constructed. The transplanted tumor cells were HSC-3 cells (human oral squamous cell carcinoma cells) used in the previous functional experiment and easy to form tumors, and the transduction construction was consistent with the previous ( Figure 6 ) that is, cells with different phosphorylation modification states of PA28γ T23 site.

[0164] From the 7th day after tumor injection, the tumor volume of nude mice was measured using a vernier caliper, once every 3 days, and the tumor was collected on the 25th day for photography ( Figure 7 A), and the change of tumor volume and the weight of tumor at the time of collection were counted, and the statistical results showed that the tumor volume and weight of the T23A, WT, T23D groups increased in turn, suggesting that PA28γ T23 site phosphorylation modification promoted the growth of OSCC subcutaneous xenograft ( Figure 7 B, Figure 7 C), which is consistent with the conclusion of the in vitro functional experiment ( Figure 6 ).

[0165] The experimental results show that PA28γ T23 site phosphorylation modification promotes the proliferation of OSCC cells. PA28γ T23 site phosphorylation modification promotes the growth of OSCC subcutaneous xenograft, and inhibiting PA28γ T23 site phosphorylation can inhibit the growth of OSCC subcutaneous xenograft.

[0166] In summary, the application provides an application of the PA28 gamma-T23 site phosphorylation antibody, and through experimental verification, the PA28 gamma T23 site phosphorylation modification promotes the proliferation of the OSCC cell. The application proves that the oral squamous cell carcinoma tumor growth speed is faster when the T23 site phosphorylation level is high than when the T23 site phosphorylation level is low, and the risk of tumors containing the PSME3 gene such as oral squamous cell carcinoma, lung adenocarcinoma or cervical cancer can be detected by detecting the PA28 gamma T23 site phosphorylation level. The application of the PA28 gamma-T23 site phosphorylation antibody provided by the application can provide an effective basis for patients to take relevant treatment measures or decisions, and has a good clinical application prospect.

[0167] The application also proves that the PA28 gamma T23 site phosphorylation promotes the proliferation of the OSCC (HSC-3 and UM1) cell through the PA28 gamma T23 site dephosphorylation and phosphorylation, and the inhibition of the PA28 gamma T23 site phosphorylation can inhibit the proliferation of the OSCC (HSC-3 and UM1) cell. Therefore, the reagent for inhibiting the PA28 gamma T23 site phosphorylation can be used for preparing a drug for treating tumors, and has a good application prospect.

Claims

1. A polypeptide phosphorylated at the PA28γ-T23 site, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A polyclonal antibody for detecting the phosphorylation level of PA28γ-T23 site, characterized in that: The invention is a polyclonal antibody prepared by taking the polypeptide shown in SEQ ID NO.1 as immunogen.

3. Use of the polyclonal antibody according to claim 2 in the preparation of a reagent for detecting the phosphorylation level of PA28γ-T23 site.

4. The use according to claim 3, characterized in that: The reagents for detecting the phosphorylation level of PA28γ-T23 site are ELISA detection reagents, dot blot detection reagents, immunoblotting detection reagents, immunohistochemistry detection reagents, and immunocytochemistry detection reagents.

5. Use of a reagent for detecting the expression level of phosphorylated PA28γ-T23 site prepared with the polyclonal antibody according to claim 2 in preparing a reagent for tumor screening, wherein the tumor is oral squamous cell carcinoma, lung adenocarcinoma and / or cervical cancer.

6. A tumor screening kit, characterized in that: The method comprises the reagent for detecting the expression level of the phosphorylated PA28γ-T23 site as claimed in claim 5.

Images