Specific anti-bridge molecule ITSN1-S protein 315 serine phosphorylation antibody and preparation method and application thereof

Abstract

The present invention discloses a specific anti-bridge molecule ITSN1-S protein 315 serine phosphorylation antibody and a preparation method and application thereof. The preparation method comprises the following steps: adding ITSN1-S-S315 phosphorylated polypeptide to activated VLP to obtain ITSN1-S-S315 phosphorylated antigen, emulsifying ITSN1-S-S315 phosphorylated antigen, immunizing animals, and isolating specific anti-ITSN1-S protein 315 serine phosphorylation antibody from the immunized animals. It is helpful to explore the mechanism of action of post-translational modification of ITSN1-S protein in tumor proliferation, migration and invasion, and provides potential action targets for the diagnosis and treatment of clinical tumor diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a specific anti-bridge molecule ITSN1-S protein 315 serine phosphorylation antibody and a preparation method and application thereof. Background Art

[0002] Intersectin 1 (ITSN1) is a highly conserved protein in the evolutionary process. There are two splice forms in the human body: ITSN1-L and ITSN1-S. The two splice forms have multiple common protein domains. The long isoform ITSN1-L is mainly expressed in neurons, participates in the endocytosis and exocytosis of neurons and the synthesis and release of neurotransmitters, and is associated with a variety of neurodegenerative diseases; while the short isoform ITSN1-S is widely distributed in various types of tissues and cells, and can participate in physiological activities such as endocytosis, exocytosis, and cell proliferation through its various domains.

[0003] Therefore, the preparation of specific antibodies against serine phosphorylation at position 315 of the bridge molecule ITSN1-S protein is of great significance for in-depth research on the regulation of its phosphorylation status on the function of ITSN1-S itself and various physiological activities. Summary of the invention

[0004] The purpose of the present invention is to effectively study the mechanism of action of the bridge molecule ITSN1-S protein in malignant tumors, and to provide a specific anti-bridge molecule ITSN1-S protein 315 site serine phosphorylation antibody and a preparation method thereof.

[0005] The technical solution adopted to achieve the purpose of the present invention is:

[0006] An ITSN1-S-S315 phosphorylated polypeptide having the amino acid sequence of SEQ ID No. 1 Ac-Arg-Val-Arg-Ser-Gly-Ser-Gly-Ile-Ser-Val-Ile-Ser-Ser-Thr-Cys-NH 2 , wherein the sixth serine from the Ac end is phosphorylated to form pSer, and its structural formula is Ac-Arg-Val-Arg-Ser-Gly-pSer-Gly-Ile-Ser-Val-Ile-Ser-Ser-Thr-Cys-NH 2 .

[0007] Another aspect of the present invention also includes an ITSN1-S-S315 phosphorylated antigen, which is formed by cross-linking the ITSN1-S-S315 phosphorylated polypeptide with virus-like particles VLP.

[0008] Another aspect of the present invention also includes a method for preparing the ITSN1-S-S315 phosphorylated antigen, comprising the following steps:

[0009] Step 1, using the peptide sequence to perform peptide synthesis to obtain ITSN1-S-S315 phosphorylated peptide;

[0010] Step 2, activating VLPs with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (Sulfo-SMCC), removing excess Sulfo-SMCC, and obtaining activated VLPs;

[0011] Step 3, dissolving the ITSN1-S-S315 phosphorylated polypeptide in HS-A, adding it to the activated VLP obtained in step 2, and standing it at room temperature in the dark to obtain the ITSN1-S-S315 phosphorylated antigen.

[0012] In the above technical solution, in step 3, the mass ratio of the activated VLP to the ITSN1-S-S315 phosphorylated polypeptide is 1:1.

[0013] Another aspect of the present invention also includes a method for preparing a specific anti-ITSN1-S protein serine phosphorylation 315 antibody, comprising the following steps: emulsifying the ITSN1-S-S315 phosphorylated antigen, immunizing an animal, and isolating the specific anti-ITSN1-S protein serine phosphorylation 315 antibody from the immunized animal.

[0014] In the above technical scheme, specific anti-ITSN1-S protein serine phosphorylation 315 antibody is isolated from the immunized animal, specifically, serum containing specific anti-ITSN1-S protein serine phosphorylation 315 antibody is isolated from the immunized animal.

[0015] The above technical solution also includes a purification step, specifically, the serum is purified by protein A affinity purification, phosphorylated polypeptide affinity purification, and non-phosphorylated polypeptide adsorption purification in sequence to obtain a specific anti-ITSN1-S protein 315 serine phosphorylated antibody.

[0016] In the above technical scheme, the animal immunization comprises the following steps: in the first week, immunizing rabbits once with a mixture of 500 μg antigen and an equal volume of Freund's complete adjuvant, and immunizing rabbits once with a mixture of 500 μg antigen and an equal volume of Freund's incomplete adjuvant in the third, fifth and seventh weeks, respectively; and one week after the last immunization, isolating specific anti-ITSN1-S protein 315 serine phosphorylated antibodies from the immunized rabbits.

[0017] In the above technical scheme, the emulsification method is: the ITSN1-S-S315 phosphorylated antigen is uniformly mixed with Freund's complete adjuvant or Freund's incomplete adjuvant in a volume ratio of 1:1 to prepare a mixture of ITSN1-S-S315 phosphorylated antigen and Freund's complete adjuvant and a mixture of antigen and Freund's incomplete adjuvant, respectively, and shaken and ultrasonicated at 4°C for 2 hours to form oil-in-water droplets.

[0018] In the above technical solution, the purification steps are as follows:

[0019] Protein A affinity purification: The protein A affinity chromatography column is washed with ultrapure water, then balanced with a buffer, and serum is loaded onto the balanced protein A affinity chromatography column; the protein A affinity chromatography column is eluted with an eluent; the protein A affinity chromatography column is eluted with an eluent, the effluent is collected, and the elution peak of protein A purification is obtained by neutralization with Tris;

[0020] Phosphorylated polypeptide affinity purification: The phosphorylated polypeptide affinity chromatography column is washed with ultrapure water, and then balanced with a buffer solution, and the elution peak purified by protein A is loaded onto the balanced phosphorylated polypeptide affinity chromatography column; the phosphorylated polypeptide affinity chromatography column is eluted with an eluent; the phosphorylated polypeptide affinity chromatography column is eluted with an eluent, the effluent is collected, and the elution peak of the phosphorylated polypeptide affinity purification is obtained by neutralization with Tris;

[0021] Non-phosphorylated polypeptide affinity purification: the non-phosphorylated polypeptide affinity chromatography column was washed with ultrapure water, and then balanced with buffer, and the elution peak purified by protein A was loaded onto the balanced non-phosphorylated polypeptide affinity chromatography column; the non-phosphorylated polypeptide affinity chromatography column was eluted with eluent; the non-phosphorylated polypeptide affinity chromatography column was eluted with eluent, the effluent was collected, and neutralized with Tris to obtain the elution peak containing the specific anti-ITSN1-S protein 315 serine phosphorylated antibody.

[0022] Another aspect of the present invention also includes a specific anti-ITSN1-S protein 315 serine phosphorylation antibody obtained by the preparation method.

[0023] Another aspect of the present invention also includes the use of the specific anti-ITSN1-S protein 315 serine phosphorylation antibody in the preparation of an anticancer drug. Preferably, the anticancer drug is an anti-breast cancer drug.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The diversity of ITSN1-S protein functions depends on the post-translational modification of its own protein, such as phosphorylation. The anti-bridge molecule ITSN1-S protein S315 phosphorylation antibody prepared by the present invention helps to explore the mechanism of post-translational modification of ITSN1-S protein in tumor proliferation, migration and invasion, and provides potential targets for the diagnosis and treatment of clinical tumor diseases.

[0026] 2. In practical applications, the antibodies provided by the present invention can specifically detect the phosphorylation modification of human ITSN1-S protein at the cellular and tissue levels.

[0027] 3. The present invention is a phosphorylated polyclonal antibody prepared against the specific 315 site of human ITSN1-S protein, which is helpful for studying the cell signal transduction pathways and interaction networks of various biological functions involved in ITSN1-S protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Electrophoresis results of the purified p-ITSN1-S-S315 antibody provided by the present invention

[0029] Figure 2 The p-ITSN1-S-S315 antibody Dot blot detection results provided by the present invention.

[0030] Figure 3 Western blot was performed using the p-ITSN1-S-S315 antibody provided by the present invention to detect the expression of exogenous S315 phosphorylated ITSN1-S-protein in human breast cancer cell lines

[0031] Figure 4 Western blot was performed using the p-ITSN1-S-S315 antibody provided by the present invention to detect the expression of ITSN1-S-protein phosphorylated at S315 in human breast invasive ductal carcinoma tissue

[0032] Figure 5 The p-ITSN1-S-S315 antibody provided by the present invention is used to perform immunohistochemistry to detect the expression of ITSN1-S-protein phosphorylated at S315 in human breast invasive ductal carcinoma tissue DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] Example 1

[0035] An ITSN1-S-S315 non-phosphorylated polypeptide having the amino acid sequence of SEQ ID No. 1 Ac-Arg-Val-Arg-Ser-Gly-Ser-Gly-Ile-Ser-Val-Ile-Ser-Ser-Thr-Cys-NH 2 .

[0036] An ITSN1-S-S315 phosphorylated polypeptide, wherein the sixth serine from the Ac end in the ITSN1-S-S315 non-phosphorylated polypeptide is phosphorylated, i.e., it carries a phosphate group (pSer), and its structural formula is Ac-Arg-Val-Arg-Ser-Gly-pSer-Gly-Ile-Ser-Val-Ile-Ser-Ser-Thr-Cys-NH 2 .

[0037] An ITSN1-S-S315 phosphorylated antigen, wherein the ITSN1-S-S315 phosphorylated polypeptide is cross-linked with a virus-like particle (VLP), and the preparation is specifically formed by the following steps:

[0038] Step 1, using the peptide sequence to synthesize peptides to obtain Ac-Arg-Val-Arg-Ser-Gly-pSer-Gly-Ile-Ser-Val-Ile-Ser-Ser-Thr-Cys-NH 2 , recorded as ITSN1-S-S315 phosphorylated polypeptide;

[0039] Step 2, take 7.2 mg of VLP, filter with 0.45 μm filter membrane, measure UV280, and calculate the amount of VLP16;

[0040] Step 3, weigh 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (Sulfo-SMCC) according to 1 mg SMCC / 5 mg VLP, dissolve it in water to a concentration of 4 mg / mL, add it to the VLP solution, and stand it at room temperature in the dark for 2 hours to obtain activated VLP;

[0041] Step 4, filter the activated VLPs through 0.2 μm and desalt them to remove excess Sulfo-SMCC, and measure the UV value;

[0042] Step 5, 6 mg of ITSN1-S-S315 phosphorylated polypeptide obtained in step 1 was dissolved in 0.6 mL HS-A (4 μL was taken out for Dot blot detection), and then added to about 6 mg of activated VLPs, and allowed to stand at room temperature in the dark for 4 h;

[0043] Step 6, after the reaction, the thiol group is determined as needed, and the absorbance result is determined.

[0044] Example 2

[0045] A method for preparing a specific anti-ITSN1-S protein serine phosphorylation antibody at position 315 (referred to as p-ITSN1-S-S315 antibody) is carried out according to the following steps:

[0046] Step 1, preparing ITSN1-S-S315 phosphorylated antigen according to the method of Example 1;

[0047] Step 2: Antigen emulsification

[0048] The ITSN1-S-S315 phosphorylated antigen prepared in step 1 was uniformly mixed with Freund's complete adjuvant and Freund's incomplete adjuvant in a volume ratio of 1:1 to prepare a mixture of antigen and Freund's complete adjuvant and a mixture of antigen and Freund's incomplete adjuvant, respectively. The mixtures were shaken and sonicated at 4°C for 2 hours to form oil-in-water droplets, and the Freund's adjuvant was added dropwise to the antigen during the operation.

[0049] Step 3: Animal immunization

[0050] Healthy male New Zealand white rabbits of about 6 weeks old were selected and kept in an animal room for one week to adapt them to the environment.

[0051] Before the experiment, 1.5 ml of blood was collected from the rabbit's ear vein as pre-immune serum.

[0052] In the first week, 500 μg of antigen mixed with an equal volume of Freund's complete adjuvant was injected at multiple points on the back of the rabbits.

[0053] In the third week, 500 μg of antigen mixed with an equal volume of Freund's incomplete adjuvant was injected at multiple points on the back of the rabbits.

[0054] In the fifth week, 500 μg of antigen mixed with an equal volume of Freund's incomplete adjuvant was injected at multiple points on the back of the rabbits.

[0055] In the seventh week, 500 μg of antigen mixed with an equal volume of Freund's incomplete adjuvant was injected at multiple points on the back of the rabbits.

[0056] In the eighth week, blood was drawn from the ear vein of rabbits to prepare serum (containing p-ITSN1-S-S315 antibody)

[0057] Step 4: Blood collection and titer testing

[0058] One week after the last immunization, 3-4 mL of blood was collected from the rabbit's ear vein, allowed to stand overnight at 4°C, and then centrifuged at 4000 rpm and 4°C for 15 min to separate the upper serum for testing.

[0059] The serum titer was tested by indirect ELISA, i.e., the binding of antiserum to antigen was tested. The titer qualification standard was (antiserum absorbance value - blank absorbance value) / (pre-immune negative control serum absorbance value - blank absorbance value)>2.1, which can be used for serum purification to prepare p-ITSN1-S-S315 antibody.

[0060] Step 5: Purification of p-ITSN1-S-S315 Antibody

[0061] The collected rabbit blood was centrifuged using a desktop centrifuge at 4200 rpm for 30 min, and the serum was collected after centrifugation. The serum was filtered using a 0.45 μM filter membrane for later use.

[0062] Rabbit serum was purified by protein A affinity purification, phosphorylated peptide affinity purification, and non-phosphorylated peptide adsorption purification in sequence to harvest p-ITSN1-S-S315 antibody. The operation steps are as follows:

[0063] Protein A affinity purification

[0064] (1) Water balance: Wash 3 column volumes (CV) with ultrapure water to replace the 25% ethanol storage solution;

[0065] (2) Equilibration of protein A chromatography column: 5-10 CV with eluent (50 mM Tris, 100 mM NaCl, pH 8.0);

[0066] (3) Sample loading: The centrifuged and filtered serum is loaded;

[0067] (4) Elution: eluent (50 mM Tris, 100 mM NaCl, pH 8.0) for 5-10 CV;

[0068] (5) Elution: elution using an elution buffer (100 mM Glycine, 10 mM NaCl, pH 3.0);

[0069] (6) Neutralization: Add 2 M Tris, pH 8.0 to neutralize the eluted antibody to obtain the elution peak of Protein A purification;

[0070] (7) Equilibration: Equilibrate the column with eluent (50 mM Tris, 100 mM NaCl, pH 8.0) for 3 CV until neutral;

[0071] (8) CIP cleaning: CIP cleaning column for more than 5CV;

[0072] (9) Alkali flushing: Wash the column with ultrapure water for 5CV;

[0073] (10) Storage: Equilibrate the column with 25% ethanol for 2CV.

[0074] Phospho-peptide affinity purification

[0075] (1) Water balance: Wash with ultrapure water for 3 CV and replace the 25% ethanol storage solution;

[0076] (2) Equilibrate the phosphorylated polypeptide chromatography column: equilibrate with eluent (50 mM Tris, 100 mM NaCl, pH 8.0) for 5-10 CV;

[0077] (3) Loading: Load the elution peak of Protein A purification;

[0078] (4) Elution: eluent (50 mM Tris, 100 mM NaCl, pH 8.0) for 5-10 CV;

[0079] (5) Elution: elution with elution buffer (100 mM Glycine, 10 mM NaCl, pH 3.0);

[0080] (6) Neutralization: Add 2M Tris (pH 8.0) to neutralize the eluted antibody to obtain the elution peak of the phosphorylated polypeptide affinity purification;

[0081] (7) Equilibration: Equilibrate the column with eluent (50 mM Tris, 100 mM NaCl, pH 8.0) for 5-10 CV until neutral;

[0082] (8) Water washing: Wash the column with ultrapure water for 5-10 CV;

[0083] (9) Storage: Equilibrate the column with 25% ethanol for 2CV.

[0084] Non-phosphorylated peptide adsorption purification

[0085] (1) Water balance: Wash with ultrapure water for 3 CV and replace the 25% ethanol storage solution;

[0086] (2) Equilibrate the non-phosphorylated peptide chromatography column: equilibrate with eluent (50 mM Tris, 100 mM NaCl, pH 8.0) for 5-10 CV;

[0087] (3) Loading: Load the elution peak of the affinity purified phosphorylated polypeptide;

[0088] (4) Elution: eluent (50 mM Tris, 100 mM NaCl, pH 8.0) for 5-10 CV;

[0089] (5) Elution: elution using an elution buffer (100 mM Glycine, 10 mM NaCl, pH 3.0);

[0090] (6) Neutralization: adding 2 M Tris, pH 8.0 to neutralize the eluted antibody to obtain an elution peak containing p-ITSN1-S-S315 antibody (purified antibody sample);

[0091] (7) Equilibration: Eluent (50 mM Tris, 100 mM NaCl, pH 8.0) was equilibrated for 5-10 CV to neutrality;

[0092] (8) Water washing: Wash the column with ultrapure water for 5-10 CV;

[0093] (9) Storage: Equilibrate the column with 25% ethanol for 2CV.

[0094] Step 6: Antibody Identification

[0095] Antibody concentration and purity detection

[0096] Start the micro-spectrophotometer, spot the sample with the corresponding buffer of the test sample, and when the absorbance value is between ±0.015 at a wavelength of 280 nm, it indicates that the instrument baseline is stable. Spot the purified antibody samples obtained in step 5 in turn, record the absorbance value, and divide the test data by the IgG extinction coefficient (1.414). The resulting value is the concentration of the test sample (in mg / mL).

[0097] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify the purity of the purified antibody sample obtained in step 5. This method separates the protein in the test sample in the electrophoresis gel according to the different molecular weights, thereby verifying the purity of the target antibody. Take 5μg of the test sample and add 5μL4R / 4N, heat it in a 100℃ water bath, and then centrifuge it at 10000rpm. Add enough 1×SDS electrode buffer to the electrophoresis tank, and use a micropipette to add the treated sample solution and protein molecular weight standard to the sample well. Connect the power supply, first electrophoresed at a constant voltage of 100V until the bromophenol blue dye enters the separation gel from the concentrated gel, then adjust the current to 140V and continue electrophoresis to the bottom of the gel plate, and turn off the power. Soak the gel in the staining solution, heat it in a microwave for 60s, and stain it at room temperature on a gently shaking platform. Replace the destaining solution to cover the gel, heat it in a microwave for 60 seconds, destain it at room temperature on a gently shaking platform, and repeat the destaining operation until a blue band and a clean background are obtained.

[0098] Purity was tested by SDS-PAGE. The purity of the purified antibody sample obtained in step 5 after purification reached 92.9%. The electrophoresis results of the purified antibody are shown in the attached figure. Figure 1 shown.

[0099] ElISA identification

[0100] The purified antibodies were subjected to ELISA binding detection and ELISA cross detection, and the results are shown in Tables 1 and 2. The results showed that the purified antibodies bound to the phosphorylated peptides, and the cross-reaction rate with the non-phosphorylated peptides was less than 10%, and the antibody specificity was high.

[0101] Table 1

[0102]

[0103] Table 2

[0104]

[0105] Example 3

[0106] In this example, the p-ITSN1-S-S315 antibody obtained in Example 2 was used to perform molecular biological experimental detection.

[0107] Dot blot test results are as attached Figure 2 As shown, 1ng, 5ng, 25ng, and 50ng of ITSN1-S-S315 non-phosphorylated peptide and ITSN1-S-S315 phosphorylated peptide were spotted on the nitrocellulose membrane in sequence, and then p-ITSN1-S-S315 antibody was added for hybridization detection. The results showed that p-ITSN1-S-S315 antibody could specifically recognize ITSN1-S-S315 phosphorylated peptide, while ITSN1-S-S315 non-phosphorylated peptide could not be recognized by the antibody.

[0108] Western blot test results of cell samples are shown in the attached Figure 3As shown, the ITSN1-S-S315 protein (wild-type protein ITSN1-S-WT) was mutated into the expression plasmid of ITSN1-S-315A (non-phosphorylated mutant protein ITSN1-S-315A) or ITSN1-S-315D (phosphorylated mutant protein ITSN1-S-315D) by point mutation technology, and the breast cancer cells with ITSN1 gene knockout were infected with exogenous HA fusion protein virus (WT, 315A, 315D). The cells were collected and lysed for protein electrophoresis and western blot hybridization experiments. Specific anti-actin and anti-HA antibodies were used as controls to detect endogenous actin and exogenous HA fusion protein in the samples. Then p-ITSN1-S-S315 antibody was used for hybridization detection. The results showed that the exogenously expressed wild-type ITSN1-S-WT-HA could be recognized by the p-ITSN1-S-S315 antibody, whereas the exogenously expressed non-phosphorylated mutant protein ITSN1-S-315A and the pseudo-phosphorylated mutant protein ITSN1-S-315D could not be recognized by the antibody due to changes in the amino acid sequence. The results showed that the p-ITSN1-S-S315 antibody could recognize ITSN1-S protein phosphorylated at serine 315 in cells.

[0109] The results of Western blot test of tissue samples are as attached. Figure 4 As shown in the figure, neutral tissue lysates from 11 patients with invasive ductal breast carcinoma were collected for protein electrophoresis and western blot hybridization experiments, and specific anti-actin and anti-ITSN1-S antibodies were used as controls to detect endogenous actin and ITSN1-S proteins in the samples. Then, p-ITSN1-S-S315 antibody was used for hybridization detection. The results showed that p-ITSN1-S-S315 antibody could recognize ITSN1-S protein phosphorylated at serine 315 in breast cancer tissues.

[0110] The results of immunohistochemistry are shown in the attached Figure 5 As shown in the figure, paraffin specimens of 5 breast cancer patients with invasive ductal carcinoma of the breast were collected, and immunohistochemistry experiments were performed using p-ITSN1-S-S315 antibody. The results showed that the ITSN1-S protein phosphorylated at serine 315 was located in the cytoplasm, and there was no nonspecific staining, which fully demonstrated that the antibody can recognize the ITSN1-S protein phosphorylated at serine 315 in a certain degree of spatial structure, and can specifically and accurately detect the expression of ITSN1-S protein phosphorylated at serine 315 in tissues.

[0111] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An ITSN1-S-S315 phosphorylated polypeptide, characterized in that It has the amino acid sequence of SEQ ID No. 1, Ac-Arg-Val-Arg-Ser-Gly-Ser-Gly-Ile-Ser-Val-Ile-Ser-Ser-Thr-Cys-NH2, wherein the sixth serine from the Ac end is phosphorylated to form pSer.

2. An ITSN1-S-S315 phosphorylated antigen, characterized in that It is formed by cross-linking the ITSN1-S-S315 phosphorylated polypeptide described in claim 1 with virus-like particles VLP.

3. The method for preparing the ITSN1-S-S315 phosphorylated antigen according to claim 2, characterized in that: The following steps are involved: Step 1, using the peptide sequence to perform peptide synthesis to obtain ITSN1-S-S315 phosphorylated peptide; Step 2, activating VLPs with 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (Sulfo-SMCC), removing excess Sulfo-SMCC, and obtaining activated VLPs; Step 3, dissolving the ITSN1-S-S315 phosphorylated polypeptide in HS-A, adding it to the activated VLP obtained in step 2, and standing it at room temperature in the dark to obtain the ITSN1-S-S315 phosphorylated antigen.

4. A method for preparing a specific anti-ITSN1-S protein 315 serine phosphorylation antibody, characterized in that: The following steps are involved: After the ITSN1-S-S315 phosphorylated antigen as claimed in claim 2 is emulsified, an animal is immunized, and a specific anti-ITSN1-S protein 315 serine phosphorylated antibody is isolated from the immunized animal.

5. The preparation method according to claim 4, characterized in that: Specific anti-ITSN1-S protein serine phosphorylation 315 antibody is isolated from the immunized animal, specifically, serum containing specific anti-ITSN1-S protein serine phosphorylation 315 antibody is isolated from the immunized animal.

6. The preparation method according to claim 4, characterized in that: The method also includes a purification step, specifically, the serum is purified by protein A affinity purification, phosphorylated polypeptide affinity purification, and non-phosphorylated polypeptide adsorption purification in sequence to obtain a specific anti-ITSN1-S protein 315 serine phosphorylated antibody.

7. The preparation method according to claim 4, characterized in that: The animal immunization comprises the following steps: in the first week, immunizing the rabbit once with a mixture of 500 μg antigen and an equal volume of Freund's complete adjuvant, and immunizing the rabbit once with a mixture of 500 μg antigen and an equal volume of Freund's incomplete adjuvant in the third week, the fifth week, and the seventh week, and isolating a specific anti-ITSN1-S protein 315 serine phosphorylated antibody from the immunized rabbit one week after the last immunization; The emulsification method comprises: mixing the ITSN1-S-S315 phosphorylated antigen with Freund's complete adjuvant or Freund's incomplete adjuvant in a volume ratio of 1:1, respectively, to prepare a mixture of ITSN1-S-S315 phosphorylated antigen and Freund's complete adjuvant, and a mixture of antigen and Freund's incomplete adjuvant, respectively, and shaking and oscillating for 2 hours at 4° C. and ultrasonicating until oil-in-water droplets are formed.

8. The preparation method according to claim 4, characterized in that: The purification steps are as follows: Protein A affinity purification: The protein A affinity chromatography column is washed with ultrapure water, then balanced with a buffer, and serum is loaded onto the balanced protein A affinity chromatography column; the protein A affinity chromatography column is eluted with an eluent; the protein A affinity chromatography column is eluted with an eluent, the effluent is collected, and the elution peak of protein A purification is obtained by neutralization with Tris; Phosphorylated polypeptide affinity purification: The phosphorylated polypeptide affinity chromatography column is washed with ultrapure water, and then balanced with a buffer solution, and the elution peak purified by protein A is loaded onto the balanced phosphorylated polypeptide affinity chromatography column; the phosphorylated polypeptide affinity chromatography column is eluted with an eluent; the phosphorylated polypeptide affinity chromatography column is eluted with an eluent, the effluent is collected, and the elution peak of the phosphorylated polypeptide affinity purification is obtained by neutralization with Tris; Non-phosphorylated polypeptide affinity purification: the non-phosphorylated polypeptide affinity chromatography column was washed with ultrapure water, and then balanced with buffer, and the elution peak purified by protein A was loaded onto the balanced non-phosphorylated polypeptide affinity chromatography column; the non-phosphorylated polypeptide affinity chromatography column was eluted with eluent; the non-phosphorylated polypeptide affinity chromatography column was eluted with eluent, the effluent was collected, and neutralized with Tris to obtain the elution peak containing the specific anti-ITSN1-S protein 315 serine phosphorylated antibody.

9. A specific anti-ITSN1-S protein serine phosphorylation antibody at position 315 obtained by the preparation method according to claim 4.

10. Use of the specific anti-ITSN1-S protein 315 serine phosphorylation antibody as claimed in claim 9 in the preparation of an anticancer drug, preferably, the anticancer drug is an anti-breast cancer drug.

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