Immunogenic peptide segment targeting human angiopoietin-like protein 3 and carrier vaccine thereof and use thereof

Abstract

The present invention relates to the field of medical biotechnology, and in particular to an immunogenic peptide segment targeting human angiopoietin-like protein 3, a carrier vaccine thereof and uses thereof; the amino acid sequence of the immunogenic peptide segment targeting human angiopoietin-like protein 3 is KPRAPRTT, as shown in SEQ ID No.1. The immunogenic carrier vaccine is prepared by coupling the ANGPTL3 immunogenic peptide segment with a carrier. The carrier is Qβ‑2aa bacteriophage virus-like particle protein. The immunogenic carrier vaccine targeting human ANGPTL3 of the present invention can effectively reduce hyperlipidemia and inhibit atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the field of medical biotechnology, and in particular to an immunogenic peptide segment targeting human angiopoietin-like protein 3, a carrier vaccine thereof and uses thereof. Background Art

[0002] The pathological basis of coronary heart disease is coronary atherosclerosis. Abnormal lipid metabolism is the most important risk factor for coronary atherosclerosis. The levels of circulating low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), TG-rich lipoproteins (TGRL) and apolipoprotein B (ApoB) are all worthy of attention. It has been proven that ApoB-containing lipoproteins are key and interventional risk factors for atherosclerosis. When the human body is fasting, 90% of ApoB-containing lipoproteins are composed of LDL, which is undoubtedly the main therapeutic target in clinical practice. However, in the case of diabetes, obesity or extremely low LDL-C, the risks brought by other ApoB-containing lipoproteins (mainly TGRL) should not be underestimated. Studies have made it clear that non-LDL ApoB-containing lipoproteins aggregated in patients with hypertriglyceridemia can cause atherosclerosis and lead to the occurrence of coronary heart disease. They are important markers and treatment targets for the residual risk after conventional lipid-lowering treatment.

[0003] In recent years, lipid-lowering drugs have made significant progress, but there are still many shortcomings. Statins and ezetimibe, as classic lipid-lowering drugs, can effectively reduce LDL-C levels and improve the prognosis of coronary heart disease. However, after treatment with statins and ezetimibe, some patients still have difficulty meeting the LDL-C standard, and are accompanied by refractory hypertriglyceridemia, and are still at high risk of cardiovascular events. Although fibrates can reduce TG, their combination with statins significantly increases the risk of myopathy and liver damage, limiting their use scenarios, and the rate of blood lipid compliance is still worrying. Although the new lipid-lowering drug proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors have partially solved the problem of LDL-C not being able to meet the standard, the means of reducing TG in clinical practice are still very limited (only fibrates). In addition, due to the lack of LDL receptors, the rare lipid metabolism genetic disease-homozygous familial hypercholesterolemia, statins and PCSK9 inhibitors that rely on LDL receptors cannot reduce the blood lipids of such patients. Although gene editing has entered the field of lipid management, it will not be applied to humans in the short term due to technical and ethical limitations. Therefore, it is necessary to find more new and effective strategies to target patients with poor lipid-lowering drug effects, high TG levels and high residual risk, in order to achieve the turning point of reducing the incidence of cardiovascular disease in my country as soon as possible.

[0004] Angiopoietin-like protein 3 (ANGPTL3) is a member of the angiopoietin protein family. It reversibly binds to lipoprotein lipase (LPL) to inhibit its activity, reducing the hydrolysis of circulating triglycerides (TG) and preventing TG-rich lipoproteins from being recognized and cleared by the liver. In the ANGPTL family, ANGPTL3, ANGPTL4 and ANGPTL8 have a high degree of sequence homology and synergistically participate in the regulation of LPL activity and TGRL metabolism. Among them, ANGPTL4 can be synthesized in the liver, fat, and multiple organs and tissues throughout the body, and tends to consume fat through oxidation. The effect of ANGPTL8 in increasing blood lipids requires the presence of ANGPTL3, which is mainly synthesized and secreted by the liver, has organ specificity, and tends to store fat. Therefore, in the angiopoietin-like family, downregulating ANGPTL3 is more valuable for lowering lipids.

[0005] From a structural analysis, the sites of ANGPTL3 related to the inhibition of LPL activity are:

[0006] ① Specific epitope (SE1): It is the region necessary for inhibiting LPL activity;

[0007] ② Linker region: The full-length ANGPTL3 has no activity and needs to be recognized by furin before hydrolysis and cleavage. The N-terminal ANGPTL3 after cleavage has stronger inhibitory effect on LPL. 221RAPR224↓T226 in the linker region is recognized by furin as a recognition point.

[0008] ③Heparin binding region: The absence of this amino acid segment will lead to a significant reduction in the inhibitory activity of ANGPTL3 on LPL in vitro.

[0009] Therefore, drug development targeting these three structural thresholds of ANGPTL3 has the potential to produce significant LPL inhibitory effects.

[0010] ANGPTL3 does not rely on LDLR to exert its effects. Currently, monoclonal antibodies and antisense oligonucleotides have entered clinical trials, but they have the disadvantages of high treatment costs, anti-antibody production, multiple injections, and poor compliance:

[0011] ①ANGPTL3 monoclonal antibody (evinacumab): This drug was approved by the FDA in 2021 for the treatment of familial hypercholesterolemia patients aged 12 years and above. It is currently the only ANGPTL3 inhibitor approved for special clinical patients. The drug must be injected once every 4 weeks, with each injection of 15 mg / kg. It costs about US$450,000 per year, which is difficult to afford even for commercial insurance;

[0012] ② Antisense nucleotide (vupanorsen): requires subcutaneous injection of 40-80 mg every 4 weeks or 20 mg per week. There is currently no approved drug available.

[0013] Therefore, it is necessary to find more effective, economical and convenient treatment methods. Summary of the invention

[0014] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an immunogenic peptide segment targeting human angiopoietin-like protein 3 (ANGPTL3) and its vector vaccine and use. After screening and experimental animal verification, the present invention has invented an immunogenic peptide segment from the ANGPTL3 connecting region and its corresponding vector vaccine. The therapeutic vaccine can effectively inhibit the effect of ANGPTL3, reduce blood lipids in animals with atherosclerosis, and alleviate the formation and progression of atherosclerosis.

[0015] To achieve the above object, the present invention designs an immunogenic peptide segment against human angiopoietin-like protein 3, and the amino acid sequence of the immunogenic peptide segment against human angiopoietin-like protein 3 is KPRAPRTT, as shown in SEQ ID No.1.

[0016] The present invention also provides an application of the above immunogenic peptide segment in the preparation of medicines for treating hyperlipidemia and atherosclerosis.

[0017] The present invention also provides an immunogenic vector vaccine for treating hyperlipidemia and atherosclerosis, wherein the immunogenic vector vaccine is prepared by coupling the above-mentioned ANGPTL3 immunogenic peptide segment with a carrier.

[0018] As a preferred embodiment, the vector is Qβ-2aa bacteriophage virus-like particle protein.

[0019] The present invention also provides an application of an immunogenic carrier vaccine in the preparation of medicines for treating hyperlipidemia and atherosclerosis.

[0020] Principle of the present invention:

[0021] Therapeutic vaccines are a new type of immunological treatment method with the advantages of high target specificity, long-lasting effect (1-3 months or even longer immunization can last for several months), high compliance and low cost. They are very suitable for the treatment and control of chronic diseases and provide new ideas for the treatment of major chronic diseases. Therapeutic lipid-lowering vaccines are a new field of research in the treatment of hyperlipidemia and atherosclerosis. If successfully developed, compared with traditional chemical synthetic drugs, they have strong specificity and long-term action. They can be administered once every 1 to 3 months or even longer and can maintain long-term stable and effective reduction of blood lipids, thereby better protecting target organs, reducing costs and improving treatment compliance. Based on short peptide screening technology and short peptide-virus-like particle (VLP) vaccine preparation platform, we have developed antihypertensive vaccines targeting angiotensin receptor type 1 (ATRQβ-001), pulmonary hypertension vaccines targeting endothelin receptor type A (ETRQβ-002), antihypertensive vaccines targeting adrenergic receptor 1D (ADRQβ-004) and their specific monoclonal antibodies, etc., and have accumulated rich experience in the development of therapeutic vaccines. At present, the ATRQβ-001 vaccine has entered the CRO pilot stage, which is the first therapeutic vaccine to enter transformation development and has great clinical application prospects. The above work provides a solid foundation for our research and development of ANGPTL3 lipid-lowering vaccines. After screening and experimental animal verification, an immunogenic peptide segment from the ANGPTL3 linker region and its corresponding vector vaccine were invented. This therapeutic vaccine can effectively inhibit the action of ANGPTL3, reduce blood lipids in animals with atherosclerosis, and alleviate the formation of atherosclerosis.

[0022] Beneficial effects of the present invention:

[0023] 1. Based on the amino acid sequence, hydrophilicity, antigenicity, accessibility and other characteristics of the human ANGPTL3 connecting region, the present invention designed a peptide segment targeting the human ANGPTL3 connecting region by integrating bioinformatics and pharmacology methods, and verified that the SEQ ID No. 1 peptide segment can effectively induce the production of specific antibodies in immune mice, wherein the sequence SEQ ID No. 1 of humans and mice is basically the same.

[0024] 2. Selecting a suitable carrier is the key to the successful development of a vaccine. The present invention uses Qβ-2aa bacteriophage virus-like particle protein.

[0025] 3. The designed immunogenic peptide segment targeting human ANGPTL3 protein was coupled with Qβ-2aa phage virus-like particle protein carrier to successfully prepare a vector vaccine. The prepared vector vaccine was used to immunize ApoE- / - mice fed with high fat to study whether the immunogenic vector vaccine targeting human ANGPTL3 protein can reduce hyperlipidemia and alleviate atherosclerosis formation in ApoE- / - mice fed with high fat. The experimental results showed that the immunogenic vector vaccine targeting human ANGPTL3 protein can effectively reduce hyperlipidemia and alleviate atherosclerotic plaques in ApoE- / - mice fed with high fat. Therefore, the immunogenic vector vaccine targeting human ANGPTL3 protein can be used to prepare and treat hyperlipidemia and atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the SDS-PAGE gel electrophoresis detection diagram of the ANGPTL3-KT8 vector vaccine in Example 2;

[0027] In the figure, lane Marker is the protein molecular weight standard, lane VLP is the Qβ-2aa bacteriophage virus-like particle protein, lane ANGPTL3-KT8 is the ANGPTL3-KT8 vector vaccine, and KD is kilodaltons.

[0028] Figure 2 This is a graph showing the antibody titer against the KT8 peptide produced after immunizing male ApoE- / - mice with the vector vaccine ANGPTL3-KT8 in Example 3.

[0029] Figure 3 This is a diagram showing the lipid-lowering effect produced after immunization of male ApoE- / - mice with the vector vaccine ANGPTL3-KT8 in Example 3;

[0030] Among them, A is the change diagram of serum triglyceride level in mice,

[0031] B is the change of total cholesterol level in mouse serum.

[0032] C is the change of serum low-density lipoprotein cholesterol level in mice.

[0033] D is a graph showing changes in high-density lipoprotein cholesterol levels in mice.

[0034] Figure 4 This is a graph showing the ratio of the aorta's total lipid plaque area calculated in Example 3;

[0035] Among them, A is the representative picture of aorta oil red O staining in each group,

[0036] B is a statistical chart of the aorta injury area ratio;

[0037] In the figure, the control group represents the aorta of ApoE- / - mice fed a high-fat diet, the vector group represents the aorta of ApoE- / - mice fed a high-fat diet and immunized with a blank vector, and the ANGPTL3-KT8 vaccine group and the vaccine group represent the aorta of ApoE- / - mice fed a high-fat diet and immunized with the vector vaccine ANGPTL3-KT8; **P<0.01vs control group, ###P<0.001vs vector group.

[0038] Figure 5 This is a graph of aortic valve annulus lipid plaque area calculated in Example 3;

[0039] Among them, A is a representative picture of the aortic valve ring oil red O staining in each group,

[0040] B is a statistical graph of the oil red staining area of ​​the aortic valve ring;

[0041] In the figure, the control group represents the aortic valve ring of ApoE- / - mice fed with high fat, the vector group represents the aortic valve ring of ApoE- / - mice fed with high fat and immunized with blank vector, and the ANGPTL3-KT8 vaccine group and the vaccine group represent the aortic valve ring of ApoE- / - mice fed with high fat and immunized with vector vaccine ANGPTL3-KT8; **P<0.01vs control group, ##P<0.01vs vector group.

[0042] Figure 6 is the aortic valve ring plaque injury area ratio calculated in Example 3;

[0043] in, Figure 6 A is a representative picture of the aortic valve ring HE in each group.

[0044] Figure 6 B is a statistical diagram of the aorta injury area;

[0045] In the figure, the control group represents the aortic valve ring of ApoE- / - mice fed with high fat, the vector group represents the aortic valve ring of ApoE- / - mice fed with high fat and immunized with blank vector, and the ANGPTL3-KT8 vaccine group and the vaccine group represent the aortic valve ring of ApoE- / - mice fed with high fat and immunized with vector vaccine ANGPTL3-KT8; ****P<0.0001vs control group, ##P<0.01vs vector group.

[0046] Figure 7 The change in serum lipoprotein lipase activity after heparin injection in male ApoE- / - mice immunized with the vector vaccine ANGPTL3-CT9 in Example 3 (unit definition: at 45°C, pH 7.5, one unit of enzyme activity is the hydrolysis of 1 nmol 4-nitrophenol per minute per milliliter of serum);

[0047] In the figure, the control group represents the post-heparin LPL activity of high-fat-fed ApoE- / - mice, the vector group represents the post-heparin LPL activity of high-fat-fed ApoE- / - mice immunized with blank vector, and the vaccine group represents the post-heparin LPL activity of high-fat-fed ApoE- / - mice immunized with vector vaccine ANGPTL3-KT8; ***P<0.001vs control group, ##P<0.01vs vector group. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0049] The immunogenic peptide segment targeting ANGPTL3 is referred to as ANGPTL3 immunogenic peptide segment, code-named KT8 herein, and the immunogenic vector vaccine targeting ANGPTL3 is referred to as ANGPTL3-KT8 vector vaccine.

[0050] Example 1 Preparation of immunogenic peptide fragments against human ANGPTL3 protein

[0051] Based on the spatial conformation, bioinformatics and pharmacological characteristics of ANGPTL3, the key structural thresholds and amino acid residues of ANGPTL3 were deduced, and an immunogenic peptide segment targeting human ANGPTL3 was designed, named ANGPTL3 immunogenic peptide segment, codenamed KT8, and the specific amino acid sequence KT8 is SEQ ID No.1.

[0052] The above-mentioned ANGPTL3 immunogenic peptide KT8 was synthesized by solid phase synthesis (Shanghai Jier Biochemical Co., Ltd. was commissioned to synthesize and inspect the quality). The purity of the synthesized peptide was analyzed by high performance liquid chromatography, and the purity was more than 98%. The obtained peptide was lyophilized and packaged, placed in cryopreservation tubes, transported with ice packs, and frozen at -20°C for future use.

[0053] Example 2 Preparation of ANGPTL3-KT8 vector vaccine

[0054] 1. Prepare the vaccine using Qβ-2aa bacteriophage virus-like particle protein as a vaccine carrier; the specific preparation process is as follows:

[0055] 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; wherein, the preparation method of Qβ-2aa VLP is:

[0056] (1a) Obtaining a recombinant strain expressing Qβ-2aa VLP: 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 Escherichia coli DH5α / pGEXQβ-A1 is CCTCC NO: M209282. For the specific preparation process, refer to Chinese patent: A method for preparing Qβ-2aa bacteriophage virus-like particle protein and its use, the authorization publication date is CN 101921733 B authorization announcement date 2013.06.05.

[0057] (1b) Inducing the expression of Qβ-2aa VLP: First, the stored Escherichia coli DH5α / pGEXQβ-A1 recombinant strain was taken out from the liquid nitrogen tank, and after activating the recombinant strain, it was spread on an LB solid culture medium plate and cultured in a 37°C incubator overnight. A single colony was picked and cultured in an LB liquid culture medium. After culturing in a 37°C constant temperature shaker for 5 hours, 0.2 M IPTG was added to induce the recombinant strain to express Qβ-2aa VLP. The induction was performed for 6 hours, and the bacterial solution was collected and ultrasonically lysed to obtain the lysate supernatant;

[0058] (1c) Purification of Qβ-2aa VLP: The lysate supernatant was subjected to ammonium sulfate precipitation, acidification, hydrophobic chromatography, and gel chromatography to obtain purified Qβ-2aa VLP;

[0059] (1d) Identification of Qβ-2aa VLP: The purified Qβ-2aa VLP was dissociated with dithiothreitol (DTT), and the molecular weight of the dissociated Qβ-2aa VLP was determined by gel electrophoresis. The morphology and particle size were observed by electron microscopy. Finally, the obtained protein was determined to be Qβ-2aa VLP based on the test results.

[0060] 2) Preparation of ANGPTL3 vector vaccine: The ANGPTL3 immunogenic peptide KT8 obtained in Example 1 was coupled with the carrier Qβ-2aa VLP using a heterobifunctional cross-linking agent (Sulfo-SMCC) to obtain an ANGPTL3-KT8 vector vaccine.

[0061] The prepared ANGPTL3-KT8 vector vaccine was subjected to reduced SDS-PAGE gel electrophoresis detection. The detection results are as follows Figure 1 As shown: By comparing with the VLP lane, it can be observed that the vector vaccine ANGPTL3-KT8 is mainly composed of 1 VLP monomer coupled with 2-3 KT8 short peptides, and the coupling efficiency is relatively high, proving that the vector vaccine was successfully prepared.

[0062] Example 3 Effects of ANGPTL3-KT8 vector vaccine on blood lipid levels and atherosclerosis formation in ApoE- / - mice fed a high-fat diet

[0063] The vector vaccine ANGPTL3-KT8 was used to immunize ApoE- / - mice fed with high-fat diet to explore whether the immunogenic vector vaccine of ANGPTL3-KT8 can reduce the blood lipids of ApoE- / - mice and improve the formation of atherosclerosis. The specific experimental process is as follows:

[0064] 1) 5-week-old male ApoE- / - mice were selected for active immunization, and the normal diet was replaced with a high-fat diet with a fat content of 21%. The mice were divided into 4 groups as follows:

[0065] Group 1: Control group (control): PBS solution was injected subcutaneously at multiple points on the back at 0, 2, 4 and 8 weeks, with the first dose of 100 μl / mouse and the subsequent booster dose of 50 μl / mouse, for a total of 10 mice;

[0066] Group 2: Vehicle group (VLP): VLP was injected subcutaneously at multiple points on the back at 0, 2, 4 and 8 weeks, with the first dose of 100 μg / mouse (100 μl system), followed by a booster dose of 50 μg / mouse (50 μl system), for a total of 10 mice;

[0067] Group 3: ANGPTL3-KT8 vector vaccine group: ANGPTL3-KT8 vector vaccine was injected subcutaneously at multiple points on the back at weeks 0, 2, 4 and 8, with the first dose of 100 μg / mouse (100 μl system), followed by a booster dose of 50 μg / mouse (50 μl system), for a total of 10 mice;

[0068] 2) Blood collection: Blood was collected from the tail of the rat at intervals of 1-4 weeks (adjusted according to the test indicators) during animal breeding, and blood was collected from the eyeball before sacrifice. The supernatant was collected by centrifugation at 3000 rpm for 10 min at room temperature and stored at -80°C for later use;

[0069] 3) ELISA experiment

[0070] Since the vaccine carrier of the immunized animals is Qβ-2aa VLP, in order to avoid cross-reaction, bovine serum albumin (BSA) was coupled with KT8 to prepare the coating substrate, and the coating plate was prepared using a high-adsorption ELISA plate. The ELISA method was used to determine the antibody titer against the immunogenic peptide KT8 of ANGPTL3;

[0071] 3a) Serial dilution: PBST buffer containing 2% BSA was used as the diluent, and a serial dilution of 1:100, 1:1000, 1:5000, 1:10000, 1:25000, 1:50000, and 1:100000 was performed;

[0072] 3b) Incubate with primary antibody: Use a pipette to add serum samples diluted at 1:100, 1:1000, 1:5000, 1:10000, 1:25000, 1:50000, 1:100000 to the coated 96-well plate, 100 μl / well, incubate at 37°C for 1.5 h;

[0073] 3c) Incubation with secondary antibody: After the primary antibody incubation is completed, discard the liquid and wash with washing solution (0.05% PBST) for 3 times, pat dry, and then add horseradish peroxidase-labeled goat anti-rat secondary antibody (1:3000 dilution, the diluent is PBST buffer containing 2% BSA), 100 μl / well, and incubate in a 37°C incubator for 0.5 h;

[0074] 3d) Color development: After the secondary antibody incubation is completed, the liquid is discarded and the cells are washed three times with washing solution (0.05% PBST), patted dry, and then TMB color development solution is added, 100 μl / well, and the color change is observed at room temperature;

[0075] 3e) Stop the reaction: When the blank control wells just begin to turn blue, add stop solution (1M dilute hydrochloric acid), 100 μl / well;

[0076] 3f) Reading: After adding the stop solution, place the sample on a microplate reader and read the absorbance (OD) value at a wavelength of 450 nm;

[0077] 3g) Result analysis: When the OD value of the blank well is 0.03-0.06, the result is more reliable. The OD value not less than 2.1 times that of the blank control group is used as the standard for the positive test sample, and the antibody titer value of the corresponding sample is calculated.

[0078] The results are as follows Figure 2 As shown: After mice were immunized with the ANGPTL3-KT8 vector vaccine, specific antibodies against the ANGPTL3 immunogenic peptide KT8 were produced. The antibody titer in the mouse serum began to increase in the first week after the third immunization (1:508,806±180,943), and maintained a high antibody titer during the subsequent experiments.

[0079] 4) Blood lipid measurement

[0080] Blood was collected as in Example 3. The corresponding test kit manufactured in Nanjing was used, and the specific detection method was as follows:

[0081] 4a) TG and TC level detection: Add 2.5 μl of distilled water, standard and sample to the blank well, standard well and sample well respectively, then quickly add 250 μl of TG or TC working solution, mix gently, incubate at 37°C for 10 minutes, take out and place in a microplate reader, adjust the absorption light wavelength to 510 nm, measure the absorbance OD value of each well, and calculate the TG and TC content.

[0082] 4b) Detection of LDL-C and HDL-C levels: Add 2.5 μl of distilled water, standard and sample to the blank well, standard well and sample well respectively, then quickly add 180 μl of LDL-C or HDL-C R1 working solution, mix gently, incubate at 37°C for 5 minutes, take out, place in the microplate reader, adjust the absorption wavelength to 546 nm, measure the absorbance value OD1 of each well, then add 60 μl of LDL-C or HDL-C R2 working solution, mix gently, incubate at 37°C for 5 minutes, take out, place in the microplate reader again, adjust the absorption wavelength to 546 nm, measure the absorbance value OD2 of each well, and calculate the LDL-C and HDL-C contents.

[0083] The results are as follows Figure 3 As shown in the figure: After feeding a high-fat diet, the four blood lipids of the three groups of mice increased rapidly. At the 5th week, the triglyceride level in the vaccine group (2.41±0.64mmol / L) was significantly lower than that in the control group (4.03±0.61mmol / L) (P<0.0001), and this reduction effect lasted until death; the same situation occurred in the total cholesterol level (control group level: 30.8±6.2mmol / L, vaccine group level: 22.2±5.4mmol / L, P=0.0 011); the effect of vaccine treatment on lowering LDL-C levels appeared at 11 weeks, when the LDL-C level of the control group rose to a peak (15.2±3.4mmol / L), and the lowering effect of the vaccine group (9.4±3.3mmol / L, P=0.0017) was reflected; finally, the HDL-C level of the vaccine group decreased at 5 weeks, but it was statistically significant only at 9 weeks (control group: 8.97±2.07mmol / L; vaccine group: 5.85±2.02mmol / L, P=0.0063). The above-mentioned reduction in blood lipids did not occur between the control group and the vector group. This shows that the simple injection of VLP vector has no lipid-lowering effect, but the injection of ANGPTL3-KT8 vaccine can effectively reduce the hyperlipidemia of ApoE- / - mice fed with high-fat diet while producing high levels of antibody titers.

[0084] 5) Measurement of aortic and aortic valve ring plaques

[0085] 5a) Sampling of aorta and aortic valve ring: After 16 weeks of feeding, mice were anesthetized with pentobarbital, blood was collected from the eyeballs, and the heart was pressed as much as possible to drain the blood. Under a microscope, microtweezers and fiber scissors were used to cut the skin in front of the xiphoid process, cut the diaphragm and sever the ribs, open the chest cavity, and perform cardiopulmonary perfusion with normal saline. The perfusion was stopped when the aorta became transparent. At this time, atherosclerotic plaques in the blood vessels could be clearly observed. The fat and connective tissue beside the heart and aorta were carefully removed to avoid touching the plaques. The heart and aorta were separated without being cut off. After washing with PBS solution, the cells were placed in a 4% paraformaldehyde solution and fixed for more than 24 hours.

[0086] 5b) Oil Red O staining of aorta: Oil Red O stock solution was diluted with distilled water at a volume ratio of 3:2, filtered and set aside, the whole aorta was placed in a 6-well plate, 1 ml of Oil Red O working solution was added, and staining was performed for 40-60 minutes; 60% isopropanol was used to wash away the excess stain until part of the aorta became white and translucent, and the plaque area was obviously stained red, then rinsed 3 times with distilled water, placed next to the ruler, and photographed under a microscope. Image Pro Plus 8.0 was used to measure the total area of ​​the aorta and the plaque area stained with Oil Red, and the plaque injury area ratio was calculated as plaque area / total aorta area.

[0087] The results are as follows Figure 4 As shown: A significant reduction in plaques was observed in the vaccine group (5.14±1.86%) compared with the control group (9.47±1.64%) (P=0.0011), while there was no significant difference between the vehicle group (9.65±3.90%) and the control group (P=0.8807).

[0088] 5c) Continuous sectioning of aortic valve ring: Quickly freeze the fixed tissue, clamp the frozen tissue block on the slicer holder, start the coarse advance and retract key, turn the knob to flatten the tissue, adjust the thickness to be cut, start slicing from the bottom of the heart, and slice continuously at intervals of 10μm. Each sample can obtain 10-15 slices for cryopreservation.

[0089] 5d) Oil red O staining of aortic valve ring: rewarm the frozen sections and dry them, fix them in the fixative for 15 minutes, wash them with tap water, and air dry them; immerse the sections in the oil red staining solution for 8-10 minutes (cover and protect from light); take out the sections, leave them for 3 seconds, and then immerse them in two cylinders of 60% isopropanol for differentiation, for 3 seconds and 5 seconds respectively; then take out the sections, and immerse them in two cylinders of pure water for 10 seconds each; take out the sections, leave them for 3 seconds, and then immerse them in hematoxylin for 3-5 minutes, and then immerse them in three cylinders of pure water. 5 seconds, 10 seconds, and 30 seconds respectively; differentiate with differentiation solution (with 60% alcohol as solvent) for 2-8 seconds, wash with 2 cylinders of distilled water for 10 seconds each, and blue with blueing solution for 1 second, gently immerse the slices in 2 cylinders of tap water for 5 seconds and 10 seconds each, and examine the staining effect under a microscope; seal the slices with glycerol gelatin mounting medium; Oil Red O dye can stain neutral fats such as glycerol sandwiches, and the distribution of fat in the plaques stained with Oil Red can be observed under a microscope, and photos are taken for preservation. Use Image Pro Plus 8.0 according to the ruler to measure the plaque damage area (i.e., neutral lipid area) of the aortic valve ring stained with Oil Red.

[0090] The results are as follows Figure 5 As shown: vaccine group (115,367±40,385μm 2 ) compared with the control group (184,242±39,564μm 2 ), it can be observed that the lipid area was significantly reduced (P = 0.002), while the vehicle group (174,176 ± 53,471 μm 2 ) had no significant difference compared with the control group (P=0.6204).

[0091] 5e) HE staining of aortic valve ring: rewarm the frozen sections and dry them, fix them in fixative for 15 minutes, cover with water: 100%, 90%, 80%, 70% alcohol for 5 minutes each, rinse with running water for 5 minutes, repeat 3 times; stain with hematoxylin for 5 minutes, and the staining time can be appropriately increased or decreased according to the staining situation, rinse with running water; differentiate with 5% acetic acid for 1 minute, rinse with running water, and use a pipette to drop acetic acid until it covers the tissue on the glass slide. After differentiation, the color becomes lighter and becomes blue; stain with eosin for 1 minute, and the staining time can be appropriately increased or decreased according to the staining situation, rinse with running water; dehydrate: 70%, 80%, 90%, 100% alcohol for 10 seconds each, xylene for 1 minute, and dry naturally in a fume hood before sealing, about 5 minutes; drip neutral gum to seal the slide, take pictures under a microscope, and use Image Pro Plus 8.0 to measure the aortic valve ring plaque area and the area of ​​the three aortic leaflets according to the ruler to obtain the percentage of plaque damage area (=plaque area / total leaflet area).

[0092] The results are as follows Figure 6As shown: The boundary between endothelial cells and lipid plaques can be observed more clearly under HE staining. Compared with the control group (60.5±4.9%), the plaques in the vaccine group (48.5±7.3%) were significantly reduced (P<0.0001), while there was no significant difference between the vector group (57.1±4.0%) and the control group (P=0.1863).

[0093] 6) Measurement of lipoprotein lipase (LPL) activity:

[0094] At 16 weeks, heparin (1 U / g) was injected into the tail vein of mice according to their body weight. The mice were killed after waiting for 15 minutes, blood was collected from the eyeballs, and the supernatant was collected after centrifugation at 3000 rpm for 15 minutes at room temperature.

[0095] Using the Beijing solarbio LPL activity kit, the specific measurement method is as follows:

[0096] 30 μl of distilled water and standard (p-nitrophenol standard solution) were added to the blank tube and the standard tube respectively. 30 μl of two serum samples from the same mouse were added to the control group and the test group respectively, and reagent 1 (120 μl, 120 μl, 108 μl, 120 μl) was added respectively. 12 μl of reagent 2 was additionally added to the control well, and the mixture was incubated in a 45°C water bath for 10 minutes. 150 μl of reagent 3 was added to each tube, and the mixture was thoroughly mixed and allowed to stand for 2 minutes. The control group and the test tube were centrifuged at 8000 g at room temperature for 10 minutes. 200 μl of the supernatant of the control tube and the test tube, the standard tube and the blank tube were placed in a microplate with a 96-well plate to measure the absorbance at 400 nm, and the activity of LPL was calculated according to the formula.

[0097] The results are as follows Figure 7 As shown: By detecting the activity of LPL after heparin, it can be found that the vector vaccine ANGPTL3-KT8 can effectively reduce the inhibitory ability of ANGPTL3 on LPL activity. Compared with the control group (98±49U / mL) and the vector group (108±50U / mL), the post-heparin LPL activity of the vaccine group (192±68U / mL) was significantly increased (P=0.0009); this is an important mechanism for the vaccine to lower blood lipids and alleviate atherosclerosis.

[0098] The entire experimental data in Example 3 shows that the ANGPTL3-KT8 immunogenic vector vaccine can be well coupled with the Qβ-2aa VLP vector, stimulating mouse B cells to produce high levels of antibodies against the ANGPTL3 connecting region, and reducing the inhibitory ability of ANGPTL3 on LPL activity, thereby increasing the LPL activity of mice, leading to increased lipolysis of circulating lipoproteins, increased uptake and absorption of lipoproteins by the liver, resulting in lower blood lipids and reduced atherosclerosis in hyperlipidemia model mice.

[0099] 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 creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. An immunogenic peptide segment against human angiopoietin-like protein 3, characterized in that: The amino acid sequence of the immunogenic peptide segment against human angiopoietin-like protein 3 is KPRAPRTT, as shown in SEQ ID No.

1.

2. Use of the immunogenic peptide segment according to claim 1 in the preparation of medicines for treating hyperlipidemia and atherosclerosis.

3. An immunogenic vector vaccine for treating hyperlipidemia and atherosclerosis, 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. Use of the immunogenic vector vaccine according to claim 3 or 4 in the preparation of medicines for treating hyperlipidemia and atherosclerosis.

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