Immunogenic peptide fragments targeting human β1-adrenergic receptor and their applications

Abstract

The present invention discloses an immunogenic peptide segment for human β1-adrenergic receptor and its application, wherein the amino acid sequence of the immunogenic peptide segment is DEARRCYND, as shown in SEQ ID NO.1. The immunogenic vector vaccine for human β1-adrenergic receptor is prepared by coupling the above-mentioned immunogenic peptide segment of human β1-adrenergic receptor with a carrier, and the carrier is Qβ-2aa bacteriophage virus-like particle protein. The present invention successfully couples the preferentially designed immunogenic peptide segment of human β1-adrenergic receptor with the Qβ-2aa bacteriophage virus-like particle protein carrier to prepare a vector vaccine. The immunogenic vector vaccine can be used to prepare drugs for treating hypertension and improving cardiac remodeling after myocardial infarction.

Description

Technical Field

[0001] The present invention relates to the field of medical biotechnology, and in particular to an immunogenic peptide segment targeting human β1-adrenergic receptor and applications thereof. Background Art

[0002] The β1-adrenergic receptor (β1-AR) is a transmembrane receptor of the cardiovascular G protein-coupled receptor family. The β1-AR is the predominant β-adrenergic receptor subtype in the human heart, accounting for 60-70% and 70-80% of β-adrenergic receptors in the atria and ventricles, respectively. It plays a key role in the regulation of cardiovascular physiology, the pathogenesis of arrhythmias, and the progression of cardiac remodeling and heart failure after myocardial infarction. Numerous lines of evidence confirm that overactivation of the sympathetic nervous system, particularly the β1-AR, plays an important role in the pathological progression of hypertension, cardiac remodeling, and heart failure. Therefore, β1-receptor blockers, as classic sympathetic nervous system inhibitors, are widely used in the treatment of hypertension and heart disease, including myocardial remodeling after myocardial infarction, heart failure, and arrhythmias.

[0003] Beta-blockers are commonly used medications for hypertension and are one of the five first-line antihypertensive drugs. Beta-blockers selectively bind to beta-receptors to produce a variety of antihypertensive effects, such as reduced cardiac output, decreased renin release, and inhibition of central sympathetic nerve impulses. Highly selective beta-1 blockers exhibit a high degree of selectivity for beta-1 receptors, effectively lowering blood pressure while also protecting target organs and reducing the risk of cardiovascular events.

[0004] Cardiac remodeling underlies the development and progression of heart failure, leading to impaired systolic and diastolic function and pressure and volume overload. Reversing ventricular remodeling is a research hotspot for the prevention and treatment of heart failure. Sufficient evidence-based medicine demonstrates that beta-blockers can significantly reduce the risk of all-cause and cardiovascular mortality in patients with acute myocardial infarction, reduce adverse myocardial remodeling, and increase survival in heart failure patients. Their mechanism is to block the sympathetic nervous system, improve cardiac function, slow heart rate, reduce myocardial oxygen consumption, improve myocardial oxygen supply and demand balance, and delay and reverse ventricular remodeling.

[0005] However, the clinical use of beta-blockers still faces numerous challenges. Hypertension treatment guidelines recommend a wide range of medications, including diuretics, calcium channel blockers, beta-blockers, angiotensin-converting enzyme inhibitors, and angiotensin II receptor blockers. These medications are diverse, with short-lived efficacy and limited stability. This undoubtedly increases the number of medications patients must take, the frequency of medications, and the financial burden, leading to poor patient compliance and low hypertension control rates. In the treatment of heart failure, the negative inotropic effects of the drugs and patient tolerance must be considered. To prevent beta-blockers from inhibiting myocardial contractility and worsening the condition, they should be used only after the condition is stabilized, and long-term use (for more than three months) is required to exert their cardioprotective effects. During clinical use, to improve patient tolerance and reduce adverse reactions caused by negative inotropic effects, the dose should be gradually increased from a low dose to the target dose. Only high doses of beta-blockers have consistently beneficial effects in patients with heart failure. Furthermore, in the treatment of ischemic heart disease, the timing and method of administration, as well as drug titration, require careful consideration. The COMMIT / CCS-2 clinical trial found that early intravenous administration of a beta-blocker (metoprolol) in acute myocardial infarction, followed by a switch to high-dose oral therapy, reduced the composite endpoint (death, recurrent myocardial infarction, cardiac arrest, and ventricular fibrillation). However, in hemodynamically unstable patients, intravenous beta-blocker use increases the risk of cardiogenic shock and mortality, particularly when used on the day of myocardial infarction or the first day. This is likely related to the timing, method, dosage, and patient tolerance of administration. Furthermore, all marketed beta-blockers have a certain degree of β2 or α-receptor blockade, which can lead to lipid metabolism disorders and, due to bronchial constriction, hinder heart disease control in patients with bronchial asthma or chronic obstructive pulmonary disease. Therefore, developing safe, stable treatments that reduce hypertension and improve cardiac remodeling after myocardial infarction is of great clinical significance.

[0006] Currently, therapeutic vaccines offer advantages over traditional chemical drugs or other biological drugs, such as high specificity, minimal side effects, long-lasting effects, and lack of drug resistance. This makes them another revolutionary class of innovative drugs developed based on the human immune system, following monoclonal antibodies. If a therapeutic vaccine targeting the β1-adrenergic receptor (β1-AR) is successfully developed, compared to traditional β-blockers, it would offer lower production costs, greater specificity, and a longer duration of action. It could be administered once every 1 to 3 months or even longer, maintaining long-term, stable, and effective effects. Its application in patients with hypertension and coronary heart disease could reduce the risk of adverse reactions and improve patient tolerance, thereby better protecting target organs, reducing the economic burden, and improving treatment compliance. Summary of the Invention

[0007] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide an immunogenic peptide segment of human β1-adrenergic receptor and its application; the present invention fully addresses the difficulties in the clinical application of β-receptor blockers and pioneeringly screens out a therapeutic vaccine specific for β1-AR. The vaccine can effectively inhibit the action of β1-AR, lower the blood pressure of hypertensive animals, and improve cardiac remodeling after myocardial infarction.

[0008] To achieve the above first purpose, the present invention designs an immunogenic peptide segment targeting β1-adrenergic receptor, characterized in that the amino acid sequence of the immunogenic peptide segment is DEARRCYND, as shown in SEQ ID NO.1.

[0009] The present invention also provides a use of the above immunogenic peptide segment in preparing a product for treating hypertension and improving cardiac remodeling after myocardial infarction.

[0010] The second object of the present invention is to provide an immunogenic vector vaccine targeting human β1-adrenergic receptor, wherein the immunogenic vector vaccine targeting human β1-adrenergic receptor is prepared by coupling an immunogenic peptide segment targeting human β1-adrenergic receptor with a carrier.

[0011] Preferably, the carrier is Qβ-2aa bacteriophage virus-like particle protein.

[0012] The third object of the present invention is to provide an immunogenic vector vaccine targeting human β1-adrenergic receptor for use in the preparation of drugs for treating hypertension.

[0013] The fourth object of the present invention is to provide an immunogenic vector vaccine targeting human β1-adrenergic receptor for use in the preparation of a drug for improving cardiac remodeling after myocardial infarction.

[0014] Beneficial effects of the present invention:

[0015] 1. Based on the extracellular loop amino acid sequence, hydrophilicity, antigenicity, accessibility, and other characteristics of the human β1-AR, and integrating bioinformatics and pharmacological methods, the present invention designed a peptide segment targeting the human β1-adrenergic receptor: SEQ ID No. 1. Animal experiments verified that the SEQ ID NO. 1 peptide segment can effectively induce the production of specific antibodies in immunized mice and rats.

[0016] 2. Selecting a suitable carrier is the key to successful vaccine development. The present invention uses Qβ-2aa bacteriophage virus-like particle protein.

[0017] 3. This study successfully prepared a vector vaccine by coupling a designed peptide targeting human β1-AR with a Qβ-2aa phage virus-like particle protein vector. The resulting vector vaccine was used to immunize SD rats induced by L-NAME hypertension to investigate whether the immunogenic vector vaccine targeting human β1-AR could reduce the L-NAME-induced blood pressure increase. The results demonstrated that the immunogenic vector vaccine targeting human β1-AR effectively reduced the L-NAME-induced blood pressure increase. Therefore, the immunogenic vector vaccine targeting human β1-AR could be used in the preparation of a drug for the treatment of hypertension.

[0018] 4. The prepared vector vaccine was used to immunize mice with a myocardial infarction model to investigate whether the immunogenic vector vaccine targeting the human β1-AR improves cardiac remodeling after myocardial infarction. The results demonstrated that the immunogenic vector vaccine targeting the human β1-AR effectively improves cardiac remodeling after myocardial infarction. Therefore, the immunogenic vector vaccine targeting the human β1-AR could be used to develop drugs to combat cardiac remodeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is an SDS-PAGE gel electrophoresis detection image of the vector vaccine ABRQβ-006 in Example 2;

[0020] In the figure, lane Qβ is Qβ-2aa bacteriophage virus-like particle protein monomer, and lane ABRQβ-006 is vector vaccine ABRQβ-006.

[0021] Figure 2 This is a graph showing the titer of anti-ABR-006 short peptide antibodies produced after male SD rats were immunized with the vector vaccine ABRQβ-006 in Example 3;

[0022] In the figure, ABRQβ-006 antibody represents anti-ABR-006 short peptide antibody, and immunization represents immunization of rats with the vector vaccine ABRQβ-006.

[0023] Figure 3 This is a graph showing the antihypertensive effect of immunizing male SD rats with the vector vaccine ABRQβ-006 and then intervening with L-NAME in Example 3;

[0024] in, Figure 3 A is a graph showing changes in the heart rate of rats. Figure 3 B is a graph showing changes in blood pressure of monitored rats;

[0025] In the figure, control represents the blank control group, L-NAME represents male SD rats receiving VLP+L-NAME intervention, ABRQβ-006 represents male SD rats receiving vector vaccine ABRQβ-006 immunization+L-NAME intervention; METO represents male SD rats receiving metoprolol gavage+L-NAME intervention; #P<0.05, ##P<0.01, ###P<0.001vs control group; *P<0.05, **P<0.01, ***P<0.001vs L-NAME group (upper: ABRQβ-006, lower: METO).

[0026] Figure 4 This is a graph showing the titer of anti-ABR-006 short peptide antibodies produced after male C57 mice were immunized with the vector vaccine ABRQβ-006 in Example 4;

[0027] In the figure, ABRQβ-006 antibody represents anti-ABR-006 short peptide antibody, and immunization represents immunization of mice with the vector vaccine ABRQβ-006.

[0028] Figure 5 This is a graph showing heart rate changes in male C57 mice immunized with the vector vaccine ABRQβ-006 and subjected to myocardial infarction modeling intervention in Example 4;

[0029] In the figure, SHAM represents the sham-operated group that received PBS; MI represents male C57 mice that received PBS and underwent myocardial infarction; ABRQβ-006 represents male C57 mice that were immunized with the vector vaccine ABRQβ-006 and underwent myocardial infarction; and METO represents male C57 mice that were orally administered with metoprolol and underwent myocardial infarction. *P<0.05, **P<0.01, ***P<0.001 vs. MI group (top: ABRQβ-006, bottom: METO) 。

[0030] Figure 6 Left ventricular M-mode ultrasound images and statistical results 2 months after immunization of C57 mice with the vector vaccine ABRQβ-006 in Example 4 and myocardial infarction modeling;

[0031] In the figure, SHAM represents the sham-operated group that received PBS, MI represents male C57 mice that received PBS and underwent myocardial infarction modeling; ABRQβ-006 represents male C57 mice that received the vector vaccine ABRQβ-006 and underwent myocardial infarction modeling; METO represents male C57 mice that received metoprolol orally and underwent myocardial infarction modeling.

[0032] Figure 7The MASSON staining diagram and statistical results of the left ventricular remodeling of the myocardial infarction model 2 months after the left ventricular MASSON staining in Example 4 were used to identify the effect of the vector vaccine ABRQβ-006.

[0033] In the figure, SHAM represents the sham-operated group that received PBS, MI represents male C57 mice that received PBS and underwent myocardial infarction modeling; ABRQβ-006 represents male C57 mice that received the vector vaccine ABRQβ-006 and underwent myocardial infarction modeling; METO represents male C57 mice that received metoprolol orally and underwent myocardial infarction modeling. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below with reference to specific embodiments so that those skilled in the art can understand.

[0035] The immunogenic peptide segment targeting human β1-adrenergic receptor is hereinafter referred to as β1-AR immunogenic peptide segment, the immunogenic vector vaccine targeting β1-adrenergic receptor is hereinafter referred to as β1-AR vector vaccine, and β1-adrenergic receptor is hereinafter referred to as β1-AR.

[0036] Example 1: Preparation of β1-AR immunogenic peptides

[0037] Based on the spatial conformation (3D structure) of β1-AR, bioinformatics, and pharmacological characteristics, the key extracellular loop domains were deduced, and a β1-AR immunogenic peptide targeting the extracellular amino acid sequence of human β1-AR was designed and named ABR-006. The specific amino acid sequence is DEARRCYND, as shown in SEQ ID NO.1.

[0038] The ABR-006 peptide was synthesized using solid-phase synthesis (synthesis and quality control performed by Shanghai Jier Biochemical Co., Ltd.). High-performance liquid chromatography analysis revealed a purity of over 98%. The resulting ABR-006 peptide was lyophilized, aliquoted, placed in cryovials, and stored at -80°C until ready for use.

[0039] Example 2: Preparation of β1-AR vector vaccine

[0040] 1. Prepare β1-AR vector vaccine ABRQβ-006 using Qβ-2aa bacteriophage virus-like particle protein. The specific preparation process is as follows:

[0041] 1) Preparation of Qβ-2aa bacteriophage virus-like particle protein: The English abbreviation of Qβ-2aa bacteriophage virus-like particle protein is: Qβ-2aa VLP, and Qβ-2aa VLP is used hereinafter to represent Qβ-2aa bacteriophage virus-like particle protein.

[0042] The preparation method of Qβ-2aa VLP is as follows:

[0043] 1a) Obtaining a recombinant strain expressing Qβ-2aa VLPs: The recombinant strain is Escherichia coli DH5α / pGEXQβ-A1, which can induce the production of Qβ-2aa virus-like particle protein. The deposit number of E. coli DH5α / pGEXQβ-A1 is CCTCCNO: M209282. For a detailed preparation process, see Chinese patent application CN 101921733 B, entitled "A Method for Preparing and Using Qβ-2aa Bacteriophage Virus-Like Particle Protein," published on June 5, 2013.

[0044] 1b) Inducing expression of Qβ-2aa VLPs: First, the stored E. coli DH5α / pGEXQβ-A1 recombinant strain was removed from a liquid nitrogen tank, activated, and plated onto LB solid medium plates. The plates were cultured in a 37°C incubator overnight. A single colony was selected and cultured in LB liquid medium. After incubation at 37°C on a shaker for 5 hours, 0.2 M IPTG was added to induce the recombinant strain to express Qβ-2aa VLPs. The induction was continued for 6 hours. The bacterial culture was collected and lysed by ultrasonication to obtain the lysate supernatant.

[0045] 1c) Purification of Qβ-2aa VLPs: The lysate supernatant was subjected to ammonium sulfate precipitation, acidification, hydrophobic chromatography, and gel chromatography to obtain purified Qβ-2aa VLPs;

[0046] 1d) Identification of Qβ-2aa VLP: The purified Qβ-2aa VLP was dissociated with dithiothreitol (DTT). The dissociated Qβ-2aa VLP was subjected to gel electrophoresis to determine its molecular weight. The morphology, size, and particle size were observed under an electron microscope. Finally, the obtained protein was confirmed to be Qβ-2aa VLP based on the test results.

[0047] 2) Preparation of β1-AR vector vaccine: The peptide obtained in Example 1 was coupled with the vector Qβ-2aa VLP using a heterobifunctional crosslinker (Sulfo-SMCC) to obtain a β1-AR vector vaccine, which is the ABRQβ-006 vector vaccine.

[0048] 3) The ABRQβ-006 vector vaccine obtained in step 2) was subjected to reduced SDS-PAGE gel electrophoresis detection, and the detection results were as follows: Figure 1 As shown: Compared with the Qβ lane, it can be observed that the vector vaccine ABRQβ-006 is mainly composed of 1 Qβ-2aa VLP monomer coupled with 2-3 ABR-006 short peptides, and the coupling efficiency is high, proving that the vector vaccine was successfully prepared.

[0049] Example 3: Effect of β1-AR vector vaccine on elevated blood pressure induced by nitric oxide synthase inhibitor L-NAME

[0050] Male Sprague-Dawley rats were immunized with the vector vaccine ABRQβ-006. The nitric oxide synthase inhibitor L-NAME was added to the rats' drinking water to investigate whether the β1-AR immunogenic vector vaccine could reduce the L-NAME-induced increase in blood pressure. The specific experimental process was as follows:

[0051] 1) Five-week-old male SD rats were actively immunized and divided into four groups as follows:

[0052] Group 1: blank control group (control): PBS was injected subcutaneously at multiple points on the back on days 0, 14, and 28, with a dose of 300 μl per animal, for a total of 10 animals;

[0053] Group 2: L-NAME intervention group (L-NAME): Qβ-2aaVLP was injected subcutaneously at multiple points on the back on days 0, 14, and 28, with a dose of 300 μg / mouse (300 μl system), for a total of 10 mice;

[0054] Group 3: ABRQβ-006 vaccine group (ABRQβ-006): 10 rats were injected with the vector vaccine ABRQβ-006 at multiple points on the back at 0, 14, and 28 days, with a dose of 300 μg / rat (300 μl system);

[0055] Group 4: Metoprolol gavage group (METO): After 14 days, metoprolol was given by gavage at a dose of 20 mg / kg / d, with a total of 10 rats;

[0056] On day 19, L-NAME was added to the drinking water of groups 2 to 4 at a dose of 50 mg / L for a total of 6 weeks;

[0057] 2) Blood collection: Blood was collected from the tail of the rats on days 7, 21, 35, 49, and 63. The supernatant was collected by centrifugation at 3000 rpm for 10 min at room temperature and stored at -80°C until use.

[0058] 3) ELISA experiment

[0059] Since the vaccine carrier for immunizing animals is Qβ-2aa VLP, in order to avoid cross-reaction, bovine serum albumin (BSA) was coupled with ABR-006 to prepare a coating substrate. The coated plate was prepared and the antibody titer against the β1-AR immunogenic peptide ABR-006 was determined by ELISA.

[0060] 3a) Serial dilution: Serum supernatant was diluted with 10% FBS in PBS buffer in a serial dilution series of 1:100, 1:1000, 1:5000, 1:10000, 1:20000, 1:40000, and 1:80000.

[0061] 3b) Incubation with primary antibody: Pipette serum samples diluted 1:1000, 1:5000, 1:10000, 1:20000, 1:40000, or 1:80000 into a coated 96-well plate at 100 μl / well and incubate at 37°C for 2 h.

[0062] 3c) Incubation with secondary antibody: After primary antibody incubation, discard the liquid and wash three times with washing buffer (0.03% PBST pH 7.4), pat dry, and then add horseradish peroxidase-conjugated goat anti-rat secondary antibody (1:3000 dilution in 10% FBS PBS buffer) at 100 μl / well and incubate at 37°C for 0.5 h.

[0063] 3d) Color development: After secondary antibody incubation, discard the solution and wash three times with washing buffer (0.03% PBST pH 7.4), pat dry, and then add TMB color development solution (100 μl / well) and observe the color change at room temperature;

[0064] 3e) Stop the reaction: When the blank control wells just begin to turn green, add 100 μl / well of stop solution (1 M dilute hydrochloric acid);

[0065] 3f) Reading: After adding the stop solution, place the tube on a microplate reader and read the absorbance (OD) at 450 nm.

[0066] 3g) Analysis of results: The OD value of the sample to be tested is not less than 2.1 times that of the blank control group as the standard for positivity, and then the antibody titer of the corresponding sample is calculated.

[0067] The results are as follows Figure 2 : After rats were immunized with the ABRQβ-006 vector vaccine, specific antibodies against the ABR-006 immunogenic short peptide were produced. The antibody titer in the rat serum began to rise one week after the second immunization and reached the highest level (1:140,000 to 1:240,000) after the third immunization. The antibody titer level remained high during the subsequent experiments.

[0068] 4) Blood pressure level measurement

[0069] The blood pressure and heart rate of the animals were monitored using a Softron BP98A rat tail sphygmomanometer on days 0, 3, 17, 25, 33, 40, 47, and 54. The specific monitoring methods are as follows:

[0070] 4a) Preheating: Place the animal to be tested in a 37°C heating cage for 15 minutes;

[0071] 4b) Fixation: After preheating, transfer the animal to be tested to a corresponding fixed cage with heating function. Pass the exposed tail through the measurement sensor and place the sensor at the base of the tail to prepare for blood pressure and heart rate measurement;

[0072] 4c) Measurement: Connect the sensor to a Softron BP98A rat tail sphygmomanometer via a data cable, and then start measuring blood pressure and heart rate. Take 15 sets of readings each time and take the average value.

[0073] The measurement was carried out between 9:00 and 11:00 in the morning. The environment was kept quiet and the room temperature was kept at 25°C during the measurement.

[0074] The results are as follows Figure 3 In this hypertension model, ABRQβ-006 had no effect on heart rate after administration of L-NAME in drinking water, whereas metoprolol reduced heart rate. Systolic blood pressure was significantly elevated in the L-NAME group compared to the control group (maximum: 23 mmHg). Compared to the L-NAME group, the ABRQβ-006 vector vaccine significantly reduced systolic blood pressure (mean: 10 mmHg), with a blood pressure-lowering effect similar to that of metoprolol. Thus, the vector vaccine ABRQβ-006 exhibits a blood pressure-lowering effect in the L-NAME-induced hypertension model.

[0075] The entire experimental data in Example 3 show that the ABRQβ-006 vector vaccine can be well coupled with the Qβ vector, stimulate rat B cells to produce high levels of antibodies against ABR-006, and has a hypotensive effect in the L-NAME-induced hypertension model.

[0076] Example 4: Effect of β1-AR immunogenic vector vaccine on cardiac remodeling after myocardial infarction

[0077] The vector vaccine ABRQβ-006 was programmed to immunize C57 mice and establish a myocardial infarction model in the mice to investigate whether the β1-AR immunogenic vector vaccine improves cardiac remodeling after myocardial infarction. The specific experimental process was as follows:

[0078] 1) Six-week-old male C57 mice were actively immunized and divided into four groups as follows:

[0079] Group 1: Sham operation group (SHAM): PBS was injected subcutaneously at multiple points on the back on days 0, 14, and 28, with a dose of 100 μl per mouse, for a total of 16 mice;

[0080] Group 2: Myocardial infarction model group (MI): PBS was injected subcutaneously at multiple points on the back on days 0, 14, and 28, with a dose of 100 μl per mouse, for a total of 21 mice;

[0081] Group 3: ABRQβ-006 vaccine + MI group (ABRQβ-006): 20 rats were injected with vector vaccine ABRQβ-006 at multiple points on the back subcutaneously on days 0, 14, and 28, respectively, at a dose of 100 μg / rat (100 μl system);

[0082] Group 4: Metoprolol gavage group (METO): Metoprolol was administered by gavage after 14 days at a dose of 20 mg / kg / d, with a total of 19 rats;

[0083] 2) Blood collection: Blood was collected from the tail of the rats on days 7, 21, 35, 56, 70, and 81. The supernatant was collected by centrifugation at 3000 rpm for 10 min at room temperature and stored at -80°C until use.

[0084] 3) ELISA experiment

[0085] Since the vaccine carrier for immunizing animals is Qβ-2aa VLP, in order to avoid cross-reaction, bovine serum albumin (BSA) was coupled with ABR-006 to prepare a coating substrate. The coated plate was prepared and the antibody titer against the β1-AR immunogenic peptide ABR-006 was determined by ELISA.

[0086] The specific implementation method of ELISA was the same as before. The result analysis used an OD value not less than 2.1 times that of the blank control group as the standard for positive test samples, and then calculated the antibody titer value of the corresponding sample.

[0087] The results are as follows Figure 4 As shown: After mice were immunized with the ABRQβ-006 vector vaccine, specific antibodies against the ABR-006 immunogenic short peptide were produced. The antibody titer in the mouse serum began to rise one week after the second immunization and reached the highest level (1:180,000-1:470,000) after the third immunization. The high antibody titer level was maintained during the subsequent experiments.

[0088] 4) Myocardial infarction model:

[0089] Four days after the second immunization of mice, myocardial infarction was induced by ligating the left anterior descending coronary artery. The model was established by the heart squeeze method. The animals were divided into four groups: the SHAM group only underwent thoracotomy without ligation, and the MI, Qβ-006, and METO groups all underwent the same coronary artery ligation model.

[0090] 4a) Anesthesia and skin preparation: Mice were anesthetized with 1% sodium pentobarbital (100 ml / kg) injected intraperitoneally. The mice were placed in the supine position on a thermostatic operating pad maintained at 37°C. The chest and abdomen were exposed, and the skin of the chest was prepared and disinfected three times with iodine-containing cotton balls.

[0091] 4b) Intubation: Illuminate the mouse's neck from the ventral side with a cold light source. Gently pull the mouse's tongue to one corner of the mouth. Use forceps or a thin cotton swab to lift the mouse's lower jaw. The tracheal opening in the middle of the throat should be visible. Gently insert the endotracheal tube. Once inserted to the appropriate depth, secure the tube. Instill a small amount of air. If the chest rises, confirm that intubation is successful. Connect the ventilator and set the tidal volume to 3 ml, the respiratory rate to 110 breaths / minute, and the inspiration-expiration ratio to 1:1.3.

[0092] 4c) Thoracotomy and heart compression: A small incision of approximately 1 cm is made on the left chest. After separating the pectoralis major and minor muscles, the third intercostal space is exposed. A small hole is created in the third intercostal space with a vascular clamp. The pleura and pericardium are then ruptured in sequence. The intercostal opening is slightly opened with a vascular clamp. Gentle pressure is applied to the chest wall, and the heart is then smoothly ejected from the opening.

[0093] 4d) Ligation of the left anterior descending artery: Use 6-0 suture to ligate the left anterior descending artery 2 mm below the left atrial appendage. Ligation is considered successful when the left ventricular wall turns white. At this time, ST segment elevation can be observed on the limb leads of the electrocardiogram.

[0094] 4e) Suturing: Reposition the heart, evacuate the chest cavity, suture the muscles and skin in sequence, and disinfect with iodine three times;

[0095] 4f) Anesthesia Recovery: The mouse was untied and placed on a heating pad in the cage. The mouse was allowed to breathe the room air freely while waiting for anesthesia recovery. During the recovery period, the mouse's body temperature, respiration, and other general conditions were monitored.

[0096] 5) Heart rate measurement

[0097] The heart rates of mice were monitored on days 0, 7, 36, 53, and 67 using a Softron BP98A rat tail sphygmomanometer. The specific monitoring method was the same as above.

[0098] The results are as follows Figure 5 After myocardial infarction (MI) modeling, the ABRQβ-006 vaccine reduced heart rate (mean, 60 bpm) compared to the MI group (571±12 vs 633±11 bpm, P<0.01), similar to the effect of metoprolol. This suggests that the vector vaccine ABRQβ-006 has a heart rate-lowering effect in the MI model.

[0099] 6) Cardiac ultrasound examination:

[0100] Echocardiography was performed 2 months after acute myocardial infarction. Mice were anesthetized with 1.5% isoflurane at 36.5–37.5°C, and images were acquired using a Vevo 3100 high-resolution imaging system (Visualsonics, Canada) equipped with a 30 MHz transducer. All measurements were analyzed offline by an observer using Vevo 3100 workstation software. Each measurement reported is an average of 10 cardiac cycles. Left ventricular views were obtained from parasternal short-axis M-mode views. Left ventricular end-diastolic dimension (LVIDD) and left ventricular end-systolic dimension (LVIDS) were measured, and fractional shortening (FS) was calculated: FS = (LVIDD - LVIDS) / LVIDD × 100%. Parasternal long-axis scans were used to provide data on left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV). Left ventricular ejection fraction (LVEF) = [(LVEDV - LVESV) / LVEDV] x 100%.

[0101] The results are as follows Figure 6 Table 1: M-mode echocardiography showed post-MI ventricular wall thinning, restricted anterior and posterior wall motion, and significant left ventricular dilatation in the MI group. The ABRQβ-006 vaccine group had relatively restricted anterior wall motion, normal posterior wall motion, and significantly smaller ventricular size than the MI group. Compared with the MI group, the ABRQβ-006 vaccine group had significantly improved ejection fraction and fractional shortening (ejection fraction 42.0±3.2% vs 25.7±3.2%, P < 0.05; fractional shortening 20.8±1.8% vs 12.4±1.6%, P < 0.05). ABRQβ-006 vaccine significantly reduced left ventricular dilatation (left ventricular end-diastolic volume 65.2±7.0 vs 91.5±7.7 μl, P < 0.05; left ventricular end-diastolic diameter 3.82±0.2 vs 4.49±0.2 mm, P < 0.05). In contrast, metoprolol did not improve cardiac function after MI. Therefore, the vector vaccine ABRQβ-006 can effectively improve cardiac function after myocardial infarction, while metoprolol did not.

[0102] Table 1 Left ventricular M-mode ultrasound results

[0103]

[0104] Note: SHAM represents the sham-operated group receiving PBS; MI represents male C57 mice receiving PBS and undergoing myocardial infarction; ABRQβ-006 represents male C57 mice receiving the vector vaccine ABRQβ-006 and undergoing myocardial infarction; METO represents male C57 mice receiving metoprolol orally and undergoing myocardial infarction. #P<0.05, ##P<0.01, ###P<0.001 vs. SHAM group; *P<0.05, **P<0.01, ***P<0.001 vs. MI group.

[0105] 7) Left ventricle MASSON staining

[0106] After the experiment, the mice were killed and the hearts were quickly isolated. After rinsing twice in PBS, a part of the left ventricle was cut out and immersed in 4% paraformaldehyde. The tissue was routinely dehydrated, paraffin-sectioned, and MASSON-stained to observe left ventricular remodeling.

[0107] like Figure 7 As shown: Myocardial fibrosis and heart area were calculated using imaging software and a photo ruler, and the percentage of myocardial fibrosis area (= myocardial fibrosis area / total heart area) was obtained. A significant decrease in the percentage of myocardial fibrosis area was observed in the ABRQβ-006 vaccine group (22.7±12.5%) compared with the MI group (50.7±17.1%) (P=0.011), while there was no significant difference between the METO group (44.0±20.6%) and the MI group (P=0.424).

[0108] The entire experimental data in Example 4 shows that the ABRQβ-006 vector vaccine can be well coupled with the Qβ vector, stimulate mouse B cells to produce high levels of antibodies against ABR-006, and has the effect of improving myocardial remodeling and cardiac function after myocardial infarction in the MI model, and is superior to metoprolol, the longest-used drug in clinical practice.

[0109] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An immunogenic peptide segment targeting β1-adrenergic receptor, characterized by: The amino acid sequence of the immunogenic peptide segment is DEARRCYND, as shown in SEQ ID NO.

1.

2. Use of the immunogenic peptide segment according to claim 1 in the preparation of a product for treating hypertension and improving cardiac remodeling after myocardial infarction.

3. An immunogenic vector vaccine targeting human β1-adrenergic receptor, characterized by: The immunogenic vector vaccine targeting human β1-adrenergic receptor is prepared by coupling the immunogenic peptide segment targeting human β1-adrenergic receptor according to claim 1 with a carrier; wherein the carrier is Qβ-2aa phage virus-like particle protein.

4. Use of the immunogenic vector vaccine targeting human β1-adrenergic receptor according to claim 3 in the preparation of a drug for treating hypertension.

5. Use of the immunogenic vector vaccine targeting human β1-adrenergic receptor according to claim 3 in the preparation of a drug for improving cardiac remodeling after myocardial infarction.

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