Immunogenic peptide fragment aiming at human sphingosine 1-phosphate receptor 2 and application of immunogenic peptide fragment

Abstract

The invention belongs to the technical field of biology, and particularly relates to an immunogenic peptide fragment aiming at a human sphingosine 1-phosphate receptor 2 and application of the immunogenic peptide fragment. The amino acid sequence of the immunogenic peptide fragment is SEQ ID No.1. The immunogenic vector vaccine aiming at the human sphingosine 1-phosphate receptor 2 is prepared by coupling the immunogenic peptide fragment of the human sphingosine 1-phosphate receptor 2 and a vector, and the vector is Q beta-2aa bacteriophage virus-like particle protein. According to the invention, the preferentially designed immunogenic peptide fragment of the human sphingosine 1-phosphate receptor 2 is coupled with the Qbeta-2aa bacteriophage virus-like particle protein carrier to successfully prepare the carrier vaccine. The immunogenic vector vaccine can be used for preparing drugs for preventing and treating abdominal aortic aneurysm.

Description

Technical Field

[0001] The present invention relates to the field of medical biotechnology, and particularly relates to an immunogenic peptide segment against human sphingosine-1-phosphate receptor 2 and its application. Background Art

[0002] Abdominal aortic aneurysm (AAA) is a serious vascular disease that poses a major threat to human health. AAA is defined as a permanent local dilation of the abdominal aorta with a diameter ≥ 50% of the normal arterial diameter. Clinically, AAA can also be diagnosed when the abdominal aortic diameter > 3 cm. AAA mostly occurs in the elderly with cardiovascular risk factors (such as smoking, hypertension, and hyperlipidemia), especially in men, and often coexists with atherosclerosis. The Global Burden of Disease Study shows that in the past 30 years, the mortality and disability-adjusted life years caused by AAA have both shown an upward trend.

[0003] Despite the progress in the research on the etiology and pathology of AAA, there is still a lack of effective drug treatment at present. Clinical trials of antihypertensive drugs, statins, doxycycline drugs, and antiplatelet drugs have not shown significant benefits, and surgical repair remains the only available treatment method for high-risk patients. Therefore, it is of great significance to find new therapeutic targets and methods.

[0004] Sphingosine-1-phosphate (S1P) is an important lipid signaling molecule, which is mainly produced by the catalysis of multiple enzymes on the cell membranes of red blood cells, platelets, and endothelial cells from sphingomyelin. Sphingosine kinase (SphK), which is divided into SphK1 and SphK2, is the rate-limiting enzyme for S1P synthesis, and SphK1 plays a major role in most cases. Inside the cell, S1P can directly bind to some intracellular targets as a second messenger and regulate the survival, proliferation, differentiation, and apoptosis of cells. Outside the cell, S1P transmits signals by binding to specific receptors. The receptors of S1P are members of the rhodopsin-like G protein-coupled receptors (GPCRs), and 5 subtypes (S1PR1-5) have been identified. Each receptor subtype is expressed in different tissues and cells and thus has different functions. For example, S1PR1 / 2 / 3 are widely distributed in most tissues, especially in the immune, cardiovascular, and central nervous systems, S1PR4 is mainly expressed in the lymphatic system, hematopoietic tissue, and lungs, and S1PR5 is mainly expressed in the brain and spleen. In terms of cardiovascular diseases, S1P / S1PR has been proven to be related to atherosclerosis, angiogenesis, ischemia-reperfusion injury, vascular tone, and myocardial fibrosis, etc. Therefore, S1P / S1PR may be a powerful candidate target.

[0005] The key pathological processes related to AAA include inflammatory cell infiltration, cytokine production, matrix metalloproteinase activation, extracellular matrix degradation, phenotypic transformation of vascular smooth muscle cells (VSMCs), VSMC death, neovascularization, and thrombosis. However, the relationship between S1PR2 and AAA remains unclear. Our team first reported in 2021 that S1PR2 was significantly highly expressed in the dilated or ruptured aortic tissues of patients with dissecting aneurysm and mice (Front Cardiovasc Med. 2021 Dec 17; 8: 748486). The specific S1PR2 antagonist JTE-013 can not only inhibit angiotensin II-induced aortic dilation, but also preserve the elastic structure of the media, reduce VSMC apoptosis, and alleviate aortic wall inflammation. Given that VSMC apoptosis, macrophage infiltration, and inflammatory factor secretion are also important mechanisms in the formation and development of AAA, we have reason to speculate that targeting S1PR2 may be a promising therapeutic strategy for AAA.

[0006] Currently, there is no specific S1PR2 antagonist on the market clinically. JTE-013 is the most widely used S1PR2 antagonist, first reported by Japanese scientists in 2001, but it is only used as a tool drug. It inhibits the specific binding of radiolabeled S1P to the cell membrane of Chinese hamster ovary cells stably transfected with human or rat S1PR2, and does not affect the binding of S1P to S1PR1 and S1PR3. However, in recent years, some studies have questioned the specificity of JTE-013, reporting its effects on other S1PRs and its role in non-S1PR-mediated biological functions. For example, in addition to S1PR2, JTE-013 also inhibits S1PR3-mediated cerebrovascular constriction in rodents and S1PR4-mediated calcium mobilization. However, JTE-013 is not targeted, and JTE-013 can also inhibit the vasoconstriction induced by prostaglandin analogue U46619, endothelin-1, and high potassium chloride (the vasoconstriction induced by high potassium chloride is not S1PR-mediated, but is related to L-type Ca 2+ channels).

[0007] Therefore, developing key molecules or receptors targeted by therapeutic vaccines has the advantages of high specificity, long-lasting effect, good compliance, and low cost, making up for the deficiencies of JTE-013 in weak specificity and multiple administrations, and providing a new idea for the treatment of AAA. Summary of the Invention

[0008] The object of the present invention is to overcome the deficiencies of the prior art, and provides an immunogenic peptide against human S1PR2 and its application. The present invention selects S1PR2 as the target to design an immunogenic peptide, and uses this immunogenic peptide to innovatively screen out a therapeutic vaccine specifically targeting S1PR2, which can effectively inhibit the formation and progression of AAA.

[0009] To achieve the above object, the technical solution designed by the present invention is as follows:

[0010] The present invention provides an immunogenic peptide against human S1PR2, and the amino acid sequence of the immunogenic peptide is TPVQWFARE, i.e., SEQ ID No.1.

[0011] The present invention also provides an application of the above immunogenic peptide in the preparation of products for preventing and treating AAA.

[0012] The present invention also provides an immunogenic carrier vaccine for preventing and treating AAA, and the immunogenic carrier vaccine is prepared by coupling the immunogenic peptide described in claim 1 with a carrier.

[0013] As a preferred embodiment, the carrier is Qβ-2aa phage virus-like particle protein.

[0014] As a preferred embodiment, the immunogenic carrier vaccine is an injection preparation.

[0015] As a preferred embodiment, the injection preparation is a freeze-dried powder injection or an aqueous solution. The beneficial effects of the present invention:

[0016] 1. According to the characteristics of the extracellular loop amino acid sequence, hydrophilicity, antigenicity, accessibility, etc. of human S1PR2, and by comprehensively using bioinformatics and pharmacological methods, the present invention designs a peptide against human S1PR2: SEQ ID No.1, and through verification, it is obtained that the SEQ ID No.1 peptide can effectively induce the production of specific antibodies in immunized mice.

[0017] 2. Selecting a suitable carrier is the key to the successful development of a vaccine. The present invention selects Qβ-2aa phage virus-like particle protein.

[0018] 3. The present invention successfully couples the designed peptide against human S1PR2 with the Qβ-2aa phage virus-like particle protein carrier to prepare a carrier vaccine. The prepared carrier vaccine is pre-immunized in C57BL / 6 mice. After high-titer specific antibodies are produced in the mice, AAA is modeled in the mice to study whether the immunogenic carrier vaccine against human S1PR2 can prevent abdominal aortic dilation in mice and reduce the incidence of AAA. The experimental results show that the immunogenic carrier vaccine against human S1PR2 can prevent abdominal aortic dilation in mice and reduce the incidence of AAA.

[0019] 4. Immunize C57BL / 6 mice with the prepared vector vaccine while establishing an AAA model in them. After the formation and stabilization of AAA (14 days), stable high-titer specific antibodies are produced in the mice. Then, study whether the immunogenic vector vaccine against human S1PR2 can inhibit abdominal aortic dilation and the progression of AAA. The experimental results show that the immunogenic vector vaccine against human S1PR2 can effectively inhibit abdominal aortic dilation, preserve the elastic fiber structure of the tunica media, and inhibit the progression of AAA.

[0020] 5. Immunize BALB / C mice with the peptide against human S1PR2. Through the preparation of hybridoma cells and subcloning, produce monoclonal antibodies against human S1PR2. While establishing an AAA model in the mice, inject the prepared monoclonal antibodies and JTE-013 into the mice respectively, and study whether the monoclonal antibodies against human S1PR2 and JTE-013 can inhibit abdominal aortic dilation and the progression of AAA. The experimental results show that both the monoclonal antibodies against human S1PR2 and JTE-013 can effectively inhibit abdominal aortic dilation and the progression of AAA.

[0021] In summary, the present invention selects S1PR2 as the target to design an immunogenic peptide segment, and based on the immunogenic peptide segment, innovatively screens out a therapeutic vaccine specifically targeting S1PR2. Pre-immunization with this vaccine can effectively inhibit abdominal aortic dilation in mice and reduce the incidence of AAA. Even after the formation of AAA, this vaccine can also effectively inhibit the progression of AAA. In addition, the monoclonal antibodies prepared against the above peptide segment have a similar therapeutic effect on abdominal aortic aneurysm as JTE-013. Compared with chemically synthesized drugs, the therapeutic vaccine has the remarkable characteristics of high treatment specificity, long action time, low cost, no need for daily administration, and good patient compliance; compared with monoclonal antibodies, it has an absolute price advantage. At the same time, compared with monoclonal antibodies that are generally administered once every two weeks, the vaccination interval of the vaccine is longer (generally 1 - 3 months for the first three times and 3 - 6 months for booster immunization), and it has more advantages in terms of compliance. Description of the Drawings

[0022] Figure 1 It is the SDS-PAGE gel electrophoresis detection diagram of the vector vaccine SPRQβ-009 in Example 2.

[0023] Figure 2 It is the diagram of the antibody titer against the SPRQβ-009 short peptide produced after pre-immunizing male C56BL / 6J mice with the vector vaccine SPRQβ-009 in Example 3.

[0024] In the figure, the arrow indicates the time point when the vector vaccine SPRQβ-009 immunizes the mice.

[0025] Figure 3 Gross and statistical charts of the abdominal aorta of male C57BL / 6J mice pre-immunized with the vector vaccine SPRQβ-009 in Example 3.

[0026] In the figure, A represents the gross picture of the mouse aorta, B represents the statistical chart of the maximum diameter of the mouse aorta (SPRQβ-009 1.31 mm vs AAA 1.70 mm), C represents the statistical chart of the incidence of mouse AAA (SPRQβ-009 63.64% vs AAA 95.45%), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.01 vs the AAA group.

[0027] Figure 4 Chart of the change in systolic blood pressure of male C57BL / 6J mice pre-immunized with the vector vaccine SPRQβ-009 in Example 3. The arrow indicates the time point when the vector vaccine SPRQβ-009 was used to immunize the mice.

[0028] Figure 5 Gross and statistical charts of the abdominal aorta sections of male C57BL / 6J mice pre-immunized with the vector vaccine SPRQβ-009 in Example 3.

[0029] In the figure, A represents the HE and EVG staining pictures of the mouse aorta, B represents the statistical chart of the elastic fiber degradation score (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs the AAA group).

[0030] Figure 6 Chart of the anti-SPRQβ-009 short peptide antibody titer of male C57BL / 6J mice immunized with the vector vaccine SPRQβ-009 and simultaneously subjected to AAA modeling in Example 4.

[0031] In the figure, the arrow indicates the time point when the vector vaccine SPRQβ-009 was used to immunize the mice.

[0032] Figure 7 Chart of the change in systolic blood pressure of male C57BL / 6J mice immunized with the vector vaccine SPRQβ-009 and simultaneously subjected to AAA modeling in Example 4.

[0033] In the figure, the arrow indicates the time point when the vector vaccine SPRQβ-009 was used to immunize the mice.

[0034] Figure 8 Gross and statistical charts of the abdominal aorta of male C57BL / 6J mice immunized with the vector vaccine SPRQβ-009 and simultaneously subjected to AAA modeling in Example 4.

[0035] In the figure, A represents the gross image of the mouse aorta, B represents the ultrasound image of the mouse aorta, C represents the statistical chart of the maximum gross diameter of the mouse aorta (SPRQβ-009 1.56 mm vs AAA 1.81 mm), D represents the statistical chart of the maximum ultrasound diameter of the mouse aorta (SPRQβ-009 1.02 mm vs AAA 1.18 mm), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.01 vs the AAA group.

[0036] Figure 9 It is the abdominal aorta section and statistical chart of immunizing male C57BL / 6J mice with the vector vaccine SPRQβ-009 and simultaneously establishing an AAA model in Example 4.

[0037] In the figure, A represents the HE and EVG staining images of the mouse aorta, B represents the statistical chart of the elastic fiber degradation score (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs the AAA group). Detailed implementation mode

[0038] The present invention will be further described in detail below in conjunction with specific embodiments for those skilled in the art to understand.

[0039] The immunogenic peptide segment targeting human S1PR2 is the SPR-009 immunogenic peptide segment (hereinafter referred to as the SPR-009 peptide segment), and the immunogenic vector vaccine targeting human S1PR2 is hereinafter referred to as the SPRQβ-009 vector vaccine.

[0040] Example 1 Preparation of SPR-009 immunogenic peptide segment

[0041] According to the spatial conformation, bioinformatics and pharmacological characteristics of S1PR2, an immunogenic peptide segment targeting the extracellular amino acid sequence of human S1PR2 was designed, named SPR-009, which is the SPR-009 immunogenic peptide segment (abbreviation: SPR-009 peptide segment). The specific amino acid sequence is TPVQWFARE, as shown in SEQ ID No.1.

[0042] The above SPR-009 peptide segment was synthesized by solid-phase synthesis (commissioned Shanghai Gil Biochemical Co., Ltd. for synthesis and quality inspection). The purity of the synthesized peptide segment was analyzed by high-performance liquid chromatography. The purity of the SPR-009 peptide segment was detected to reach more than 98%. The obtained SPR-009 peptide segment was freeze-dried, aliquoted and placed in a cryotube, and stored at -80 °C for later use.

[0043] Example 2 Preparation of SPRQβ-009 vector vaccine

[0044] 1. Prepare the SPRQβ-009 vector vaccine using the Qβ-2aa phage virus-like particle protein. The specific preparation process is as follows:

[0045] 1) Prepare the Qβ-2aa phage virus-like particle protein: The English abbreviation of the Qβ-2aa phage virus-like particle protein is Qβ-2aa VLP. Hereinafter, Qβ-2aa VLP is used to represent the Qβ-2aa phage virus-like particle protein.

[0046] The preparation method of Qβ-2aa VLP is as follows:

[0047] 1a) Obtain the recombinant strain expressing Qβ-2aa VLP: This recombinant strain is Escherichia coli DH5α / pGEXQβ-A1, which can induce the production of Qβ-2aa virus-like particle protein. The preservation number of Escherichia coli DH5α / pGEXQβ-A1 is CCTCC NO: M209282. For the specific preparation process, refer to the Chinese patent: A preparation method and use of Qβ-2aa phage virus-like particle protein, and the authorized publication date is the authorization announcement date of CN 101921733 B on June 5, 2013.

[0048] 1b) Induce the expression of Qβ-2aa VLP: First, take out the preserved Escherichia coli DH5α / pGEXQβ-A1 recombinant strain from the liquid nitrogen tank. After activating this recombinant strain, coat it on the LB solid medium plate and culture it overnight in a 37°C incubator. Pick a single colony and culture it in LB liquid medium. After culturing in a 37°C constant temperature shaker for 5 h, add 0.2 M IPTG to induce the recombinant strain to express Qβ-2aa VLP, induce for 6 hours, collect the bacterial liquid and perform ultrasonic lysis to obtain the lysate supernatant;

[0049] 1c) Purification of Qβ-2aa VLP: The lysate supernatant is subjected to ammonium sulfate precipitation, acidification treatment, hydrophobic chromatography, and gel chromatography to obtain purified Qβ-2aa VLP;

[0050] 1d) Identification of Qβ-2aa VLP: Treat the purified Qβ-2aa VLP with dithiothreitol (DTT) for dissociation, perform gel electrophoresis on the dissociated Qβ-2aa VLP to identify its molecular weight, and observe its morphological size and particle diameter by electron microscopy; finally, determine through the test results that the obtained protein is Qβ-2aa VLP;

[0051] 2) Preparation of the SPRQβ-009 vector vaccine: Couple the SPR-009 peptide segment obtained in Example 1 with the vector Qβ-2aaVLP. The coupling reaction uses a heterobifunctional cross-linking agent (Sulfo-SMCC) to obtain the SPRQβ-009 vector vaccine.

[0052] 3) The SPRQβ-009 vector vaccine obtained in step 2) was detected by reducing SDS-PAGE gel electrophoresis.

[0053] The detection results are as Figure 1 shown: By comparing with the VLP lane, it can be observed that the vector vaccine SPRQβ-009 mainly consists of 1 Qβ conjugated with 3-4 SPR-009 short peptides, and the conjugation efficiency is relatively high, proving the successful preparation of the vector vaccine.

[0054] Example 3 Preventive effect of pre-immunization with SPRQβ-009 vector vaccine on elastase-induced AAA

[0055] The C57BL / 6J mice were pre-immunized with the vector vaccine SPRQβ-009. After high-titer specific antibodies were produced in the mice, the mice were subjected to AAA modeling to explore whether the SPRQβ-009 immunogenic vector vaccine could prevent abdominal aortic dilation in mice and reduce the incidence of AAA. The specific experimental process was as follows:

[0056] 1) Male C57BL / 6J mice at 4 weeks of age were selected. After 1 week of adaptive feeding, they were divided into 3 groups as follows:

[0057] The first group: Control group (Control, n = 15): No treatment was given.

[0058] The second group: AAA model group (AAA, n = 22): PBS was injected subcutaneously at multiple points on the back at 0, 14, and 28 days of the experiment, with a dose of 100 μg / mouse, and the first dose was doubled.

[0059] The third group: SPRQβ-009 vaccine group (SPRQβ-009, n = 22): The vector vaccine SPRQβ-009 was injected subcutaneously at multiple points on the back at 0, 14, and 28 days of the experiment, with a dose of 100 μg / mouse, and the first dose was doubled.

[0060] 2) Blood collection: Blood was collected from the mouse tails at 7, 21, 35, and 49 days respectively. The supernatant was collected by centrifugation at 3000 rpm for 10 min at room temperature and stored at -80 °C for later use.

[0061] 3) ELISA experiment:

[0062] Since the vaccine vector for immunized animals is Qβ-2aa VLP, in order to avoid cross-reaction, bovine serum albumin (BSA) was conjugated with the SPR-009 peptide segment to prepare a coating substrate, and a coated plate was prepared. The antibody titer of the immunogenic peptide SPR-009 of S1PR2 was measured by ELISA;

[0063] 3a) Serial dilution: Use PBS buffer with 10% FBS as the diluent to serially dilute the serum supernatant at gradients of 1:100, 1:1000, 1:5000, 1:10000, 1:20000, 1:40000, and 1:80000;

[0064] 3b) Incubate with primary antibody: Use a pipette to add the serially diluted serum specimens at 1:1000, 1:5000, 1:10000, 1:20000, 1:40000, and 1:80000 to the coated 96-well plate, 100 μl per well, and incubate in an incubator at 37 °C for 2 h;

[0065] 3c) Incubate with secondary antibody: After the incubation with the primary antibody is completed, discard the liquid and wash 3 times with the washing solution (0.03% PBST, pH 7.4), pat dry, and then add the secondary antibody of horseradish peroxidase-labeled goat anti-rat (diluted at 1:3000, with the diluent being PBS buffer with 10% FBS), 100 μl per well, and incubate in an incubator at 37 °C for 0.5 h;

[0066] 3d) Color development: After the incubation with the secondary antibody is completed, discard the liquid and wash 3 times with the washing solution (0.03% PBST, pH 7.4), pat dry, and then add the TMB color development solution, 100 μl per well, and observe the color change at room temperature;

[0067] 3e) Terminate the reaction: After the color of the blank control well just begins to turn green, add the termination solution (1 M dilute hydrochloric acid), 100 μl per well;

[0068] 3f) Reading: After adding the termination solution, read the absorbance (OD) value at a wavelength of 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader;

[0069] 3g) Result analysis: Take the OD value not less than 2.1 times that of the blank control group as the standard for a positive test specimen, and then calculate the antibody titer value of the corresponding specimen.

[0070] The results are as Figure 2 : After immunizing C57BL / 6J mice with the SPRQβ-009 vector vaccine, specific antibodies against the SPR-009 immunogenic peptide were produced, and the antibody titer in the mouse serum reached the highest level (1:420 * 10 3 ) 1 week after the second immunization, and maintained a relatively high antibody titer level during the subsequent experiment.

[0071] 4) AAA modeling:

[0072] One week after the third immunization of mice, porcine pancreatic elastase (Sigma-Aldrich, E1250) was used to induce AAA in mice. The Control group was treated with an equal volume of 0.9% saline, and the same modeling was performed in the AAA group and the SPRQβ-009 group.

[0073] 4a) Anesthesia and skin preparation: Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 100 ml / kg. The mice were fixed in the supine position on a thermostatic operating pad with the temperature adjusted to 37°C. The chest and abdomen were exposed, and the abdomen was prepared for skin, and the area was disinfected three times with iodophor cotton balls.

[0074] 4b) Exposure of the abdominal aorta: A 2.5-cm longitudinal incision was made on the skin along the midline of the lower abdomen with surgical scissors. The underlying muscles were gently lifted, and a 2.5-cm longitudinal incision was made along the white line to enter the abdominal cavity. The intestines and stomach were moved to the right side of the mouse with a moistened cotton swab (ideally, this would expose the lower renal segment of the aorta. If the aorta is difficult to locate, the right kidney and the right renal artery can be used to identify the aorta because the anatomical position of the right kidney is slightly lower than that of the left kidney). The connective tissue covering the abdominal aorta and the inferior vena cava was gently removed with forceps (the abdominal aorta and the inferior vena cava are located within the same vascular sheath, and gentle actions should be taken to avoid damaging the two blood vessels). The muscles behind the abdominal aorta and the inferior vena cava were gently dissected with forceps (the tip of the forceps should enter the back of the sheath horizontally to form a hole in the fascia connecting the sheath and the underlying muscles. Once the hole is made, slowly release the forceps to expand the size of the hole), so as to fully expose the lower renal segment of the abdominal aorta.

[0075] 4c) Drug incubation: For the AAA group and the SPRQβ-009 group, 10 μL of porcine pancreatic elastase was dropped onto a 5 cm × 5 mm gauze with a pipette, and the gauze was wrapped around the abdominal aorta (0.5 cm from the right renal artery). The Control group was treated with an equal volume of 0.9% saline. After 30 minutes, the gauze was removed with forceps.

[0076] 4d) Irrigation of the abdominal cavity: The aorta and the abdominal cavity were irrigated with 500 μL of 0.9% sterile saline. The remaining saline was absorbed with a 10 cm × 10 cm gauze.

[0077] 4e) Suture: The abdominal organs were repositioned, and the muscles and skin were sutured successively with 6-0 non-absorbable monofilament sutures, and the area was disinfected three times with iodophor.

[0078] 4f) Anesthesia recovery: The mice were removed and placed on a heating pad in the cage, allowed to breathe the indoor air freely, waited for anesthesia recovery, and the general conditions such as body temperature and respiration of the mice were monitored during the recovery period.

[0079] 5) Blood pressure monitoring:

[0080] On the 0th, 14th, 28th, 35th, and 49th days of mouse immunization, the tail artery blood pressure of each group of mice was measured using a non-invasive tail-cuff blood pressure monitor BP-2010A (Softron, Japan). The mice were placed in a dark environment and heated at 37°C for about 15 minutes, and then blind measurements were started. During the measurement process, the mice were kept in a quiet and peaceful state. Each mouse was measured about 15 times, and the average value was statistically analyzed. All measurements were completed between 9:00 and 11:00 in the morning.

[0081] The results were as Figure 3 follows: There were no significant changes in systolic blood pressure in all groups during the entire experimental observation period, and there were no significant differences among the groups.

[0082] At the end of the experiment, all mice were euthanized, the heart and the entire aorta were exposed, perfused with phosphate-buffered saline, the perimembranous tissue was dissected, and then quickly excised and photographed.

[0083] The results were as Figure 4 follows: Compared with the AAA group, the abdominal aortic diameter in the SPRQβ-009 vaccine group was significantly reduced (1.70 mm vs 1.31 mm, P < 0.0001), and the incidence of AAA was significantly decreased (95.45% vs 63.64%, P < 0.01). Thus, the vector vaccine SPRQβ-009 can effectively prevent abdominal aortic dilation in mice and reduce the incidence of AAA.

[0084] The tissues were fixed in 4% paraformaldehyde overnight and then embedded in paraffin. Serial sections were cut at 5 μm intervals. The sections were stained with hematoxylin and eosin (HE) and elastic van Gieson (EVG) to observe the degradation of elastic fibers in the media. The degree of degradation of elastic fibers was graded from 1 to 4 (grade 1, intact elastic layer and good tissue; grade 2, reduced fiber density, linear; grade 3, occasional fiber breakage; grade 4, severe degradation of elastin, visible rupture sites).

[0085] As Figure 5 shown: Compared with the AAA group, the elastin score in the SPRQβ-009 vaccine group was significantly improved (4.00 vs 2.67, P = 0.038). Thus, the vector vaccine SPRQβ-009 can effectively inhibit the degradation of elastic fibers in the media and thereby inhibit the formation of AAA.

[0086] Example 4 Therapeutic effect of SPRQβ-009 vector vaccine on elastase-induced AAA

[0087] The prepared SPRQβ-009 vector vaccine was used to immunize C57BL / 6 mice while establishing the AAA model. After the formation and stabilization of AAA (14 days), stable high-titer specific antibodies were produced in the mice. At this time, it was studied whether the immunogenic vector vaccine against human S1PR2 could inhibit the further progression of AAA after its formation. The specific experimental procedure was as follows:

[0088] 1) Male C57BL / 6J mice at 6 weeks of age were selected. After 1 week of adaptive feeding, they were divided into 3 groups as follows:

[0089] The first group: control group (Control, n = 15): Treated with 0.9% saline on the 0th day of the experiment according to the above-mentioned modeling method.

[0090] The second group: AAA model group (AAA, n = 22): Modeled according to the above method on the 0th day of the experiment, and at the same time, PBS was injected subcutaneously at multiple points on the back on the 0th, 14th, and 28th days, with a dose of 100 μg / mouse, and the first dose was doubled.

[0091] The third group: SPRQβ-009 vaccine group (SPRQβ-009, n = 22): Modeled according to the above method on the 0th day of the experiment, and at the same time, the vector vaccine SPRQβ-009 was injected subcutaneously at multiple points on the back on the 0th, 14th, and 28th days, with a dose of 100 μg / mouse, and the first dose was doubled.

[0092] 2) Blood sampling: Blood was collected from the mouse tails on the 7th, 21st, 35th, 49th, and 56th days respectively. The supernatant was collected by centrifugation at 3000 rpm for 10 min at room temperature and stored at -80 °C for later use.

[0093] 3) ELISA experiment:

[0094] The specific implementation method of ELISA was the same as before. The result analysis was based on the standard that the OD value was not less than 2.1 times that of the blank control group as the positive standard for the test sample, and then the antibody titer value of the corresponding sample was calculated.

[0095] The results were as Figure 6 : After immunizing C57BL / 6J mice with the SPRQβ-009 vector vaccine, specific antibodies against the SPR-009 immunogenic peptide segment were produced. The antibody titer in the mouse serum reached the highest level (1:410*10 3 ) 1 week after the third immunization, and maintained a relatively high antibody titer level during the subsequent experimental process.

[0096] 4) Blood pressure monitoring:

[0097] The specific implementation method of blood pressure measurement was the same as before.

[0098] The results were as Figure 7: There were no significant changes in blood pressure in all groups during the entire experimental observation period, and there were no significant differences among the groups.

[0099] 5) Ultrasound detection:

[0100] Echocardiography was performed on day 56. The mice were anesthetized with 1.5% isoflurane at a temperature of 36.5 - 37.5 °C, and images were obtained 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 the Vevo 3100 workstation software.

[0101] The results were as Figure 8 : Compared with the AAA group, the abdominal aortic dilation in the SPRQβ-009 vaccine group was significantly reduced (1.18 mm vs 1.02 mm, P < 0.0001). Thus, the vector vaccine SPRQβ-009 can effectively reduce the abdominal aortic dilation rate and inhibit the progression of AAA after the formation of AAA.

[0102] At the end of the experiment, all mice were euthanized, the heart and the entire aorta were exposed, perfused with phosphate-buffered saline, the perivascular tissue was dissected, and then quickly excised and photographed.

[0103] The results were as Figure 8 : Compared with the AAA group, the abdominal aortic dilation in the SPRQβ-009 vaccine group was significantly reduced (1.81 mm vs 1.56 mm, P < 0.0001). Thus, the vector vaccine SPRQβ-009 can effectively reduce the abdominal aortic dilation rate and inhibit the progression of AAA after the formation of AAA.

[0104] The tissues were fixed in 4% paraformaldehyde overnight and then embedded in paraffin. Serial sections were cut at 5 μm intervals. The sections were stained with hematoxylin and eosin (HE) and elastic van Gieson (EVG) to observe the degradation of elastic fibers in the media of blood vessels.

[0105] As Figure 9 shown: Compared with the AAA group, the elastin score in the SPRQβ-009 vaccine group was significantly improved (4.00 vs 3.00, P = 0.021). It can be seen from this that the vector vaccine SPRQβ-009 can effectively retain the elastic structure in the media after the formation of AAA, thereby inhibiting the progression of AAA.

[0106] As can be seen from the above: The entire experimental data in this example show that the SPR-009 immunogenic peptide segment can be well conjugated with the Qβ vector to prepare the SPRQβ-009 immunogenic vector vaccine, stimulate mouse B cells to produce high levels of antibodies against the SPR-009 immunogenic peptide segment, and has the effects of reducing the incidence of AAA, retaining the elastic structure in the media, and inhibiting the progression of AAA in the AAA model.

[0107] Other parts not described in detail are all prior arts. Although the above embodiments have described the present invention in detail, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An immunogenic peptide segment targeting human sphingosine 1-phosphate receptor 2, characterized in that: The amino acid sequence of the immunogenic peptide segment is TPVQWFARE, i.e., SEQ ID No.

1.

2. Use of the immunogenic peptide segment according to claim 1 in the preparation of a product for preventing and treating abdominal aortic aneurysm.

3. An immunogenic vector vaccine for preventing and treating abdominal aortic aneurysm, characterized in that: The immunogenic vector vaccine is prepared by coupling the immunogenic peptide segment described in claim 1 with a carrier.

4. The immunogenic vector vaccine according to claim 3, characterized in that: The vector is Qβ-2aa bacteriophage virus-like particle protein.

5. The immunogenic vector vaccine according to claim 3 or 4, characterized in that: The immunogenic vector vaccine is an injectable preparation.

6. The immunogenic vector vaccine according to claim 5, characterized in that: The injection preparation is a freeze-dried powder injection or an aqueous solution.

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