Antigen peptides targeting ser572 phosphorylation of drp1 protein, antibodies and applications thereof
By preparing antigenic peptides and antibodies targeting the phosphorylation of DRP1 protein Ser572, the problem of the inability to identify DRP1 Ser572 phosphorylation in existing technologies has been solved, enabling specific identification of this site and research on disease mechanisms, and providing diagnostic and therapeutic tools.
Patent Information
- Application Number
- CN202610833339.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-10
- Publication Date
- 2026-08-25
AI Technical Summary
There is a lack of commercially available antibodies that can specifically identify the Ser572 phosphorylation site of the DRP1 protein, making it impossible to accurately quantify the modification level at this site, which affects the research and treatment of diseases such as cardiovascular disease and sepsis.
We designed and synthesized an antigenic peptide targeting Ser572 phosphorylation of the DRP1 protein, prepared a specific antibody, and obtained an antibody that can recognize Ser572 phosphorylation through animal immunization and affinity purification techniques. This antibody was then used to prepare ELISA kits and pharmaceutical compositions.
It achieves highly specific recognition of DRP1Ser572 phosphorylation, can stably recognize the modification at this site at the cellular and animal levels, reveals its molecular mechanism in disease, supports research on the basic mechanisms of disease and clinical translation, and provides diagnostic and therapeutic tools.
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Figure CN122629022A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to antigenic peptides, antibodies, and their applications that target phosphorylation of the DRP1 protein Ser572. Background Technology
[0002] Mitochondria are the core energy metabolism organelles of eukaryotic cells, and their division-fusion homeostasis is regulated by dynamics-associated protein 1.
[0003] As a member of the large GTPase family, DRP1 mediates mitochondrial constriction by assembling higher-order structures in the outer mitochondrial membrane. Its abnormal function is closely related to a variety of major diseases such as cardiovascular disease and neurodegenerative diseases.
[0004] The variable domain of DRP1 is the core region for its activity regulation. The two phosphorylation sites, Ser616 and Ser637, have been studied most extensively. They regulate mitochondrial dynamics by promoting and inhibiting the mitochondrial translocation of DRP1, respectively.
[0005] Recent studies have found that DRP1 can also alter its function by forming protein condensates through liquid-liquid phase separation. Previous studies in this application have confirmed for the first time that phosphorylation at the Ser572 site of the variable domain of DRP1 is the key to regulating its liquid-liquid phase separation. Abnormal modification of this domain can directly lead to myocardial mitochondrial damage and cardiac dysfunction.
[0006] However, there are currently no commercially available antibodies or related technologies that can specifically identify the DRP1 Ser572 phosphorylation site. Existing DRP1 antibodies can only detect the phosphorylation status of total protein or Ser616 and Ser637 sites, and cannot accurately quantify the modification level of the Ser572 site. Summary of the Invention
[0007] The main objective of this invention is to provide antigenic peptides, antibodies, and their applications that target the phosphorylation of DRP1 protein Ser572, which can effectively solve the problems mentioned above.
[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An isolated antigenic peptide having immunogenicity targeting the Ser572 phosphorylation site of the DRP1 protein, wherein the amino acid sequence of the antigenic peptide is RETKNVA{piSer}GGGGVGC, where {piSer} indicates that the serine residue at this site is phosphorylated.
[0009] An antigen construct comprising the antigenic peptide and a carrier for loading or coupling the antigenic peptide.
[0010] Preferably, the carrier is selected from either keyhole hemocyanin or bovine serum albumin.
[0011] Preferably, the application of the antigenic peptide or the antigen construct is any one of the following: (1) Application in the preparation of Ser572 phosphorylation-specific antibodies against DRP1 protein; (2) Application in the preparation of vaccines for diseases related to DRP1 protein Ser572 phosphorylation.
[0012] An antibody that specifically binds to the antigenic peptide or the antigen construct.
[0013] Preferably, the method for preparing the antibody includes the steps of immunizing an animal with the antigen peptide or the antigen construct, collecting antiserum, and purifying it. The purification process employs an affinity separation-affinity purification cycle technique. First, non-phosphorylated specific antibodies in the antiserum are removed using a chromatography column coupled with a non-phosphorylated DRP1Ser572 antigen peptide. Then, the target specific antibody is purified using a chromatography column coupled with a phosphorylated DRP1Ser572 antigen peptide.
[0014] Preferably, the animal is selected from rabbits or mice.
[0015] An ELISA kit comprising the antibody described herein or an antibody obtained by the preparation method described herein.
[0016] A pharmaceutical composition comprising the antibody described herein or an antibody prepared by the method described herein, and a pharmaceutically acceptable excipient or carrier.
[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes a specific antigenic peptide targeting the Ser572 phosphorylation site of the DRP1 protein. The antibody prepared based on this antigenic peptide has extremely strong site specificity, can recognize DRP1 protein phosphorylated at the Ser572 site, and does not cross-bind with non-phosphorylated DRP1 or phosphorylated DRP1 at the Ser616 / Ser637 sites.
[0018] The antibody prepared by this invention can stably recognize DRP1Ser572 phosphorylation modification at the cellular and animal levels, and can clearly reveal the molecular mechanism by which phosphorylation at this site regulates DRP1 liquid-liquid phase separation and mediates myocardial mitochondrial damage. It can not only support the basic mechanism research of diseases such as sepsis, but also be used to develop specific ELISA kits and drug compositions, filling the technical gap in the research and clinical translation of DRP1Ser572 phosphorylation, and providing new technical support for the diagnosis and treatment of major diseases such as cardiovascular diseases and sepsis. Attached Figure Description
[0019] Figure 1 This is a sequencing alignment diagram of the Ser572 site of the DRP1 protein in this invention; a DRP1 protein expression vector containing DRP1 protein and a DRP1 protein expression vector with Ser572 dephosphorylation were constructed; Figure 2 Lanes 1, 2, and 3, arranged vertically in the middle, are all Western Blot results of cell lysates obtained from AC16 cells lacking DRP1 transfected with pCMV-3xFLag-DRP1-WT. Lane 4 shows the Western Blot results of cell lysates obtained from AC16 cells lacking DRP1 transfected with pCMV-3xFLag-DRP1-Ser572Mutant. In DRP1-deficient AC16 cells, DRP1-WT and DRP1-Ser572 mutants were reintroduced; detection was performed using a self-made antibody targeting Ser572 phosphorylation. Figure 2 As shown, after LPS stimulation, phosphorylation of DRP1 at the Ser572 site in the WT group increased significantly, while histone expression in the S572A mutant decreased, indicating that the S572A mutation was successful and the mutation effect was clear. Figure 3 This is a schematic diagram of Western Blot detection of the phosphorylation signal of DRP1 protein Ser572 site in mouse myocardial tissue using the self-made antibody of the present invention. Figure 4 In myocardial-specific DRP1 knockout (DRP1^KO) mice, different DRP1-related constructs were reintroduced via tail vein injection of the pAAV vector: from left to right, pAAV-cTNTo-mCherry-3xFLAG-tWPA, pAAV-cTNTo-mCherry-linker-Dnm1I-3xFLAG-tWPA, and pAAV-cTNTo-mCherry-linker-Dnm1I(S548A)-3xFLAG-tWPA; (the mouse Ser548 site is a homologous site to the human Ser572 site). In vivo imaging of the small animals showed the localization signal of the virus in myocardial tissue, indicating that the relevant gene reintroduction was successful. Figure 5Mid-A position characteristic M-mode echocardiography images, expressing pAAV-cTNTo-mCherry-3xFLAG-tWPA, pAAV-cTNTo-mCherry-linker-Dnm1I-3xFLAG-tWPA, and pAAV-cTNTo-mCherry-linker-Dnm1I(S548A)-3xFLAG-tWPA, respectively; (B, C) quantitative analysis of left ventricular ejection fraction and left ventricular shortening fraction; data are expressed as mean SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Compared with the control group, LPS stimulation of Drp1-S548A mutant rescue alleviated the downregulation of myocardial function indicators such as LVEF and LVFS induced by Drp1-WT; Figure 6 This is a schematic diagram of the complementation experiment in myocardial-specific DRP1 knockdown mice stimulated by LPS. Mice were injected via tail vein with AAV expressing empty vector (control), DRP1-WT complementation, and DRP1-S548A mutant complementation. H&E staining showed that myocardial cells in the WT complementation group exhibited significant swelling and necrosis under LPS stimulation, while S548A mutant complementation alleviated cell damage, suggesting that the S548A mutation has a partial protective effect on the myocardium. Figure 7 This diagram illustrates the morphological changes of mitochondria in myocardial-specific DRP1 knockout mice under LPS stimulation. Mice were administered AAV expression empty vector via tail vein injection as a control, DRP1-WT complementation, and DRP1-S548A mutant complementation. Transmission electron microscopy revealed significant disorder of the inner cristae of myocardial mitochondria in the WT complementation group, while S548A mutant complementation alleviated the damage to the inner cristae structure, suggesting that the S548A mutation partially protects mitochondrial structure. Figure 8 This diagram illustrates how the Ser572 loss-of-function mutation alleviates DRP1-mediated mitochondrial membrane potential. In AC16 cardiomyocytes, DRP1 knockout was followed by reintroduction of DRP1-WT and DRP1-S572A mutants, respectively. Mitochondrial membrane potential was assessed using TMRE staining. Confocal microscopy showed that the mitochondrial membrane potential was significantly reduced in the WT reintroduction group, while the membrane potential remained at a higher level in the S572A mutant reintroduction group, suggesting that the Ser572 loss-of-function mutation can partially offset the damage of DRP1 to mitochondrial membrane potential. Figure 9This diagram illustrates the regulation of maximum mitochondrial respiratory capacity in AC16 human cardiomyocytes by DRP1-Ser572 phosphorylation. In AC16 cells with DRP1 knockdown, DRP1-WT and DRP1-S572A mutants were reintroduced, and the changes in extracellular acidification rate and oxygen consumption rate over time were measured. The right figure shows the bar chart quantification of maximum respiratory capacity. Results showed that WT reintroduction significantly restored mitochondrial respiratory function, while the S572A mutant reintroduction group partially restored respiratory function. Data are expressed as mean SEM. Statistical analysis was performed using one-way ANOVA with Tukey post-hoc test: **P<0.01, ***P<0.001; Figure 10 To observe the purified DRP1 protein under a 60x microscope using fluorescence microscopy. The image shows DRP1 spontaneously forming fluorescent spots of varying sizes in solution (green signal), indicating phase separation and droplet formation. The yellow arrows indicate representative droplet structures. Scale bar: 20m; Figure 11 To express Empty-eGFP, Drp1-FL-eGFP, and Drp1-IDR-deletion-eGFP in live cells, their dynamic characteristics were analyzed using fluorescence microscopy combined with fluorescence recovery after photobleaching (FRAP) experiments. Pre-bleach represents the state before bleaching, Bleach (0s) represents the instantaneous local photobleaching, and Recovery (4min) represents the recovery status after bleaching. Compared to Empty-eGFP, Drp1-FL-eGFP forms distinct spot-like or droplet-like structures within cells (indicated by yellow arrows), and its fluorescence signal gradually recovers over time after bleaching, suggesting dynamic exchange characteristics. In contrast, the structure formed by the Drp1-IDR deletion mutant exhibits significantly reduced fluorescence recovery ability. The right side shows the FRAP quantitative analysis results, indicating that the fluorescence recovery degree of Drp1-FL-eGFP is significantly higher than that of Drp1-IDR-deletion-eGFP. These results indicate that DRP1 protein can undergo LLPS within cells, and its IDR region plays an important role in this process. Figure 12To express Drp1-FL-eGFP and Drp1-dri3-deletion-eGFP intracellularly, respectively, their molecular dynamics were analyzed using fluorescence recovery photobleaching (FRAP) assays. The left side shows representative fluorescence images: Pre-bleach (before bleaching), Bleach (0s) (instantaneous photobleaching), and Recovery (4 min 30s) (after bleaching). Drp1-FL-eGFP formed a regular droplet-like structure, and its fluorescence signal recovered significantly after bleaching, indicating a strong molecular dynamic exchange capacity. In contrast, the mutant lacking the driving residues-3 region within the IDR (Drp1-dri3-deletion-eGFP) exhibited an irregular aggregate-like structure, and its fluorescence recovery was significantly restricted. The right side shows the FRAP quantitative analysis results, showing that the fluorescence recovery level of Drp1-FL-eGFP was significantly higher than that of Drp1-dri3-deletion-eGFP. The above results indicate that the drivingresidues-3 region within the IDR plays a crucial driving role in the liquid-liquid phase separation of the DRP1 protein. Scale bar: 5m; Figure 13 The Ser572 mutation weakens the liquid-liquid phase separation ability of the DRP1 protein;
[0020] Drp1-FL-eGFP and Drp1-Ser572Ala-eGFP were expressed intracellularly, and their molecular dynamics were analyzed using fluorescence recovery after photobleaching (FRAP) assays. The left side shows representative fluorescence images: Pre-bleach (before bleaching), Bleach (0s) (instantaneous photobleaching), and Recovery (4 min 30s) (after bleaching). Drp1-FL-eGFP formed a typical droplet-like structure in the cell (indicated by the yellow arrow), and the fluorescence signal recovered significantly over time after bleaching, suggesting a strong molecular dynamic exchange capacity. In contrast, the Drp1-Ser572Ala-eGFP mutant formed an irregular structure with significantly reduced fluorescence recovery. The right side shows the FRAP quantitative analysis results, indicating that the fluorescence recovery level of Drp1-Ser572Ala-eGFP was significantly lower than that of Drp1-FL-eGFP. These results indicate that the Ser572 site plays an important regulatory role in the phase separation ability and dynamic characteristics of the DRP1 protein. Scale: 5m; Figure 14 This diagram illustrates the screening of the DRP1Ser572 site and the construction of related plasmids. Detailed Implementation
[0021] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with experimental data. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.
[0022] Previous research in this application revealed that DRP1 (Dynamics-associated protein 1), as a core large GTPase regulating mitochondrial division, has a key site at the Ser572 site within its variable domain that regulates protein liquid-liquid phase separation. Under pathological conditions such as sepsis, phosphorylation modification at this site significantly alters the aggregation state and functional activity of DRP1, thereby mediating myocardial mitochondrial damage and dysfunction. Therefore, this application discloses an antigenic peptide targeting the Ser572 phosphorylation site of DRP1 protein and a specific antibody prepared therefrom, capable of accurately identifying the phosphorylation form of DRP1 at the Ser572 site in cells and tissues, providing a core tool for exploring the mechanism of action of this modification in diseases.
[0023] like Figure 1-14 As shown, antigenic peptides, antibodies, and their applications target phosphorylation of the DRP1 protein at Ser572.
[0024] Example 1: Preparation of an antibody targeting Ser572 phosphorylation of DRP1 protein; Step 1: Antigen peptide design and synthesis Based on the amino acid sequence of the human DRP1 protein, a phosphorylated antigenic peptide was designed and synthesized from the Ser572 site and the 67 amino acid residues upstream and downstream of it. The sequence is RETKNVA{piSer}GGGGVGC, where {piSer} represents phosphorylation of serine residue 572. Simultaneously, a control peptide without phosphorylation at the corresponding site was synthesized for subsequent antibody purification and specificity detection. All peptides were prepared using solid-phase synthesis and purified by high-performance liquid chromatography (HPLC) to a purity of over 95%.
[0025] The antigenic peptides used in this experiment and the corresponding information on the immunized animals are summarized below: polypeptide number Sequence information Immunized animal numbering TP61389 RETKNVA{piSer}GGGGVGC RB12167, RB12168, RB12169 Step 2: The antigen peptide is coupled to the carrier protein; Phosphorylated antigenic peptides and non-phosphorylated control peptides were conjugated to carrier proteins, respectively. The product conjugated with keyhole hemocyanin (KLH) was used as an immunogen for animal immunization, while the product conjugated with bovine serum albumin (BSA) was used for subsequent ELISA detection and affinity chromatography purification. The specific conjugation procedure is as follows: 1. Dissolve 20 mg KLH in 2 mL of an aqueous solution containing 5 mM EDTA, and set aside. 2. Weigh 8 mg of Sulfo-SMCC crosslinking agent and dissolve it completely in 50 LDMSO. Add 150 L PBS and mix well. Then add it dropwise to KLH solution and gently shake at room temperature for 1 hour. 3. Place the activated KLH solution into a dialysis bag and dialyze in 2L PBS at 4°C. Change the dialysate every 2 hours for a total of 3 dialysis cycles. 4. Weigh 4 mg of the synthesized peptide and dissolve it in 50 LDMSO. Add 200 L PBS and mix quickly. Add activated KLH at a ratio of peptide:KLH = 1 mg: 680 g and react at 4°C overnight. 5. After the reaction is complete, dialyze again to remove uncoupled free peptides. Aliquot the coupling product and store at -20°C for later use. The same method is used to couple the non-phosphorylated control peptide to BSA.
[0026] Step 3: Animal immunization and antiserum collection; Three healthy SPF-grade New Zealand White rabbits (numbers RB12167, RB12168, and RB12169) were selected and vaccinated after two weeks of pre-fostering. The specific immunization procedure is as follows: 1. Initial immunization: The phosphorylated antigen peptide-KLH conjugate and Freund's complete adjuvant are mixed at a 1:1 volume ratio and repeatedly aspirated with a double syringe until completely emulsified (it does not diffuse when dropped into 37°C water). The mixture is then injected subcutaneously at multiple points in the neck, back, and buttocks, with a total injection volume of 0.5 mL per syringe and an antigen dose of approximately 0.5 mg. 2. Booster immunization: The second immunization is given on day 14 after the first immunization, and the third immunization is given on day 21. Freund's incomplete adjuvant is used for both immunizations, the antigen dose is halved, and the injection method is the same as for the first immunization. 3. Blood Test and Immunization: On day 7 after the third immunization, 8 mL of blood was collected from the central artery of the rabbit's ear. After separating the serum, the titer was detected by ELISA. Once the titer reached the target level, a final immunization was performed on day 28, and whole blood was collected 7 days later using the cardiac blood sampling method. 4. Serum separation: Place the collected blood in a 37°C water bath for 30 minutes, let it stand overnight at 4°C to allow the blood clots to shrink, centrifuge at 4000 rpm for 10 minutes, and collect the supernatant as antiserum. Add 0.02% thimerosal for preservation, aliquot and store at -20°C.
[0027] Step 4: Preliminary detection of antiserum titer The antiserum titer was detected by indirect ELISA. The experimental conditions were uniformly set as follows: coating amount 1 g / ml 50 μL, coating conditions 37℃ for 2 h; blocking buffer 1% BSA-PBST, blocking volume 150 μL, blocking conditions 37℃ for 1 h; primary antibody serially diluted test serum, volume 50 μL, incubation conditions 37℃ for 30 min; secondary antibody 1 / 35000 diluted GoatAntiRabbitIgG-HRP, volume 50 μL, incubation conditions 37℃ for 45 min; and color development time 37℃ for 10 min.
[0028] The results of the highest-potency rabbit antiserum RB12167 are as follows: Antiserum dilution OD450 value of coated phosphorylated peptide OD450 value of coated nonphosphorylated peptide 1% BSA blank control OD450 value 1:250 2.595 2.483 0.023 1:1000 2.503 2.105 0.015 1:4000 2.063 1.618 0.008 1:16000 1.826 0.832 0.007 1:64000 1.293 0.261 0.007 1:256000 0.592 0.079 0.007 1:1024000 0.256 0.026 0.007 The test results showed that the antiserum from rabbit RB12167, at a dilution of 1:64000, still maintained an OD450 value of 1.293 for the phosphorylated antigen peptide, while the binding signal for the non-phosphorylated control peptide was significantly reduced (OD450=0.261), indicating that the antiserum produced by immunization had good phosphorylation specificity. The antiserum titers from the other two rabbits were slightly lower, but both met the requirements for subsequent purification.
[0029] Example 2: Affinity purification of the antibody and detection of its titer after purification; The antiserum was purified using an affinity separation-affinity purification cycle to remove non-specific antibodies and non-phosphorylated specific antibodies. The specific steps are as follows: 1. Pretreatment of chromatography columns: Affinity chromatography columns coupled with non-phosphorylated peptide-BSA and phosphorylated peptide-BSA were prepared respectively, and thoroughly washed with 20 mL of pure water and 20 mL of 1 PBS (pH 7.4) at a flow rate of 70 mL / h. 2. Sample loading: Take 10 mL of antiserum from rabbit RB12167, filter it through a 0.45 μm microporous membrane, and then pass it through a non-phosphorylated peptide-BSA chromatography column at a flow rate of 40 mL / h. Collect the eluent. This step can remove the antibody that recognizes non-phosphorylated DRP1 in the antiserum. 3. Purification of target antibody: Pass the above effluent through a phosphorylated peptide-BSA chromatography column at the same flow rate, wash away unbound contaminating proteins with 20 mL of 1 PBS until the absorbance reading of the protein detector stabilizes at the baseline; 4. Antibody elution: Elute the bound antibody with 0.2M glycine-hydrochloric acid buffer (pH 2.7) at a flow rate of 40 mL / h. Start collecting when the absorbance begins to increase, collecting 1 mL from each tube, and immediately adjust the pH to about 7.0 with 1M Tris-HCl. 5. Concentration and storage: Combine the antibody solutions corresponding to the elution peaks, concentrate to a suitable concentration using an ultrafiltration concentrator, sterilize through a 0.22 μm filter membrane, add 25% glycerol, aliquot, and store at -80°C.
[0030] After purification, the titer of the purified antibody was detected using the same ELISA experimental conditions as in Example 1. The detection results of the purified antibody from rabbit RB12167 are as follows: antibody dilution OD450 value of coated phosphorylated peptide OD450 value of coated nonphosphorylated peptide 1% BSA blank control OD450 value 1:250 2.903 2.430 0.008 1:1000 2.467 1.376 0.010 1:4000 2.289 0.619 0.036 1:16000 2.042 0.189 0.032 1:64000 1.431 0.070 0.032 1:256000 0.744 0.040 0.032 1:1024000 0.287 0.033 0.032 Comparing the data before and after purification, it can be seen that the purified antibody slightly improved its binding ability to phosphorylated antigen peptides, while significantly decreasing its binding ability to non-phosphorylated control peptides. At a dilution of 1:16000, the OD450 value of the purified antibody for non-phosphorylated peptides decreased from 0.832 to 0.189, indicating that the phosphorylation site specificity of the purified antibody was greatly enhanced.
[0031] Example 3, Identification of antibody specificity; Cellular-level specificity verification 1. Plasmid construction: Wild-type DRP1 expression plasmid carrying the 3FLAG tag (pCMV-3xFLAG-DRP1-WT) and mutant plasmid with Ser572 mutated to alanine (pCMV-3xFLAG-DRP1-S572A) were constructed respectively. 2. Cell transfection and treatment: Human AC16 cardiomyocytes were seeded in 6-well plates and cultured to 60%-70% confluence. Cells were then transfected with the two plasmids described above. Twenty-four hours after transfection, cells were stimulated with 10 ng / mL LPS for 24 hours. A group of cells transfected with the WT plasmid was treated with protein phosphatase (PPase) as a negative control. 3. Western blot assay: Cells were collected, and total protein was extracted using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. SDS-PAGE electrophoresis was performed, followed by membrane transfer. The DRP1Ser572 phosphorylation antibody prepared in this invention was used as the primary antibody (1:1000 dilution), and the cells were incubated overnight at 4°C. The next day, HRP-labeled goat anti-rabbit secondary antibody was added, and ECL chemiluminescence imaging was performed.
[0032] The results showed that a clear, specific band of approximately 70 kDa was detected in cell samples transfected with DRP1-WT and stimulated with LPS; however, no band was detected in samples transfected with the DRP1-S572A mutant; and the band signal completely disappeared in WT samples treated with PPase. This indicates that the antibody of the present invention recognizes only phosphorylated DRP1 protein at the Ser572 site and does not recognize unphosphorylated or mutant forms of DRP1, exhibiting extremely high site specificity.
[0033] Animal-level specificity verification 1. Establishment of a mouse model of sepsis: Male C57BL / 6J mice aged 8-10 weeks were selected and a sepsis model was established by intraperitoneal injection of LPS (10 mg / kg). The control group was injected with an equal volume of physiological saline. 2. Tissue sample preparation: 24 hours after modeling, mice were sacrificed, myocardial tissue was collected, and total protein was extracted by cryogenic grinding with liquid nitrogen and then added to pre-cooled RIPA lysis buffer. 3. Western blot assay: The same method as at the cellular level is used for the assay.
[0034] The results showed that a significant DRP1Ser572 phosphorylation band could be detected in the myocardial tissue of mice in the sepsis model group, while the band signal in the control group mice was extremely weak. This indicates that the antibody of the present invention can effectively recognize the phosphorylation modification of the DRP1 homologous site (Ser548) in mice and can be used for in vivo experimental studies at the animal level.
[0035] Example 4: Application of antibodies in the study of the mechanism of myocardial injury in sepsis; To verify the practical application value of the antibody of this invention, we used it to detect the effect of the DRP1Ser572 mutation on myocardial function in septic mice. First, myocardial-specific DRP1 knockout mice were constructed. Then, adeno-associated virus (AAV) was reinjected via tail vein injection into wild-type DRP1 (WT group) and S548A mutant DRP1 (mutant group), respectively. Four weeks later, the mice were subjected to LPS stimulation to establish a DRP1 knockout model. Cardiac function was assessed using small animal ultrasound, and the phosphorylation level of DRP1Ser572 in myocardial tissue was detected using the antibody of this invention.
[0036] The results showed that, compared with the WT group, the mutant group mice had significantly increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), significantly reduced cardiomyocyte swelling and necrosis, and improved mitochondrial cristae structure disorder. Simultaneously, antibody detection results showed that the phosphorylation level of DRP1Ser572 in the myocardium of WT group mice was significantly increased, while no phosphorylation signal was detected in the mutant group. This indicates that phosphorylation at the DRP1Ser572 site plays an important role in septic myocardial injury, and the antibody of this invention provides a reliable detection tool for further research on this mechanism.
[0037] Example 5: Experimental study on the liquid-liquid phase separation mechanism of DRP1 and the regulatory role of the Ser572 site; 1. Experimental Materials and Methods (1) Plasmid construction: Based on DRP1 protein sequence analysis, a series of eGFP fusion expression plasmids were screened and constructed, including: Empty-eGFP empty vector control, full-length DRP1-eGFP fusion plasmid, intrinsic disorder region deletion mutant plasmid, IDR driving residues-3 region deletion mutant plasmid, and a mutant plasmid with a Ser572 site mutation to alanine. All plasmids were sequenced to verify their correct sequence.
[0038] (2) In vitro expression and purification of DRP1 protein: Recombinant DRP1 protein was expressed and purified using a prokaryotic expression system. High-purity protein was obtained by Ni-NTA affinity chromatography and gel filtration chromatography. The protein concentration was adjusted to an appropriate range for in vitro phase separation and observation.
[0039] (3) Cell culture and transfection: Human HEK293T cells or AC16 cardiomyocytes were cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator. Cells were seeded in confocal culture dishes, and when the confluence reached 50%-60%, the plasmids were transfected using Lipofectamine 3000 transfection reagent.
[0040] (4) Fluorescence microscopy observation: 24-48 hours after transfection, observation was performed using a laser confocal scanning microscope. The in vitro purified protein samples were placed on a glass slide and the spontaneous aggregation of DRP1 protein was observed under a 60x oil immersion microscope; the fluorescence distribution pattern of live cell samples was observed directly under a microscope.
[0041] (5) Photobleaching experiment after fluorescence recovery: The experiment was conducted using the FRAP module of a laser confocal microscope. Typical DRP1 condensate structures were selected from the cells, and the selected areas were locally photobleached using a high-power laser. Subsequently, fluorescence images were continuously acquired, and the fluorescence recovery process of the bleached areas was recorded for 4-4.5 minutes. The experiment was repeated at least 3 times, and representative results were selected for quantitative analysis.
[0042] 2. Experimental Procedures and Results Analysis (1) Observation of spontaneous phase separation of DRP1 protein in vitro: The in vitro purified DRP1 protein was placed in physiological buffer and observed under a 60x fluorescence microscope. DRP1 protein spontaneously formed fluorescent spot-like structures of varying sizes in solution; the yellow arrows indicate representative droplet structures. This result directly demonstrates that DRP1 protein can spontaneously undergo liquid-liquid phase separation and form droplet-like aggregates in vitro without any other cofactors. Scale bar: 20m.
[0043] (2) Phase separation characteristics of DRP1 in living cells and the role of the IDR region: Empty-eGFP, Drp1-FL-eGFP, and Drp1-IDR-deletion-eGFP plasmids were transfected into live cells, and their dynamic characteristics were observed using fluorescence microscopy and analyzed using FRAP experiments.
[0044] Experimental results are as follows Figure 11 As shown: Pre-bleach represents the state before bleaching, Bleach represents the instantaneous local photobleaching, and Recovery represents the recovery status after bleaching. Compared with the Empty-eGFP control group, Drp1-FL-eGFP forms obvious spot-like or droplet-like structures in cells, and the fluorescence signal gradually recovers over time after bleaching, suggesting that it has dynamic exchange characteristics unique to liquid-liquid phase separation.
[0045] In contrast, while the Drp1-IDR deletion mutant can also form aggregates, its fluorescence recovery ability is significantly reduced. FRAP quantitative analysis on the right side shows that the fluorescence recovery of Drp1-FL-eGFP is significantly higher than that of Drp1-IDR-deletion-eGFP. These results indicate that DRP1 protein can undergo liquid-liquid phase separation within the cell, and its intrinsically disordered region (IDR) plays an important role in this process.
[0046] (3) The key driving role of the driving residues-3 segment within the IDR: To further locate the key sequences regulating phase separation in the IDR region, Drp1-FL-eGFP and Drp1-dri3-deletion-eGFP were expressed in cells, and their molecular dynamics were analyzed using FRAP experiments.
[0047] Experimental results are as follows Figure 12 As shown: The left side displays representative fluorescence images, including three time points: Pre-bleach, Bleach, and Recovery. Drp1-FL-eGFP forms a regular droplet-like structure, and its fluorescence signal recovers significantly after bleaching, suggesting a strong molecular dynamic exchange capacity. In contrast, the mutant lacking the driving residues-3 region within the IDR exhibits an irregular aggregate-like structure, and its fluorescence recovery is significantly limited. The FRAP quantitative analysis results on the right show that the fluorescence recovery level of Drp1-FL-eGFP is significantly higher than that of Drp1-dri3-deletion-eGFP. These results indicate that the driving residues-3 region within the IDR plays a crucial driving role in the liquid-liquid phase separation of the DRP1 protein. Scale bar: 5m.
[0048] (4) Effect of the Ser572 mutation on the phase segregation ability of DRP1: To verify the regulatory role of the Ser572 site in DRP1 phase separation, Drp1-FL-eGFP and Drp1-Ser572Ala-eGFP were expressed in cells, and their molecular dynamics were analyzed using FRAP assays. Experimental results are as follows Figure 13 As shown: The left side displays representative fluorescence images, including three time points: Pre-bleach, Bleach, and Recovery. Drp1-FL-eGFP forms a typical droplet-like structure within the cell, and the fluorescence signal recovers significantly over time after bleaching, suggesting that it has a strong molecular dynamic exchange capacity.
[0049] In contrast, the Drp1-Ser572Ala-eGFP mutant exhibits an irregular morphology and significantly reduced fluorescence recovery. FRAP quantitative analysis on the right side shows that the fluorescence recovery level of Drp1-Ser572Ala-eGFP is significantly lower than that of Drp1-FL-eGFP. These results indicate that the Ser572 site plays a crucial regulatory role in the phase separation ability and dynamic properties of the DRP1 protein; mutations at this site can significantly weaken the liquid-liquid phase separation ability of DRP1 (scale bar: 5m).
[0050] 3. Experimental Conclusions This embodiment demonstrates through systematic in vitro and in vivo experiments that: (1) DRP1 protein has spontaneous liquid-liquid phase separation properties and can form droplet-like aggregates in vitro and in living cells; (2) The IDR region of DRP1 is the key structural domain that mediates its phase separation, among which the driving residues-3 segment plays a core driving role; (3) The Ser572 site is a key phosphorylation site that regulates the phase separation of DRP1. Mutations at this site can significantly weaken the phase separation ability and dynamic exchange characteristics of DRP1.
[0051] The above findings provide direct experimental evidence for understanding the molecular mechanism by which DRP1Ser572 phosphorylation regulates mitochondrial function, and also lay a theoretical foundation for the application of the antibody of this invention in the study of the mechanism of DRP1 phase separation-related diseases.
[0052] The above description is merely 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. An isolated antigenic peptide, characterized in that: The antigenic peptide has immunogenicity targeting the Ser572 phosphorylation site of the DRP1 protein, wherein the amino acid sequence of the antigenic peptide is RETKNVA{piSer}GGGGVGC, and {piSer} indicates that the serine residue at this site is phosphorylated.
2. An antigen construct, characterized in that: The antigen construct comprises the antigenic peptide of claim 1 and a carrier for loading or coupling the antigenic peptide of claim 1.
3. The antigen construct according to claim 2, characterized in that: The carrier is selected from either keyhole hemocyanin or bovine serum albumin.
4. The application of the antigenic peptide according to claim 1 or the antigen construct according to any one of claims 2-3, characterized in that, The application is any one of the following: (1) Application in the preparation of Ser572 phosphorylation-specific antibodies against DRP1 protein; (2) Application in the preparation of vaccines for diseases related to DRP1 protein Ser572 phosphorylation.
5. An antibody, characterized in that: The antibody specifically binds to the antigenic peptide of claim 1 or the antigen construct of any one of claims 2 to 3.
6. The method for preparing the antibody according to claim 5, characterized in that: The steps include immunizing animals with the antigen peptide of claim 1 or the antigen construct of any one of claims 2 to 3, collecting antiserum, and purifying it; The purification process employs an affinity separation-affinity purification cycle technique. First, non-phosphorylated specific antibodies in the antiserum are removed using a chromatography column coupled with a non-phosphorylated DRP1Ser572 antigen peptide. Then, the target specific antibody is purified using a chromatography column coupled with a phosphorylated DRP1Ser572 antigen peptide.
7. The method for preparing the antibody according to claim 6, characterized in that, The animal is selected from rabbits or mice.
8. An ELISA kit, characterized in that, The ELISA kit includes the antibody as described in claim 5 or the antibody prepared by any one of claims 6 to 7.
9. A pharmaceutical composition, characterized in that, It includes the antibody as described in claim 5 or the antibody prepared by any one of claims 6 to 7, as well as pharmaceutically acceptable excipients or carriers.