Preparation method and application of anti-PPP2R5A protein Ser41 site phosphorylated antibody

Abstract

The invention provides a preparation method of an antibody for resisting PPP2R5A protein Ser41 site phosphorylation and an application of the antibody. Phosphorylated proteomics analysis is carried out on beta 1-AA positive mouse myocardial tissue, and it is found that the phosphorylation level of the S41 site of PPP2R5A is remarkably increased; by constructing related cell and animal models and detecting autophagy indexes, it is proved that PPP2R5A-S41 site phosphorylation can promote occurrence and development of heart failure. The PPP2R5A-S41 site phosphorylated antibody obtained in the invention provides a brand new action target and a prognosis evaluation index for clinical treatment of heart failure, and also expands a new scene and direction for application of the PPP2R5A-S41 site phosphorylated antibody.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the preparation method and application of an antibody against phosphorylation at the Ser41 site of the PPP2R5A protein. Background Technology

[0002] Heart failure is a cardiovascular disease with high morbidity and mortality worldwide. Its complex pathophysiological mechanisms remain incompletely understood, directly leading to a lack of clinical treatment options and poor overall patient prognosis. In recent years, the academic community has conducted extensive research on the regulatory role of autoimmune responses in the occurrence and development of heart failure, and related attention continues to increase. Clinical studies have confirmed that various autoantibodies can be detected in the serum of heart failure patients, among which only β1-adrenergic receptor autoantibody (β1-AA) has been clearly verified to be independently associated with poor patient prognosis. This antibody can persistently bind to β1-adrenergic receptors (β1-AR) on the surface of cardiomyocyte membranes, thereby inducing progressive damage to cardiomyocytes and ultimately leading to irreversible deterioration of cardiac function.

[0003] Protein phosphorylation is a core element in the precise regulation of autophagy occurrence and development. However, the key phosphorylation events mediating autophagy inhibition under the influence of β1-AA remain unclear. The specific molecular mechanism by which β1-AA regulates downstream signaling pathways through phosphorylation modification, thereby downregulating autophagic flux in cardiomyocytes, remains a crucial scientific question that urgently needs to be elucidated in this field. Therefore, in-depth analysis of the downstream phosphorylation modification signaling network regulated by β1-AA is of significant scientific and clinical value for identifying novel therapeutic targets for heart failure and developing innovative intervention strategies. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a method for preparing an antibody against Ser41 phosphorylation of the PPP2R5A protein and its application.

[0005] This study performed phosphorylated proteomics analysis on myocardial tissue from β1-AA-positive mice and found that the phosphorylation level of autophagy-related protein PPP2R5A-S41 was significantly increased. PPP2R5A is the regulatory subunit of the major serine / threonine phosphatase-protein phosphatase 2A (PP2A) in cells and is responsible for guiding the holoenzyme to specific substrates.

[0006] The present invention provides a polypeptide phosphorylated at the PPP2R5A-S41 site, having an amino acid sequence as shown in SEQ ID NO.1; the serine at the third site of the polypeptide is phosphorylated.

[0007] This invention provides a polyclonal antibody for detecting phosphorylation levels at the PPP2R5A-S41 site, which is prepared by immunizing animals with the polypeptide as the antigenic peptide.

[0008] The present invention also provides the application of the polyclonal antibody as described above in the preparation of a reagent for detecting the phosphorylation level of the PPP2R5A-S41 site.

[0009] Furthermore, the reagents include any one of the following: ELISA detection reagents, dot blot hybridization detection reagents, immunoblotting detection reagents, immunohistochemical detection reagents, and immunocytochemical detection reagents.

[0010] The present invention also provides a kit for detecting phosphorylation levels at the PPP2R5A-S41 site, the kit comprising the aforementioned polyclonal antibody.

[0011] This invention provides the use of the polyclonal antibody in the preparation of pharmaceutical compositions or reagents for screening, alleviating, and determining the prognosis of heart failure.

[0012] The present invention also provides a pharmaceutical composition or reagent for screening, alleviating and prognosticating heart failure, including the polyclonal antibody; by injecting mice with a virus mutated at the PPP2R5A-S41 site, it was found that blocking phosphorylation at the PPP2R5A-S41 site can effectively alleviate the deterioration of cardiac function caused by β1-AA and improve heart failure.

[0013] The present invention also provides a method for preparing the polyclonal antibody, comprising the following steps: S1: Synthesize a polypeptide with the amino acid sequence shown in SEQ ID NO.1, phosphorylate its third serine residue to obtain an antigenic peptide, and couple it with a hapten conjugate to obtain a complete antigen; the hapten conjugate is hemocyanin. S2: The complete antigen of S1 is combined with an adjuvant and used to immunize animals, and the serum is collected to obtain antiserum; the adjuvant is Freund's complete adjuvant and Freund's incomplete adjuvant. S3: Purify the antiserum from S2 to obtain the PPP2R5A-S41 phosphorylated polyclonal antibody.

[0014] Furthermore, the adjuvant is any one or more of Freund's complete adjuvant, Freund's incomplete adjuvant, and aluminum adjuvant, wherein Freund's complete adjuvant is for basic immunization and Freund's incomplete adjuvant is for booster immunization.

[0015] Furthermore, the immunized animal is a New Zealand white rabbit; the antigen is administered 3-5 times.

[0016] In summary, compared with the prior art, the present invention achieves the following technical effects: The antibody obtained in this invention can specifically recognize phosphorylation at the PPP2R5A-S41 site, exhibiting high specificity and affinity. It can precisely bind to this target site, avoiding off-target effects, and efficiently regulate related pathological processes, significantly improving pathological indicators. Furthermore, it demonstrates strong stability, wide applicability, low toxicity, and high safety. The antibody provided by this invention offers a new treatment option for the detection and treatment of heart failure, and also provides a novel and effective means for the prevention, treatment, diagnosis, and basic research of diseases related to PPP2R5A-S41 phosphorylation, possessing significant clinical, research, and industrialization value. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 In Example 1 of this invention, Western Blot was used to verify the specificity of positive antibody 1 and positive antibody 2 for detecting PPP2R5A-S41 phosphorylation. Figure 2 This is a diagram showing the results of immunofluorescence detection of phosphorylation at the PPP2R5A-S41 site in cardiomyocytes in Example 1 of the present invention. Figure 3 The following are the results of Western Blot detection of PPP2R5A-S41 phosphorylation in Example 2 of this invention: (A) Western Blot detection of protein expression levels of PPP2R5A-S41, PPP2R5A, and GAPDH in mouse cells of the Ctrl and β1-AA groups; (B) Statistical graph of relative expression levels of P-PPP2R5A-S41 / PPP2R5A in H9c2 cardiomyocytes; (C) Western Blot detection of protein expression levels of PPP2R5A-S41, PPP2R5A, and GAPDH in cardiomyocytes of the Ctrl and β1-AA groups; (D) Statistical graph of relative expression levels of P-PPP2R5A-S41 / PPP2R5A in cardiomyocytes. Figure 4This is a graph showing the results of Western Blot detection of autophagy levels in cardiomyocytes transfected with PPP2R5A-S41 phosphorylated plasmid (S41D) in Example 3 of the present invention; (A) shows the protein expression levels of LC3-II, p62 and internal control GAPDH in the WT, β1-AA, S41D and S41D+β1-AA groups detected by Western Blot; (B) and (C) are statistical graphs of the relative expression levels of LC3-II and p62 proteins, respectively. Figure 5 This is a graph showing the results of Western Blot detection of autophagy levels in cardiomyocytes transfected with PPP2R5A-S41A dephosphorylated plasmid (S41A) in Example 4 of the present invention; (A) shows the protein expression levels of LC3-II, p62 and internal control GAPDH in the WT, S41A, β1-AA and S41A+β1-AA groups detected by Western Blot; (B) and (C) are statistical graphs of the relative expression levels of LC3-II and p62 proteins, respectively. Figure 6 This is a graph showing the results of Western Blot detection of autophagy levels in mice with PP2R5A-S41A dephosphorylated plasmid (S41A) in Example 4 of the present invention; (A) shows the protein expression levels of LC3-II, p62 and internal reference GAPDH in the WT, S41A, β1-AA and S41A+β1-AA groups detected by Western Blot; (B) and (C) are statistical graphs of the relative expression levels of LC3-II and p62 proteins, respectively. Figure 7 Example 5 of the present invention shows a small animal ultrasound image of a WT mouse heart. Figure 8 This is an echocardiogram of a WT mouse injected with β1-AR-ECⅡ, as shown in Example 5 of the present invention. Figure 9 This is an echocardiogram of a mouse injected with PP2R5A-S41A dephosphorylated, as shown in Example 5 of the present invention. Figure 10 This is a cardiac ultrasound image of a mouse injected with β1-AR-ECⅡ after being injected with PP2R5A-S41A dephosphorylated using small animal ultrasound in Example 5 of the present invention. Figure 11 Example 5 of this invention shows the statistical results of cardiac function index analysis in four groups of mice; (A) Ejection fraction (%); (B) Fractional shortening (%); (C) Left ventricular end-systolic diameter (LVIDs); (D) Left ventricular end-diastolic diameter (LVIDd); (E) End-diastolic volume (EDV); (F) End-systolic volume (ESV). Detailed Implementation

[0019] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0020] Example 1: Preparation of PPP2R5A-S41 site antigen and antibody I. Immunogen Preparation (1) Antigen peptide design: In this study, phosphorylated proteomics analysis of myocardial tissue from β1-AA positive mice revealed that the phosphorylation level of autophagy-related protein PPP2R5A-S41 was significantly increased. Based on the PPP2R5A protein sequence SEQ ID NO.4 and targeting the S41 phosphorylation modification site, two positive and one negative peptides were designed to study the phosphorylation modification-specific antibody at this site.

[0021] Positive polypeptide sequence 1 SEQ ID NO.1 S41-phosphorylated(p-S41): QG (pS) SQFRSQ-C, Positive polypeptide sequence 2 SEQ ID NO.2 S41-phosphorylated(p-S41): C-KRSQG (pS) SQ, Negative polypeptide sequence SEQ ID NO.3 S41-unphosphorylated(unphos): CKRSQGSSQFRSQ.

[0022] (2) Synthesis method: Organic chemical solid phase synthesis method (Fmoc protected amino acid, solid phase carrier-resin) was adopted. The three-channel peptide automatic synthesizer (CS360) produced by CS Company, USA, was used to synthesize from the base end to the amino end of the peptide to obtain peptide resin. Then, the peptide was cut off from the resin by TFA method and crude product was obtained.

[0023] (3) Purification method: The waters high performance liquid chromatograph from the United States was used to separate and purify the material using a C18 reversed phase chromatography column, and then the material was freeze-dried.

[0024] (4) The polypeptide fragments (SEQ ID NO.1 and SEQ ID NO.2) were linked to the carrier protein-hemocyanin (KLH): 10 mg of the purified polypeptide and 15 mg of hemocyanin were condensed under the catalysis of a condensing agent to obtain the polypeptide-hemocyanin conjugate (immunogen).

[0025] II. Preparation of PPP2R5A-S41 phosphorylated antibody Four New Zealand white rabbits were selected and purchased from Qingdao Kangda Biotechnology Co., Ltd. All were clean-grade laboratory animals that had passed quarantine and were free of pathogens, with each rabbit weighing approximately 2.5 kg. All rabbits underwent an acclimatization period of one week, during which they remained active, had glossy fur, and ate normally, meeting the health requirements for subsequent immunization experiments.

[0026] The conjugated modified peptides shown in SEQ ID NO.1 and SEQ ID NO.2 were emulsified with equal volumes of Freund's complete adjuvant (basal immunization) and Freund's incomplete adjuvant (booster immunization), respectively, and then injected intradermally at multiple points on the back of experimental rabbits. After three and four immunizations, the antibody titers were found to be low; therefore, the immunization frequency was increased to five times. The specific procedure is as follows:

[0027] III. ELISA Testing (1) Coat the enzyme-linked reaction plate with polypeptide fragments (uncoupled to hemocyanin), 0.2 μg / well.

[0028] (2) Dilute the rabbit antiserum to: 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000.

[0029] (3) The secondary antibody was horseradish peroxidase-labeled goat anti-rabbit IgG, the colorimetric reagent was tetramethylbenzidine, the detection wavelength was 450nm, and the optical density value was measured.

[0030] (4) The test results are as follows: Antibody obtained from antigen peptide SEQ ID NO.1:

[0031] Antibody obtained from antigen peptide SEQ ID NO.2:

[0032] IV. Western Blot Detection of Phosphorylation Specificity of Antibodies Prepared from Different Antigenic Peptides 1) Cell lysis: The total protein of the sample was obtained by lysing cardiomyocytes and the protein concentration was determined by BCA method.

[0033] 2) Protein electrophoresis and transfer: Select the appropriate separation gel concentration according to the molecular weight of the target protein. Load 20-40 μg of sample per well, and perform electrophoresis at 80V for the stacking gel and 120V for the separating gel. Pre-cool the transfer buffer before transfer. Arrange the gel, membrane, and filter paper in the transfer buffer in a sandwich structure to avoid air bubbles. The transfer voltage is 80V-120V.

[0034] 3) Blocking: Add blocking solution to the transferred membrane and block at room temperature for 60 min. Wash with 1×TBST for 5 min after incubation.

[0035] 4) Primary antibody incubation: Dilute the antibodies obtained from different antigenic peptides, incubate at room temperature for 2 hours, and then wash three times with 1×TBST for 5 minutes each time.

[0036] 5) Secondary antibody incubation: Select the corresponding mouse or rabbit antibody according to the properties of the primary antibody; add the secondary antibody at a dilution ratio of 1:1000, incubate at room temperature for 1 hour, and then wash 3 times with 1×TBST for 5 minutes each time.

[0037] 6) Expose the washed film after adding a developing substrate.

[0038] like Figure 1 As shown, the antibody obtained from antigen peptide 1 can specifically recognize the PPP2R5A-S41 protein, while the antibody corresponding to SEQ ID NO.2 has poor specificity and strong background band signal. The results indicate that the antibody obtained from antigen peptide 1 has better affinity than the antibody from antigen peptide 2. In subsequent experiments, the antigen peptide of SEQ ID NO.1 was used to prepare the antibody.

[0039] V. Specificity of Immunofluorescence Detection of PPP2R5A-S41 Phosphorylated Antibody 1) Cell slides: Using small forceps, place clean, sterile cell slides into a six-well plate. Add 1 mL of sterile PBS solution to each well and wash three times to remove impurities. Then, aspirate any remaining PBS solution and add 1 mL of complete culture medium to each well.

[0040] 2) Cell seeding: Seed the digested cell suspension into a six-well plate containing 1 mL of culture medium, ensuring uniform cell distribution.

[0041] 3) Drug treatment: The next day, β1-AA was added and treated for 24 hours before proceeding to the next step.

[0042] 4) Fixation and permeabilization: Remove the six-well plate and wash the cell slides three times with PBS buffer preheated to 37°C. Fix and permeabilize the H9c2 cardiomyocytes with 200 μL of methanol and acetone in a 1:1 ratio for 15-20 min. After the permeabilization, wash the cells three times with PBS solution for 5 min each time.

[0043] 5) Blocking: Prepare 5% BSA solution, add 500 μL / well to a six-well plate, and block at room temperature for 1-2 hours.

[0044] 6) Primary antibody incubation: Wash with PBST solution for 5 min each time, for a total of 3 washes. Place the six-well plate in a humidified chamber, drop the prepared primary antibody onto the cell slide, and incubate overnight at 4°C.

[0045] 7) Secondary antibody incubation: Recover the primary antibody, discard the PBST solution, and wash again with PBST solution for 5 min / time, for 3 times. Add the prepared fluorescent secondary antibody to the slide, 200 μL / well, and then place it in a humidified chamber at 37℃ for 1-2 h, washing with PBST solution for 5 min / time, for 3 times.

[0046] 8) Fluorescence detection: Place 50 μL of DAPI solution on the cell slide, stain for about 10 seconds, wash with PBST for 5 minutes each time, for a total of 3 washes, and observe the staining of cell nuclei under a fluorescence microscope. Add an anti-fluorescence attenuation agent to the slide, place the cell side of the slide facing the slide, seal the slide with clear nail polish along the edge of the slide, and take pictures as soon as possible using a laser confocal microscope to prevent fluorescence attenuation.

[0047] like Figure 2 As shown, the antibody detected β1-AA-induced phosphorylation modification of PPP2R5A-S41 at the PPP2R5A-S41 site in H9c2 cardiomyocytes, mainly located in the cytoplasm, indicating the successful preparation of the PPP2R5A-S41 phosphorylation antibody.

[0048] Example 2: Establishment of a β1-AR Active Immune Model I. Establishment of β1-AR Active Immune Model An antigenic peptide was synthesized targeting the amino acid sequence SEQ ID NO.4 (197aa-223aa, HWWRAESDEA-RR-CYNDPKCCDFVTNRA) corresponding to the extracellular second loop β1-AR-ECII of the β1-adrenergic receptor.

[0049] Male C57BL / 6 mice aged 6-8 weeks were randomly divided into Ctrl and β1-AA groups. Myocardial tissue was collected for subsequent in vivo experiments. Male SD rats aged 6-8 weeks were selected, and blood was collected from the abdominal aorta. Positive rat serum was screened using SA-ELISA (sandwich enzyme-linked immunosorbent assay), purified, and concentrated for subsequent cell experiments. The model construction process was as follows: A 10 mg / mL β1-AR-ECⅡ peptide stock solution was prepared using 100 mM Na2CO3 solution, diluted with physiological saline, and the diluted antigen peptide was mixed with an equal volume of Freund's adjuvant. After emulsification, the mixture was injected subcutaneously at multiple points on the back of both rats and mice. After the initial active immunization with complete Freund's adjuvant, booster immunizations were performed every 2 weeks, for a total of 4 weeks for mice and 6 weeks for rats. After immunization, mouse myocardial cells were collected to detect phosphorylation at the PPP2R5A-S41 site.

[0050] II. Phosphorylated Proteomics (1) Extraction of tissue protein A suitable amount of left ventricular myocardial tissue was weighed, thoroughly ground with liquid nitrogen, and then lysed using an 8M urea solution. To ensure the stability of the experimental conditions, the centrifuge was pre-cooled to 4°C. After lysis, the tissue was centrifuged at 12000g for 10 minutes, the supernatant was collected, and protein quantification was performed to provide accurate protein concentration data for subsequent experiments.

[0051] (2) Enzymatic hydrolysis with trypsin TCA was added to the myocardial tissue protein sample, and the mixture was incubated on ice for 2 hours to allow for complete protein precipitation. Subsequently, the sample was centrifuged at 10,000 g for 10 minutes, the supernatant was removed, and the cell pellet was collected. The pellet was washed with acetone to remove impurities and then air-dried at room temperature. Next, TEBA solution was added, and the pellet was vortexed to fully disperse it. Finally, trypsin was added for overnight enzymatic digestion. During the digestion process, the protein was first reduced with DTT for 30 minutes to break disulfide bonds and allow for complete protein development; then, IAA was added at room temperature and the mixture was reacted in the dark for 15 minutes for alkylation to protect cysteine ​​residues and prevent oxidation in subsequent experiments.

[0052] (3) Peptide TMT labeling The enzymatically digested peptides were desalted and dried to remove impurities and moisture, thus improving labeling efficiency. Subsequently, the peptides were mixed with TMT labeling reagents, and the labeling reaction was performed following the instructions of the TMT kit. TMT labeling is a peptide quantification technique that allows for quantitative comparison of protein expression in different samples by labeling peptides from different samples with different isotope tags, enabling subsequent mass spectrometry analysis.

[0053] (4) HPLC fractionation High-pH reversed-phase HPLC was used to fractionate labeled peptides. High-pH reversed-phase HPLC is a highly efficient separation technique that enables the separation of peptides under high pH conditions, improving separation efficiency and resolution. Fractionation allows complex peptide mixtures to be separated into multiple components, facilitating subsequent mass spectrometry analysis.

[0054] (5) Liquid chromatography-mass spectrometry analysis The fractionated peptides were further separated using an ultra-high performance liquid chromatography (UHPLC) system to ensure more complete separation within the chromatographic column. Subsequently, the separated peptides were introduced into a mass spectrometer for ionization and mass spectrometry analysis. Mass spectrometry analysis can accurately determine the molecular weight and structural information of peptides, providing crucial data for protein identification and quantification.

[0055] (6) Database search and bioinformatics analysis Raw mass spectrometry data were analyzed using specialized database searching software. The data was then compared with protein databases to identify the types and sequence information of proteins. Bioinformatics analysis was employed to perform functional annotation and pathway analysis on the identified proteins, further exploring their biological significance and potential functions in myocardial tissue.

[0056] III. Phosphorylated proteomics detection in β1-AR active immunization model mice Tissue protein extraction: A suitable amount of mouse left ventricular myocardial tissue was weighed, thoroughly ground with liquid nitrogen, and then lysed using an 8M urea solution. To ensure the stability of experimental conditions, the centrifuge was pre-cooled to 4°C. After lysis, the tissue was centrifuged at 12000g for 10 minutes, the supernatant was collected, and protein quantification was performed to provide accurate protein concentration data for subsequent experiments.

[0057] Trypsin digestion: TCA was added to the myocardial tissue protein sample, and the mixture was incubated on ice for 2 hours to allow for complete protein precipitation. Subsequently, the sample was centrifuged at 10000g for 10 minutes, the supernatant was removed, and the cell pellet was collected. The pellet was washed with acetone to remove impurities and then air-dried at room temperature. Next, TEBA solution was added, and the pellet was vortexed to ensure complete dispersion. Finally, trypsin was added for overnight enzymatic digestion. During the digestion process, the protein was first reduced with DTT for 30 minutes to break disulfide bonds and allow for complete protein development; then, IAA was added at room temperature and the mixture was reacted in the dark for 15 minutes for alkylation to protect cysteine ​​residues and prevent oxidation in subsequent experiments.

[0058] TMT labeling of peptides: After enzymatic digestion, the peptides are desalted and dried to remove impurities and moisture, thereby improving labeling efficiency. Subsequently, the peptides are mixed with TMT labeling reagents, and the labeling reaction is performed according to the TMT kit instructions. TMT labeling is a peptide quantification technique that allows for quantitative comparison of protein expression in different samples by labeling peptides from different samples with different isotope tags, enabling subsequent mass spectrometry analysis.

[0059] HPLC fractionation: Labeled peptides were fractionated using high-pH reversed-phase HPLC. High-pH reversed-phase HPLC is a highly efficient separation technique that can separate peptides under high pH conditions, improving separation efficiency and resolution. Fractionation allows complex peptide mixtures to be separated into multiple components, facilitating subsequent mass spectrometry analysis.

[0060] Liquid chromatography-mass spectrometry (LC-MS) analysis: Fractionated peptides are further separated using an ultra-high performance liquid chromatography (UHPLC) system, ensuring more complete separation within the chromatographic column. Subsequently, the separated peptides are introduced into a mass spectrometer for ionization and mass spectrometry analysis. Mass spectrometry analysis can accurately determine the molecular weight and structural information of peptides, providing crucial data for protein identification and quantification.

[0061] Database search and bioinformatics analysis: Utilizing specialized database search software, raw mass spectrometry data is analyzed and compared with protein databases to identify protein types and sequence information. Through bioinformatics analysis, functional annotation and pathway analysis are performed on the identified proteins to further explore their biological significance and potential functions in myocardial tissue.

[0062] IV. Purification of β1-AA by Affinity Chromatography (1) Take out the rat serum that was positive in the SA-ELISA test, mix it in a sterile 50mL centrifuge tube, and take out the affinity chromatography column.

[0063] (2) Wash the column twice with 3-4 mL of high-pressure triple-distilled water, and rinse the chromatography column with 3-4 mL of Binding Buffer.

[0064] (3) Use a 5mL syringe connected to a 0.45μm filter to slowly filter the serum through the chromatography column. Repeat 2-3 times. After each 10mL of serum is filtered, wash twice with 5mL Binding Buffer to remove non-specific binding.

[0065] (4) Add elution buffer to the chromatography column and wash the column slowly. Repeat the washing twice and collect the washing solution. Add the corresponding amount of neutralization buffer to the EP tube according to the ratio of 1 mL elution buffer to 75 μL neutralization buffer.

[0066] (5) Rinse the chromatography column with Binding Buffer, and finally inject 20% ethanol into the chromatography column and store at 4°C.

[0067] (6) After concentration using an ultrafiltration tube, the concentration was measured by BCA for subsequent cell experiments.

[0068] V. β1-AA stimulation of H9c2 cardiomyocytes H9c2 cardiomyocytes were divided into a Ctrl group and a β1-AA group. The Ctrl group was treated with 1 μmol / L IgG, which was obtained by injecting rats with a mixture of Na2CO3 and adjuvant, followed by affinity chromatography and concentration. The β1-AA group was treated with concentrated 1 μmol / L β1-AA, and the phosphorylation level at the PPP2R5A-S41 site was measured after 24 hours.

[0069] Cardiomyocytes were divided into two groups. One group, the Ctrl group, received an equal volume of Na₂CO₃ and adjuvant mixture subcutaneously in the back of rats at the same time and frequency as the active immunization group. Negative IgG was purified using affinity chromatography, concentrated using a concentration column, and then injected into H9c2 cardiomyocytes (1 μmol / L). The other group received a 0.4 μg / g dose of a 10 mg / mL β1-AR-ECⅡ peptide stock solution prepared with Na₂CO₃ solution, which was then emulsified and injected subcutaneously at multiple sites in the back of SD rats. Booster immunizations were performed every 2 weeks for a total of 6 weeks. Serum from successfully immunized rats was selected using ELISA, and β1-AA was purified using affinity chromatography and concentrated using a concentration column. H9c2 cardiomyocytes were treated with 1 μmol / L β1-AA for 24 hours, and phosphorylation at the PPP2R5A-S41 site was detected according to the Western blot assay protocol.

[0070] Experimental results like Figure 3 As shown in AB, the phosphorylation of PPP2R5A and PPP2R5A-S41 sites in mice with β1-AA and Ctrl was detected. The expression level of PPP2R5A-S41 phosphorylation protein increased in mice treated with β1-AA. Figure 3 CD showed that β1-AA treatment of H9c2 cardiomyocytes for 24 h significantly increased the phosphorylation level of PPP2R5A-S41. These in vivo and in vitro experimental results indicate that β1-AA induces phosphorylation of PPP2R5A-S41.

[0071] Example 3: Transfection with the PPP2R5A-S41 phosphorylation-mimicking plasmid (S41D) inhibited autophagy levels in cardiomyocytes. I. Construction and Validation of Cell Mutation Models A mutant carrying wild-type PPP2R5A (PPP2R5A-WT-OE), a mutant simulating sustained phosphorylation (PPP2R5A-S41D), and a mutant simulating dephosphorylation (PPP2R5A-S41A) were constructed. The plasmids were synthesized by Hanheng Biotechnology Co., Ltd. H9c2 cardiomyocytes were transfected with a multiplicity of infection (MOI) of 30. Stable transfected cells were obtained through puromycin selection, and overexpression efficiency was verified using Western blotting.

[0072] II. Autophagy Level Detection Western blotting was used to detect the protein expression levels of autophagy-related markers (such as LC3-II / I and p62) in PPP2R5A-WT / S41D / S41A stable cells to assess changes in autophagy flux.

[0073] Experimental results like Figure 4 As shown in AC, compared with the normal group, cells transfected with PPP2R5A-S41D and β1-AA showed decreased LC3-II protein content and increased p62 protein expression. Furthermore, the addition of β1-AA to PPP2R5A-S41D transfected cells significantly increased the decrease in LC3-II protein and the increase in p62 protein, indicating that both PPP2R5A-S41D transfection and β1-AA treatment can promote autophagy. When transfected with dephosphorylated PPP2R5A-S41, there were no significant changes in LC3-II and p62 protein levels compared with the normal group. Moreover, after transfecting dephosphorylated PPP2R5A-S41 cells, treatment with β1-AA also showed no significant changes in LC3-II and p62 protein levels. Figure 5 As shown in AC. Detection of autophagy markers in cells transfected with phosphorylated and dephosphorylated PPP2R5A-S41 and cells treated with β1-AA showed that phosphorylation of β1-AA and PPP2R5A-S41 promotes autophagy.

[0074] Example 4: Construction of a mouse PPP2R5A-S41A mutant model Function of PPP2R5A-S41 phosphorylation: In this study, serine at position 41 (Ser41) was mutated to alanine (Ala). The following adeno-associated virus (AAV9) vectors were constructed by Shanghai Jikai Gene Medical Technology Co., Ltd.: AAV9-PPP2R5A-RNAi (knockdown of endogenous PPP2R5A expression), AAV9-PPP2R5A(WT)-OE (overexpression of wild-type PPP2R5A), and AAV9-PPP2R5A(S41A)-OE (overexpression of S41A mutant PPP2R5A). C57BL / 6 mice were divided into four groups: the WT group (injected with AAV9-PPP2R5A-RNAi-PPP2R5A(WT)-OE), the WT+β1-AA group (injected with AAV9-PPP2R5A-RNAi-PPP2R5A(WT)-OE and actively immunized with β1-AA), the S41A mutant group (injected with AAV9-PPP2R5A(S41A)-OE), and the S41A+β1-AA group (injected with AAV9-PPP2R5A(S41A)-OE and actively immunized with β1-AA). The corresponding viruses were injected via the tail vein two weeks prior to active immunization.

[0075] Experimental results like Figure 6 As shown in AC, after introducing endogenous PPP2R5A knockdown, PPP2R5A overexpression, and mutant PPP2R5A into mice, the autophagy markers LC3-II and p62 proteins showed no significant changes. However, after active immunization with β1-AA, the expression of LC3-II and p62 proteins changed significantly. In contrast, the addition of β1-AA to the S41 mutant group did not significantly change LC3-II and p62 proteins, indicating that β1-AA affects autophagy through the phosphorylation of PPP2R5A-S41.

[0076] Example 5: Assessment of cardiac function in mice Cardiac function parameters of mice in the WT group, WT+β1-AA group, S41A mutant group, and S41A+β1-AA group were detected and analyzed using a small animal ultrasound imaging system. These parameters included ejection fraction (EF%), fractional shortening (FS%), left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), end-diastolic volume (EDV), and end-systolic volume (ESV). Ejection fraction and fractional shortening were core indicators of cardiac systolic function; left ventricular end-diastolic / end-systolic diameter and end-diastolic / end-systolic volume were indicators of cardiac remodeling and chamber expansion.

[0077] Experimental results according to Figure 7-10The four groups of mice shown have echocardiographic images. Analysis of cardiac function and structure was performed to obtain various cardiac parameters. The results are as follows: Figure 11 As shown in the figure, compared with the WT group, the WT+AA group mice showed significantly decreased EF and FS, while LVIDs, LVIDd, EDV, and ESV were significantly increased, suggesting that active β1-AA immunization can cause a decline in cardiac contractile function in wild-type mice, induce left ventricular dilation, cardiac pathological remodeling, and significantly worsen cardiac function. Compared with the WT group, there were no statistically significant differences in EF, FS, left ventricular diameter, and cardiac chamber volume in the S41A group, indicating that PPP2R5A-S41 dephosphorylation does not alter the normal basic cardiac structure and contractile function in mice. Compared with the WT+AA group, the S41A+AA group mice showed significantly increased EF and FS, and significantly decreased LVIDs, LVIDd, EDV, and ESV; moreover, there were no significant differences in any indicators between the S41A+AA group and the WT group, indicating that PPP2R5A-S41A dephosphorylation can completely block the β1-AA-induced decline in cardiac contractile function and pathological remodeling.

[0078] These results indicate that β1-AA significantly reduces cardiac systolic function in wild-type mice and exacerbates left ventricular remodeling and chamber dilation. Dephosphorylation of PPP2R5A-S41A completely reverses β1-AA-mediated cardiac dysfunction and cardiac remodeling, suggesting that β1-AA exerts its cardiotoxic effect by regulating phosphorylation at the PPP2R5A-S41 site, thereby inducing cardiac dysfunction. Blocking phosphorylation at the PPP2R5A-S41 site can effectively alleviate cardiac dysfunction caused by β1-AA.

[0079] sequence list SEQ ID NO.1: QGSSQFRSQC SEQ ID NO.2: CKRSQGSSQ SEQ ID NO.3: CKRSQGSSQFRSQ SEQ ID NO.4: MSSPPSPAVACAAISASEKVDGFTRKSVRKAQRQKRSQGSSQFRSQGSQAELHPLPQLKDATSNEQQELFCQKLQQCCVLFDFMDSVSDLKSKEIKRATLNELVEYVSTNRGVIVESAYS DIVKMISANIFRTLPPSDNPDFDPEEDEPTLEASWPHIQLVYEFFLRFLESPDFQPSIAKRYIDQKFVQQLLELFDSEDPRERDFLKTVLHRIYGKFLGLRAFIRKQINNIFLRFIYETEHF NGVAELLEILGSIINGFALPLKAEHKQFLMKVLIPMHTAKGLALFHAQLAYCVVQFLEKDTTLTEPVIRGLLKFWPKTCSQKEVMFLGEIEEILDVIEPTQFKKIEEPLFKQISKCVSSSH FQVAERALYFWNNEYILSLIEENIDKILPIMFASLYKISKEHWNQTIVALVYNVLKTLMEMNGKLFDDLTSSYKAERQREKKKELEREELWKKLEELQLKKALEKQNNAYNMHSIRSSTSAK SEQ ID NO.5:HWWRAESDEARRCYNDPKCCDFVTNRA The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 polypeptide phosphorylated at the PPP2R5A-S41 site, characterized in that, It has the amino acid sequence shown in SEQ ID NO.1; the serine at the third site of the polypeptide is phosphorylated.

2. A polyclonal antibody for detecting phosphorylation levels at the PPP2R5A-S41 site, characterized in that, The polyclonal antibody was prepared by immunizing animals with the polypeptide described in claim 1 as the antigenic peptide.

3. The use of the polyclonal antibody as described in claim 2 in the preparation of a reagent for detecting the phosphorylation level of the PPP2R5A-S41 site.

4. The application according to claim 3, characterized in that, The reagents include any one of the following: ELISA detection reagents, dot blot hybridization detection reagents, immunoblotting detection reagents, immunohistochemical detection reagents, and immunocytochemical detection reagents.

5. A kit for detecting phosphorylation levels at the PPP2R5A-S41 site, characterized in that, The kit contains the polyclonal antibody as described in claim 2.

6. The use of the polyclonal antibody as described in claim 2 in the preparation of a pharmaceutical composition or reagent for screening, alleviating and prognosticating heart failure.

7. A pharmaceutical composition or reagent for screening, alleviating, and determining the prognosis of heart failure, characterized in that, It includes the polyclonal antibody as described in claim 2.

8. A method for preparing the polyclonal antibody as described in claim 2, comprising the following steps: S1: Synthesize a polypeptide with the amino acid sequence shown in SEQ ID NO.1, phosphorylate its third serine residue to obtain an antigenic peptide, and couple it with a hapten-coupled vector to obtain a complete antigen. S2: The complete antigen of S1 is combined with an adjuvant and used to immunize animals, and the serum is collected to obtain antiserum; S3: Purify the antiserum from S2 to obtain the PPP2R5A-S41 phosphorylated polyclonal antibody.

9. The preparation method according to claim 8, characterized in that, The adjuvant is any one or more of Freund's complete adjuvant, Freund's incomplete adjuvant, and aluminum adjuvant.

10. The preparation method according to claim 8, characterized in that, The immunized animal was a New Zealand white rabbit.

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