Methylation-modified STAT1 protein and its application

Abstract

The present invention relates to the field of biotechnology, and more particularly to a methylated STAT1 protein and its application. The present invention provides a STAT1 protein. Through bioinformatics prediction, key sites that regulate the transcriptional regulatory activity and cell transformation function of the STAT1 protein are identified. Furthermore, it is found that the 494th Arg site of the STAT1 protein is methylated. The methylated site is used as an artificial antigen to immunize animals to obtain a methylated antibody against the STAT1 protein. The methylated antibody can specifically recognize the methylation of the R494 site of the STAT1 protein in tumor cells. The methylation level of the STAT1 protein in tumor tissues is further detected, and the antibody is used to assess and detect anti-tumor drug resistance, thereby providing certain assistance for clinical screening of tumor therapeutic drugs, preparation of drugs for diagnosing tumors, or preparation of drugs for treating tumors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a methylated STAT1 protein and applications thereof. Background Art

[0002] STAT1 (signal transducer and activator of transcription 1) is the first member of the STATs family to be discovered. Its encoding gene is located on chromosome 2 and consists of 750 amino acid residues. It is activated by tyrosine kinase (JAK) and mitogen-activated protein kinases (MAPK) to phosphorylate the conserved tyrosine and serine residues in its C-terminal activation region. After forming a dimer, it translocates into the cell nucleus and regulates target genes. Its main signaling pathway is dependent on the interferon (IFN) signaling pathway.

[0003] The biological functions of STAT1 include the following aspects: (1) Inhibiting tumorigenesis. In STAT1 / p53 knockout mice, the rate of spontaneous or induced tumor formation is higher than that in mice with only p53 knockout, and the anti-tumor activity of IFN-α disappears. At the same time, in human tumors, such as breast cancer and Wilms' tumor, STAT1 expression is associated with better prognosis. However, some studies have shown that STAT1 can also promote the occurrence of blood tumors unrelated to IFN. (2) Regulating the immune system. STAT1 is involved in all major histocompatibility complex (MHC)-dependent antigen presentation processes. In addition, STAT1 is involved in the early development of B cells. The loss of STAT1 or mutation of the STAT1 gene increases the body's sensitivity to parasites, bacteria, viruses, etc. In addition, STAT1 can also play a role by promoting cell apoptosis, inhibiting cell proliferation, and negatively regulating the cell cycle.

[0004] Protein methylation is a key mechanism of post-translational modification, involved in the regulation of diverse biological processes and functions. The activity of transcription factors is often regulated by multiple methylation sites, which modulate their DNA binding ability, protein stability, and interactions with other transcription-related proteins, thereby regulating their transcriptional activity. Currently, there is a lack of understanding of the functional and molecular mechanisms of STAT1 and its antibodies in the screening or preparation of anti-tumor drugs, and this area of ​​research urgently needs to be addressed. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a methylated STAT1 protein and its application. The present invention provides a technical solution for methylating the STAT1 protein to obtain an antigen and preparing an antibody that specifically detects the methylated modification of the STAT1 protein.

[0006] The present invention provides the use of the STAT1 protein R494 site as a target in the following I and / or II:

[0007] Ⅰ. Screening of tumor therapeutic drugs;

[0008] II. Application in the preparation of drugs for diagnosing and / or treating tumors.

[0009] Through bioinformatics prediction, the present invention discovered that the 494th Arg site of the STAT1 protein is its new methylation site. After the site is mutated, the transcriptional regulatory activity and cell transformation function of STAT1 are significantly changed, thereby affecting the occurrence and development of tumors.

[0010] In some embodiments, the tumor comprises a colorectal tumor.

[0011] The present invention provides a STAT1 protein having an amino acid sequence as shown in SEQ ID NO: 1, wherein position 494 of the STAT1 protein is a methylated arginine.

[0012] The present invention provides an artificial antigen obtained by coupling a carrier protein with the STAT1 protein.

[0013] In some embodiments, the carrier protein is keyhole limpet hemocyanin.

[0014] The present invention provides a methylated antibody against STAT1 protein, wherein the methylated antibody is obtained by immunizing an animal with the STAT1 protein or the artificial antigen;

[0015] The methylation antibody of STAT1 protein includes monoclonal antibody and / or polyclonal antibody.

[0016] The present invention provides a method for preparing the methylated antibody of STAT1 protein, comprising immunizing an animal with the STAT1 protein or the artificial antigen, and then isolating the methylated antibody of STAT1 protein from the serum of the immunized animal.

[0017] In some embodiments, the animal comprises a rabbit, and the number of immunizations is ≥ 3 times.

[0018] The present invention provides the following applications of ① and / or ② in screening tumor therapeutic drugs or preparing drugs for diagnosing and / or treating tumors:

[0019] 1. The methylation antibody of STAT1 protein;

[0020] ②. The methylated antibody against STAT1 protein prepared by the preparation method.

[0021] The present invention provides a method for screening tumor therapeutic drugs or preparing drugs for diagnosing and / or treating tumors, characterized by comprising the following (1) and / or (2):

[0022] (1) The methylation antibody of STAT1 protein;

[0023] (2) The methylated antibody against STAT1 protein prepared by the preparation method.

[0024] The present invention provides STAT1 protein. Through bioinformatics prediction, key sites that regulate the transcriptional regulatory activity and cell transformation function of STAT1 protein are found. It is further found that after the 494th Arg site of STAT1 protein is methylated, it is used as an artificial antigen to immunize animals to obtain methylated antibodies of STAT1 protein. The methylated antibodies can specifically recognize the methylation of the R494 site of STAT1 protein in tumor cells. The methylation level of STAT1 protein in tumor tissue is further detected and used for the evaluation and detection of anti-tumor drug resistance, providing certain assistance for the screening of tumor treatment drugs, the preparation of drugs for diagnosing tumors, or the preparation of drugs for treating tumors in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shows the results of immunogenicity, hydrophilicity and surface accessibility analysis of STAT1 protein;

[0026] Figure 2 Shows the results of species-specific analysis of STAT1 protein;

[0027] Figure 3 Shows the analysis results of the conserved domain of STAT1 protein;

[0028] Figure 4 Shows the results of post-translational modification analysis of STAT1 protein;

[0029] Figure 5 Shows the analysis results of the transmembrane domain of STAT1 protein;

[0030] Figure 6 Shows the signal peptide analysis results of STAT1 protein;

[0031] Figure 7 Anti-STAT1 (R494 me ) Immunodot assay results using polyclonal antibodies;

[0032] Figure 8 Anti-STAT1 (R494 me ) Western-Blot identification results of polyclonal antibodies;

[0033] Figure 9 Anti-STAT1 (R494 me ) Results of polyclonal antibody immunohistochemical staining for STAT1 R494 monomethylation in tissue microarrays. Figure a shows the immunohistochemical detection of STAT1 R494 methylation levels in paired colorectal cancer (CRC) patient tissue and adjacent tissues in the tissue microarray. The tissue microarray was constructed by selecting one point from each paraffin block of colon cancer and its paired adjacent tissue, and then inserting a 3 mm diameter core of each point from the cancer / adjacent tissue into pre-designed locations on the tissue array. Figure b shows the quantitative statistical analysis of the immunohistochemical results.

[0034] Figure 10 Anti-STAT1 (R494 me ) Immunohistochemical identification results of polyclonal antibodies in tumor tissues of different CRC patients. Figure a shows immunohistochemical detection of STAT1 R494 methylation levels in tumor tissues of colorectal cancer patients with different chemotherapy effects before bevacizumab treatment, and Figure b shows the correlation analysis between STAT1 R494 methylation levels and the efficacy of bevacizumab targeted therapy.

[0035] Figure 11 Shows the ROC curve analysis results of STAT1 R494 methylation level and the efficacy of bevacizumab targeted therapy in patients with colorectal cancer;

[0036] Figure 12 Figure 4 shows the Kaplan-Meier survival analysis of STAT1 R494 methylation level and the survival outcomes of colorectal cancer patients treated with bevacizumab targeted therapy. DETAILED DESCRIPTION

[0037] The present invention provides methylated STAT1 proteins and their applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It should be noted that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0038] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention will be further described below with reference to the examples.

[0039] Example 1 Peptide Synthesis

[0040] The antigen polypeptide sequence for specifically recognizing STAT1 R494 methylation was designed as follows.

[0041] 1. STAT1 amino acid sequence

[0042] The amino acid sequence of STAT1 protein was obtained from the following website (>sp|P42224|STAT1_HUMAN Signal transducer and activator of transcription 1-alpha / beta OS=Homo sapiens OX=9606 GN=STAT1 PE=1 SV=2). Specifically, the STAT1 protein was synthesized by methylating arginine at position 494.

[0043] (SEQ ID NO: 1)

[0044] The characteristics of human STAT1 protein were analyzed using DNAstar software ( Figure 1 ). Analyze the species specificity of human STAT1 protein ( Figure 2 ), conserved domains ( Figure 3 ), post-translational modifications ( Figure 4 )、Transmembrane domain analysis( Figure 5 )、Signal peptide( Figure 6 ).

[0045] 2. STAT1 synthetic peptide sequence:

[0046] After the above analysis, the antigen polypeptide sequence selected for specific recognition of R494 methylation is TPPCAR(me)WAQLS (SEQ ID NO: 2)

[0047] Example 2 Experimental process for preparing polyclonal antibodies

[0048] 1. Antigen preparation

[0049] 1.1 Peptide-coupled KLH

[0050] 1.1.1 Dissolve 20 mg of KLH in 2 mL of 5 mM EDTA aqueous solution.

[0051] 1.1.2 Weigh 8 mg of Sulfo-SMCC and completely dissolve it in 50 μL of DMSO. Then add 150 μL of 1× PBS and mix thoroughly.

[0052] 1.1.3 Add the Sulfo-SMCC solution dropwise to the KLH solution, shaking gently (vigorous shaking will produce precipitation), and let it stand at room temperature for 1 h.

[0053] 1.1.4 Place the activated KLH solution into a dialysis bag, secure with a dialysis clamp, and dialyze in 2 L of 1× PBS at 4°C under magnetic stirring for 1 h.

[0054] 1.1.5 Replace with fresh 1× PBS and dialyze for 2 hours. Repeat once. Place the activated, dialyzed KLH into a 15 mL centrifuge tube. Label the tube with the reagent name, time, and concentration. Store in a refrigerator at 4°C.

[0055] 1.1.6 Weigh 4 mg of peptide and dissolve it in 50 μL of DMSO. Add 200 μL of 1× PBS and mix quickly. Immediately add KLH at a ratio of 1 mg peptide: 680 μg KLH. Incubate at 4°C overnight or at room temperature for 2 h.

[0056] 1.1.7 Place the cross-linked KLH-peptide complex into a dialysis bag, secure with a dialysis clamp, and dialyze overnight in 4 L of 1× PBS at 4°C under magnetic stirring.

[0057] 1.1.8 Remove the dialyzed KLH-peptide into clean 1.5 mL centrifuge tubes, divide into aliquots according to the immunization dose, and store in a -20°C refrigerator.

[0058] 2 Animal immunization plan

[0059] 2.1 Animal Selection: Select healthy animals with lustrous fur and free mobility. Pre-breed the selected animals for approximately two weeks to eliminate any unqualified animals and ensure smooth progress in subsequent experiments.

[0060] 2.2 Preparation before the experiment: Mark the animals.

[0061] 2.3 Antigen preparation:

[0062] 2.3.1 Remove the antigen from the -20°C freezer and dissolve it at room temperature to avoid repeated freezing and thawing. Label the syringe with the project number and animal number.

[0063] 2.3.2 Extract the antigen (mix the antigen completely). The first dose is 0.1-1.0 mg (depending on the type and weight of the immunized animal), 0.5 mL / animal. The amount of antigen for the second to fourth immunizations should be halved.

[0064] 2.3.3 Draw the adjuvant at a 1:1 volume ratio of adjuvant to antigen. Use complete adjuvant for the first dose and incomplete adjuvant for the second, third, fourth, and fourth doses. Thoroughly mix the adjuvant before drawing it into the syringe.

[0065] 2.3.4 After connecting the two syringes with a syringe connecting tube, complete emulsification is carried out. The emulsification standard is: the emulsified immunogen does not disperse when dropped into 37°C water.

[0066] 2.4 Immunization: Animals were injected subcutaneously at multiple sites, with 0.2 mL injected at each site.

[0067] Immunization time: The second vaccination is carried out 14 days after the first vaccination, and the interval between the second and third vaccinations is 7 days. On the 7th day after the third vaccination, a small sample of serum is collected from the middle ear artery of the animal for testing. If the test is qualified, an additional vaccination is carried out 7 days later. Whole blood can be collected 7 days after the additional vaccination.

[0068] 2.4.1 Steps for collecting serum samples

[0069] Secure the rabbit in a holding frame and gently tap the ear to dilate the central auricular artery. Disinfect the rabbit's ear with 75% alcohol. Hold the rabbit's ear with your left hand and hold the syringe with your right hand. Insert the needle about one-third of the way from the distal end of the central auricular artery, parallel to the artery and centripetally. Draw 8 mL of blood at a time. After collection, apply pressure with a cotton ball to stop the bleeding.

[0070] 2.4.2 Whole blood collection steps

[0071] 2.4.2.1 Catch the animals to be immunized, check the ear tag number, weigh them, and anesthetize them by intravenous injection of 1 mL of 3% sodium pentobarbital per kilogram.

[0072] 2.4.2.2 After anesthesia, the immunized animal is placed with its abdomen upwards and its limbs fixed on a stainless steel grid. Blood is collected using the cardiac method.

[0073] 2.4.2.3 After blood collection, place the centrifuge tube containing the blood in a 37°C water bath for 15-30 minutes. Remove the tube, cool it, and place it in a 4°C refrigerator. Wait for the blood to separate automatically and remove the supernatant into a clean 50 mL centrifuge tube.

[0074] 2.2.4.2.4 Centrifuge at 12000 rpm for 2 min, transfer the supernatant to a clean centrifuge tube, add 100 μL of 10% sodium thimerosal solution (final concentration 0.02%) to 50 mL of supernatant, mix well, and store at -20°C.

[0075] 3. ELISA test (indirect method)

[0076] 3.1 Coating: Dilute the known antigen to 1 μg / mL using coating buffer (Na2CO3 and NaHCO3 buffer), add 50 μL to each reaction well of the polystyrene plate, and incubate at 4°C overnight. The next day, discard the solution in the wells and wash once with 1× PBST wash buffer, using 180 μL per well.

[0077] 3.2 Blocking: Add 150 μL of 1% BSA (prepared in PBST) to each well for blocking and incubate at 37°C for 1 hour. Discard the blocking solution.

[0078] 3.3 Sample Addition: Add 50 μL of the sample to be tested (dilute the sample to be tested according to the specified ratio) to the blocked reaction wells. Also set up a negative control well (1% BSA). Incubate at 37°C for 30 minutes. Wash three times with 150 μL of 1× PBST wash buffer per well.

[0079] 3.4 Add enzyme-labeled antibody: Add freshly diluted secondary antibody-HRP (diluted with 1% BSA) to the wells of the enzyme-labeled plate at 50 μL / well, incubate at 37°C for 45 min, and wash three times with 1× PBST buffer at 150 μL per well.

[0080] 3.5 Add substrate solution for color development: Add 50 μL of the temporarily prepared TMB substrate solution to each reaction well and react at 37°C for 5 min.

[0081] 3.6 Terminate the reaction: Add 50 μL of 1 M sulfuric acid to each reaction well.

[0082] 3.7 Reading the plate: Place the plate in a preheated microplate reader (450 nm) for reading, save the data, and analyze.

[0083] 4 Antibody Purification

[0084] 4.1 The affinity chromatography column was thoroughly cleaned with 20 mL of pure water and 1× PBS (pH = 7.4) at a flow rate of 70 mL / h.

[0085] 4.2 Take 10 mL of serum to be purified and place it in a 50 mL centrifuge tube. Filter it through a microporous filter membrane with a pore size of 0.45 μm and a diameter of 25 mm.

[0086] 4.3 Load the filtered serum sample at a flow rate of 40 mL / h and repeat once.

[0087] 4.4 Wash the column with 20 mL of 1× PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 minutes, connect the column to the protein detector. During the washing process, adjust the instrument transmittance (T position) to 100.

[0088] 4.5 Adjust the protein detector absorbance (1 A position) to 0. Now open the HD-A computer collector on the computer desktop and adjust the full screen range to 5. Elute the antibody with glycine solution (pH = 2.7, 0.2 M) at a rate of 40 mL / h. Press the green elution record button to start elution. Start collecting the antibody when the instrument reading starts to rise.

[0089] 4.6 During the antibody collection process, adjust the pH value of the antibody to around 7 with 1 M sodium bicarbonate and record the highest peak of the elution peak.

[0090] 4.7 After collecting the antibodies, adjust the pH to around 7 and record the volume of eluted antibodies. Then rinse the rubber tube connected to the collector with pure water.

[0091] 4.8 Wash the affinity chromatography column with 20 mL of 1× PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal the column, and store in a refrigerator at 4°C.

[0092] 4.9 The purified antibodies are sent for testing according to different requirements. (When purifying modified antibodies, the serum is first passed through a modified purification column, and the eluted antibodies are then passed through a non-modified purification column to obtain specific modified antibodies.)

[0093] 4.10 After the antibody quantity required for delivery is purified and the semi-finished product has passed the titer test, all antibodies are mixed and concentrated using ultrafiltration tubes to reach a certain concentration and volume.

[0094] 4.11 Place the concentrated antibody in 1 L of 0.01 M PBS (pH = 7.4) and dialyze at room temperature, changing the solution every 3 hours for a total of 3 times (for overnight dialysis, place in a refrigerator at 2-8°C).

[0095] 4.12 Remove the dialyzed antibody to a clean centrifuge tube and filter it using a 0.22 μm disposable low-adsorption filter in a clean bench. Take a small sample for testing and another 5 μL for concentration determination.

[0096] 4.13 Measure the concentration using the Protein A280 application on a denovix DS-11 ultra-micro-volume spectrophotometer.

[0097] 4.14 After all tests are passed, fill in the report.

[0098] 5. Test results

[0099] 5.1 Antigen information is shown in Table 1 below.

[0100] Table 1

[0101]

[0102] 5.2 The results of serum ELISA are shown in Table 2 below.

[0103] Table 2

[0104]

[0105] 5.3 The ELISA test results of the finished antibodies are shown in Table 3 below.

[0106] Table 3

[0107]

[0108]

[0109] 5.4 The volume and concentration of the finished antibody are shown in Table 4 below.

[0110] Table 4

[0111]

[0112] Example 3. Anti-STAT1 (R494 me ) Application of polyclonal antibodies

[0113] 1. Anti-STAT1 (R494 me ) Polyclonal antibody immunospot detection

[0114] Reagents: TBST buffer, NaCl 187.6 g, 20 mM Tris-HCl buffer pH = 7.5100 mL, Tween 205 mL, ddH20 to 1000 mL.

[0115] Weigh 1 mg of each of the R494 methylated modified peptide and the unmodified peptide and dissolve them in 1 mL of 0.01 M PBS buffer. Take 5 μL of each dissolved peptide and spot it on the PVDF membrane. Block the PVDF membrane with TBST blocking solution containing 5% skim milk powder for 1 h. Add the purified anti-STAT1 (R494 me ) polyclonal antibody (dilution factor 1:4000), incubated at room temperature for 1 hour, and then washed five times (8 min / time). IRDye700-labeled goat anti-rabbit secondary antibody (dilution factor 1:5000) was added, and incubated at room temperature for 1 hour, and then washed five times (8 min / time). The final data were collected by Jena UVP Chemstudio touch multifunctional imager.

[0116] The results are as follows Figure 7 : Anti-STAT1 (R494) of the present invention is affinity-purified me ) The polyclonal antibody was able to detect the pR494 and R494 polypeptides on the PVDF membrane.

[0117] 2. Anti-STAT1 (R494 me ) Western-Blot identification of polyclonal antibodies

[0118] pcDNA3.0 / Flag-STAT1 wild-type and pcDNA3.0 / Flag-STAT1(R494A) mutant expression vectors were constructed (the mutant had arginine 494 mutated). The two expression vectors were then transiently transfected into colorectal cancer LoVo cells. HEK293T cells were harvested 48 hours after transfection, lysed with lysis buffer, and subjected to 12% SDS-PAGE electrophoresis. After electrophoresis, the cells were transferred to a PVDF membrane, blocked with skim milk powder, and then purified anti-STAT1 (R494 me ) polyclonal antibody (dilution factor 1:4000), incubated at room temperature for 1 hour, and then washed five times (8 min / time). IRDye700-labeled goat anti-rabbit secondary antibody (dilution factor 1:5000) was added and incubated at room temperature for 1 hour, and then washed five times (8 min / time). The final data were collected by Jena UVP Chemstudio touch multifunctional imager.

[0119] The results are as follows Figure 8 : Anti-STAT1 (R494 me ) polyclonal antibodies detected a protein band of approximately 88 kDa in lysates from HEK293T, LoVo, and HCT116 cells, consistent with the molecular weight of STAT1. STAT1 expressed in cells transfected with the wild-type plasmid showed significant methylation at position 494, whereas STAT1 expressed in cells transfected with the mutant plasmid showed no significant methylation.

[0120] 3. Anti-STAT1 (R494 me ) Immunohistochemical identification of polyclonal antibodies

[0121] Main reagents: environmentally friendly transparent agent, anhydrous ethanol, 1× PBS, DAB staining solution (polymer method) kit, hematoxylin, neutral gum, EDTA (1 mM, pH = 8.0) antigen retrieval solution 8 mL, ddH2O dilute to 400 mL.

[0122] Experimental procedures

[0123] a. A tissue core with a diameter of 3 mm was obtained from one point in each paraffin block of colon cancer and its paired adjacent paracancerous tissue to create a tissue microarray.

[0124] b. Place the pathological sections in a 60°C oven for 2 hours, soak in an environmentally friendly transparent agent three times for 10 minutes each time, and then hydrate and debenzene with 100%, 95%, 80%, and 70% graded alcohol for 5 minutes each time. Soak in deionized water for 3 minutes.

[0125] c. Thermal antigen retrieval: Add 1× EDTA antigen retrieval solution to a microwave oven and heat in a microwave until boiling. Place the dewaxed and hydrated paraffin sections on a high-temperature resistant plastic slide rack and slowly place them in boiling buffer. Microwave on medium speed for 20 minutes. Remove from the microwave oven and allow to cool naturally to room temperature. Remove the slides from the buffer and soak them twice in distilled water, then three times in PBS for 3 minutes each time.

[0126] d. Block the activity of endogenous peroxidase: shake off and dry the liquid around the tissue (do not let the tissue dry), place it flat in a humidified chamber, add endogenous peroxidase blocker, incubate at room temperature in the dark for 15 minutes, wash three times with PBS, 5 minutes each time, shake off and dry the liquid around the tissue, and place it flat in a humidified chamber.

[0127] e. Primary antibody incubation: add anti-STAT1 (R494 me ) 100 μL of polyclonal antibody (1:100 diluted with 1× PBS) and 100 μL of 1× PBS for negative control were added, placed in a humidified chamber, refrigerated at 4°C overnight, reheated at 37°C for 30 min, washed with PBS 5 times, 5 min each time, and the liquid around the tissue was shaken off and dried, and placed flat in a humidified chamber.

[0128] f. Secondary antibody incubation: Add 100 μL of specific secondary antibody working solution to the tissue, place in a humidified chamber, incubate at room temperature for 20 min, wash with PBS five times, 5 min each time, and shake off and dry the liquid around the tissue.

[0129] g. Add 100 μL of the pre-prepared color developer DAB working solution and monitor the color development under a light microscope. After complete color development, immerse the plate in distilled water to terminate the color development.

[0130] h. Counterstain with hematoxylin for 5 min, soak in 1% hydrochloric acid alcohol for 30-60 s for differentiation, and rinse with tap water.

[0131] i. Soak in 5% ammonia water for 20-30 seconds until it turns blue, then rinse with tap water for 10 minutes.

[0132] Dehydrate the slides in a gradient of 70%, 80%, 95%, and 100% alcohol, 3 minutes per level. Soak in an environmentally friendly transparent agent three times, 5 minutes each time. Mount the slides with neutral gum and observe under a microscope.

[0133] The results are as follows Figure 9 : Anti-STAT1 (R494 me ) polyclonal antibody can detect STAT1 R494 monomethylation in tissue specimens. Furthermore, the level of STAT1 R494 monomethylation was found to be significantly higher in colorectal cancer tissues than in corresponding adjacent tissues. These results suggest that STAT1 R494 monomethylation is significantly higher in colorectal cancer tissues than in adjacent tissues, indicating that STAT1 R494 monomethylation may play an important role in the tumor microenvironment.

[0134] 4. Immunohistochemical detection of STAT1 R494 me Relationship between expression level in CRC patients and the efficacy of bevacizumab treatment ( Figure 10 ).

[0135] Endoscopic tumor tissue samples and corresponding clinical data of pathologically confirmed colorectal cancer patients were collected, including chemotherapy regimens, targeted therapy regimens, and efficacy evaluation: divided into complete remission (CR), partial remission (PR), progressive disease (PD), and stable disease (SD), to screen out patients treated with bevacizumab.

[0136] The collected endoscopic tumor tissues were fixed with formalin, embedded in paraffin and sectioned; anti-STAT1 (R494 me ) polyclonal antibodies for immunohistochemical staining, and the staining results were observed under a microscope. The methylation level of STAT1 R494 was quantitatively scored (0-12 points). Figure 10 As shown in the results, the methylation level of STAT1 R494 in colorectal cancer tissues was positively correlated with resistance to bevacizumab targeted therapy. The tumor tissues in the resistant group had higher STAT1 R494 methylation levels. Low methylation levels indicated better targeted therapy effects (no complete remission cases were received). High STAT1 R494 methylation levels were positively correlated with disease stability and disease progression after targeted therapy.

[0137] The ROC curve analysis was further used to evaluate the diagnostic efficacy of the immunohistochemical score of STAT1 R494 methylation level in predicting the efficacy of bevacizumab targeted therapy in patients with colorectal cancer. Figure 11 As shown: The area under the curve (AUC) is 0.8125, indicating that the diagnostic model has certain accuracy.

[0138] In addition, we used Kaplan-Meier survival analysis to analyze the relationship between STAT1 R494 methylation level and the survival outcomes of colorectal cancer patients treated with bevacizumab targeted therapy. Figure 12 As shown, STAT1 R494 methylation level is associated with the survival outcome of colorectal cancer patients, and high STAT1 R494 methylation level is associated with poor prognosis.

[0139] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of the STAT1 protein R494 site as a target in the following I and / or II: Ⅰ. Screening of drugs for the treatment of colorectal cancer; II. Preparation of drugs for treating colorectal cancer.

2. STAT1 protein, characterized in that It has an amino acid sequence as shown in SEQ ID NO: 1, wherein the 494th position of the STAT1 protein is a methylated arginine.

3. Artificial antigen, characterized in that The STAT1 protein is obtained by coupling a carrier protein with the STAT1 protein according to claim 2.

4. The artificial antigen according to claim 3, characterized in that The carrier protein is keyhole limpet hemocyanin.

5. A methylation antibody against STAT1 protein, characterized in that: The methylated antibody is obtained by immunizing an animal with the STAT1 protein according to claim 2 or the artificial antigen according to claim 3 or 4; The methylation antibody of STAT1 protein includes monoclonal antibody and / or polyclonal antibody.

6. The method for preparing the methylated antibody against STAT1 protein according to claim 5, characterized in that: The method comprises immunizing an animal with the STAT1 protein according to claim 2 or the artificial antigen according to claim 3 or 4, and then isolating and obtaining the methylated antibody of the STAT1 protein from the serum of the immunized animal.

7. The preparation method according to claim 6, characterized in that The animal includes a rabbit, and the number of immunizations is ≥ 3 times.

8. Use of the following ① and / or ② in screening for drugs for treating colorectal cancer or preparing drugs for treating colorectal cancer:

1. The methylated antibody against STAT1 protein according to claim 5; ②. A methylated antibody against STAT1 protein obtained by the preparation method according to claim 6 or 7.

9. Screening for drugs for treating colorectal cancer or preparing drugs for treating colorectal cancer, characterized in that: Including the following (1) and / or (2): (1) The methylated antibody against STAT1 protein according to claim 5; (2) The methylated antibody against STAT1 protein prepared by the preparation method according to claim 6 or 7.

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