WSTF-K181me3 as well as polyclonal antibody and application thereof

Abstract

The invention provides WSTF-K181me3 as well as a polyclonal antibody and application thereof, and relates to the technical field of biology. The invention further provides a group of immunogen polypeptides, the group of immunogen polypeptides comprises a polypeptide A and a polypeptide B. The polyclonal antibody of the WSTF-K181me3 is prepared by mixing the immunogen polypeptides with immune animals, and the polyclonal antibody can specifically recognize endogenous WSTF-K181me3 protein through an ELISA / Dot blot / Western blot method and is used for preparing tumor and Williams syndrome detection products.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to WSTF-K181me3 and its polyclonal antibody and application. Background Art

[0002] WSTF (Williams syndrome transcription factor) is a transcription factor identified in patients with Williams-Beuren syndrome, a developmental disorder caused by a deletion of human chromosome 7. WSTF is frequently deleted in individuals with this syndrome and its functions are implicated in chromatin remodeling, cell cycle function, apoptosis, and vitamin metabolism regulation. Studies have revealed that WSTF is a previously unknown novel histone tyrosine kinase. The primary kinase activity of WSTF is located in the amino-terminal WAC (WSTF / Acf1 / cbpq46) catalytic domain, which bears no homology to any known kinase domain. WSTF regulates the DNA damage response by phosphorylating tyrosine residue (Y) at position 142 of H2A.X. Following DNA damage, phosphorylation of H2A.X determines whether the damaged cell undergoes repair or must die. If phosphorylation at Y142 is promptly removed after DNA damage, the cell can successfully initiate repair. Conversely, if this site is not dephosphorylated, the damaged cell undergoes apoptosis.

[0003] Traditionally, WSTF is a transcription factor, and its links to normal growth and development, as well as pathological changes in disease, are indirectly mediated through the regulation of the expression of specific target genes. While WSTF has been increasingly recognized for its important functions in promoting human tumor cell proliferation, migration, and energy metabolism, research has primarily focused on its regulation of target gene expression. Against this backdrop, its novel kinase activity has immediately captured the attention of both basic researchers and clinicians, becoming a rapidly emerging area of ​​research with implications far beyond DNA damage repair. Given WSTF's crucial roles in chromatin remodeling, cell cycle, and metabolic regulation, researchers have hypothesized that it may phosphorylate additional substrates, the identification of which may shed light on WSTF's regulatory role in tumorigenesis and the clinical manifestations of Williams-Beuren syndrome. Furthermore, other proteins may also contain WAC domains and possess kinase activity. The investigation of these kinases is expected to provide new insights and insights into normal development and disease progression. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an application of WSTF-K181me3 in the preparation of a product for assessing tumor risk, and the risk of tumor can be assessed by detecting the expression level of WSTF-K181me3.

[0005] The present invention also provides an application of WSTF-K181me3 in the preparation of a tumor detection product.

[0006] Preferably, the tumor is human breast ductal carcinoma.

[0007] Preferably, the product includes antibodies, drugs and kits.

[0008] The present invention also provides an immunogenic polypeptide for preparing a WSTF-K181me3 polyclonal antibody, wherein the polypeptide comprises a polypeptide A having a sequence of KETVV-Tri-Methyl K-EDEGRRE and a polypeptide B having a sequence of ETVV-Tri-Methyl K-EDEGRRES.

[0009] The present invention also provides a polyclonal antibody against WSTF-K175me3, which is obtained by immunizing an animal with a mixture of the immunogenic polypeptides according to claim 5.

[0010] Preferably, polypeptide A and polypeptide B are separately coupled to KLH and then mixed for immunization.

[0011] Preferably, the coupled polypeptide A solution and the coupled polypeptide B solution are mixed at a volume ratio of 1-2:1-2.

[0012] The present invention also provides a method for preparing the polyclonal antibody, comprising the following steps: coupling the immunogenic polypeptides described in claim 5 with KLH respectively, mixing the coupled polypeptide solutions in a volume ratio of 1-2:1-2 and using them for animal immunization, collecting the serum of the immunized animals and purifying them to obtain a polyclonal antibody against WSTF-K181me3.

[0013] Preferably, the animal is immunized 3 to 5 times.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The immunogenic polypeptide provided by the present invention has good immunogenicity. The polyclonal antibody against WSTF-K181me3 obtained by immunizing animals using the polypeptide as an antigen has good affinity and can specifically recognize endogenous WSTF-K181me3 protein by ELISA / Dotblot / Westernblot methods. It has high specificity, high sensitivity and high titer, and can be used to prepare WSTF-K181me3 protein-related disease detection reagents.

[0016] The present invention found through Western blot detection that there is a significant difference in the expression level of WSTF-K181me3 protein in tumor cells and in normal cells. By detecting the expression level of WSTF-K181me3, the risk of tumor can be assessed. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the mass spectrometry detection result of WSTF-K181me3 peptide A;

[0018] Figure 2 This is the mass spectrometry detection result of WSTF-K181me3 peptide B;

[0019] Figure 3 This is the mass spectrometry detection result of WSTF-K181me3 non-modified peptide;

[0020] Figure 4 This is the serum Western blot initial screening result;

[0021] Figure 5 This is a flow chart for the purification of antibodies of the present invention;

[0022] Figure 6 This is the result of ELISA test of the antibody of the present invention;

[0023] Figure 7 This is the Dotblot test result of the antibody of the present invention;

[0024] Figure 8 The Western blot test results of the antibody of the present invention are shown in FIG.

[0025] Figure 9 The results of Western blot analysis of WSTF-K181me3 expression levels in mouse tumor tissues induced by EZH2 knockdown HCC38 cells. DETAILED DESCRIPTION

[0026] The present invention provides an application of WSTF-K181me3 in the preparation of a product for assessing tumor risk.

[0027] The present invention provides an application of WSTF-K181me3 in the preparation of a tumor detection product.

[0028] In the present invention, the tumor is preferably human mammary ductal carcinoma. In a specific embodiment of the present invention, the human mammary ductal carcinoma is more preferably HCC38 cells.

[0029] In the present invention, the products preferably include antibodies, drugs and kits.

[0030] The present invention also provides an immunogenic polypeptide for preparing a WSTF-K181me3 polyclonal antibody, comprising a polypeptide A having a sequence of KETVV-Tri-Methyl K-EDEGRRE and a polypeptide B having a sequence of ETVV-Tri-Methyl K-EDEGRRES. Polypeptides A and B are designed and synthesized based on the WSTF protein sequence and modification type (methylation modification). The amino acid sequence of polypeptide A is: C KETVV-(Tri-Methyl)K-EDEGRRE ("C" represents the carboxyl-terminal COOH group of the peptide segment), with a molecular weight of 1719.84. The "K" at position 6 of the amino acid sequence is trimethylated, and the amino acid linker sequence is shown in SEQ ID NO. 1. The amino acid sequence of polypeptide B is: CETVV-Tri-Methyl K-EDEGRRES ("C" represents the carboxyl-terminal COOH group of the peptide segment), with a molecular weight of 1678.74. The "K" at position 5 of the amino acid sequence is trimethylated, and the amino acid linker sequence is shown in SEQ ID NO. 2.

[0031] The present invention also provides the use of the above immunogenic polypeptide in preparing polyclonal antibodies against WSTF-K181me3. By coupling the immunogenic polypeptide with KLH, the polyclonal antibodies against WSTF-K181me3 can be prepared by animal immunization.

[0032] The present invention also provides a polyclonal antibody against WSTF-K181me3, obtained by immunizing an animal with a mixture of the immunogenic polypeptides. Polypeptide A and polypeptide B are separately conjugated to KLH and then mixed for immunization. The conjugated polypeptide A solution and the conjugated polypeptide B solution are preferably mixed in a volume ratio of 1-2:1-2, more preferably in a volume ratio of 1:1.

[0033] The present invention also provides a method for preparing the polyclonal antibody, which preferably comprises the following steps: coupling the above-mentioned immunogenic polypeptides with KLH respectively, mixing the coupled polypeptide solutions in a volume ratio of 1-2:1-2 and then using them for animal immunization, collecting the serum of the immunized animals and purifying them to obtain a polyclonal antibody against WSTF-K181me3.

[0034] In the present invention, the animal is preferably immunized 3 to 5 times, more preferably 4 times.

[0035] In the present invention, as one possible implementation, the antigen and adjuvant are thoroughly mixed to form a stable emulsion. The antigen mixture is then drawn up with a syringe and injected into the rabbits' shoulders at two subcutaneous points and the hind leg muscles at two points. Each injection site preferably receives 1 / 4 of the immunogen emulsion volume to ensure the long-lasting presence of the immunogen, thereby enhancing the immune response. Preferably, the antigen polypeptide is diluted with physiological saline at a 10- to 20-fold, more preferably 12- to 15-fold, dilution factor; the adjuvant is Freund's adjuvant; and the immunogen-to-adjuvant mixing ratio is 1:1.

[0036] The immunogenic polypeptide or the polyclonal antibody or the method for preparing the polyclonal antibody of the present invention can be used to prepare a detection reagent for WSTF-K181me3 protein-related diseases. Preferably, the related diseases include tumors and Williams syndrome.

[0037] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0039] Example 1

[0040] According to the WSTF protein sequence and modification type, two modified antigen peptides were designed and synthesized for animal immunization, purification and detection; at the same time, a non-modified peptide (SEQ ID NO.3) was designed and synthesized for purification and detection.

[0041] Table 1 Primer and probe information

[0042]

[0043] The synthetic peptides were detected by mass spectrometry, e.g. Figures 1 to 3 As shown, the sequences of the three peptides are accurate. The vertical axis represents the intensity of the ion peak, and the horizontal axis represents the mass-to-charge ratio. The difference between the measured and theoretical masses of the three peptides is within 10 ppm, and the mass spectrometry results confirm that the sequences are correct.

[0044] Example 2

[0045] This example uses 4 healthy New Zealand rabbits to conduct animal immunization experiments. The experiment was commissioned by Hangzhou Jingjie Biotechnology Co., Ltd. The specific experimental steps are as follows:

[0046] 1. Peptide coupling

[0047] The two modified polypeptides (polypeptide A and polypeptide B) from Example 1 were conjugated to KLH and used for rabbit immunization. The polypeptide conjugation steps were as follows:

[0048] (1) Dissolve 20 mg of SMCC in 2 mL of DMF.

[0049] (2) Add 0.8 mL of KLH to a 25 mL round-bottom flask and add 1× PBS (pH 7.2) to make the final protein concentration 15 mg / mL.

[0050] (3) The dissolved SMCC solution was slowly added dropwise to the 120 mg KLH protein system and stirred at room temperature for 1 h.

[0051] (4) The cell suspension was dialyzed against 1 L of 1×PBS (pH 7.4) at 4°C for 6 h to remove free SMCC.

[0052] (5) Pour the dialyzed KLH protein into a 50 mL centrifuge tube. Determine its volume using the scale on the centrifuge tube. Calculate the concentration of the dialyzed protein based on the amount of KLH protein added before the reaction. Then, transfer 2.5 mg of the KLH-SMCC solution to a 5 mL centrifuge tube based on its concentration.

[0053] (6) Dissolve 3.0 mg of the polypeptide in 0.6 ml of 1×PBS (pH 7.2) solution.

[0054] (7) The peptide solution was added dropwise to the KLH-SMCC tube and mixed using a vertical mixer at room temperature for 4 h.

[0055] 2. Rabbit Immunization

[0056] (1) Prepare immune materials

[0057] The modified peptides conjugated in step 1 were mixed in a 1:1 ratio to obtain the immunogen. The immunogen was diluted 15-fold with saline and then mixed with the corresponding adjuvant (Freund's adjuvant) in a 1:1 ratio. The antigen and adjuvant were completely mixed to form a stable emulsion. The antigen mixture was withdrawn with a syringe and used to immunize rabbits.

[0058] (2) Immunity

[0059] Antigen was injected subcutaneously at two points on both shoulders and at two points in the muscles of both hind legs. Approximately 1 / 4 volume of immunogen was administered to each area. A complex of 0.5 mg of peptide and 0.5 mL of complete Freund's adjuvant (CFA) was used for the first immunization, and a complex of 0.25 mg of peptide and 0.5 mL of incomplete Freund's adjuvant (IFA) was used for subsequent immunizations. Except for the first immunization, the injection volume for all subsequent immunizations was the same. Each rabbit was immunized a total of six times, on days 1, 7, 14, 21, 28, and 40.

[0060] (3) Blood collection

[0061] First blood draw: On day 28, 2 mL of blood was collected and centrifuged. The supernatant was collected and sent to the laboratory for serum screening tests, including ELISA or Western blot.

[0062] Blood was drawn three times (2nd, 3rd, and 4th times): on the 35th, 47th, and 52nd day, respectively, with 30 mL of blood drawn each time. After centrifugation, the supernatant was collected and sent to the laboratory for serum screening tests, including ELISA or Western blot.

[0063] Rabbits immunized with WSTF-K181me3 peptide A were designated R1 and R2, and rabbits immunized with WSTF-K181me3 peptide B were designated R3 and R4. The results of the initial ELISA test on serum collected on day 28 are shown in Table 2. The titers of R1, R2, R3, and R4 recognizing the modified peptide were all greater than or equal to 1:16K (OD>1).

[0064] Table 2 Serum ELISA test results

[0065]

[0066] Endogenous WSTF-K181me3 protein was detected by WB in the sera of immunized rabbits. Figure 4 The WB results showed that R3 and R4 could detect a band with a theoretical molecular weight of about 170 kDa for the target protein.

[0067] Based on the test results of ELISA and WB, the sera of four rabbits, R1, R2, R3 and R4, were selected for purification.

[0068] 3. Antibody Purification

[0069] (1) Prepare protein A affinity column:

[0070] Usually, 5 mL or 10 mL of protein A filler is selected, and equal volumes of filler and PBS buffer solution are mixed and stirred, and air is pumped out to remove bubbles in the filler.

[0071] Prepare the column by slowly adding Protein A filler to the glass column. Be careful not to dry out the column. After filling, equilibrate the column with 10 volumes of pre-chilled PBS buffer.

[0072] (2) Protein A affinity chromatography:

[0073] After filtering the serum with a filter, load it onto a well-equilibrated protein A chromatography column. In order to detect the binding efficiency of the antiserum to the filler, the load effluent needs to be retained.

[0074] The column was washed with PBS buffer solution and then eluted with 150 mM glycine buffer solution. The eluate was collected and adjusted to pH 7 by adding neutralization buffer solution.

[0075] (3) Enrichment of target antibodies:

[0076] The crude IgG obtained after protein A purification is loaded onto a well-balanced antigen polypeptide affinity chromatography column to specifically enrich the target antibody.

[0077] (4) Removal of non-specific antibodies:

[0078] The target antibody obtained in the previous step is loaded onto a non-modified affinity chromatography column, and the flow-through (FT3) is directly collected to remove non-specific antibody components.

[0079] (5) Antibody preservation:

[0080] Determine the protein content. Add 10% glycerol to preserve the antibody. The purified antibody is divided and stored at -20°C. The specific purification process is shown in Figure 5 .

[0081] Example 3

[0082] Sufficient rabbit serum was collected and affinity purified using Protein A and the immunogen polypeptide column according to the steps in Example 2. The purified antibodies from R1, R2, R3, and R4 were labeled Ab1, Ab2, Ab3, and Ab4, respectively. The prepared purified polyclonal antibodies against WSTF-K181me3 were assayed by ELISA, Dotblot, and Western blot, respectively.

[0083] 1. ELISA test:

[0084] (1) Antigen coating:

[0085] Dilute the antigen with coating solution (1×PBS) and add it to the ELISA plate in the amount of 50 μg / well. Incubate in a refrigerator at 4°C overnight or in a 37°C oven for 2 hours.

[0086] (2) Washing plate:

[0087] The ELISA plate coated the day before was taken out and washed three times with 1×TBST.

[0088] (3) Closure:

[0089] The cleaned ELISA plate was added with 1% BSA blocking solution, incubated at 37°C for 1 hour, and then washed 1-3 times.

[0090] (4) Primary antibody incubation:

[0091] Prepare a 3-fold serial dilution of the antibody starting at 1:1K. Adjust the dilution volume based on the actual situation. Add the antibody to the ELISA plate in sequence, incubate at 37°C for 1.5 hours, and then wash 1-3 times.

[0092] (5) Secondary antibody incubation:

[0093] 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.

[0094] (6) Color development:

[0095] In a 96-well microtiter plate coated with antigen-modified peptide and control peptide, antibodies are added at different dilution ratios for incubation (e.g. 1:54K, 1:162K). Then, enzyme-labeled secondary antibodies and TMB substrates are applied to detect the binding of peptide and antibody. Figure 6 The ELISA test results showed that Ab2, Ab3, and Ab4 had recognition abilities for at least one modified peptide at a dilution of no less than 1:50K, and did not recognize the non-modified negative peptide.

[0096] 2. Dotblot detection:

[0097] (1) Sample:

[0098] Uncross-linked antigen polypeptides were spotted onto the PVDF membrane according to a gradient of 1 ng, 4 ng, 16 ng, and 64 ng.

[0099] (2) Closed:

[0100] After the membrane surface is dry, add blocking solution and block at room temperature for 60 minutes.

[0101] (3) Washing:

[0102] Wash with 1× TBST for 10 min.

[0103] (4) Primary antibody incubation:

[0104] The antibody was diluted with 2.5-5% skim milk powder, incubated at room temperature for 2 h, and then washed three times with 1×TBST, each time for 5-10 min.

[0105] (5) Secondary antibody incubation:

[0106] 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 45 minutes to 1 hour, and then wash three times with 1×TBST, each time for 5-10 minutes.

[0107] (6) Add a color developing substrate to the washed membrane and then expose it to light.

[0108] Different doses (such as 4ng, 16ng) of modified peptide and non-modified peptide are fixed on the solid phase membrane, incubated with antibodies, and then enzyme-labeled secondary antibodies and chemiluminescent substrates are added to detect the binding of peptide and antibody. Figure 7 The Dotblot results showed that Ab3 and Ab4 had almost no binding to the negative non-modified peptide and had strong binding to the positive peptide.

[0109] 3. Western blot detection:

[0110] (1) Lysis of cells:

[0111] Select cells or tissues for testing 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.

[0112] (2) Protein electrophoresis and membrane transfer:

[0113] Protein electrophoresis: Select a separating gel with the appropriate concentration based on the molecular weight of your target protein. Add 10% ammonium persulfate and TEMED at the end of the gel preparation, with a volume of 4.7 mL per plate. Load 20–40 μg of sample per well and perform electrophoresis. Electrophoresis conditions: stacking gel at 80 V, separating gel at 120 V.

[0114] Wet transfer: Cool the transfer solution before transferring. Place the gel, membrane, and filter paper in the transfer solution to form a sandwich structure to avoid air bubbles. The transfer voltage is 80V to 120V.

[0115] (3) Closure:

[0116] Add blocking solution to the transferred membrane and block at room temperature for 60 minutes. After incubation, wash with 1× TBST for 10 minutes.

[0117] (4) Primary antibody incubation:

[0118] The antibody was diluted with 2.5-5% skim milk powder, incubated at room temperature for 2 h, and then washed three times with 1×TBST, each time for 5-10 min.

[0119] (5) Secondary antibody incubation:

[0120] 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 45 minutes to 1 hour, and then wash three times with 1×TBST, each time for 5 to 10 minutes.

[0121] (6) Add a color developing substrate to the washed membrane and then expose it to light.

[0122] The positive antibodies Ab3 and Ab4 in Dot / ELISA results were detected by Western Blot using HeLa and MCF-7 cell lysates. Figure 8 Western Blot test results show that Ab4 can detect a band of about 170 kDa in HeLa lysate, which is the target protein WSTF-K181me3.

[0123] Example 4

[0124] This example examined the methylation modification at the K181 site of the purified WSTF-K181me3 polyclonal antibody prepared in Example 2. This example validated the specificity and efficacy of this antibody using an EZH2 knockdown model, combined with Western blot analysis. This study not only validated the antibody's performance but also explored the potential role of methylation modification at the WSTF K181 site in tumor cells. The experimental procedure is as follows:

[0125] 1. Establishment of stable cell lines and tumorigenesis experiments:

[0126] (1) HCC38 breast cancer cells were transfected with a control plasmid (sh-scr) and a knockdown plasmid targeting EZH2 (shEZH2), stable cell lines were constructed through drug screening, and the cells were injected into mice to form tumors.

[0127] 2. Western blot detection:

[0128] Total cellular proteins were extracted from nude mouse tumors and analyzed by Western blot using the following antibodies:

[0129] (1) WSTF and WSTF S158p were used to detect the total expression level and activity of WSTF.

[0130] (2) H3 and H3K27me3 were used as references for the knockdown effect of EZH2.

[0131] (3) WSTF-K181me3 newly prepared antibody, used to detect the methylation level of WSTF K181 site.

[0132] 3. Result analysis, such as Figure 9 As shown:

[0133] (1) EZH2 knockdown validation:

[0134] The H3K27me3 level was significantly decreased in the EZH2 knockdown group, indicating successful knockdown.

[0135] (2) Detection of WSTF activity and expression:

[0136] WSTF total protein levels and WSTF S158p activity markers showed that EZH2 knockdown inhibited WSTF activity. EZH2 knockdown also affected WSTF methylation modification at this site, suggesting that this modification is involved in the regulation of WSTF protein function.

[0137] (3) Validation of WSTF-K181me3 Antibody:

[0138] Detection with the WSTF-K181me3 antibody revealed a significant decrease in trimethylation levels at the WSTF K181 site in the EZH2 knockdown group. This suggests that EZH2 can inhibit WSTF K181 function by regulating its methylation. Methylation, as an important epigenetic regulatory factor, plays a role in the regulation of tumor cells. EZH2, as an epigenetic regulator, influences WSTF K181 methylation levels, suggesting that this WSTF modification is associated with the development and progression of breast cancer.

[0139] The above results indicate that, through detection of the prepared WSTF-K181me3 antibody, EZH2 knockdown significantly reduced methylation modification at the WSTF K181 site. This provides a new research direction for the function of WSTF in tumors and demonstrates the high specificity of the antibody in detecting methylation modification at the WSTF K181 site, providing strong support for the antibody's patent application.

[0140] The above is only a preferred embodiment 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 within the scope of protection of the present invention.

Claims

1. Application of WSTF-K181me3 in the preparation of tumor risk assessment products.

2. Application of WSTF-K181me3 in the preparation of tumor detection products.

3. The use according to claim 1 or 2, characterized in that The tumor is human breast ductal carcinoma.

4. The use according to claim 1 or 2, characterized in that The products include antibodies, drugs and kits.

5. Preparation of immunogenic polypeptides for WSTF-K181me3 polyclonal antibodies, characterized in that: The polypeptides include polypeptide A with a sequence of KETVV-Tri-Methyl K-EDEGRRE and polypeptide B with a sequence of ETVV-Tri-Methyl K-EDEGRRES.

6. A polyclonal antibody against WSTF-K175me3, characterized in that The polyclonal antibody is obtained by immunizing an animal with a mixture of the immunogenic polypeptides according to claim 5.

7. The polyclonal antibody according to claim 6, characterized in that Peptide A and polypeptide B were coupled with KLH respectively and then mixed for immunization.

8. The polyclonal antibody according to claim 6, characterized in that The coupled polypeptide A solution and the coupled polypeptide B solution are mixed in a volume ratio of 1-2:1-2.

9. The method for preparing a polyclonal antibody according to any one of claims 6 to 8, characterized in that: The method comprises the following steps: coupling the immunogenic polypeptide according to claim 5 with KLH respectively, mixing the coupled polypeptide solutions in a volume ratio of 1-2:1-2 and then using them for animal immunization, collecting the serum of the immunized animals and purifying them to obtain a polyclonal antibody against WSTF-K181me3.

10. The preparation method according to claim 9, characterized in that The animal is immunized 3 to 5 times.

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