Application of PA28gamma-T23 site phosphorylated antibody

Abstract

The invention provides application of a PA28gamma-T23 site phosphorylated antibody, and belongs to the field of biomedicine. According to the invention, a new phosphorylation site of PA28gamma is found and is related to tumors, and a PA28gamma-T23 site phosphorylation modification detection antibody is prepared on the basis of the new phosphorylation site, so that more possibilities are provided for analyzing a specific mechanism of a cancer promoting function of PA28gamma and finding new tumor treatment targets and prognosis indexes.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the preparation and application of a PA28γ-T23 site phosphorylation antibody. Background Art

[0002] Tumor diseases are a major category that threatens human health. The main treatment methods include surgery, radiotherapy, chemotherapy, immunotherapy and molecular targeted therapy. Among them, molecular targeted therapy has attracted close attention due to its advantages such as good efficacy and few toxic side effects. Therefore, exploring the mechanism of action of new molecules that mark the occurrence and development of tumors is of great significance for screening new therapeutic targets and prognostic indicators, improving the survival rate of tumor patients and saving social resources.

[0003] Proteasome activator 28 subunit gamma (PA28γ) is a 11S-20S-11S proteasome activator that can mediate peptide and protein degradation without relying on ubiquitin and adenosinetriphosphate (ATP). Currently, a variety of substrate molecules that rely on the PA28γ proteasome system have been discovered. The gene name of PA28γ is PSME3.

[0004] Many studies have shown that PA28γ expression is closely related to tumor diseases. Its high expression 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, pancreatic cancer, etc., and is associated with the poor prognosis of patients, but its detailed regulatory 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 the post-translational modification of PA28γ, especially the most widely studied phosphorylation modification. So far, PA28γ has only been reported to be phosphorylated at the S247 site in human osteosarcoma cells, which is related to cell apoptosis.

[0006] In order to analyze the specific mechanism of PA28γ's cancer-promoting function and provide more possibilities for finding new tumor treatment targets and prognostic indicators, more specific identification of the phosphorylation sites of PA28γ protein is needed. Summary of the invention

[0007] The purpose of the present invention is to provide a preparation and application of a PA28γ-T23 site phosphorylation antibody.

[0008] The invention provides a novel PA28γ protein, which is a PA28γ protein with phosphorylation modification at the T23 site.

[0009] The present invention also provides a use of a reagent for inhibiting the phosphorylation level of the PA28γ-T23 site in preparing a drug for preventing and / or treating tumors.

[0010] Furthermore, 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 present invention also provides a PA28γ-T23 site phosphorylated polypeptide, characterized in that: its amino acid sequence is shown in SEQ ID NO.1.

[0012] The present invention also provides an antibody for detecting the phosphorylation level of PA28γ-T23 site, characterized in that it is an antibody prepared using the polypeptide shown in SEQ ID NO.1 as an immunogen.

[0013] The antibody is used in preparing a reagent for detecting the phosphorylation level of the PA28γ-T23 site.

[0014] Furthermore, the reagent for detecting the phosphorylation level of the PA28γ-T23 site is an ELISA detection reagent, a dot hybridization detection reagent, an immunoblotting detection reagent, an immunohistochemistry detection reagent, or an immunocytochemistry detection reagent.

[0015] The present invention also provides the use of a reagent for detecting the expression level of the phosphorylated PA28γ-T23 site in preparing a reagent for tumor screening.

[0016] Furthermore, 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 present invention also provides a tumor screening kit, characterized in that it comprises an optional reagent for detecting the expression level of the phosphorylated PA28γ-T23 site.

[0018] The PA28γ-T23 site refers to the 23rd amino acid site of the PA28γ protein, and the 23rd amino acid is threonine (T).

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

[0020] So far, there has been no report on antibodies that specifically recognize the phosphorylation modification of the T23 site of PA28γ protein. The invention prepares antibodies for detecting the phosphorylation modification of the PA28γ-T23 site, which will provide more possibilities for analyzing the specific mechanism of PA28γ's cancer-promoting function and for finding new tumor treatment targets and prognostic indicators.

[0021] The experimental results show that the present invention provides an application of a PA28γ-T23 site phosphorylation antibody, and through experimental verification, the phosphorylation modification of the PA28γT23 site 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 phosphorylation levels of the T23 site is faster than the growth rate of tumors with low phosphorylation levels of the T23 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 by detecting the phosphorylation level of the PA28γT23 site. 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 decisions, and has good prospects for clinical application.

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

[0023] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Preparation of antigen peptides designed for anti-PA28γ-T23 site phosphorylation-modified antibodies and sequence accuracy testing.

[0026] Figure 2 Dot hybridization detection results of positive and negative modified peptides at the PA28γ-T23 site.

[0027] Figure 3 Immunoblotting was used to detect the phosphorylation modification of PA28γ-T23 site in oral squamous cell carcinoma cell lines.

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

[0029] Figure 5 Immunocytochemistry was used to detect the phosphorylation modification of PA28γ-T23 site in HeLa cells.

[0030] Figure 6 Effect of phosphorylation modification of PA28γT23 site on OSCC cell growth A. CCK8 experiment was performed on HSC-3 and UM1 cells that stably knocked down endogenous PA28γ and overexpressed exogenous PA28γT23 site with different phosphorylation modification states or control EV. (**P<0.01, ***P<0.001, 3 independent repeated experiments are expressed as mean ± standard deviation).

[0031] Figure 7 Figure Nude mouse subcutaneous transplant tumor model verifies the effect of PA28γT23 site phosphorylation on OSCC tumor growth A. Nude mouse subcutaneous transplant tumor model was constructed by HSC-3 cells that stably knocked down endogenous PA28γ and overexpressed exogenous PA28γT23 site with different phosphorylation states or control EV, and the tumors in each group grown to day 25 were photographed (n=6 per group); B. The changes in the volume of OSCC tumors in each group in Figure A were statistically analyzed (measured once every 3 days). **P<0.01, 6 mice in each group are expressed as mean ± standard deviation; C. The weight of OSCC tumors in each group in Figure A was statistically analyzed on day 25. **P<0.01, 6 mice in each group are expressed as mean ± standard deviation. DETAILED DESCRIPTION

[0032] The raw materials and equipment used in the present invention are all known products, which are 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 peptide and negative peptide to develop specific antibody 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 PA28γ protein T23 phospho-site antibody (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) Prepare immune materials: dilute the immunogen with saline and then mix it with Freund's adjuvant in a 1:1 ratio. The antigen and Freund's adjuvant are completely mixed to form a stable emulsion. Use a syringe to extract the antigen mixture and inject the antigen into two points under the skin of the rabbit's shoulders and two points in the muscles of both hind legs. Each area is about 1 / 4 of the volume of the immunogen. In this way, the immunogen can persist and thus enhance the immune response.

[0047] (2) Immunization: Each rabbit was immunized 4 times, on day 1, day 21, day 28, and day 35;

[0048] (3) Blood collection: The first blood collection: On the 45th day, collect 30 mL of whole blood and centrifuge it. Take the supernatant after centrifugation and send it to the laboratory for serum screening tests, including ELISA or Dot blot. The second / third / 4th blood collection: On the 50th day, 65th day, and 70th day, collect whole blood three times, 20 mL each time, centrifuge it, take the supernatant and send it to the laboratory for serum screening tests, including ELISA or Dot blot. Take the serum with positive test results for purification.

[0049] 3) Antibody purification

[0050] (1) Prepare the protein A affinity column: Usually select 5mL or 10mL of protein A filler, mix and stir equal volumes of filler and PBS buffer solution, and remove bubbles in the filler by vacuuming. Slowly add protein A filler into the glass column to prepare the chromatography column. Avoid column drying during this process. After filling, equilibrate the column with 10 times the volume of pre-cooled PBS buffer solution.

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

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

[0053] (4) Removal of non-specific antibodies: The target antibody obtained in the previous step is loaded onto a non-modified affinity chromatography column and the effluent is directly collected to remove non-specific antibody components.

[0054] (5) Antibody preservation: Determine the protein content. Add 10% glycerol to preserve the antibody, and store the purified antibody in aliquots at -20°C.

[0055] (II) Verification of the detection of the phosphorylation modification site of T23 of PA28γ protein using the aforementioned antibodies 1. ELISA test:

[0056] 1) Antigen coating: dilute the antigen with coating solution, add 50ug / well of the ELISA plate, and incubate in a refrigerator at 4°C overnight or in a 37°C oven for 2h.

[0057] 2) Washing: Take out the ELISA plate coated the day before and add 1×TBST to wash three times.

[0058] 3) Blocking: Add 1% BSA blocking solution to the cleaned ELISA plate, incubate at 37°C for 1 hour, and then wash 1-3 times.

[0059] 4) Primary antibody incubation: dilute the antibody in a 3-fold gradient starting from 1:1K (1:1000). Adjust the dilution volume according to the actual situation. Add to the ELISA plate in sequence, incubate at 37°C for 1.5 hours, and then wash 1-3 times.

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

[0061] 6) Color development: Add TMB colorimetric solution and then add 1M sulfuric acid to terminate the display reaction 5-10 minutes later, and read the data using an ELISA reader.

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

[0063]

[0064] The antibody of the present invention 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 the ability to recognize the positive modified polypeptide, and the antibody has no ability to recognize the negative modified polypeptide.

[0065] 2. Dot hybridization detection:

[0066] 1) Spotting: Spot the uncross-linked antigen peptides onto the PVDF membrane according to the gradient of 1ng, 4ng, 16ng, and 64ng.

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

[0068] 3) Washing: Wash with 1× TBST for 10 min.

[0069] 4) Primary antibody incubation: dilute the antibody with 2.5-5% skim milk powder, incubate at room temperature for 2 hours, and then wash three times with 1×TBST, each time for 5-10 minutes.

[0070] 5) Secondary antibody incubation: Select the corresponding mouse or rabbit antibody according to the properties of the primary antibody; add the secondary antibody at a dilution ratio of 1:10K, incubate at room temperature for 45min-1h, and then wash 3 times with 1×TBST, each time for 5-10min.

[0071] 6) Add a color developing substrate to the washed membrane and then expose it to light.

[0072] 7) Dot blot detection results (Dot blot positive detection limit reached 4ng, and there was no obvious cross signal with negative non-modified peptides):

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

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

[0075] The above ELISA test results and dot hybridization test results prove that the antibody of the present invention can effectively detect positive polypeptides and can accurately detect whether the T23 site of PA28γ protein is phosphorylated.

[0076] (III) Using the aforementioned antibodies to detect the phosphorylation modification site of PA28γ protein T23 in the tissue to be tested

[0077] 1. Western blot detection:

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

[0079] 2) Protein electrophoresis and 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 the gel preparation. 4.7ml of separation gel per plate. Load 20-40ug sample per well and perform electrophoresis; the electrophoresis conditions are: 80v for concentrated gel and 120v for separation gel. Wet transfer: Cool the transfer solution before transfer, and lay the gel, membrane, and filter paper in the transfer solution into a sandwich structure to avoid bubbles. The transfer voltage is 80v-120v.

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

[0081] 4) Primary antibody incubation: dilute the antibody with 2.5-5% skim milk powder, incubate at room temperature for 2 hours, and then wash three times with 1×TBST, each time for 5-10 minutes.

[0082] 5) Secondary antibody incubation: Select the corresponding mouse or rabbit antibody according to the properties of the primary antibody; add the secondary antibody at a dilution ratio of 1:10K, incubate at room temperature for 45min-1h, and then wash 3 times with 1×TBST, each time for 5-10min.

[0083] 6) Add a color developing substrate to the washed membrane and then expose it to light.

[0084] Western blot detection results show that the antibody can detect phosphorylation modification of PA28γ-T23 site in oral squamous cell carcinoma cell lines. Figure 3 Phosphorylation modification of PA28γ-T23 site in mesenchymal cells.

[0085] 2. Immunohistochemistry:

[0086] 1) Dewaxing / rehydration

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

[0088] ② Dewax / hydrate using the following procedure: 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 three times with distilled water.

[0089] 2) For antigen retrieval, heat-induced antigen retrieval using a pressure cooker is recommended.

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

[0091] ② Immerse the slide in a staining dish containing antigen retrieval solution, and then place the staining dish in a pressure cooker.

[0092] ③Operate the pressure cooker at 125°C for 30 seconds or at 110°C for 15-30 minutes.

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

[0094] ⑤Perform staining step.

[0095] 3) Dyeing

[0096] ① Wash the slides three times with distilled water (if using a shaker, 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, 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, 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 in 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), rinse with 95% ethanol (3 minutes), rinse twice with absolute ethanol (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 to the slides using mounting medium.

[0112] The results of immunohistochemistry 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) Cultivate cells in a 12-well cell culture plate, allowing the cells to adhere to and grow on the cover glass at the bottom of the 12-well cell culture plate. When the cell density reaches about 80%, the cell culture plate can be removed for cell immunofluorescence experiments.

[0117] b) Remove the culture medium from the 12-well plate and wash once with 500 μl 1× PBS.

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

[0119] d) Wash three times with 1× PBS (500 μl, 5 min each time, room temperature).

[0120] e) Discard PBS and add at least 500 μl of cell storage solution. The cell plate can be stored at 2℃~8℃

[0121] Store for 2 months.

[0122] ②Suspension cells

[0123] a) Adjust the cell density to 1×106 cells / mL. Transfer 1 mL of the cell suspension into a 1.5 mL eppendorf tube and centrifuge at 1000 rpm for 3 minutes.

[0124] b) Remove the supernatant, wash the cells three times with 1 ml 1× PBS, and centrifuge at 1000 rpm for 3 minutes.

[0125] c) Remove the supernatant and fix the cells with 1 ml of 4% paraformaldehyde at room temperature for 20 minutes or 1 ml of 100% ice-cold methanol for 5 minutes.

[0126] d) Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, and wash the cells twice with 1 ml 1× PBS. Centrifuge at 1000 rpm for 3 minutes.

[0127] e) Discard the supernatant and resuspend the cells in 1 ml of cell storage solution. The cell suspension can be stored at 2℃~8℃

[0128] Store for 1 month.

[0129] f) Use a hydrophobic pen to draw circles on the slide. Add 10-15 μl of cell suspension to each circle and then use the tip of a pipette to spread the cell suspension over the entire circle.

[0130] g) Dry the slides at 37°C for 20 minutes and use them after they are completely dry.

[0131] 2) Cell permeabilization

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

[0133] ② At room temperature, permeabilize the cells with 200 μl of 0.1% Triton X-100 for 5 minutes.

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

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

[0136] 4) Antibody staining

[0137] ① Dilute the primary antibody according to the instructions of the antibody.

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

[0139] ③ Wash the cells three times with washing buffer (500 μl, 5 min each time, 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 at room temperature in the dark for 45 minutes.

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

[0142] ⑥ Seal the slides with fluorescent mounting medium and store away from light.

[0143] 5) Immunocytochemistry test results

[0144] The antibody detected the phosphorylation modification of PA28γ-T23 site in HeLa cells (cervical cancer cells), which was mainly located in the cell nucleus.

[0145] The experimental results show that the phosphorylation of PA28γ-T23 site can be detected by ELISA, dot hybridization, immunoblotting, immunohistochemistry, immunocytochemistry and other methods to screen tumors such as oral squamous cell carcinoma, lung adenocarcinoma and cervical cancer.

[0146] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0147] Experimental Example 1: Phosphorylation modification of the PA28γT23 site of the present invention promotes OSCC cell growth

[0148] (I) CCK8 experimental detection

[0149] 1. The cells to be tested were plated in a 96-well plate, with 3000 to 5000 cells per well, and 5 to 6 replicates. A blank control group with only culture medium was set up, and ddH 2 O, prepare 4 identical plates, and place the 96-well plate in a cell culture incubator (37°C, 5% CO 2 ) overnight.

[0150] 2. Carry out CCK8 experiment at 24h, 48h, 72h and 96h after plating.

[0151] 3. Add 10 μL of CCK8 solution to each well (do not generate bubbles) and place in a cell culture incubator (37°C, 5% CO 2 ) for 1 to 4 h.

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

[0153] (II) Construction of nude mouse OSCC xenograft tumor model

[0154] 1. Prepare the cell line HSC-3 human oral squamous cell carcinoma cells to be inoculated.

[0155] 2. Digest the cells in the logarithmic growth phase with trypsin, collect them into a clean BD tube, resuspend and wash twice with PBS, add serum-free and antibiotic-free culture medium, count the cells after resuspending and adjust the cell concentration.

[0156] 3. BALB / c-nu nude mice were selected, with 6 mice in each group. After disinfecting the back skin of the nude mouse to be injected with 75% alcohol, 100 μL of cell suspension was inoculated subcutaneously using an insulin needle (gently mix the cell suspension before inoculation).

[0157] 4. On the 7th day after inoculation, the volume of the transplanted tumor was measured with a vernier caliper and measured every 3 days. On the 25th day, the transplanted tumor was collected, the volume and weight were measured, and photos were taken and statistics were collected.

[0158] In order to further explore whether the phosphorylation of PA28γT23 site has an effect on OSCC cell proliferation, CCK8 and plate clone formation experiments were performed.

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

[0160] First, CCK8 was performed on HSC-3 (human oral squamous cell carcinoma) and UM1 cells (human oral squamous cell carcinoma) that stably knocked down endogenous PA28γ and overexpressed exogenous PA28γWT, T23A, T23D or control EV. The results showed that the cell growth rates in the T23A, WT, and T23D groups increased in sequence, indicating that phosphorylation of PA28γT23 site promoted OSCC cell proliferation.

[0161] The experimental results showed that the cell growth rates of T23A, WT, and T23D groups increased successively, indicating that PA28γT23 site phosphorylation promoted OSCC (HSC-3 and UM1) cell proliferation, and that inhibiting PA28γT23 site phosphorylation could inhibit OSCC (HSC-3 and UM1) cell proliferation.

[0162] (III) Phosphorylation modification of PA28γT23 site can promote the growth of OSCC subcutaneous transplanted tumors

[0163] In order to detect the effect of PA28γT23 phosphorylation modification on OSCC growth in vivo, a nude mouse subcutaneous xenograft tumor model was constructed. The transplanted tumor cells were selected from HSC-3 cells (human oral squamous cell carcinoma cells) used in the previous functional experiments and prone to tumorigenesis. The transduction construction was the same as above ( Figure 6 ) are consistent with the cells with different phosphorylation modification states of PA28γT23 site.

[0164] The tumor volume of nude mice transplanted tumors was measured using a vernier caliper starting from the 7th day after tumor injection, and was measured once every 3 days. Tumors were collected and photographed on the 25th day ( Figure 7 A), and the changes in tumor volume and tumor weight at the time of collection were statistically analyzed. The statistical results showed that the tumor volume and weight of the T23A, WT, and T23D groups increased in sequence, suggesting that the phosphorylation modification of the PA28γT23 site promoted the growth of OSCC subcutaneous transplanted tumors ( Figure 7 B, Figure 7 C), which is consistent with the conclusion of in vitro functional experiments ( Figure 6 ).

[0165] The experimental results showed that PA28γT23 phosphorylation promoted the proliferation of OSCC cells. PA28γT23 phosphorylation promoted the growth of OSCC subcutaneous transplanted tumors, and inhibition of PA28γT23 phosphorylation could inhibit the growth of OSCC subcutaneous transplanted tumors.

[0166] In summary, the present invention provides an application of a PA28γ-T23 site phosphorylation antibody, and experimentally verified that the phosphorylation modification of the PA28γT23 site promotes the proliferation of OSCC cells. The present invention proves that 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. The risk of suffering from tumors containing the PSME3 gene such as oral squamous cell carcinoma, lung adenocarcinoma or cervical cancer can be detected by detecting the phosphorylation level of the PA28γT23 site. 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 decisions, and has good prospects for clinical application.

[0167] The present invention also proves that phosphorylation of the PA28γT23 site promotes the proliferation of OSCC (HSC-3 and UM1) cells by pseudo-dephosphorylation and pseudo-phosphorylation of the PA28γT23 site; and inhibiting the phosphorylation of the PA28γT23 site can inhibit the proliferation of OSCC (HSC-3 and UM1) cells. Therefore, the reagent that inhibits the phosphorylation of the PA28γT23 site can be used to prepare drugs for treating tumors, and has good application prospects.

Claims

1. A novel PA28γ protein, characterized in that: It is a PA28γ protein that is phosphorylated at the T23 site.

2. Use of an agent for inhibiting the phosphorylation level of PA28γ-T23 site in the preparation of a drug for preventing and / or treating tumors.

3. The use according to claim 2, characterized in that: 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.

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

1.

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

6. Use of the antibody according to claim 5 in preparing a reagent for detecting the phosphorylation level of PA28γ-T23 site.

7. The use according to claim 6, characterized in that: The reagents for detecting the phosphorylation level of the PA28γ-T23 site are ELISA detection reagents, dot hybridization detection reagents, immunoblotting detection reagents, immunohistochemistry detection reagents, and immunocytochemistry detection reagents.

8. Use of a reagent for detecting the expression level of phosphorylated PA28γ-T23 site in the preparation of a reagent for tumor screening.

9. The use according to claim 8, characterized in that: 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.

10. A tumor screening kit, characterized in that: It includes, optionally, a reagent for detecting the expression level of the phosphorylated PA28γ-T23 site.

Images