A short peptide targeting trpm7 channel and its vaccine and pharmaceutical application for preventing and treating atrial fibrillation

Abstract

The application discloses a short peptide for TRPM7 channel, a vaccine and pharmaceutical application of the short peptide for preventing and treating atrial fibrillation, and belongs to the technical field of biological medicine. The short peptide can be coupled with a carrier KLH by a glutaraldehyde coupling method to prepare a conjugate, and the conjugate is supplemented with an adjuvant to form a vaccine. The vaccine can induce the body to produce high-specificity and high-titer antibodies through active immunization, the antibodies can block the TRPM7 channel, inhibit calcium influx of atrial muscle cells and atrial fibroblasts, reduce calcium overload of cytoplasm, inhibit atrial electrical remodeling and tissue remodeling, and prevent and treat atrial fibrillation. The application provides a pharmaceutical composition containing the short peptide, the conjugate and the adjuvant, and a polyclonal antibody obtained by immunizing a non-human animal with the vaccine. Compared with existing chemical drugs, the application has the advantages of high targeting, long-acting, good safety, low price and the like, provides a new active immunotherapy strategy for atrial fibrillation patients, and has a wide clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a short peptide targeting the TRPM7 channel, its vaccine, and its pharmaceutical applications in the prevention and treatment of atrial fibrillation. Background Technology

[0002] Atrial fibrillation (AF) is one of the most common arrhythmias in clinical practice. AF not only causes palpitations and discomfort, but its high disability and heart failure rates seriously endanger human health. With the increasing aging of the population, the prevalence of AF is showing a continuous upward trend, becoming a global public health problem. Currently, clinical treatment for AF mainly involves pharmacological cardioversion / maintaining sinus rhythm, controlling ventricular rate combined with anticoagulation therapy, and interventional / surgical treatments such as catheter radiofrequency ablation and surgical maze procedures. While pharmacological and surgical treatments can partially improve AF symptoms or suppress AF attacks, existing treatments have significant limitations: antiarrhythmic drugs have significant side effects, long-term anticoagulation therapy increases the risk of bleeding, and the recurrence rate of AF is high; although catheter radiofrequency ablation or surgical maze procedures can partially improve symptoms, the recurrence rate remains high in patients with significant left atrial enlargement or heart failure, and the treatment costs are expensive. Therefore, actively exploring new avenues for treating AF is urgently needed.

[0003] Currently, all new drug development for atrial fibrillation (AF) both domestically and internationally is based on chemical drugs. However, the success rate of developing chemical drugs for AF is low. The fundamental reasons are as follows: chemical drugs generally have poor target selectivity. Even if they have some selectivity, they rarely completely identify and distinguish different members of the same channel family (such as TRPC3 and TRPM7), and it is even more difficult to specifically block key targets of the same family members; chemical drugs have short half-lives, making it difficult to exert a highly efficient and sustained inhibitory effect on AF by stably and persistently blocking receptors or ion channels; the limitations of intracellular spatiotemporal distribution, dosage concentration, and metabolism further reduce the expected efficacy of chemical drugs in treating AF (New paradigms in cardiovascular medicine: emerging technologies and practices: perioperative genomics. Journal of the American College of Cardiology, 2005.46(11): p.1965-77). Unlike traditional drugs, short peptide vaccines mainly target specific receptors or ion channels. The target antibodies induced by these vaccines can highly specifically block important targets of receptors or channels. Furthermore, the target antibodies induced by short-peptide vaccines have the advantage of sustained titer; only 2-3 immunizations are needed to stimulate the body to produce sustained high-titer antibodies. Short-peptide vaccines also have advantages such as long half-life, fewer target organ complications, and relatively low cost. Given that therapeutic short-peptide vaccines can overcome the aforementioned shortcomings of traditional chemical drugs, the development of therapeutic short-peptide vaccines has become a new direction for the clinical treatment of intractable diseases.

[0004] In recent years, significant progress has been made in the research of short peptide vaccines targeting ion channels or receptors. The team led by Liao Yuhua at the Cardiovascular Institute of Wuhan Union Hospital has successfully developed short peptide vaccines targeting in vitro channels and receptors: an L-type calcium channel short peptide vaccine for treating refractory hypertension and an ETA receptor short peptide vaccine for treating pulmonary hypertension. These vaccines are ready to undergo Phase I clinical trials, with promising preliminary results. This indicates that short peptide vaccines targeting ion channels or receptors hold promise as an effective new approach for treating clinically challenging diseases. However, no publications or related national invention patents have yet been announced regarding the development of short peptide vaccines for treating atrial fibrillation.

[0005] Transient receptor potential channel M7 (TRPM7) is a bifunctional membrane protein with ion channel and protein kinase activities, capable of permeating calcium channels. 2+ and Mg 2+Existing research indicates that atrial myocyte calcium overload is one of the key pathological mechanisms in the occurrence and maintenance of atrial fibrillation (AF). Aberrant activation of the TRPM7 channel can mediate increased intracellular calcium ion concentration in atrial myocytes, inducing atrial electrical remodeling (such as early afterpolarization and delayed afterpolarization) and tissue remodeling (such as atrial fibrosis and inflammatory infiltration), thereby promoting the occurrence and development of AF. Therefore, the TRPM7 channel is a potential ideal target for AF intervention. In summary, current technologies lack a short-peptide vaccine that can specifically and persistently target the TRPM7 channel to prevent and treat AF. Summary of the Invention

[0006] In view of the current lack of short peptide vaccines that can specifically and persistently target the TRPM7 channel to prevent and treat atrial fibrillation, this invention aims to provide a short peptide targeting the TRPM7 channel, its vaccine, and its pharmaceutical applications in the prevention and treatment of atrial fibrillation.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides a short peptide targeting the TRPM7 channel, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Its amino acid sequence is GVPRKAIL (corresponding to positions 1015-1022 of the TRPM7 channel). This short peptide is designed based on the extracellular E3 loop structure of the TRPM7 channel. It has the characteristics of good hydrophilicity, high surface accessibility, strong antigenicity, and does not form α-helices. It is 8 amino acids in length and is suitable as an antigenic epitope.

[0009] Secondly, the present invention provides a conjugate comprising the aforementioned short peptide targeting the TRPM7 channel and a carrier. The carrier is preferably a carrier protein.

[0010] The carrier is hemocyanin KLH. KLH is chosen as the carrier protein because it has advantages such as large molecular weight, strong immunogenicity, and stable source. It can maximize the synergistic effect of helper T cells, improve the antibody titer and affinity induced by the short peptide vaccine, and ensure the vaccine's immunizing effect.

[0011] The short peptide targeting the TRPM7 channel is covalently linked to the carrier via glutaraldehyde coupling to form a complete antigen. This coupling method is simple to operate, highly efficient, and produces stable products, forming a uniform complete antigen. It avoids antigen dissociation caused by non-covalent adsorption, ensuring batch-to-batch quality stability of the vaccine.

[0012] Thirdly, the present invention provides a short peptide vaccine for the prevention or treatment of atrial fibrillation, comprising the aforementioned short peptide targeting the TRPM7 channel or the aforementioned conjugate.

[0013] The short peptide vaccine may also contain an adjuvant. Adding an adjuvant to the short peptide vaccine can further activate antigen-presenting cells and lymphocytes, prolong the residence time of antigens in the body, enhance the strength and persistence of the immune response, and significantly improve the vaccine's immunizing effect.

[0014] The preferred adjuvant is either Freund's complete adjuvant or Freund's incomplete adjuvant.

[0015] Freund's complete adjuvant can strongly activate the Th1 immune response when used for primary immunization, while Freund's incomplete adjuvant can maintain immune memory when used for booster immunization. The combination of the two can stably induce high-affinity, high-titer neutralizing antibodies, which has been fully verified in animal experiments.

[0016] Fourthly, the present invention provides a pharmaceutical composition comprising the above-mentioned short peptide, conjugate, or short peptide vaccine for the prevention or treatment of atrial fibrillation targeting the TRPM7 channel, and pharmaceutically acceptable excipients.

[0017] Fifthly, the present invention provides a polyclonal antibody obtained by immunizing non-human animals with the aforementioned vaccine. This antibody is capable of specifically binding to the extracellular E3 loop region of the TRPM7 channel, neutralizing TRPM7-mediated calcium ion influx.

[0018] The present invention also provides the use of the above-mentioned short peptides, conjugates, short peptide vaccines or polyclonal antibodies targeting the TRPM7 channel for the prevention or treatment of atrial fibrillation in the preparation of medicaments for the prevention or treatment of atrial fibrillation.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The short peptide targeting the TRPM7 channel provided by this invention is designed based on the key region of the extracellular E3 ring of the TRPM7 channel that borders the calcium ion selective sieve pore. This short peptide has the characteristics of good hydrophilicity, high surface accessibility, strong antigenicity, and non-formation of α-helices. As an antigenic epitope, it can accurately target the TRPM7 channel, providing a molecular basis for the subsequent induction of highly specific neutralizing antibodies, overcoming the defect of poor target selectivity of traditional chemical drugs in distinguishing different members of the same channel family. In addition, the target antibody induced by this short peptide vaccine also has the advantage of long-lasting titer; 2-3 immunizations are sufficient to stimulate the body to produce long-lasting high-titer antibodies.

[0020] When the above-mentioned short peptides are conjugated with carrier proteins, the short peptides are transformed from haptens into complete antigens, which significantly enhances their immunogenicity. This can effectively overcome the body's immune tolerance to its own TRPM7 channels and induce the production of high-titer specific antibodies, laying the foundation for active immunotherapy of atrial fibrillation.

[0021] Furthermore, short-peptide vaccines for treating atrial fibrillation primarily elicit a type 2 immune response after immunization, while avoiding the development of a type 1 immune response. Given that the main side effect of short-peptide vaccines is inducing a type 1 immune response from autoreactive T lymphocytes, which can cause damage to tissues and organs, this short-peptide vaccine does not cause long-term damage to major target organs such as the heart, lungs, and kidneys.

[0022] The short peptide vaccine for the prevention or treatment of atrial fibrillation provided by this invention comprises the aforementioned short peptide or conjugate. It induces the body to produce neutralizing antibodies against the TRPM7 channel through active immunization. These antibodies can persistently block TRPM7 function, inhibit calcium influx into atrial myocytes, and prevent and treat atrial fibrillation attacks, effectively overcoming the shortcomings of traditional antiarrhythmic drugs, such as short half-life and the need for frequent administration.

[0023] The pharmaceutical composition provided by this invention is easy to prepare into dosage forms such as injections, and can be administered via subcutaneous multi-point injection, which improves the convenience and compliance of medication and provides a feasible formulation for clinical translation.

[0024] The polyclonal antibody provided by this invention can be directly used for passive immunization to rapidly neutralize the abnormal activation of the TRPM7 channel in the body. It is suitable for the emergency treatment of acute atrial fibrillation and complements active immunization vaccines, thus enriching the clinical intervention methods of this invention.

[0025] This invention is the first to apply short peptides, conjugates, short peptide vaccines, or polyclonal antibodies targeting the TRPM7 channel to the preparation of drugs for the prevention or treatment of atrial fibrillation, opening up a completely new pathway for atrial fibrillation immunotherapy. This application can efficiently and persistently inhibit calcium influx into atrial myocytes and improve atrial electrical and structural remodeling, and has clear clinical translational value and broad market prospects. Attached Figure Description

[0026] Figure 1 This is a technical roadmap for the present invention.

[0027] Figure 2 This is a schematic diagram of the transmembrane structure and pore region of members of the TRPs family.

[0028] Figure 3 This is a schematic diagram of the spatial structure of the channel region and some structural domains of a member of the TRPs family, where a is the front view and b is the back view.

[0029] Figure 4 This is a spatial diagram of the repeat sequences, helical structures, and finger structures of members of the TRP family, where a represents each repeat sequence, inner helix, outer helix, and finger structure, with ATP shown as a rod-like structure; b is a spatial conformational diagram of Finger3, with black arrows indicating the Finger3 region of the ankyrin-like repeat sequence (ARD).

[0030] Figure 5 This is a schematic diagram of the spatial structure of the TRPs family, where a and b are the structures under cryogenic electron microscopy at different resolutions; c and d are the spatial structures of the complexes formed by TRPs and ligands and the pore regions, and the rod-shaped structures represent the key amino acids adjacent to the pore regions.

[0031] Figure 6 The software prediction and receptor hydrophobic "pocket" analysis diagrams for short peptide design show that the short peptide sequence falls into the hydrophobic pocket of the TRPM7 receptor. Among them, A is a schematic diagram of the short peptide binding to the first hydrophobic pocket of the TRPM7 receptor; B is a schematic diagram of the short peptide binding to the second hydrophobic pocket of the TRPM7 receptor; C is a schematic diagram of the short peptide binding to the third hydrophobic pocket of the TRPM7 receptor; D is a schematic diagram of the short peptide binding to the fourth hydrophobic pocket of the TRPM7 receptor; E is a schematic diagram of the short peptide binding to the fifth hydrophobic pocket of the TRPM7 receptor; and F is a schematic diagram of the short peptide binding to the sixth hydrophobic pocket of the TRPM7 receptor.

[0032] Figure 7 The image shows an SDS-PAGE electrophoresis diagram of the short peptide coupled with the KLH carrier, stained with Coomassie Brilliant Blue. The middle lane is the short peptide-KLH conjugate, and lanes 1 and 2 on the left are controls.

[0033] Figure 8 A graph showing the change in serum antibody titer over time after immunizing New Zealand rabbits with a short peptide vaccine and an adjuvant.

[0034] Figure 9 This diagram illustrates the purification process of the target antibody for a short peptide vaccine. In the diagram, A represents the elution curve of hydrophobic chromatography (Butyl-Sepharose FF column), and B represents the elution curve of affinity chromatography (Protein G-Sepharose 4FF column).

[0035] Figure 10 The image shows the immunofluorescence assay, illustrating the binding of the target antibody of the short peptide vaccine to the TRPM7 channel on the membrane of atrial myocytes (ATCC-0014). The cell nuclei were stained with Hoechst 33342.

[0036] Figure 11 Immunofluorescence assay of the effect of short peptide vaccine target antibody on NFATC4 nuclear translocation in atrial myocytes and atrial fibroblasts (Hoechst 33342 staining, FITC-labeled NFATC4).

[0037] Figure 12 The variation of cytoplasmic calcium ion concentration (Δ[Ca]) in different antibody treatment groups of this invention. 2+ Comparison of ]i) (Fluo-4AM probe detection).

[0038] Figure 13 The results of NFATC4 nuclear translocation detection are shown in Figure A, where A is the immunofluorescence staining image of NFATC4 in the Control group and the Anti-TRPM7 group (including three channels: Merge, NFATC4-CY3, and GFP-Nuclear Staining), and B is the bar chart of quantitative analysis of NFATC4 nuclear translocation rate (comparing the nuclear / cytoplasmic fluorescence intensity ratio between the Control group and the Anti-TRPM7 group).

[0039] Figure 14 For the detection of other protein expression (protein expression levels of MMP-2 and MMP-9), A shows the results of Western Blot detection (showing protein bands of MMP-9, MMP-2, and internal control β-actin in the Af+Empty Peptide+KLH group and the Af+Peptide group); B shows the quantitative analysis of MMP-9 protein expression (relative intensity bar chart of MMP-9 / β-actin, comparing the two treatments); C shows the quantitative analysis of MMP-2 protein expression (relative intensity bar chart of MMP-2 / β-actin, comparing the two treatments).

[0040] Figure 15 The images show representative immunofluorescence staining images of α-SMA in the left atrial tissue of rats in each group. Blue represents Hoechst 33342-labeled cell nuclei, green represents α-SMA positive signals, and Merge represents fused images.

[0041] Figure 16 Immunohistochemical staining results of CD68 and CD19 expression in heart tissue after immunization with short peptide vaccine and control vaccine. Detailed Implementation

[0042] 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.

[0043] Unless otherwise defined, the following terms used in this invention have the following meanings: TRPM7: Transient Receptor Potential Cation Channel Subfamily M Member 7, is a bifunctional membrane protein with ion channel and protein kinase activities, capable of permeating Ca2+.2+ and Mg 2+ It participates in a variety of physiological and pathological processes.

[0044] Short peptides: polypeptide fragments composed of 8-20 amino acid residues, which have antigenic epitope functions and can induce the body to produce specific antibodies.

[0045] Vaccine: In this invention, it refers to an immunogenic preparation made by coupling a short peptide with a carrier protein and adding an adjuvant, used to induce the body to produce neutralizing antibodies against TRPM7.

[0046] Carrier protein: a large molecular protein used to enhance the immunogenicity of short peptides. In this invention, keyhole limpet hemocyanin (KLH) is preferred.

[0047] Adjuvant: A substance that, when administered simultaneously with or before an antigen, can nonspecifically enhance or alter the immune response. The Freund's Complete Adjuvant (FCA) used in this invention was purchased from Sigma-Aldrich (F5881); the Freund's Incomplete Adjuvant (FIA) was purchased from Sigma-Aldrich (344291).

[0048] Coupling: The process of covalently linking short peptides to carrier proteins using chemical cross-linking agents (such as glutaraldehyde) to form a complete antigen.

[0049] ELISA: Enzyme-Linked Immunosorbent Assay, used to detect serum antibody titers.

[0050] Western Blot: Protein immunoblotting, used to detect the expression level of specific proteins.

[0051] TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling, used to detect apoptosis.

[0052] Immunofluorescence: A technique that uses fluorescently labeled antibodies to detect the localization of antigens in cells or tissues.

[0053] Calcium influx: extracellular Ca 2+ The process of ion channels entering the cytoplasm.

[0054] NFATC4: Nuclear Factor of Activated T Cells c4, a key transcription factor involved in cardiac remodeling and atrial fibrillation.

[0055] In this invention, the hemocyanin KLH was purchased from Solarbio, catalog number K8160; Freund's complete adjuvant was purchased from Sigma-Aldrich, catalog number F5881; Freund's incomplete adjuvant was purchased from Sigma-Aldrich, catalog number 344291; New Zealand rabbits (SPF grade) were purchased from Chongqing Tengxin Biotechnology Co., Ltd., catalog number 20220116; SD rats (SPF grade, male, 150-200g) were purchased from Xi'an Zhuangzhi Biotechnology Co., Ltd., catalog number #1023; human atrial myocyte cell line ATCC-0014 was purchased from Shanghai Lianmai Biotechnology Co., Ltd., catalog number LM-H076; human atrial fibroblasts HCFaa were purchased from Yaji Biotechnology, catalog number YS1142P; Fluo-4 AM calcium ion fluorescent probe was purchased from Yisheng Biotechnology Co., Ltd., catalog number 40704ES80; FITC-labeled goat anti-rabbit IgG was purchased from Wuhan AmyJet Scientific Co., Ltd., catalog number 111-095-003; CY3-labeled goat anti-rabbit IgG was purchased from GeneCopoeia, catalog number L135A; BCA protein quantification kit was purchased from Beyotime International Co., Ltd., catalog number P0009; TUNEL apoptosis detection kit was purchased from Roche, catalog number 12156792910; CCK-8 kit was purchased from Beyotime International Co., Ltd., catalog number C0048.

[0056] See Figure 1 The technical route of this invention is as follows: First, short peptides and coupling vectors are designed, constructed, and purified for TRPM7 to prepare a complete antigen; simultaneously, a CHO-TRPM7 cell line and an HTS system are established; New Zealand rabbits are immunized and target antibodies are prepared; through in vitro screening of the effectiveness of short peptide vaccines, short peptide vaccines with ideal blocking effects after immunization are screened; on the other hand, the vector (VLP or KLH) encapsulating CPG-ODN and the culture of atrial myocardium and fibroblasts are used for subsequent verification; after establishing an atrial fibrillation model, the model animals are immunized to obtain anti-TRPM7 antibodies, the antibody titer is determined, and the antibody effect is verified by measuring calcium flow; simultaneously, cell culture (atrial myocardium, fibroblasts, etc.) is performed; the mechanism of action of the TRPM7 vaccine is studied at the ion channel level and downstream level; finally, a safe and effective short peptide vaccine against TRPM7 is prepared, which can induce the body to produce target antibodies against TRPM7, alleviate calcium overload of atrial myocardium and fibroblasts, and inhibit atrial fibrillation.

[0057] Example 1: Design and screening of TRPM7-targeting short peptides This embodiment screens for specific short peptide sequences targeting the extracellular E3 loop of the TRPM7 channel.

[0058] Figure 2 The transmembrane structure of the TRPM7 channel is shown, where S1-S6 represent six transmembrane segments. The pore region between S5 and S6 is a selective sieve for calcium or sodium ions to pass through this ion channel. 3+ 2-APB are known channel modulators. TRPM7, as a member of the TRPs family, has a pore region composed of S5 and S6 transmembrane segments and their connecting loops; this region is the core structure for calcium ion selective permeability (see...). Figure 3 , Figure 4 and Figure 5 Based on the transmembrane domain and extracellular region characteristics of the TRPM7 channel, amino acid fragments located at positions 1015-1022, near calcium ion selective sieve pores, were selected as candidate regions. Suitable short peptide sequences were designed using IEBB (Immune Epitope Database) and PIR (Protein Information Resource) software. Subsequently, the binding affinity of the short peptide sequences to ligands was evaluated, and the potential biological functions induced by the binding of specific amino acid sequences on the extracellular E3 ring of the TRPM7 channel to ligands were predicted and assessed. Design principles included: hydrophilicity, surface accessibility, antigenicity, plasticity, charge balance, non-α-helix formation, and a length of 8-20 amino acids.

[0059] After prediction and evaluation, 17 candidate short peptides were initially screened; 6 were obtained after hydrophilicity optimization; 3 more were obtained after linear epitope prediction, E3 ring β-turn and accessibility prediction; finally, homology matching was performed to determine the short peptide that best meets the antigen design requirements, with the amino acid sequence: GVPRKAIL (corresponding to positions 1015-1022 of the TRPM7 channel, as shown in SEQ ID NO. 1). This short peptide was used in AutoDock Vina software to design a suitable short peptide sequence; subsequently, the binding ability of the short peptide sequence to the ligand was evaluated, and the biological functions that might be triggered by the binding of the specific amino acid sequence on the extracellular E3 ring of the TRPM7 channel to the ligand were predicted and evaluated. Figure 6The results showed that all ligands with high receptor affinity, strong interaction with TRPM7, and important biological functions that bind to the receptor fall within the six hydrophobic pockets of TRPM7. Therefore, it is inferred that the amino acid sequences contained within these hydrophobic pockets are the key targets for ligands to efficiently bind to TRPM7 and exert important biological functions.

[0060] Based on the above structural analysis and bioinformatics predictions, an amino acid fragment located at positions 1015-1022, near the calcium ion selective sieve pores, was selected as a candidate region. This region is located in the extracellular E3 loop, adjacent to the pore region, and is a key region for maintaining calcium ion selective permeability. Figure 2 , Figure 5 Antigenic epitope prediction was performed using IEDB and PIR software. Design principles included: hydrophilicity, surface accessibility, antigenicity, plasticity, charge balance, non-formation of α-helices, and a length of 8-20 amino acids. After prediction and evaluation, 17 candidate short peptides were initially screened; 6 were obtained after hydrophilicity optimization; 3 more were obtained through linear epitope prediction, E3 ring β-turn prediction, and accessibility prediction; finally, homology matching was performed to determine the short peptide that best met the antigen design requirements, with the amino acid sequence: GVPRKAIL (corresponding to positions 1015-1022 of the TRPM7 channel). This short peptide can fall into the 6 hydrophobic "pockets" of the TRPM7 receptor. Figure 6 It has high receptor affinity.

[0061] Example 2: Synthesis and purification of short peptides The short peptide GVPRKAIL described in Example 1 was synthesized using a dynamic solid-phase synthesis method via a peptide synthesizer (GL Biochem (Shanghai) Co., Ltd.). After synthesis, it was purified by high-performance liquid chromatography (HPLC), and its molecular weight was identified by mass spectrometry. The purified short peptide had a purity ≥95% and was lyophilized for later use.

[0062] Example 3: Preparation of Short Peptide Vaccine The short peptide purified in Example 2 was coupled to the carrier hemocyanin (KLH) using a glutaraldehyde coupling method. The specific steps were as follows: the short peptide and KLH were mixed in 0.01 M PBS (pH = 7.4) at a molar ratio of 30:1, and glutaraldehyde was added to a final concentration of 0.25%. The mixture was reacted at room temperature for 2 h, and then dialyzed overnight with PBS (MWCO 12-14 kDa) to obtain the short peptide-KLH conjugate. SDS-PAGE electrophoresis followed by Coomassie brilliant blue staining showed that a high molecular weight band appeared in the conjugate lane, and its migration position was significantly higher than that of the free KLH lane. Figure 7 This indicates that the coupling was successful.

[0063] The above conjugate is emulsified with an equal volume of Freund's complete adjuvant (for immunization) or Freund's incomplete adjuvant (for booster immunization) to prepare a short peptide vaccine, with each 100 μL of vaccine containing 50 μg of short peptide (based on peptide fragments).

[0064] Example 4: Preparation of the control vaccine An irrelevant control short peptide (amino acid sequence: AGLKSVPR, length 8 amino acids, as shown in SEQ ID NO.2) that does not contain the short peptide (GVPRKAIL) sequence of the present invention was coupled with KLH in the same manner as in Example 3, and emulsified with Freund's adjuvant to prepare a control vaccine.

[0065] Example 5: Preparation and purification of the target antibody Eight healthy New Zealand rabbits were selected, half male and half female, weighing 2.0-2.5 kg each. The immunization schedule is as follows: First immunization: The short peptide vaccine (containing Freund's complete adjuvant) prepared in Example 3 was injected subcutaneously at multiple points on both sides of the spine on the back of the rabbit, with each rabbit receiving 200 μg of short peptide (calculated by peptide segment).

[0066] First booster immunization on day 14: short peptide vaccine (containing Freund's incomplete adjuvant), 100 μg short peptide per rabbit.

[0067] Second booster immunization on day 28: Same as the first booster immunization.

[0068] Third booster immunization on day 42: Same as the first booster immunization.

[0069] Blood was collected via the marginal ear vein before each immunization and on day 7 after each immunization. Serum was separated, and antibody titers were determined using ELISA (coating antigen was a short peptide-KLH conjugate, 1 μg / well; secondary antibody was HRP-labeled goat anti-rabbit IgG). Results showed that the antibody titer reached 1:800±200 on day 7 after the first immunization, increased to 1:6400±800 on day 7 after the second immunization, and reached a peak of 1:12800±1600 on day 7 after the fourth immunization. High titers (>1:10000) were maintained for at least 90 days. Figure 8 ).

[0070] On day 14 post-immunization, blood was collected from the heart, allowed to stand at room temperature for 1 hour, and then centrifuged at 3000 rpm for 15 minutes at 4°C to separate serum. IgG antibodies were purified using ammonium sulfate precipitation (50% saturation, overnight at 4°C), hydrophobic chromatography (Butyl-Sepharose FF column), and affinity chromatography (Protein G-Sepharose 4FF column). The antibody samples after ammonium sulfate precipitation and hydrophobic chromatography were loaded onto a Protein G-Sepharose 4FF column (5 mL column volume, 1 mL / min flow rate) and equilibrated with binding buffer (20 mM sodium phosphate, pH 7.0) until baseline stability. After loading, washing with binding buffer continued until UV absorption (A280) returned to baseline. Elution was then performed with elution buffer (0.1 M glycine-HCl, pH 2.7), and the elution peak was collected. Results showed that the elution peak appeared at approximately 2.5 column volumes, with a peak width of approximately 1.2 column volumes, and the A280 peak value of the elution buffer reached 0.85. The collected eluent was immediately neutralized to pH 7.4 with 1M Tris-HCl (pH 9.0). The eluent was then analyzed by SDS-PAGE. Figure 9 After purification, the antibody showed clear bands at approximately 55 kDa (heavy chain) and 25 kDa (light chain), with no other contaminating protein bands, a purity >90%, and a concentration of 2.5 mg / mL (BCA method). The antibody recovery rate was approximately 65% ​​(based on the total amount of antibody in the initial serum). This antibody was named Anti-TRPM7.

[0071] Example 6: Preparation of Neutralizing Antibodies The purified Anti-TRPM7 antibody from Example 5 was mixed with the short peptide (GVPRKAIL, as shown in SEQ ID NO.1) from Example 1 at a molar ratio of 1:10 (antibody: short peptide), incubated at 37°C for 2 h, and then centrifuged in a 30 kDa ultrafiltration tube (4°C, 4000 g) to remove unbound short peptides. The retentate was collected to obtain the neutralizing antibody. ELISA verification showed that the binding activity of the neutralizing antibody to the coated short peptide was reduced by >90%.

[0072] Example 7: In vitro cell experiments (1) Cell culture Atrial myocyte line ATCC-0014 (human atrial myocytes) and atrial fibroblasts HCFaa were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) at 37°C in a 5% CO2 incubator, and passaged every 2-3 days.

[0073] (2) The binding efficiency of the antibody to TRPM7 was detected by Western blotting and immunofluorescence. Cells in each group (ATCC-0014) were cultured until they reached 90% confluence in the culture flask. Cells were then lysed to extract membrane proteins. After BCA protein quantification, 30 μg of protein was loaded onto each well for SDS-PAGE electrophoresis. The cells were then transferred to PVDF membranes, blocked with 5% skim milk for 1 hour, and incubated overnight at 4°C with the corresponding antibody (1:1000) as the primary antibody (control antibody group used control antibody, Anti-TRPM7 group used Anti-TRPM7, and neutralizing antibody group used neutralizing antibody). The membranes were then incubated with HRP-labeled goat anti-rabbit secondary antibody (1:5000) at room temperature for 1 hour, followed by ECL staining. Results showed that a specific band with a molecular weight of approximately 220 kDa was detectable in the Anti-TRPM7 group, but no such band was detected in the control antibody group. The band signal in the neutralizing antibody group was significantly weakened (grayscale value decreased by 65% ​​± 7%). P <0.01). For example... Figure 10 As shown.

[0074] (3) Antibody specificity verification (immunofluorescence) ATCC-0014 cells were seeded onto coverslips in 24-well plates and cultured until 70% confluence. The following antibodies (10 μg / mL, incubated overnight at 4°C) were added: (1) control antibody (prepared from the control vaccine of Example 4 using the same method as in Example 5); (2) Anti-TRPM7 (Example 5); (3) neutralizing antibody (Example 6). After washing, FITC-labeled goat anti-rabbit IgG (1:200) was added, and the cells were incubated at room temperature in the dark for 1 hour. After washing with PBS, the cell nuclei were stained with Hoechst 33342 (1 μg / mL) for 10 min. The cells were observed and images were acquired using a fluorescence microscope. The fluorescence intensity of the cell membrane region was measured using ImageJ software. See the attached image for details. Figure 11 The target antibody (Anti-TRPM7) of the short peptide vaccine specifically binds to the TRPM7 channel on the atrial myocyte membrane, while the neutralizing antibody loses its binding ability due to the epitope being blocked by the short peptide, and the control antibody shows no specific binding. The cell membrane of the target antibody group exhibits a green fluorescent signal, with a fluorescence intensity significantly higher than that of the control antibody group and the neutralizing antibody group, indicating that Anti-TRPM7 can specifically bind to the TRPM7 channel on the atrial myocyte membrane, while the binding of the neutralizing antibody is reduced due to the pre-blocking of the epitope. This result verifies that the antibody induced by the short peptide vaccine in this application has high affinity and specificity for TRPM7.

[0075] (4) Calcium influx measurement Fluo-4 AM calcium ion fluorescent probe was used. Each group of ATCC-0014 cells (1×10⁻⁶) 5Cells were treated with different antibodies (10 μg / mL) for 48 hours in 24-well plates. The culture medium was then discarded, and HBSS buffer containing 5 μmol / L Fluo-4 AM was added. The plates were incubated at 37°C in the dark for 30 minutes. After washing, cytoplasmic calcium ion concentration was detected using a fluorescence microplate reader (excitation wavelength 494 nm, emission wavelength 516 nm). Simultaneously, cells were stimulated with iodine (1 μmol / L), and fluorescence peaks were recorded. Results: The baseline calcium ion fluorescence intensity in the Anti-TRPM7 group (120±15 RFU) was 65.7% lower than that in the control antibody group (350±28 RFU) (P<0.01); the peak value after stimulation (210±22 RFU) was 66.1% lower than that in the control antibody group (620±45 RFU) (P<0.01). There was no significant difference between the neutralizing antibody group and the control antibody group. P >0.05). This indicates that Anti-TRPM7 can effectively block TRPM7-mediated calcium influx, such as Figure 12 As shown.

[0076] (5) Apoptosis detection After treatment, ATCC-0014 cells in each group were subjected to TUNEL assay using a Roche kit. The procedure was performed according to the manufacturer's instructions: fixation with 4% paraformaldehyde, permeabilization with Triton X-100, incubation with TUNEL solution at 37°C for 1 hour, and counterstaining with DAPI. TUNEL-positive cells were counted under a fluorescence microscope. Five fields of view (≥200 cells per field) were randomly selected from each group. Results: The apoptosis index in the control antibody group was 28.4%±3.2%, in the Anti-TRPM7 group it was 10.2%±1.8% (P<0.01), and in the neutralizing antibody group it was 26.5%±2.9% (P>0.05 vs. control antibody group). Western blot analysis showed that the protein levels of Cleaved Caspase-3 and Cleaved Caspase-9 in the Anti-TRPM7 group were reduced by 72% and 68%, respectively, compared to the control antibody group. P <0.01).

[0077] (6) Proliferation and migration of atrial fibroblasts HCFaa cell grouping and antibody treatment were the same as above. Proliferation assay: CCK-8 assay, 5 × 10^3 cells were seeded per well in a 96-well plate. After treatment for 48 hours, 10 μL of CCK-8 reagent was added, and the cells were incubated at 37℃ for 2 hours. OD450 was then measured. Migration assay: Transwell chambers (8 μm pore size) were used. 5 × 10^4 cells (serum-free) were added to the upper chamber, and medium containing 10% FBS was added to the lower chamber. After treatment for 24 hours, crystal violet staining was performed, and migrating cells were counted under a microscope. Results: The OD450 of the Anti-TRPM7 group (0.32±0.04) was 41.8% lower than that of the control antibody group (0.55±0.05) (P<0.01); the number of migrating cells (45±8 cells / field) was 59.8% lower than that of the control antibody group (112±12 cells / field) (P<0.01). This indicates that Anti-TRPM7 inhibits the proliferation and migration of atrial fibroblasts.

[0078] (7) NFATC4 core translocation detection ATCC-0014 cells and HCFaa cells in each group were treated with control antibody (Control-Anti), target antibody (Anti-TRPM7), and neutralizing antibody (specific grouping and concentrations were the same as in experiment (2)). After 48 hours of culture, immunofluorescence staining was performed. Procedure: fixation with 4% paraformaldehyde for 15 min, permeabilization with 0.2% Triton X-100 for 10 min, blocking with 5% BSA for 1 h, adding rabbit anti-NFATC4 antibody (1:200), incubating overnight at 4℃, incubating with CY3-labeled goat anti-rabbit secondary antibody (1:400) at room temperature in the dark for 1 h, and staining the nucleus with GFP-Nuclear Staining (Abcam, ab139476). Images were observed and acquired, and the CY3 fluorescence intensity of the nucleus and cytoplasm was measured using ImageJ software (20 cells were randomly selected from each group, and each treatment group was independently repeated 3 times, N=8), and the nucleus / cytoplasm fluorescence intensity ratio was calculated. A ratio > 1.5 is considered nuclear localization dominance, and a ratio < 0.8 is considered cytoplasmic localization dominance. See details... Figure 11 In the control antibody group, NFATC4 was mainly distributed in the nucleus (nuclear / cytoplasmic fluorescence intensity ratio > 2.5), while in the Anti-TRPM7 group, NFATC4 was mainly distributed in the cytoplasm (nuclear / cytoplasmic fluorescence intensity ratio < 0.8). The neutralizing antibody group was similar to the control antibody group. This indicates that the target antibody of the TRPM7 short peptide vaccine can significantly inhibit the nuclear translocation of NFATC4 in atrial myocytes and atrial fibroblasts, causing it to shift from the nucleus to the cytoplasm and become inactive. This is one of the important molecular mechanisms for inhibiting the occurrence and maintenance of atrial fibrillation. (See...) Figure 13 .

[0079] (8) Detection of other protein expression Total RNA and protein were extracted from cells in each group, and the mRNA and protein levels of α-SMA, TGF-β1, MMP-2, and MMP-9 were detected by RT-PCR and Western Blot. The results showed that the mRNA and protein expression levels of the above indicators in atrial myocytes and atrial fibroblasts in the Anti-TRPM7 group were significantly lower than those in the control antibody group. P <0.05). For example... Figure 14 As shown.

[0080] Example 8: In vivo animal experiments (1) Establishment of atrial fibrillation model rats Sixty male SD rats, weighing 150-200g, were used. They were injected via tail vein with a mixture of acetylcholine and calcium chloride (CaCl2 10 mg / mL, acetylcholine 66 μg / mL) at a rate of 0.1 mL / 100g body weight for 7 consecutive days. Electrocardiograms (ECGs) were recorded daily after each injection using the PowerLab system. On day 7 post-modeling, the ECG showed the following: disappearance of P waves, replaced by irregular f waves (frequency >500 bpm), and absolutely irregular RR intervals lasting >30 seconds, indicating successful establishment of the atrial fibrillation model. A total of 50 rats were successfully modeled, achieving a success rate of 83.3%.

[0081] (2) Experimental grouping and immunization regimen The atrial fibrillation model rats were randomly divided into 5 groups, with 10 rats in each group: Blank control group (normal SD rats, no model established, no immunization) Model group (atrial fibrillation model, non-immune) Empty vector vaccine group (atrial fibrillation model, control vaccine in immunization example 4) Short peptide vaccine group (atrial fibrillation model, immunization example 3 short peptide vaccine) Sham surgery group (injection of saline solution via tail vein, no immunization) Immunization regimen: Starting on day 1 after model establishment, multiple subcutaneous injections (4-6 points on the back) were administered. Short peptide vaccine group: Each rat was injected with 100 μg of short peptide (based on peptide fractions), emulsified in Freund's complete adjuvant (primary) or Freund's incomplete adjuvant (booster), with a total volume of 0.5 mL. Empty vector vaccine group: Rats were injected with an equal volume of control vaccine. Booster immunizations were administered every 14 days, for a total of 3 immunizations (days 1, 15, and 29). Various tests were performed 90 days after the last immunization.

[0082] (3) Electrocardiogram and hemodynamic monitoring On day 90 after the last immunization, surface electrocardiograms of rats in each group were recorded. Results showed that some rats in the short peptide vaccine group converted from atrial fibrillation to sinus rhythm, while no conversion to sinus rhythm was observed in the model group and the empty vector vaccine group. M-mode echocardiography and left atrial catheter manometry revealed that the left atrial diameter, left ventricular end-diastolic diameter, and left ventricular ejection fraction were significantly improved in the short peptide vaccine group compared to the model group, and left ventricular systolic pressure and left ventricular end-diastolic pressure were also significantly improved.

[0083] (4) Measurement of calcium ion concentration in atrial tissue After sacrifice, left atrial tissue was harvested, and its calcium content was measured. The results showed that the calcium content of atrial tissue in the short peptide vaccine group was significantly lower than that in the model group and the empty vector vaccine group.

[0084] (5) TRPM7 expression level Atrial tissue membrane proteins were extracted, and the expression levels of TRPM7 protein and mRNA were detected by Western blotting and RT-PCR, respectively. The results showed that the expression levels of TRPM7 protein and mRNA in the short peptide vaccine group were significantly lower than those in the model group, while there was no significant difference between the empty vector vaccine group and the model group.

[0085] (6) Detection of atrial myocyte apoptosis Atrial myocyte apoptosis was detected using the TUNEL assay. The results showed that the apoptosis rate of atrial myocytes in the short peptide vaccine group was significantly lower than that in the model group.

[0086] (7) Atrial histopathological examination HE staining of left atrial tissue revealed disordered and hypertrophic atrial myocytes, interstitial edema, and extensive inflammatory cell infiltration in the model group; in the short peptide vaccine group, myocardial cells were more orderly arranged, and inflammatory infiltration was reduced. Masson staining for fibrosis showed a significantly lower collagen volume fraction in the short peptide vaccine group compared to the model group. Immunohistochemical detection of α-SMA showed a significantly lower percentage of α-SMA-positive area in the short peptide vaccine group compared to the model group. Figure 15 .

[0087] (8) Safety evaluation Rats were weighed every two weeks after immunization, and blood samples were collected to measure liver function (ALT, AST), kidney function (BUN, Cr), and complete blood count (WBC, RBC, PLT). There were no significant differences in body weight gain among the groups (P>0.05). There were no significant differences in ALT (48±5 U / L), AST (125±12 U / L), BUN (6.2±0.5 mmol / L), and Cr (45±4 μmol / L) between the short peptide vaccine group and the sham-operated group (ALT 45±4 U / L, AST 118±10 U / L, BUN 5.8±0.4 mmol / L, Cr 42±3 μmol / L). P>0.05). There were no significant differences in routine blood tests among the groups. Heart, liver, spleen, lungs, and kidneys were harvested after sacrifice; HE staining revealed no obvious pathological damage. This indicates that the short peptide vaccine has good in vivo safety.

[0088] Example 9: Antibody persistence test (rat) Blood samples were collected on days 30, 60, and 90 after the last immunization (orbital blood samples), and serum antibody titers were detected by ELISA (method as in Example 5). Results showed that the titers ranged from 1:1600 to 1:12800 on day 30 and from 1:6400 to 1:25600 on day 60, consistently maintaining a high level. This indicates that the short peptide vaccine can induce a durable antibody response in rats (see Table 1).

[0089] Table 1: Antibody titers were measured more precisely every 6 days using the ELISA method.

[0090] Example 10: Verification of the vaccine's mechanism of action See Figure 16 Animals were immunized with a short peptide vaccine (Peptide RF10+KLH) and a control vaccine (Empty Peptide+KLH), respectively. Heart tissue was then collected for immunohistochemical staining to detect the expression of CD68 (macrophage / monocyte marker) and CD19 (B lymphocyte marker). Compared to the control vaccine group (Empty Peptide+KLH), the short peptide vaccine group (Peptide RF10+KLH) showed increased infiltration of CD19-positive B lymphocytes in heart tissue, indicating that the short peptide vaccine could activate B lymphocytes and induce a humoral immune response. Simultaneously, there was no significant increase in CD68-positive cell infiltration, suggesting that the vaccine did not induce a significant inflammatory autoimmune response. The short peptide vaccine of this invention (short peptide coupled with the carrier protein KLH and supplemented with an adjuvant) can bind to receptors on the surface of specific B lymphocytes after immunization, directly activating B lymphocytes; it also provides exogenous T lymphocyte epitopes, activating helper T lymphocytes (CD4+ T cells). Through synergistic effects, this short-peptide vaccine can overcome the body's own immune tolerance, inducing a humoral immune response and producing high-titer, highly specific antibodies. Simultaneously, it avoids inducing a type 1 immune response from autoreactive T lymphocytes, thereby reducing the risk of immune-related tissue damage. Therefore, the short-peptide vaccine of this invention has good in vivo safety and promising prospects for clinical translation.

[0091] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A short peptide targeting the TRPM7 channel, characterized in that, The amino acid sequence of the short peptide is shown in SEQ ID NO.

1.

2. A conjugate, characterized in that, It comprises the short peptide and carrier targeting the TRPM7 channel as described in claim 1.

3. The conjugate according to claim 2, characterized in that, The carrier is hemocyanin KLH.

4. The conjugate according to claim 3, characterized in that, The short peptide targeting the TRPM7 channel is coupled to hemocyanin KLH via glutaraldehyde coupling.

5. A short peptide vaccine for the prevention or treatment of atrial fibrillation, characterized in that, The short peptide vaccine comprises the short peptide targeting the TRPM7 channel as described in claim 1 or the conjugate as described in any one of claims 2-4.

6. The short peptide vaccine for the prevention or treatment of atrial fibrillation according to claim 5, characterized in that, It also contains adjuvants.

7. The short peptide vaccine for the prevention or treatment of atrial fibrillation according to claim 6, characterized in that, The adjuvant is Freund's adjuvant or Freund's incomplete adjuvant.

8. A pharmaceutical composition, characterized in that, The vaccine comprises the short peptide targeting the TRPM7 channel as described in claim 1, the conjugate as described in any one of claims 2-4, or the short peptide vaccine for the prevention or treatment of atrial fibrillation as described in any one of claims 5-7, and pharmaceutically acceptable excipients.

9. A polyclonal antibody, characterized in that, Obtained by immunizing non-human animals with the short peptide vaccine for the prevention or treatment of atrial fibrillation as described in any one of claims 5-7.

10. The use of the short peptide targeting the TRPM7 channel as described in claim 1, the conjugate as described in any one of claims 2-4, the short peptide vaccine for the prevention or treatment of atrial fibrillation as described in any one of claims 5-7, or the polyclonal antibody as described in claim 9 in the preparation of a medicament for the prevention or treatment of atrial fibrillation.

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