PPP2R5C-S497 site phosphorylated antibody and application thereof

Abstract

According to the PPP2R5C-S497 site phosphorylated antibody and the application thereof, phosphorylated proteomics analysis is carried out on myocardial tissue of a beta 1-AA positive mouse, and it is found that the phosphorylation level of autophagy related protein PPP2R5C-S497 is remarkably increased. And the phosphorylated and dephosphorylated plasmids of the PPP2R5C-S497 site are further constructed, and the myocardial cells are transfected, so that the dephosphorylation of the PPP2R5C-S497 site is proved to be capable of obviously reducing the death rate of the myocardial cells. According to the PPP2R5C-S497 phosphorylation specific antibody provided by the invention, the blank in the prior art is filled, and meanwhile, a new diagnosis target and a new treatment strategy are provided for heart failure.

Description

Technical Field

[0001] This invention relates to the field of antibody preparation, and in particular to a PPP2R5C-S497 site phosphorylated antibody and its application. Background Technology

[0002] Heart failure is one of the leading causes of cardiovascular disease-related morbidity and mortality worldwide. Its complex pathophysiological mechanisms are not yet fully understood, resulting in limited treatment options and poor prognosis. In recent years, the role of autoimmunity in the progression of heart failure has received increasing attention. Clinical data show that various autoantibodies can be detected in the serum of heart failure patients. Among them, only β1-adrenergic receptor autoantibodies (β1-AA) have been proven to be independently associated with poor prognosis. β1-AA can persistently bind to β1-adrenergic receptors (β1-AR) on the surface of cardiomyocyte membranes, causing persistent damage to cardiomyocytes and ultimately leading to deterioration of cardiac function.

[0003] Protein phosphorylation is a core molecular mechanism regulating cardiac function, influencing the progression of heart failure by rapidly altering protein activity and functional networks. However, the key phosphorylation events mediated by β1-AA remain unclear. The specific molecular mechanisms by which phosphorylation modulates downstream signaling pathways and subsequently downregulates autophagic flux in cardiomyocytes remain crucial scientific questions that urgently need to be elucidated. Therefore, understanding the downstream phosphorylation modification signals mediated by β1-AA is of great significance for developing novel therapeutic targets and strategies for heart failure. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a PPP2R5C-S497 site phosphorylation antibody 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 PPP2R5C-S497 was significantly increased. PPP2R5C is the regulatory subunit of the major intracellular serine / threonine phosphatase-protein phosphatase 2A (PP2A), which is responsible for guiding the holoenzyme to specific substrates.

[0006] The present invention provides a polypeptide phosphorylated at the PPP2R5C-S497 site, the amino acid sequence of which is shown in SEQ ID NO.1; the serine at the seventh site of the polypeptide is phosphorylated.

[0007] This invention provides a method for preparing a polyclonal antibody for detecting the phosphorylation level of the PPP2R5C-S497 site, comprising the following steps: S1: Synthesize a polypeptide with the amino acid sequence shown in SEQ ID NO.1, phosphorylate the serine at position 7 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 immunized animals are rabbits; S3: The antiserum from S2 was purified to obtain the PPP2R5C-S497 phosphorylated polyclonal antibody.

[0008] Furthermore, the adjuvant is any one or more of Freund's complete adjuvant, Freund's incomplete adjuvant, and aluminum adjuvant.

[0009] Furthermore, the immunized animals were immunized 3-5 times. The results of antiserum testing after the third and fourth immunizations showed that the antibody titer was low, while the antibody titer increased significantly after the fifth immunization.

[0010] This invention provides a polyclonal antibody prepared by the aforementioned method for detecting phosphorylation levels at the PPP2R5C-S497 site.

[0011] This invention provides the application of the polyclonal antibody described above in the preparation of a reagent for detecting the phosphorylation level of the PPP2R5C-S497 site.

[0012] 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.

[0013] This invention provides a kit for detecting phosphorylation levels at the PPP2R5C-S497 site, the kit comprising the aforementioned polyclonal antibody.

[0014] Furthermore, the kit can be used for screening and prognostic detection of heart failure.

[0015] This invention provides a pharmaceutical composition for relieving heart failure, the pharmaceutical composition comprising the aforementioned polyclonal antibody. In some embodiments, phosphorylation and dephosphorylation of the PPP2R5C-S497 site have demonstrated that phosphorylation of the PPP2R5C-S497 site promotes cell death, and inhibition of phosphorylation of the PPP2R5C-S497 site can effectively alleviate cell death. Therefore, the PPP2R5C-S497 site phosphorylation antibody provided by this invention can be used to prepare drugs for relieving heart failure and has good application prospects.

[0016] In summary, compared with the prior art, the present invention achieves the following technical effects: The highly specific and high-affinity PPP2R5C-S497 phosphorylated antibody prepared in this invention helps to elucidate the specific mechanism by which β1-AA regulates downstream signaling pathways through phosphorylation modification, thereby downregulating autophagic flux in cardiomyocytes. The PPP2R5C-S497 phosphorylated antibody provides a new target for the clinical diagnosis and treatment of heart failure, and also offers possibilities for prognostic detection of heart failure. 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 This is a graph showing the phosphorylation fluorescence results of the PPP2R5C-S497 site in cardiomyocytes detected by immunofluorescence in Example 1 of the present invention. Figure 2 This is a diagram showing the results of Western Blot detection of phosphorylation at the PPP2R5C-S497 site in mouse cardiomyocytes in Example 2 of the present invention. Figure 3 This is a graph showing the results of Western Blot detection of phosphorylation at H9c2 cardiomyocyte sites in Example 2 of the present invention; Figure 4 In Example 3 of this invention, CCK-8 was used to detect the inhibition of cardiomyocyte survival rate by transfection of PPP2R5C-S497-mimicked phosphorylated plasmid (S497D) and dephosphorylated plasmid (S497A). 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 PPP2R5C-S497 antibody I. Immunogen Preparation (1) Antigen peptide design: In this study, phosphorylated proteomics analysis of myocardial tissue from β1-AA positive mice was performed, and the phosphorylation level of autophagy-related protein PPP2R5C-S497 was found to be significantly increased. Based on the PPP2R5C protein sequence (SEQ ID NO.2) and targeting the S497 site, SEQ ID NO.1 was obtained. Based on SEQ ID NO.1, PPP2R5C-S497 phosphorylation positive and negative peptides were synthesized to study PPP2R5C-S497 phosphorylation modification-specific antibodies.

[0021] Positive peptide: S497-phosphorylated (p-S497): PLARRK(pS)EL, Negative peptide: S497-unphosphorylated(unphos): PLARRKSEL.

[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) Linking of positive fragment with carrier protein-hemocyanin (KLH): Take 10 mg of purified polypeptide and 15 mg of hemocyanin and carry out condensation reaction under the catalysis of condensing agent to obtain polypeptide-hemocyanin conjugate (immunogen).

[0025] II. Preparation of PPP2R5C-S497 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] Positive polypeptides were conjugated with modified peptides and emulsified thoroughly with equal volumes of Freund's complete adjuvant (basal immunization) and Freund's incomplete adjuvant (booster immunization), respectively. These emulsions were then administered via intradermal injection at multiple sites on the back of experimental rabbits. After three and four immunizations, 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; (2) Dilute the rabbit antiserum to: 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000; (3) The secondary antibody was horseradish peroxidase-labeled goat anti-rabbit IgG, the colorimetric reagent was tetramethylbenzidine, the detection wavelength was 450 nm, and the optical density value was measured. (4) The test results are as follows:

[0028] IV. Function of Immunofluorescence Detection of PPP2R5C-S497 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.

[0029] 2) Cell seeding: Seed the digested cell suspension into a six-well plate containing 1 mL of culture medium, ensuring uniform cell distribution.

[0030] 3) Drug treatment: The next day, β1-AA was added and treated for 24 hours before proceeding to the next step.

[0031] 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.

[0032] 5) Blocking: Prepare 5% BSA solution, add 500 μL / well to a six-well plate, and block at room temperature for 1-2 hours.

[0033] 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.

[0034] 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.

[0035] 8) Fluorescence detection: Place 50 μL of DAPI solution on the cell slide, stain for about 10 seconds, rinse with PBST for 5 min each time, and wash 3 times. 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.

[0036] like Figure 1 As shown, the phosphorylation level of PPP2R5C-S497 is mainly expressed in the cell nucleus, indicating the successful preparation of the PPP2R5C-S497 phosphorylation antibody.

[0037] Example 2: β1-AA induces phosphorylation at the PPP2R5C-S497 site in cardiomyocytes. I. Establishment of β1-AR Active Immune Model An antigenic peptide was synthesized targeting the amino acid sequence SEQ ID NO.3 (197aa-223aa, HWWRAESDEA-RR-CYNDPKCCDFVTNRA) corresponding to the extracellular second loop β1-AR-ECII of the β1-adrenergic receptor.

[0038] 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; blood was collected from the abdominal aorta, and positive rat serum was screened using SA-ELISA. Serum from these rats was 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 emulsified solution 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, myocardial cells from mice were collected to detect phosphorylation at the PPP2R5C-S497 site.

[0039] like Figure 1 As shown, compared with unimmunized mouse cardiomyocytes, the expression level of PPP2R5C-S497 was increased in cardiomyocytes of actively immunized β1-AR mice, indicating that β1-AR promotes phosphorylation of mouse cardiomyocytes.

[0040] II. 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] III. 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.

[0047] (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.

[0048] (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.

[0049] (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.

[0050] (5) Rinse the chromatography column with Binding Buffer, and finally inject 20% ethanol into the chromatography column and store at 4°C.

[0051] (6) After concentration using an ultrafiltration tube, the concentration was measured by BCA for subsequent cell experiments.

[0052] IV. β1-AA stimulation of 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 PPP2R5C-S497 site in the cardiomyocytes was measured after 24 hours.

[0053] like Figure 2 As shown, compared with cardiomyocytes treated with IgG antibody, the phosphorylation level of PPP2R5C-S497 in H9c2 cells after active immunization with β1-AA increased.

[0054] Example 3: Transfection with PPP2R5C-S497-mimicking phosphorylated plasmid (S497D) inhibited cardiomyocyte survival. To simulate persistent phosphorylation and dephosphorylation at the PPP2R5C-S497 site, Hanheng Biotechnology Co., Ltd. was commissioned to synthesize plasmids carrying the following genes: wild-type PPP2R5C (PPP2R5C-WT-OE), a mutant simulating persistent phosphorylation (PPP2R5C-S497D), and a mutant simulating dephosphorylation (PPP2R5C-S497A), which were then transfected into H9c2 cardiomyocytes. Cell viability after transfection was assessed using CCK-8 assay.

[0055] Test results as follows Figure 4 As shown, the cell survival rates of cells actively immunized with β1-AA and those transfected with mutants simulating sustained phosphorylation were significantly lower than those of the control group, while overexpression of PPP2R5C and S497A mutations did not significantly alter cell survival. Phosphorylation at the S497 site of PPP2R5C is a key factor regulating H9c2 cell survival, and β1-AA affects cell survival by regulating phosphorylation at the S497 site of PPP2R5C.

[0056] sequence list SEQ ID NO.1: PLARRKSEL SEQ ID NO.2: MLTCNKAGSGMVVDAASSNGPFQPVALLHIRDVPPADQEKLFIQKLRQCCVLFDFVSDPLSDLKWKEVKRAALSEMVEYITHNRNVITEPIYPEAVHMFAVNMFRTLPPSSNPTGAEFDPEEDEPTLEAAW PHLQLVYEFFLRFLESPDFQPNIAKKYIDQKFVLQLLELFDSEDPRERDFLKTTLHRIYGKFLGLRAYIRKQINNIFYRFIYETEHHNGIAELLEILGSIINGFALPLKEEHKIFLLKVLLPLHKVKSLSV YHPQLAYCVVQFLEKDSTLTEPVVMALLKYWPKTHSPKEVMFLNELEEILDVIEPSEFVKIMEPLFRQLAKCVSSPHFQVAERALYYWNNEYIMSLISDNAAKILPIMFPSLYRNSKTHWNKTIHGLIYNA LKLFMEMNQKLFDDCTQQFKAEKLKEKLKMKEREEAWVKIENLAKANPQYAVYSQASAVSIPVAMETDGPQFEDVQMLKKTVSDEARQAQKELKKDRPLVRRKSELPQDPHTEKALEAHCRASELLSQDGR SEQ ID NO.3: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 PPP2R5C-S497 site, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.1; the serine residue at the seventh site of the polypeptide is phosphorylated.

2. A method for preparing a polyclonal antibody for detecting the phosphorylation level of PPP2R5C-S497, characterized in that, Includes the following steps: S1: Synthesize a polypeptide with the amino acid sequence shown in SEQ ID NO.1, phosphorylate the serine at position 7 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: The antiserum from S2 was purified to obtain the PPP2R5C-S497 phosphorylated polyclonal antibody.

3. The preparation method according to claim 2, characterized in that, The adjuvant is any one or more of Freund's complete adjuvant, Freund's incomplete adjuvant, and aluminum adjuvant.

4. The preparation method according to claim 2, characterized in that, The immunized animals are immunized 3-5 times.

5. A polyclonal antibody for detecting the phosphorylation level of the PPP2R5C-S497 site, obtained by the preparation method according to any one of claims 2-4.

6. The use of the polyclonal antibody as described in claim 5 in the preparation of a reagent for detecting the phosphorylation level of the PPP2R5C-S497 site.

7. The application according to claim 6, 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.

8. A kit for detecting phosphorylation levels at the PPP2R5C-S497 site, characterized in that, The kit contains the polyclonal antibody as described in claim 5.

9. The detection kit according to claim 8, characterized in that, The kit can be used for screening and prognostic detection of heart failure.

10. A pharmaceutical composition for relieving heart failure, characterized in that, The pharmaceutical composition comprises the polyclonal antibody of claim 5.

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