Ripk3 protein phosphorylation antigen peptide, specific antibody and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a Ripk3 protein phosphorylation antigen peptide, a specific antibody and a preparation method and application thereof. It is found for the first time that a lipid can induce phosphorylation modification of a Ripk3 protein Ser176 site (corresponding to a mouse Ser181 site), and the site is a key new target for triggering atherosclerosis inflammation. Based on this, a phosphorylation modification antigen polypeptide for the site is designed and synthesized, and a polyclonal antibody specifically recognizing Ripk3 Ser176 / Ser181 phosphorylation is prepared by immunizing animals with the polypeptide. ELISA detection shows that the antibody can efficiently and specifically recognize the phosphorylation antigen polypeptide and does not recognize a non-phosphorylation control peptide. In addition, it is found that sodium hydrogenated alpogenide can play an anti-inflammatory role by inhibiting Ripk3 Ser176 / Ser181 phosphorylation, and animal experiments prove that it can significantly reduce the aortic plaque area of atherosclerosis model mice, reduce the necrotic core and reduce the inflammation level. The application provides a new target, an antibody tool and a candidate drug for the diagnosis and treatment of atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to Ripk3 protein phosphorylated antigenic peptides, specific antibodies, their preparation methods, and applications. Background Technology

[0002] Atherosclerosis (AS) is a chronic, progressive immune disease caused by the deposition of lipids, cholesterol, and other substances in the inner walls of arteries, forming plaques that thicken and harden the vessel walls, narrow the lumen, and ultimately affect blood flow. It is the main pathological basis for cardiovascular diseases such as coronary heart disease, myocardial infarction, and stroke. The main causes of AS include endothelial damage caused by factors such as hypertension, high cholesterol, smoking, and diabetes; lipid deposition due to the accumulation of lipids such as low-density lipoprotein cholesterol (LDL-C); and inflammatory responses caused by the aggregation of immune cells. In addition, aging, unhealthy diet, smoking, lack of exercise, obesity, and diabetes are also risk factors for AS.

[0003] Although lipid-lowering therapies such as statins, ezetimibe, and PCSK9 inhibitors have significantly reduced the risk of cardiovascular events in recent years, plaque progression and persistent inflammation activation can still be observed in a large number of patients, suggesting significant residual risks beyond lipid factors, with residual inflammation being the most critical. In broad-spectrum anti-inflammatory treatment of ankylosing spondylitis (AS), only a few studies have shown benefits due to limitations such as infection risk and indication selection. Therefore, identifying specific targets that can both explain the persistence of inflammation in the context of lipotoxicity in AS and support early identification and intervention is a key focus of current research in this field.

[0004] Necroptosis, a regulated cell death process with pro-inflammatory properties, plays an increasingly prominent role in atherosclerosis (AS). Receptor-interacting protein kinase 3 (Ripk3) has received considerable attention in recent years. Activated Ripk3, stimulated by necroptosis inducers, drives macrophage necroptosis, constituting the primary cellular source of the necrotic core in atherosclerotic plaques.

[0005] Besides necroptosis, Ripk3 also plays a crucial role in the inflammatory response, promoting the production and release of inflammatory factors such as IL-1β, IL-6, and TNF-α. These inflammatory factors also play a central role in the pathogenesis of atherosclerosis. Although the role of Ripk3 in necroptosis and the inflammatory response has been extensively studied, there are currently no reports of inhibiting Ripk3-induced inflammation to alleviate atherosclerosis. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides Ripk3 protein phosphorylated antigenic peptides, specific antibodies, their preparation methods, and applications.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0008] The first aspect of this invention discloses a phosphorylated modified antigen polypeptide, characterized in that the amino acid sequence of the polypeptide is selected from any of the following: (1) KSQSGTG(pS)GE; (2) TG(pS)GEPGGTC; (3) SG(pS)RDSGGTL-C; (4) GGSSG(pS)RD; Where (pS) represents phosphorylated serine residues.

[0009] A second aspect of the present invention discloses a polyclonal antibody that specifically recognizes phosphorylation sites of the Ripk3 protein, characterized in that the antibody can specifically bind to the phosphorylation-modified antigen polypeptide described above.

[0010] A third aspect of this invention discloses a method for preparing the aforementioned polyclonal antibody, characterized by comprising the following steps: (1) The phosphorylated modified antigen polypeptide of claim 1 is coupled with a carrier protein to prepare an immunogen; (2) Immunize the experimental animals with the immunogen and adjuvant emulsified; (3) Collect antiserum from immunized animals and purify it to obtain the specific polyclonal antibody.

[0011] The fourth aspect of this invention discloses the application of the aforementioned polyclonal antibody in the preparation of reagents or kits for detecting the phosphorylation level of Ripk3 protein in biological samples.

[0012] The fifth aspect of this invention discloses the application of the aforementioned polyclonal antibody in the preparation of products for the auxiliary diagnosis of atherosclerosis.

[0013] Furthermore, the product contains the polyclonal antibody of claim 2, which is used to specifically recognize the phosphorylated Ripk3 protein at the Ser176 or Ser181 site.

[0014] The sixth aspect of this invention discloses the use of phosphorylation inhibitors of the Ripk3 protein at the Ser176 or Ser181 sites in the preparation of medicaments for the treatment of atherosclerosis.

[0015] Furthermore, the phosphorylation inhibitor of the Ripk3 protein at the Ser176 or Ser181 site is GSK872 or fasudil hydrochloride.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] This invention explores how targeted inhibition of Ripk3 phosphorylation in macrophages can exert an anti-inflammatory effect under lipotoxic conditions of atherosclerosis, thereby reducing the degree of atherosclerosis.

[0018] Discovery of a new target: This invention reveals that the Ser176 site of the Ripk3 protein is a key target for alleviating atherosclerosis, providing a new research direction for the development of other Ripk3 inhibitors.

[0019] Targeting the inflammatory response: This invention inhibits the phosphorylation of Ripk3 Ser176 by fasudil hydrochloride, thereby inhibiting the release of downstream inflammatory factors. This mechanism effectively reduces the inflammatory response of the arterial wall and is a powerful complement to traditional lipid-lowering treatments.

[0020] Improvement of atherosclerosis symptoms: In the AAV-PCSK9 atherosclerosis mouse model, treatment with fasudil hydrochloride to inhibit phosphorylation of Ripk3 Ser181 (homogeneous Ser176 site) showed significant improvement in atherosclerosis symptoms, including inhibiting the release of pro-inflammatory factors, reducing the area of ​​plaque necrosis core, enhancing plaque stability, and improving serum lipid levels, indicating that it has a significant effect in slowing down disease progression. Attached Figure Description

[0021] Figure 1 These are the results of mass spectrometry identification of the accuracy of modified antigen peptide sequences; where A is the mass spectrum of the Ripk3 Ser176 phosphorylated modified antigen peptide; and B is the mass spectrum of the Ripk3 Ser181 phosphorylated modified antigen peptide.

[0022] Figure 2Figure 1 shows the results of identifying novel sites and validating the mechanism of lipid-induced Ripk3 phosphorylation. A represents the Western blot results of Ripk3 pathway-related protein expression in the THP-1 human monocyte-macrophage cell line under different treatment conditions; B represents the immunoprecipitation and Western blot results of Ripk3 panphosphorylation levels under PA stimulation; C represents the mass spectrometry identification of secondary fragment ions after phosphorylation modification at the Ripk3 Ser176 site; D represents the Western blot results of p-Ripk3 Ser176-specific expression under PA stimulation; and E represents the immunoprecipitation and Western blot results of Ripk3 panphosphorylation and expression levels after transfection with Ripk3 wild-type (WT) and Ser176A mutant plasmids under PA stimulation.

[0023] Figure 3 This is a graph showing the results of in vitro cell experiments verifying the inhibition of Ripk3 phosphorylation and macrophage polarization by drugs. Specifically, A shows the Western blot results of the effect of GSK872 intervention on p-Ripk3 Ser181 expression in PA-induced RAW264.7 mouse macrophages; B shows a representative scatter plot of CD86 expression in macrophages (BMDMs) of each group detected by flow cytometry; C shows the binding energy score table for FDA-approved drugs based on the crystal structure of Ripk3 protein; D shows the Western blot results of the effect of fasudil hydrochloride intervention on p-Ripk3 Ser181 expression in PA-induced RAW264.7 cell lines; E shows a representative scatter plot of CD86 expression in macrophages (BMDMs) under fasudil hydrochloride intervention detected by flow cytometry; and F shows a statistical graph of the relative expression levels of IL-10, IL-1β, and TNF-α mRNA in macrophages of each group detected by qPCR.

[0024] Figure 4 This is a graph showing the effects of drug intervention on the pathological characteristics of the aorta in a mouse model of atherosclerosis. A shows gross Oil Red O staining photographs of the aorta in each group of mice; B shows a statistical analysis of the percentage of aortic plaque area to the total vascular area; C shows histological photographs of aortic root sections stained with Oil Red O, H&E, and Masson staining in each group of mice; D shows immunofluorescence staining photographs of p-Ripk3 Ser181 expression in the aortic root; E shows a statistical graph of the percentage of Oil Red O positive area in the aortic root; F shows a statistical graph of the percentage of plaque necrosis core area; G shows a statistical graph of the percentage of plaque collagen content; and H shows a statistical graph of the percentage of p-Ripk3 positive area in the aortic root.

[0025] Figure 5This is a graph showing the effects of drug intervention on serum biochemical indicators in a mouse model of atherosclerosis. Among them, A is the serum TNF-α concentration detection graph; B is the serum IL-1β concentration detection graph; C is the serum IL-10 concentration detection graph; D is the serum triglyceride (TG) level detection graph; E is the serum total cholesterol (T-CHO) level detection graph; and F is the serum low-density lipoprotein (LDL) level detection graph. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0027] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0028] Example 1: Preparation of antigenic peptides modified by Ripk3 phosphorylation sites.

[0029] 1. Design of modified antigen peptides.

[0030] This invention experimentally reveals that phosphorylation at the human Ripk3 Ser176 and mouse Ripk3 Ser181 sites is crucial for inflammation in atherosclerosis. Based on the Ripk3 protein sequence and modification type, modified antigen peptides were designed and synthesized.

[0031] Synthesis method: Organic chemical solid-phase synthesis (Fmoc protected amino acids, solid-phase carrier-resin) was adopted. A three-channel automated peptide synthesizer (CS360 model) manufactured by CS Corporation of the United States was used to synthesize peptides from the carboxyl terminus to the amino terminus to obtain peptide resin. Then, the peptides were cleaved from the resin by TFA method to obtain crude product.

[0032] Purification method: The material was separated and purified using a Waters high-performance liquid chromatograph with a C18 reversed-phase column, followed by freeze-drying. (Company: Beijing Biosen)

[0033] Specifically, the following modified antigen peptide was designed targeting the Ripk3 Ser176 site: .

[0034] Specifically, the following modified antigen peptide was designed targeting the Ripk3 Ser181 site: .

[0035] Wherein, (pS) represents phosphorylated serine residues; the cysteine ​​residue (C) at the end of sequence (3) is an amino acid added at the C-terminus to facilitate thiol-maleimide coupling with carrier proteins (such as KLH, BSA). This addition does not change the spatial conformation and immunogenicity of the core phosphorylated epitope and is a conventional coupling strategy in this field.

[0036] Identification of modified antigen peptides. Antigen mass spectrometry analysis confirmed the accuracy of the modified antigen peptide sequences obtained in this invention. Figure 1 As shown.

[0037] Example 2: Preparation of Ripk3 Ser176 specific antibody.

[0038] 1. Antibody preparation.

[0039] The modified antigen peptides, such as modified peptide A and modified peptide B obtained in Example 1, were used as immunogens. After multiple immunizations of four SPF experimental-grade New Zealand white rabbits, a small amount of serum was taken for ELISA detection to preliminarily evaluate the titer and specificity of the antiserum. The specific process is as follows.

[0040] (1) Preparation of immunization materials: Take the conjugated modified peptides A and B and emulsify them with equal amounts of Freund's complete adjuvant (basic immunization) or Freund's incomplete adjuvant (boost immunization), and inject them intradermally at multiple points on the back of the rabbit.

[0041] Peptide fragments (A and B) were conjugated with the carrier protein-hemocyanin (KLH): 10 mg of purified peptides and 15 mg of hemocyanin were reacted under MBS-mediated reaction to obtain peptide-hemocyanin conjugates (immunogens).

[0042] (2) Immunization: Each rabbit was immunized 5 times, on day 1, day 14, day 28, day 42 and day 56 respectively.

[0043] .

[0044] (3) Antibody purification: Antiserum was collected after the third blood collection and purified using Protein A affinity chromatography column to obtain four groups of antibodies AbA-1, AbA-2, AbB-1 and AbB-2.

[0045] 2. Detection of purified antibodies. For ELISA detection, the polypeptide fragment (unconjugated to hemocyanin) was coated onto the enzyme-linked reaction plate. Rabbit antiserum was diluted at 0.2 μg / well to the following concentrations: 1:500; 1:1000; 1:2000; 1:4000; 1:8000; 1:16000; 1:32000. The secondary antibody was horseradish peroxidase-labeled goat anti-rabbit IgG. The chromogenic reagent was tetramethylbenzidine, and the detection wavelength was 450 nm. The measured optical density values ​​are shown in the table below.

[0046] Table 1. ELISA results of antibodies AbA-1 and AbA-2.

[0047] Table 2. ELISA results of antibodies AbB-1 and AbB-2.

[0048] Example 3: Preparation of Ripk3 Ser181 specific antibody.

[0049] 1. Antibody preparation. The modified antigen polypeptide obtained in Example 1, as shown in Ripk3 Ser181, was used as an immunogen. The preparation method was the same as in Example 2. After purification (using a Protein A affinity chromatography column), four groups of antibodies, AbA-3, AbA-4, AbB-3, and AbB-4, were obtained.

[0050] 2. Detection of purified antibodies.

[0051] (1) ELISA detection. The detection method is the same as in Example 2.

[0052] The ELISA results are shown in Tables 3 and 4. In the tables, peptide A is Ripk3 Ser181 peptide A, and peptide B is Ripk3 Ser181 peptide B. The results show that the four antibodies have excellent recognition ability for antigen-modified peptides and do not recognize unmodified negative peptides.

[0053] Table 3. ELISA results of antibodies AbA-3 and AbA-4.

[0054] Table 4. ELISA results for antibodies AbB-3 and AbB-4.

[0055] Example 4: Validation of the effect of targeting and inhibiting phosphorylation of mouse Ripk3 Ser181 (corresponding to human Ser176) site in an atherosclerosis model.

[0056] 1. Identify novel sites and functions of lipid-induced Ripk3 phosphorylation modification.

[0057] We identified that saturated fatty acids, represented by palmitic acid (PA), can induce phosphorylation of the Ripk3 protein at the Ser176 site in the THP-1 human monocyte-macrophage cell line. We prepared antibodies specifically recognizing neophosphorylation modifications in human and mouse Ripk3 and constructed a Ripk3 Ser176A mutant plasmid for subsequent experimental detection. To further verify the role of Ripk3 neophosphorylation activation in inflammation regulation, we examined the expression levels of inflammatory factors such as IL-1β in BMDMs (mouse bone marrow-derived macrophages) stimulated by PA.

[0058] (1) THP-1 cells were treated with palmitic acid (PA, final concentration 0.3 mM) and Z-VAD-FMK alone or in combination. Western blot was used to detect the levels of Ripk3 and p-Ripk3 S227 [p-Ripk3 S227 (CST, #93654, 1:1000); Ripk3 (CST, #95702S, 1:1000)]. This confirmed that Ripk3 can undergo phosphorylation under lipid stress, and that novel phosphorylation sites exist. Specific conditions were: BSA conjugation ratio 2.77:1, treatment for 24 hours. Figure 2 A).

[0059] (2) The Flag-Ripk3 WT plasmid was transfected into the HEK293T cell line and the cells were stimulated with PA. The changes in Ripk3 panphosphorylation were detected by IP assay. Combined with the results of phosphorylation modification mass spectrometry, human Ser176 (corresponding to mouse Ser181) was screened as a potential modification site. Figure 2 BC). p-Ripk3 Ser176 antibody was prepared and Western blot experiments were performed under PA stimulation. Figure 2 D); In the HEK293T cell line, Flag-Ripk3 WT plasmid and Flag-Ripk3 Ser176A plasmid were transfected. IP assays were performed to detect changes in Ripk3 panphosphorylation, confirming that Ser176 is a novel site for Ripk3 phosphorylation activation under lipid stress. Figure 2 E).

[0060] 2. Drug screening and in vitro efficacy validation based on the phosphorylation target of Ripk3 Ser181.

[0061] GSK872 has been reported as an effective phosphorylation inhibitor of Ripk3 at the Ser227 site. In our experiments, we found that GSK872 also significantly inhibited PA-induced activation of Ripk3 at the Ser181 site (corresponding to the human Ser176 site) in the RAW264.7 cell line. Furthermore, in mouse BMDMs, GSK872 treatment reduced the expression of the PA-induced pro-inflammatory macrophage marker CD86, suggesting that GSK872 can effectively inhibit Ripk3 phosphorylation and exert an anti-inflammatory effect. Based on this, we selected fasudil hydrochloride from the FDA's drug library as a potential treatment for AS, and experimentally confirmed that fasudil hydrochloride can significantly inhibit PA-stimulated Ripk3 Ser181 phosphorylation in the RAW264.7 cell line and exert an anti-inflammatory effect.

[0062] 2.1 GSK872 validates the effectiveness of Ripk3 Ser181 phosphorylation as an anti-inflammatory target.

[0063] WT mice were treated with BMDMs of GSK872 (final concentration of GSK872 2.5 μM, treated for 24 hours). Western blot analysis of p-Ripk3 Ser181 levels was performed under PA stimulation. The results showed that GSK872 significantly inhibited PA-induced Ripk3 Ser181 phosphorylation. WT mice were treated with GSK872 (BMDMs). Under PA stimulation, flow cytometry [F-4 / 80 (#123108, Biolegend) APC-CD86 (#105012), antibody dilution (1:100), 4℃, 30 min surface staining, protected from light throughout, washed once with 1×PBS, 300g, 5 min, instrument BD Accuri] was used to detect the expression level of CD86, a macrophage M1 polarization marker, in each group. qPCR was used to detect the mRNA levels of cytokines (TNF-α, IL-1β, IL-10) (pre-incubation 180s; two-step amplification 85℃ 15s, 58℃ 60s, 45 cycles; melting 95℃ 10s, 65℃ 60s, 97℃ 1s; cooling 37℃ 30s)). This demonstrated that GSK872 can effectively inhibit PA-induced macrophage inflammatory responses. Figure 3 AB, F).

[0064] The PCR primer sequences are shown in the table below: .

[0065] 2.2 Virtual screening based on Ripk3 protein crystal structure to lock fasudil hydrochloride.

[0066] The crystal structure of the Ripk3 protein that binds to GSK872 was selected, and a virtual screening was performed on 2809 compounds in the FDA's drug database. Based on molecular docking scores, subsequent literature review and database searches were conducted to exclude drugs with reported cardiotoxicity or other serious adverse reactions, ensuring the clinical safety of the candidate drugs. Furthermore, using our previously established macrophage lipid stress model, the remaining candidate drugs were evaluated in vitro to exclude compounds that did not significantly interfere with Ser181 phosphorylation modification under these experimental conditions. Through this step-by-step screening, fasudil hydrochloride was ultimately selected as the preferred drug. Figure 3 C).

[0067] 2.3 In vitro efficacy verification of fasudil hydrochloride.

[0068] WT mouse BMDM cells were treated with fasudil hydrochloride (15 μM, for 24 hours). Under PA stimulation, the p-Ripk3 Ser181 level was detected by Western blot. WT mouse BMDM cells were also treated with fasudil hydrochloride. Under PA stimulation, the expression level of the macrophage M1 polarization marker CD86 was detected by flow cytometry, and the mRNA levels of cytokines (TNF-α, IL-1β, IL-10) were detected by qPCR. This demonstrated that fasudil hydrochloride regulates macrophage inflammatory responses by inhibiting Ripk3 Ser181 phosphorylation. Figure 3 DF).

[0069] 3. Animal experiments were conducted to verify the therapeutic effects of GSK872 and fasudil hydrochloride on atherosclerosis.

[0070] Based on the above findings, a mouse AS model was constructed and treated with GSK872 and fasudil hydrochloride. AS mouse samples were collected and related indicators were detected.

[0071] C57BL / 6 mice (8 weeks old, male) were injected with 2×10 11 Mice were given vg / mouse of AAV8-D377Y-mPCSK9 adeno-associated virus (AS model group and drug intervention group) or an equivalent amount of AAV8-Vector Control (solvent control group), and all groups were fed a high-fat diet for 14 weeks. Starting from week 4 after the start of the high-fat diet, mice were randomly divided into 6 groups (n=5 per group): (1) Wild-type control group: AAV8-Vector Control was injected, and an equal volume of normal saline was injected intraperitoneally once a day; (2) AS model group: AAV8-D377Y-mPCSK9 was injected, and an equal volume of normal saline was injected into the peritoneum once a day; (3) GSK872 solvent control group: AAV8-Vector Control was injected, and an equal amount of GSK872 solvent was injected intraperitoneally once a day; (4) Fasudil hydrochloride solvent control group: AAV8-Vector Control was injected, and an equal volume of fasudil hydrochloride solvent was injected intraperitoneally once a day; (5) GSK872 intervention group: AAV8-D377Y-mPCSK9 was injected, and GSK872 (90μg / kg) was injected intraperitoneally once a day. (6) Fasudil hydrochloride intervention group: AAV8-D377Y-mPCSK9 was injected, and fasudil hydrochloride (50mg / kg) was injected intraperitoneally once a day.

[0072] Mice were administered the drug continuously for up to week 14, for a total dosing period of 2.5 months. During the dosing period, mouse body weight was measured weekly; aortic ultrasound was performed using an ultrasound machine to record plaque formation; gross Oil Red O staining was performed on the mouse aorta to compare plaque area and number; frozen sections of the aortic sinus were stained with Oil Red O, HE, and Masson staining to assess the degree of lesions and lipid deposition; immunofluorescence was used to detect macrophage infiltration and p-Ripk3 Ser181 expression; serum levels of inflammatory factors (TNF-α, IL-1β, IL-10) and total cholesterol (TC), triglycerides (TG), and high-density and low-density lipoprotein cholesterol (HDL-C, LDL-C) were measured. Figure 4-5 ).

[0073] Compared with the AS model group, the GSK872 and fasudil hydrochloride intervention group mice showed a significant reduction in aortic plaque area, a smaller necrotic core area, enhanced plaque stability, and decreased serum inflammatory factor levels, suggesting that GSK872 and fasudil hydrochloride have therapeutic effects on AS.

[0074] Statistical analysis. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software, Inc.). In vitro experimental data are expressed as mean ± standard deviation (SD) from three independent experiments; animal experimental data are expressed as mean ± standard deviation (SD) for each group of five animals. One-way ANOVA was used for comparisons among multiple groups, and Tukey's post-hoc test was used for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A phosphorylated modified antigenic polypeptide, characterized in that, The amino acid sequence of the polypeptide is selected from any of the following: (1) KSQSGTG(pS)GE; (2) TG(pS)GEPGGTC; (3) SG(pS)RDSGGTL-C; (4) GGSSG(pS)RD; Where (pS) represents phosphorylated serine residues.

2. A polyclonal antibody that specifically recognizes phosphorylation sites of the Ripk3 protein, characterized in that, The antibody can specifically bind to the phosphorylated modified antigen polypeptide of claim 1.

3. A method for preparing the polyclonal antibody according to claim 2, characterized in that, Includes the following steps: (1) The phosphorylated modified antigen polypeptide of claim 1 is coupled with a carrier protein to prepare an immunogen; (2) Immunize the experimental animals with the immunogen and adjuvant emulsified; (3) Collect antiserum from immunized animals and purify it to obtain the specific polyclonal antibody.

4. The use of the polyclonal antibody of claim 2 in the preparation of a reagent or kit for detecting the phosphorylation level of Ripk3 protein in biological samples.

5. The use of the polyclonal antibody according to claim 2 in the preparation of a product for the auxiliary diagnosis of atherosclerosis.

6. The application according to claim 5, characterized in that, The product contains the polyclonal antibody of claim 2, which is used to specifically recognize the phosphorylated Ripk3 protein at the Ser176 or Ser181 site.

7. Application of Ripk3 protein Ser176 or Ser181 phosphorylation inhibitors in the preparation of drugs for the treatment of atherosclerosis.

8. The application according to claim 7, characterized in that, The phosphorylation inhibitor of the Ripk3 protein at the Ser176 or Ser181 site is GSK872 or fasudil hydrochloride.

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