Methylation modified STAT1 protein and application thereof

Abstract

The invention relates to the technical field of biology, in particular to methylation modified STAT1 protein and application thereof. According to the STAT1 protein provided by the invention, a key site for regulating the transcriptional regulation activity and the cell transformation function of the STAT1 protein is found through bioinformatics prediction, and further it is found that after being methylated, the 494th Arg site of the STAT1 protein serves as an artificial antigen to be used for immunizing animals to obtain the methylated antibody of the STAT1 protein. The methylated antibody can specifically recognize methylation of the R494 site of the STAT1 protein in tumor cells, and further detect the methylation level of the STAT1 protein in tumor tissues, so that the methylated antibody is used for evaluating and detecting the drug resistance of anti-tumor drugs. Certain help is provided for clinical screening of tumor treatment 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 particularly to a methylated STAT1 protein and its applications. Background Art

[0002] STAT1 (signal transducer and activator of transcription 1) is the first discovered member of the STATs family. Its encoding gene is located on chromosome 2 and consists of 750 amino acid residues. It is activated by tyrosine kinase (janus kinase, JAK) and mitogen-activated protein kinases (MAPK), which phosphorylate the conserved tyrosine and serine residues in its C-terminal activation region. After forming a dimer, it translocates into the nucleus to regulate 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 spontaneous or induced tumor formation rate is higher than that of 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 nephroblastoma, the expression of STAT1 is associated with better prognosis. However, some studies have shown that STAT1 can also promote the occurrence of blood tumors independent of 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 deletion of STAT1 or the 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 apoptosis, inhibiting cell proliferation, and negatively regulating the cell cycle.

[0004] Protein methylation is one of the important mechanisms of post-translational protein modification and is involved in the regulation of various biological processes and functions. The activity of transcription factors is usually regulated by multiple methylation sites, which regulate the DNA binding ability, protein stability, and interaction with other transcription-related proteins of transcription factors, thereby regulating their transcriptional activity. Currently, the related functional analysis and molecular mechanism of STAT1 and its antibodies in the screening or preparation of anti-tumor drugs are still blank, and this part of the research urgently needs to be solved. 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 obtaining an antigen by methylating the STAT1 protein and preparing an antibody for specifically detecting the methylation modification of the STAT1 protein.

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

[0007] I. Screening antitumor drugs;

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

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

[0010] In some embodiments, the tumor includes colorectal tumors.

[0011] The present invention provides a STAT1 protein, which has the amino acid sequence shown in SEQ ID NO: 1, wherein the 494th position of the STAT1 protein is arginine with methylation modification.

[0012] The present invention provides an artificial antigen obtained by conjugating a carrier protein and the above-mentioned STAT1 protein.

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

[0014] The present invention provides a methylation antibody of the STAT1 protein, which is obtained by immunizing an animal with the above-mentioned STAT1 protein or the artificial antigen;

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

[0016] The present invention provides a method for preparing the methylation antibody of the STAT1 protein, including separating the methylation antibody of the STAT1 protein from the serum of the immunized animal after immunizing the animal with the above-mentioned STAT1 protein or the artificial antigen.

[0017] In some embodiments, the animal includes rabbits, and the number of immunizations is ≥3 times.

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

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

[0020] ②. The methylation antibody of the STAT1 protein obtained by the preparation method described above.

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

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

[0023] (2). The methylation antibody of the STAT1 protein obtained by the preparation method described above.

[0024] The present invention provides a STAT1 protein. Through bioinformatics prediction, the key sites regulating the transcriptional regulatory activity and cell transformation function of the STAT1 protein are found. Further, it is found that after the Arg site at position 494 of the STAT1 protein is methylated, it is used as an artificial antigen to immunize animals to obtain the methylation antibody of the STAT1 protein. This methylation antibody can specifically recognize the methylation of the R494 site of the STAT1 protein in tumor cells, and further detect the level of STAT1 protein methylation in tumor tissues, which is used for the evaluation and detection of anti-tumor drug resistance, and provides certain help for screening anti-tumor drugs, preparing drugs for diagnosing tumors or preparing drugs for treating tumors in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0026] Figure 2 Showing the analysis results of the specificity of the STAT1 protein in species;

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

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

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

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

[0031] Figure 7 Showing the immunoblotting detection results of the anti-STAT1 (R494 me ) polyclonal antibody;

[0032] Figure 8 Showing the Western-Blot identification results of the polyclonal antibody against STAT1 (R494 me );

[0033] Figure 9 Showing the results of immunohistochemical staining of the polyclonal antibody against STAT1 (R494 me ) for detecting STAT1 R494 monomethylation in tissue microarrays. Among them, Figure a shows the immunohistochemical detection results of the methylation levels of STAT1 R494 in cancer tissues and adjacent tissues of paired colorectal cancer (CRC) patients in the tissue microarray. The tissue microarray was constructed by taking one point from each of the paraffin blocks of each colon cancer and its paired adjacent tissue, and punching a 3-mm diameter cancer / adjacent tissue core at each point into the pre-designed positions in the tissue array. Figure b shows the quantitative statistical analysis of the immunohistochemical results;

[0034] Figure 10 Showing the immunohistochemical identification results of the polyclonal antibody against STAT1 (R494 me ) for detecting tumor tissues of different CRC patients. Among them, Figure a shows the methylation levels of STAT1 R494 in tumor tissues of colorectal cancer patients before bevacizumab treatment with different chemotherapy effects detected by immunohistochemistry. Figure b shows the correlation analysis between the methylation level of STAT1 R494 and the therapeutic effect of bevacizumab targeted therapy;

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

[0036] Figure 12 Showing the Kaplan-Meier survival analysis of the methylation level of STAT1 R494 and the survival results of colorectal cancer patients treated with bevacizumab targeted therapy. Detailed implementation mode

[0037] The present invention provides a methylated STAT1 protein and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications in this article 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 ordinary commercially available products and can be purchased in the market. The present invention will be further described below in conjunction with embodiments.

[0039] Example 1 Polypeptide 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 the STAT1 protein was obtained from the following website (>sp|P42224|STAT1_HUMAN Signaltransducer and activator of transcription 1-alpha / beta OS=Homo sapiens OX=9606 GN=STAT1 PE=1 SV=2), as shown below. When synthesizing the following STAT1 protein, we performed methylation modification on the 494th arginine.

[0043] MSQWYELQQLDSKFLEQVHQLYDDSFPMEIRQYLAQWLEKQDWEHAANDVSFATIRFHDLLSQLDDQYSRFSLENNFLLQHNIRKSKRNLQDNFQEDPIQMSMIIYSCLKEERKILENAQRFNQAQSGNIQSTVMLDKQKELDSKVRNVKDKVMCIEHEIKSLEDLQDEYDFKCKTLQNREHETNGVAKSDQKQEQLLLKKMYLMLDNKRKEVVHKIIELLNVTELTQNALINDELVEWKRRQQSACIGGPPNACLDQLQNWFTIVAESLQQVRQQLKKLEELEQKYTYEHDPITKNKQVLWDRTFSLFQQLIQSSFVVERQPCMPTHPQRPLVLKTGVQFTVKLRLLVKLQELNYNLKVKVLFDKDVNERNTVKGFRKFNILGTHTKVMNMEESTNGSLAAEFRHLQLKEQKNAGTRTNEGPLIVTEELHSLSFETQLCQPGLVIDLETTSLPVVVISNVSQLPSGWASILWYNMLVAEPRNLSFFLTPPCARWAQLSEVLSWQFSSVTKRGLNVDQLNMLGEKLLGPNASPDGLIPWTRFCKENINDKNFPFWLWIESILELIKKHLLPLWNDGCIMGFISKERERALLKDQQPGTFLLRFSESSREGAITFTWVERSQNGGEPDFHAVEPYTKKELSAVTFPDIIRNYKVMAAENIPENPLKYLYPNIDKDHAFGKYYSRPKEAPEPMELDGPKGTGYIKTELISVSEVHPSRLQTTDNLLPMSPEEFDEVSRIVGSVEFDSMMNTV (SEQ ID NO:1)

[0044] Analysis of the characteristics of human STAT1 protein using DNAstar software ( Figure 1 ). Analyze the specificity of human STAT1 protein in species ( Figure 2 ), conserved domains ( Figure 3 ), post-translational modifications ( Figure 4 ), transmembrane domain analysis ( Figure 5 ), signal peptides ( Figure 6 ).

[0045] 2. STAT1 synthetic polypeptide 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 procedure for preparing polyclonal antibodies

[0048] 1 Antigen preparation

[0049] 1.1 Polypeptide conjugation with 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 dissolve it completely in 50 μL of DMSO, then add 150 μL of 1×PBS and mix well.

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

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

[0054] 1.1.5 Replace with fresh 1×PBS and dialyze for 2 h, repeat once. Place the activated and dialyzed KLH in a 15 mL imported centrifuge tube, mark the reagent name, time and concentration on the tube, and store it in a 4°C refrigerator.

[0055] 1.1.6 Weigh 4 mg of polypeptide, dissolve it in 50 μL of DMSO, then add 200 μL of 1×PBS, mix quickly, and then immediately add KLH according to the ratio of polypeptide:KLH = 1 mg:680 μg, and leave it in a 4°C refrigerator overnight or react at room temperature for 2 h.

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

[0057] 1.1.8 Take out the dialyzed KLH-peptide and transfer it to a clean 1.5 mL centrifuge tube, dispense it according to the immunization dose, and store it in a -20°C refrigerator.

[0058] 2 Animal immunization protocol

[0059] 2.1 Animal selection: Select healthy animals with shiny fur and free movement. After selecting the animals, pre-raise them for about 2 weeks. The purpose is to eliminate some unqualified animals and ensure the smooth progress of subsequent experiments.

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

[0061] 2.3 Antigen preparation:

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

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

[0064] 2.3.3 Draw the adjuvant. The adjuvant and antigen are drawn at a volume ratio of 1:1. Complete adjuvant is used for the initial immunization, and incomplete adjuvant is used for the second to fourth immunizations. When drawing, the adjuvant should be fully mixed evenly before being drawn into the syringe.

[0065] 2.3.4 Connect two syringes with a syringe connecting tube and perform complete emulsification. The emulsification standard is: the emulsified immunogen dropped into 37 °C water does not disperse, which is qualified.

[0066] 2.4 Immunization: The animals are immunized by multi-point subcutaneous injection, 0.2 mL per point.

[0067] Immunization time: The second immunization is carried out 14 days after the initial immunization. The interval between the second and third immunizations is 7 days. Small sample serum is collected from the middle ear artery of the animals 7 days after the third immunization. If the detection is qualified, a booster immunization is carried out 7 days later, and whole blood can be collected 7 days after the booster immunization.

[0068] 2.4.1 Steps for collecting small sample serum

[0069] Fix the experimental rabbit in the fixing rack, gently pat the ear to dilate the central artery in the ear. Disinfect with 75% alcohol. Fix the rabbit ear with the left hand and hold the syringe with the right hand. Insert the needle at 1 / 3 from the end of the central artery in the ear, parallel to the artery, and pierce the artery in the centripetal direction. The blood collection volume at one time is 8 mL. After blood collection, compress the blood vessel with a cotton ball to stop bleeding.

[0070] 2.4.2 Steps for collecting whole blood

[0071] 2.4.2.1 Grasp the required immunized animal, check the ear tag number and then weigh it. Anesthetize it by intravenous injection of 1 mL of 3% sodium pentobarbital per kilogram.

[0072] 2.4.2.2 Place the anesthetized immunized animal with its abdomen upward, fix its four limbs on the stainless steel wire rack, and use the cardiac blood collection method.

[0073] 2.4.2.3 After blood collection, place the centrifuge tube containing blood into a 37 °C water bath for 15 - 30 minutes, then take it out, cool it, and place it into a 4 °C refrigerator. Wait for the blood to separate automatically, and take out 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 the supernatant, mix well, and store at -20 °C.

[0075] 3 ELISA Detection (Indirect Method)

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

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

[0078] 3.3 Sample Addition: Add a certain dilution of the sample to be tested (dilute the sample to be tested according to a certain ratio), 50 μL to the above - blocked reaction wells. At the same time, set up negative control wells (1% BSA). Incubate at 37 °C for 30 min and wash each well 3 times with 150 μL of 1×PBST washing buffer.

[0079] 3.4 Enzyme - labeled Antibody Addition: Add freshly diluted secondary antibody - HRP (diluted with 1% BSA), 50 μL per well to the enzyme - labeled plate wells, incubate at 37 °C for 45 min, and wash 3 times with 150 μL of 1×PBST buffer per well.

[0080] 3.5 Substrate Solution Addition for Color Development: Add 50 μL of freshly prepared TMB substrate solution to each reaction well and react at 37 °C for 5 min.

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

[0082] 3.7 Reading the Plate: Place the enzyme - labeled plate into a pre - heated microplate reader (450 nm) for reading, save the data, and perform analysis.

[0083] 4 Antibody Purification

[0084] 4.1 Wash the affinity chromatography column successively with 20 mL of pure water and 1×PBS (pH = 7.4) at a flow rate of 70 mL / h for sufficient cleaning.

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

[0086] 4.3 Load the filtered serum sample onto the column 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 min, connect the protein detector and adjust the transmittance (T scale) reading of the instrument to 100 during the washing process.

[0088] 4.5 Adjust the absorbance reading (1 A scale) of the protein detector to 0. At this time, turn on 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 speed of 40 mL / h. At this time, press the green elution record button to start elution, and start collecting the antibody when the reading of the instrument begins to increase.

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

[0090] 4.7 After the antibody collection is completed, adjust the pH value to about 7, record the volume of the eluted antibody, and then rinse the rubber tube connecting the collector with pure water.

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

[0092] 4.9 Send the purified antibody for inspection according to different requirements. (When purifying modified antibodies, the serum first passes through a modified purification column, and the eluted antibody then passes through an unmodified purification column to obtain specific modified antibodies.)

[0093] 4.10 After purifying enough antibodies for delivery and the titer of the semi-finished product is qualified, mix all the antibodies and concentrate them using an ultrafiltration concentrator tube 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 it at room temperature, changing the solution every 3 h for a total of 3 times (for overnight dialysis, place it in a 2 - 8℃ refrigerator).

[0095] 4.12 Take out the dialyzed antibody into a clean centrifuge tube, filter the antibody with a 0.22 μm disposable low-binding filter head in a laminar flow hood, take a small sample for inspection, and take another 5 μL to detect the concentration.

[0096] 4.13 Detect the concentration on the Protein A280 application of a ultra-micro spectrophotometer (denovix DS-11).

[0097] 4.14 After all inspections are qualified, fill out the report.

[0098] 5. Test Results

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

[0100] Table 1

[0101]

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

[0103] Table 2

[0104]

[0105] 5.3 The ELISA test results of the finished antibody 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. Application of Polyclonal Antibody Against STAT1 (R494 me )

[0113] 1. Immunoblotting Detection of Polyclonal Antibody Against STAT1 (R494 me )

[0114] Reagents: TBST buffer, 87.6 g of NaCl, 100 mL of 20 mM Tris-HCl buffer pH = 7.5, 5 mL of Tween 20, made up to 1000 mL with ddH20.

[0115] Weigh 1 mg each of the R494-methylated modified polypeptide and the non-modified polypeptide, dissolve them in 1 mL of 0.01M PBS buffer, respectively take 5 μL of the dissolved polypeptide and spot it on the PVDF membrane, and block the PVDF membrane with a TBST blocking solution containing 5% skim milk powder for 1 h. Add the purified anti-STAT1 (R494 me )-polyclonal antibody (dilution factor 1:4000), incubate at room temperature for 1 h and then wash the membrane five times (8 min each time), add the IRDye700-labeled goat anti-rabbit secondary antibody (dilution factor 1:5000), incubate at room temperature for 1 h and then wash the membrane five times (8 min each time). The final result is collected by the Jena UVP Chemstudio touch multifunctional imager.

[0116] The results are as Figure 7 : The polyclonal antibody against STAT1 (R494 me ) affinity-purified in the present invention can detect the pR494 and R494 polypeptides on the PVDF membrane.

[0117] 2. Western-Blot identification of the anti-STAT1 (R494 me ) polyclonal antibody

[0118] Construct the pcDNA3.0 / Flag-STAT1 wild-type and pcDNA3.0 / Flag-STAT1(R494A) mutant expression vectors (the mutant mutates the arginine at position 494). Then transiently transfect the colorectal cancer LoVo cells with the two expression vectors respectively. After 48 hours of transfection, collect the HEK293T cells. After lysing the cells with the lysis buffer, perform 12% SDS-PAGE electrophoresis. After electrophoresis, transfer to the PVDF membrane. After blocking with skim milk powder, add the purified anti-STAT1 (R494 me ) polyclonal antibody (dilution factor 1:4000), incubate at room temperature for 1 h and then wash the membrane five times (8 min each time), add the IRDye700-labeled goat anti-rabbit secondary antibody (dilution factor 1:5000), incubate at room temperature for 1 h and then wash the membrane five times (8 min each time). The final result is collected by the Jena UVP Chemstudio touch multifunctional imager.

[0119] The results are as Figure 8 : The anti-STAT1 (R494 me ) polyclonal antibody in the present invention can detect a protein band of about 88 kDa in the cell lysates of HEK293T, LoVo and HCT116 cells, which is consistent with the molecular weight of the STAT1 protein. The STAT1 protein expressed in the cells transfected with the wild-type plasmid shows obvious methylation at the 494th site, while the STAT1 expressed in the cells transfected with the mutant plasmid shows no obvious methylation phenomenon.

[0120] 3. Immunohistochemical Identification of Anti-STAT1 (R494 me ) Polyclonal Antibody

[0121] Main reagents: Environmentally friendly clearing agent, absolute ethanol, 1×PBS, DAB staining solution (polymer method) kit, hematoxylin, neutral gum, EDTA (1 mM, pH = 8.0) antigen retrieval solution 8 mL, made up to 400 mL with ddH2O.

[0122] Experimental procedures

[0123] a. Select 1 point from each paraffin block of colon cancer and its paired adjacent tissues, punch a tissue core with a diameter of 3 mm, and make a tissue microarray.

[0124] b. Place the pathological sections in an oven at 60°C and bake for 2 h, soak in the environmentally friendly clearing agent 3 times, 10 min each time, hydrate and remove benzene with 100%, 95%, 80%, 70% gradient alcohol in turn, 5 min each time, and soak in deionized water for 3 min.

[0125] c. Heat antigen retrieval: Add 1×EDTA antigen retrieval solution to the microwave box, heat to boiling in the microwave, place the dewaxed and hydrated paraffin sections on a heat-resistant plastic slide rack, slowly place them into the boiling buffer solution, microwave at medium power for 20 min, take out the microwave box and let it cool to room temperature naturally, take out the slides from the buffer solution, soak them in distilled water twice first, and then soak them in PBS 3 times, 3 min 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 wet box, add endogenous peroxidase blocker, incubate at room temperature in the dark for 15 min, wash with PBS 3 times, 5 min each time, shake off and dry the liquid around the tissue, and place it flat in a wet box.

[0127] e. Primary antibody incubation: Add 100 μL of anti-STAT1 (R494 me ) polyclonal antibody (diluted 1:100 with 1×PBS), add 100 μL of 1×PBS for negative control, place in a wet box, incubate overnight in a 4°C refrigerator, rewarm and repair at 37°C for 30 min, wash with PBS 5 times, 5 min each time, shake off and dry the liquid around the tissue, and place it flat in a wet box.

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

[0129] g. Add 100 μL of the pre-prepared chromogenic reagent DAB working solution, control the chromogenesis under a light microscope, and after complete chromogenesis, immerse it in distilled water to terminate the chromogenesis.

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

[0131] i. Immerse in 5% ammonia water for 20 - 30 s for bluing, and rinse with tap water for 10 min.

[0132] j. Dehydrate and dry with gradient alcohols of 70%, 80%, 95%, and 100%, 3 min for each grade, immerse in an environmental clearing agent 3 times, 5 min each time. Mount with neutral balsam and observe the results under a microscope.

[0133] The results were as Figure 9 : The anti-STAT1 (R494 me ) polyclonal antibody can detect the STAT1 R494 monomethylation in tissue specimens. At the same time, it was found that in colorectal cancer tissues, the STAT1 R494 monomethylation level was significantly higher than that in the corresponding adjacent tissues. These results suggest that the STAT1 R494 monomethylation in colorectal cancer tissues is significantly higher than that in its adjacent tissues, indicating that STAT1 R494 monomethylation may play an important role in the tumor microenvironment.

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

[0135] Collect endoscopic tumor tissue samples and corresponding clinical data of colorectal cancer patients confirmed by pathology, including chemotherapy regimens, targeted therapy regimens, and efficacy evaluations: divided into complete remission (CR), partial remission (PR), disease progression (PD), and disease stability (SD), and screen out patients treated with bevacizumab.

[0136] Fix the collected endoscopic tumor tissues with formalin, embed them in paraffin and section; perform immunohistochemical staining with the anti-STAT1 (R494 me ) polyclonal antibody, observe the staining results under a microscope, and quantitatively score the STAT1 R494 methylation level (0 - 12 score system). The results were as Figure 10 shown: The STAT1 R494 methylation level in colorectal cancer tissues was positively correlated with the resistance to bevacizumab targeted therapy. The tumor tissues in the resistant group had a higher STAT1 R494 methylation level. A low methylation level predicted a better targeted therapy effect (no complete remission cases were received). A high STAT1 R494 methylation level was positively correlated with disease stability and disease progression after targeted therapy.

[0137] Furthermore, through ROC curve analysis, the diagnostic efficacy of the immunohistochemical score of STAT1 R494 methylation level in predicting the efficacy of bevacizumab targeted therapy for colorectal cancer patients was evaluated. The results are as Figure 11 shown: the area under the curve (AUC) was 0.8125, indicating that the diagnostic model had a certain degree of accuracy.

[0138] In addition, we analyzed the survival outcome of STAT1 R494 methylation level and colorectal cancer patients treated with bevacizumab targeted therapy through Kaplan-Meier survival analysis. The results are as Figure 12 shown. The STAT1 R494 methylation level was related to the survival outcome of colorectal cancer patients, and a high STAT1 R494 methylation level was associated with poor prognosis.

[0139] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of STAT1 protein R494 site as a target in the following Ⅰ and / or Ⅱ: Ⅰ. Screening of tumor therapeutic drugs; II. Application in the preparation of drugs for diagnosing and / or treating tumors.

2. The use according to claim 1, characterized in that: The tumors include colorectal tumors.

3. 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.

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

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

6. 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 3 or the artificial antigen according to claim 4 or 5; The methylation antibody of STAT1 protein includes monoclonal antibody and / or polyclonal antibody.

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

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

9. Application of ① and / or ② below in screening tumor therapeutic drugs or preparing drugs for diagnosing and / or treating tumors: ①. The methylated antibody against STAT1 protein according to claim 6; ②. The methylated antibody of STAT1 protein prepared by the preparation method according to claim 7 or 8.

10. Screening tumor therapeutic drugs or preparing drugs for diagnosing and / or treating tumors, characterized in that: This includes (1) and / or (2): (1) The methylated antibody against STAT1 protein according to claim 6; (2) A methylated antibody against STAT1 protein prepared by the preparation method according to claim 7 or 8.

Images