Immunogenic peptide segments of metalloprotease ADAMTS-7, and its use in anti-atherosclerotic arteriosclerosis and related diseases

Abstract

Immunogenic peptide segments of the metalloprotease ADAMTS-7 and their use in the prevention and treatment of atherosclerosis and related diseases. Provided are short peptides containing any of the short peptides whose amino acid sequence is set forth in Sequence 1 of the Sequence Listing, the short peptides whose amino acid sequence is set forth in Sequence 2 of the Sequence Listing, the short peptides whose amino acid sequence is set forth in Sequence 3 of the Sequence Listing, and the short peptides whose amino acid sequence is set forth in Sequence 4 of the Sequence Listing. Conjugates containing the above short peptides, vaccines containing the conjugates, and uses thereof. Vaccines containing the short peptides significantly suppress neointima formation in a mouse model of vascular restenosis and the development of atherosclerosis in high-fat-fed mice, and are useful for the prevention or treatment of atherosclerosis and / or vascular restenosis.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and medicine, and in particular, to immunogenic peptide segments of metalloprotease ADAMTS-7 and their use in anti-atherosclerotic arteriosclerosis and related diseases.

Background Art

[0002] Coronary heart disease is called the "number one killer of humans" because it seriously endangers human health due to its high incidence and high mortality rate. The main cause of coronary heart disease is coronary atherosclerosis, and its pathological process is the formation of plaques protruding into the lumen due to damage to vascular endothelial cells, proliferation and migration of vascular smooth muscle cells, and infiltration of inflammatory cells caused by abnormal deposition of lipids on the vascular wall. The formation of plaques leads to lumen stenosis and insufficient blood supply.

[0003] Currently, there are mainly the following three treatment methods for coronary heart disease: (1) Drug treatment. The aim is to relieve symptoms, reduce angina attacks and myocardial infarctions, delay the progression of coronary atherosclerotic arteriosclerosis, and reduce deaths due to coronary heart disease. Lipid-lowering drugs such as statins can reduce cardiovascular events by 25%, but they increase the risk of new-onset diseases such as diabetes. Although newly introduced drugs such as ezetimibe and PCSK9 monoclonal antibodies are promising, they are still strategies designed for lipid-lowering. However, at the current medical level, there is no drug that can cure coronary heart disease. In fact, the treatment for coronary heart disease mainly aims to control the condition of coronary heart disease and avoid complications caused by the deterioration of the condition. (2) Percutaneous coronary intervention (PCI). In percutaneous transluminal coronary angioplasty (PTCA), a special balloon catheter is used to be sent to the coronary artery stenosis through the peripheral artery (femoral artery or radial artery). When the balloon is inflated, the stenosis is expanded to improve blood flow, and a stent can be placed in the expanded stenosis to prevent restenosis. However, currently, there is still a risk of stent failure, such as in-stent thrombosis (ST) and in-stent restenosis (ISR). In-stent thrombosis is a relatively serious clinical complication. In severe cases, it may cause ascending myocardial infarction in the thrombosis part, and the mortality rate can reach 20% - 40%. The occurrence of in-stent restenosis has a very significant impact on the long-term prognosis after PCI. The arterial wall can avoid lipid deposition under the normal endothelial cell barrier. However, drug-eluting stents may cause damage to the structure and function of endothelial cells, leading to delayed endothelial healing and a tendency to form neoatherosclerotic arteriosclerosis. Furthermore, in surgical retreatment, both arterial bypass grafting and minimally invasive intervention techniques are not suitable for the treatment of most vascular restenoses due to their high operation difficulty and severe damage. Therefore, new means are needed to prevent and treat restenosis and thrombosis after dilation.

[0004] Since stents and bypass surgeries do not achieve completely satisfactory effects for clinicians and patients, medical scientists from various countries have conducted research on the causes that induce coronary heart disease and lead to vascular stenosis. In recent years, attention has been drawn to the development of anti-atherosclerotic vaccines. Professor Dr. Horvath Istvan of Hungary first discovered the existence of cholesterol antibodies in the bodies of normal people in 1983, developed a cholesterol antigen vaccine, and entered human clinical trials in 2002 after receiving approval from the Hungarian National Healthcare Authority. After about 10 years until today, more than 40,000 patients have already received treatment, and the effect is remarkable, taking an important step in the field of completely curing cardiovascular diseases. Since Professor Istvan developed the cholesterol immune vaccine in 1983, scientists at the Karolinska University in Sweden have developed a cholesterol vaccine that can prevent cholesterol from harming arterial blood vessels and reduce the incidence of heart disease by two-thirds. In addition, monoclonal antibodies or short-chain peptide vaccines targeting pathogenic LDL apolipoprotein ApoB100 and the LDLR-degrading enzyme PCSK9 have been developed one after another, and preliminary animal model experiments and pre-clinical verifications have been carried out. Recently, the monoclonal antibody (REGN727) against proprotein convertase subtilisin / kexin type 9 (PCSK9), which is most expected to receive approval for use in human treatment, in the results of phase I clinical trials, low-dose REGN727 can lower the LDL level of the subjects by more than 30%. For patients who cannot tolerate treatment with statins, REGN727 can be expected to have a wider clinical application. Furthermore, as a result of designing a short-chain peptide vaccine that acts on apolipoprotein ApoB100, it showed a certain anti-atherosclerotic effect in experimental animals. From these things, all have shown that the research, development, design of short-chain peptide vaccines against specific targets and the production of corresponding monoclonal antibodies have potential significance for disease prevention and treatment.

[0005] ADAMTS-7 (a disintegrin-like and metalloprotease-7, containing a type I thrombospondin motif disintegrin-like and metalloprotease) is a novel metalloprotease cloned in 2004 and belongs to the Zn 2+ -dependent metalloprotease family. The human disintegrin-like and metalloprotease (ADAMTS) family containing type I thrombospondin (TSP) motifs consists of 19 secreted multi-domain zinc finger metalloproteases that degrade extracellular matrix components (ECM) including procollagen, proteoglycan, and cartilage oligomeric matrix protein (COMP). ADAMTS proteases play important roles in the turnover of extracellular matrix components in various tissues, and abnormal expression of these enzymes is usually closely related to inflammatory pathological processes such as tumors and rheumatoid arthritis. According to methods such as small interfering RNA silencing, overexpression of ADAMTS-7 by adenovirus, and construction of ADAMTS-7 gene knockout animal models, it has been discovered that the metalloprotease ADAMTS-7 promotes the migration of vascular smooth muscle cells and neointima formation after vascular injury through the degradation of cartilage oligomeric matrix protein (COMP) (Wang, L. et al. Adamts-7 mediates vascular smooth muscle cell migration and neointima formation in balloon-injured rat arteries. Circ Res. 2009;104:688-698), and it has also been found that ADAMTS-7 inhibits endothelial repair through TSP-1, thus promoting neointima formation through a dual mechanism (Kessler T, Zhang L. et al. Adamts-7 inhibits re-endothelialization of injured arteries and promotes vascular remodeling through cleavage of thrombospondin-1. Circulation. 2015;131:1191-1201).

[0006] However, currently, there has been no report on an effective short-chain peptide vaccine related to ADAMTS-7.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The technical problem to be solved by the present invention is how to obtain an immunogenic peptide segment of metalloprotease ADAMTS-7 in order to prevent or treat atherosclerosis and / or vascular restenosis.

Means for Solving the Problems

[0008] In order to solve the above technical problem, the present invention first provides a short peptide which is any one of A1) to A4). A1) A short peptide whose amino acid sequence is shown in SEQ ID NO: 1 in the Sequence Listing, A2) A short peptide whose amino acid sequence is shown in SEQ ID NO: 2 in the Sequence Listing, A3) A short peptide whose amino acid sequence is shown in SEQ ID NO: 3 in the Sequence Listing, A4) A short peptide whose amino acid sequence is shown in SEQ ID NO: 4 in the Sequence Listing.

[0009] Specifically, the short peptide is the following A1) or A2). A1) A short peptide whose amino acid sequence is shown in SEQ ID NO: 1 in the Sequence Listing, A2) A short peptide whose amino acid sequence is shown in SEQ ID NO: 2 in the Sequence Listing.

[0010] More specifically, the short peptide is a short peptide whose amino acid sequence is shown in SEQ ID NO: 1 in the Sequence Listing.

[0011] The use of the above short peptide is also within the scope of the present invention.

[0012] The use of the above short peptide of the present invention includes any one of the following B1) to B4). Use in the manufacture of a product for preventing or treating atherosclerosis Use in the manufacture of a product for preventing or treating vascular restenosis Use in the manufacture of a product for reducing the neointimal area of an injured artery Use in the manufacture of a product for reducing the ratio of the intimal area to the medial area of an injured artery

[0013] In the above use, the product may be a pharmaceutical, a reagent, a vaccine, or the like

[0014] The present invention further provides a conjugate of the short peptide, which is a complete antigen obtained by conjugating the short peptide with a carrier protein

[0015] In the above conjugate, the carrier protein may be keyhole limpet hemocyanin, recombinant Qβ phage particle protein, recombinant bovine papillomavirus protein, recombinant hepatitis B virus protein, or tetanus toxoid

[0016] In the above conjugate, the linker used for the conjugation is a hetero-bifunctional crosslinker. Specifically, the hetero-bifunctional crosslinker may be SMCC, Sulfo-SMCC, or LC-SMCC

[0017] The use of the above conjugate is also within the protection scope of the present invention

[0018] The use of the conjugate of the present invention includes any one of the following B1) to B4) Use in the manufacture of a product for preventing or treating atherosclerosis Use in the manufacture of a product for preventing or treating vascular restenosis Use in the manufacture of a product for reducing the neointimal area of an injured artery Use in the manufacture of a product for reducing the ratio of the intimal area to the medial area of an injured artery

[0019] In the above use, the product may be a pharmaceutical, a reagent, a vaccine, or the like.

[0020] The present invention further provides a vaccine for preventing or treating atherosclerosis, and / or preventing or treating vascular restenosis, and / or reducing the neointimal area of an injured artery, and / or reducing the ratio of the neointimal area to the neo-medial area of the injured artery.

[0021] The above vaccine contains a conjugate in which a short peptide described in any of the following A1) to A4) is conjugated with a carrier protein. A1) A short peptide whose amino acid sequence is shown in SEQ ID NO: 1 in the Sequence Listing, A2) A short peptide whose amino acid sequence is shown in SEQ ID NO: 2 in the Sequence Listing, A3) A short peptide whose amino acid sequence is shown in SEQ ID NO: 3 in the Sequence Listing, A4) A short peptide whose amino acid sequence is shown in SEQ ID NO: 4 in the Sequence Listing.

[0022] In the above vaccine, the carrier protein may be keyhole limpet hemocyanin (KLH), recombinant Qβ phage particle protein, recombinant bovine papillomavirus protein, recombinant hepatitis B virus protein, or tetanus toxoid.

[0023] The above vaccine further contains an immunoadjuvant.

[0024] In the above vaccine, the immunoadjuvant may be an aluminum hydroxide adjuvant, a potassium alum adjuvant, or a Freund's adjuvant.

[0025] The use of the above vaccine in the manufacture of a product for preventing or treating atherosclerosis, and / or preventing or treating vascular restenosis, and / or reducing the neointimal area of an injured artery, and / or reducing the ratio of the neointimal area to the neo-medial area of the injured artery is also within the protection scope of the present invention.

[0026] In the above use, the product may be a pharmaceutical or a reagent.

[0027] The present invention further provides a method for preventing or treating atherosclerosis and / or preventing or treating vascular restenosis and / or reducing the neointimal area of an injured artery and / or reducing the ratio of the neointimal area to the medial area of an injured artery.

[0028] The method of the present invention for preventing or treating atherosclerosis and / or preventing or treating vascular restenosis and / or reducing the neointimal area of an injured artery and / or reducing the ratio of the neointimal area to the medial area of an injured artery includes administering the above conjugate or the above vaccine to an animal in order to prevent or treat atherosclerosis and / or prevent or treat vascular restenosis and / or reduce the neointimal area of an injured artery and / or reduce the ratio of the neointimal area to the medial area of an injured artery.

[0029] The short peptide of the present invention includes a first linking site that can be linked to a second linking site of a carrier protein via a covalent bond. Recombinant Qβ phage particle protein, recombinant bovine papillomavirus protein, recombinant hepatitis B virus protein, keyhole limpet hemocyanin, and tetanus toxoid are used as carrier proteins, and these carrier proteins have at least one second linking site. Preferably, the second linking site includes an amino group, a carboxyl group, and a mercapto group. Specifically, it is selected from lysine residue, arginine residue, glutamic acid residue, aspartic acid residue, and cysteine residue. A heterobifunctional crosslinking agent is used to link the ADAMTS-7 short peptide to the above carrier protein to form a regular and repetitive short peptide-vector vaccine. The short peptide is repeated and arranged and displayed on the vector or surface at a certain density to form an array display with high antigenicity, which is advantageous for the production of efficient and specific antibodies against ADAMTS-7 immunogenic peptide segments in humans and mice. It can effectively block the hydrolytic enzyme function of ADAMTS-7 and significantly suppress the intimal hyperplasia in the vascular restenosis mouse model and the occurrence of atherosclerosis in high-fat diet-fed mice.

Brief Description of Drawings

[0030]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0031] Hereinafter, the present invention will be described in more detail with reference to specific embodiments. The examples shown are not intended to limit the scope of the present invention, but merely to clarify the present invention.

[0032] Unless otherwise specified, all the experimental methods in the following examples are ordinary methods.

[0033] Unless otherwise specified, all the materials, reagents, etc. used in the following examples can be obtained from commercial channels.

[0034] Example 1, Production and Titer Detection of ADAMTS-7 Vaccine 1. Screening of Immunogenic Peptide Segments of Metalloprotease ADAMTS-7 ADAMTS-7 promotes the migration of VSMCs and the formation of neointima mainly by binding to and degrading COMP. In the present invention, the catalytic domain and 4 TSP-1-like domains were selected as target regions for screening short peptides. The amino acid sequences of the catalytic domain and 4 TSP-1-like domains were respectively input into the dialog box of IEDB B Cell Epitope Prediction. Based on the predicted scores, short peptides with high comprehensive scores were selected from those with high scores, with 8, 9, 10, 11, or 12 amino acids as units. The amino acid sequences of the above two regions were input into the dialog box of IEDB, and epitope prediction was performed using the existing Crystal Structure Of Adamts4 With Inhibitor Bound, which has the highest similarity to ADAMTS-7, as a model. Combining the results of both, and based on the comparison results of the human and mouse sequences of the ADAMTS-7 molecule, the first short peptide sequence was selected on the basis that there is no difference in two or more amino acids. Furthermore, the screened short peptide sequences were aligned with the sequences of protein molecules of the ADAMTS family and other protein sequences using tools such as PIR Peptide Match and BLAST, and short peptide sequences with the lowest possible homology were selected. Combining the above principles, four immunogenic peptide segments (four short peptides) were screened, and the results are shown in Fig. 1A and are as follows respectively. DP9: DCEPVGKRP (Sequence 1) CD9: CLDDPPAKD (Sequence 2) CP9: CNMKGDAHP (Sequence 3) CD8: CSSGRDED (Sequence 4).

[0035] 2. Preparation of conjugates Using a heterobifunctional crosslinker Sulfo-SMCC (purchased from Thermofisher, product number 22122), each of the four immunogenic peptide segments was conjugated to keyhole limpet hemocyanin (KLH), and the steps were as follows. (1) 2 mg of Sulfo-SMCC reagent was weighed and completely dissolved in 400 μL of pure water, then added to 20 mg of KLH (10 mg / mL) and mixed uniformly, and left at room temperature for 30 minutes to obtain an activated vector. (2) The activated vector was added to a 100K TFF concentration column (Merck-Millipore), 50 mM PBS (containing 1 mM EDTA, pH 7.2) was added, and centrifuged at 5000 g three times to remove the free Sulfo-SMCC and obtain a purified vector. (3) Approximately 5 mg of a short peptide (DP9, CD9, CP9 or CD8) was weighed and completely dissolved in 1 mL of 50 mM PBS (containing 1 mM EDTA, pH 7.2), the purified vector was added, and reacted at room temperature for 1 hour, gently shaken every 20 minutes to obtain a vaccine mixture. (4) The vaccine mixture was added to a 100K TFF concentration column and centrifuged at 5000 g three times to remove the free unreacted corresponding short peptide.

[0036] Through the above conjugation reaction, finally, four types of conjugate dry powders of KLH-CP9 (conjugate of CP9 and KLH), KLH-DP9 (conjugate of DP9 and KLH), KLH-CD9 (conjugate of CD9 and KLH), and KLH-CD8 (conjugate of CD8 and KLH) were obtained.

[0037] 3. Detection of titer A total of 40 six-week-old C57BL / 6 male mice weighing 21 - 23 g (purchased from the Animal Department of Peking University Health Science Center) were randomly divided into five groups: experimental group 1, experimental group 2, experimental group 3, experimental group 4, and control group, with 8 mice in each group.

[0038] The four types of conjugate dry powders obtained in Step 2 were each dissolved in sterile physiological saline to obtain four types of conjugate solutions with a concentration of 1 mg / mL. Each of the four types of conjugate solutions was mixed with aluminum hydroxide adjuvant (purchased from HEART, product number BF040) at a volume ratio of 10:1 to obtain four types of vaccines, namely Vaccine CP-9, Vaccine DP-9, Vaccine CD-9, and Vaccine CD-8. They were injected into the mice of Experimental Group 1, Experimental Group 2, Experimental Group 3, and Experimental Group 4 by subcutaneous injection at 3-4 sites respectively. KLH was dissolved in sterile physiological saline to obtain a KLH solution with a concentration of 1 mg / mL. This KLH solution was mixed with aluminum hydroxide adjuvant (purchased from HEART, product number BF040) at a volume ratio of 10:1 to obtain a KLH mixture, which was administered to the mice in the control group by subcutaneous injection at 3-4 sites. The injection dose per mouse in Experimental Groups 1 to 4 was 50 μg in terms of conjugate, and the injection dose per mouse in the control group was 50 μg in terms of KLH. As shown in B of Figure 1, a total of two injections were given. The first vaccine injection was performed at 6 weeks of age (designated as week 0, i.e., 0W). Blood was collected from the tails of the mice for the first titer detection. The second vaccine injection was performed at week 2. Blood was collected from the tails of the mice at weeks 3, 6, 8, 10, 12, 14, and 16 for titer detection. The titer detection results are shown in C of Figure 1. As can be seen from this figure, after immunizing the mice with the four types of vaccines, antibody titers that persisted for more than 12 weeks could be generated.

[0039] The method for detecting the vaccine titer was as follows. (1) 100 μl of mouse sera from Experimental Groups 1 to 4 with different dilution ratios were added to each well of the coated ELISA plate, and the mouse serum sample from the control group was added. They were incubated at 37°C for 1.5 hours. (2) They were washed with PBST, gently tapped 5 times, and dried. 100 μl of the diluted HRP-labeled secondary antibody was added and incubated at 37°C for 0.5 hours. (3) They were washed with PBST, gently tapped 5 times, and dried. 100 μl of TMB (purchased from Biolegend, product number 421106) was added to each well. When the control well turned blue, 100 μl of dilute hydrochloric acid was added to each well to stop the reaction, and the absorbance A was measured with a microplate reader. 450The absorbance value was read, and the maximum dilution multiple at which the absorbance values of the sera of the mice in experimental groups 1 to 4 were 2.1 times greater than those of the sera of the mice in the control group was calculated. Here, the coated ELISA plates were of four types: those coated with BSA-CP9 as the coating antigen, those coated with BSA-DP9 as the coating antigen, those coated with BSA-CD9 as the coating antigen, and those coated with BSA-CD8 as the coating antigen. BSA-CP9, BSA-DP9, BSA-CD9, and BSA-CD8 are conjugates obtained by binding a short peptide (CP9, DP9, CD9, or CD8) and bovine serum albumin (BSA) by the binding method in step 2. The coating method of the ELISA plate was as follows. (1) The coating antigen was dissolved in a carbonate coating buffer at pH 9.6 so that the concentration of the coating antigen was 15 μg / ml, and 100 μl / well was added to a 96-well ELISA plate (purchased from Biolegend, product number 423501) and left overnight at 4°C. (2) The next day, the coating solution was discarded, the plate was gently tapped and dried, 120 μl of 1% BSA (purchased from Solarbio, product number A8020-100) was added to each well, blocked at 37°C for 1.5 hours, discarded, and gently tapped and dried.

[0040] Example 2, Inhibition of Neointimal Formation by Vaccines DP-9 and CD-9 in a Ligation Model 1. Establishment of the Left Common Carotid Artery Ligation Model in Immunized Mice A total of 40 six-week-old C57BL / 6 male mice weighing 21 - 23 g (purchased from the Animal Department of Peking University Health Science Center) were randomly divided into five groups: experimental group 1, experimental group 2, experimental group 3, experimental group 4, and the control group, with 8 mice in each group.

[0041] The four types of conjugate dry powders (KLH-CP9, KLH-DP9, KLH-CD9, KLH-CD8) obtained in step 2 of Example 1 were each dissolved in sterile physiological saline to obtain four types of conjugate solutions with a concentration of 1 mg / mL. Each of the four types of conjugate solutions was mixed with an aluminum hydroxide adjuvant (purchased from HEART, product number BF040) at a volume ratio of 10:1 to obtain four types of vaccines (vaccine CP-9, vaccine DP-9, vaccine CD-9, and vaccine CD-8), which were injected into the mice in experimental groups 1, 2, 3, and 4 by subcutaneous injection at 3 - 4 sites respectively. KLH was dissolved in sterile physiological saline to obtain a KLH solution with a concentration of 1 mg / mL. This KLH solution and the aluminum hydroxide adjuvant (purchased from HEART, product number BF040) were mixed at a volume ratio of 10:1 to obtain a KLH mixture, which was administered to the mice in the control group by subcutaneous injection at 3 - 4 sites. The injection dose per mouse in experimental groups 1 - 4 was 50 μg in terms of conjugate, and the injection dose per mouse in the control group was 50 μg in terms of KLH. As shown in A of Figure 2, the first vaccine injection (week 0, denoted as 0W) was performed at 6 weeks of age, blood was collected from the tails of the mice to measure the first titer and body weight, the second vaccine injection was performed at 2 weeks, and blood was collected from the tails of the mice at 3 weeks to perform the second titer detection. Here, the titer detection method was the same as that in Example 1. As shown in B of Figure 2, the mice that had an immune response at 3 weeks were ready for left common carotid artery ligation experiment (i.e., model establishment). That is, before the experiment, 150 μl of 1% pentobarbital was intraperitoneally injected, and ligation was performed near the bifurcation of the common carotid artery using a 6-0 suture (purchased from HARVEYBIO, product number FX-6-0). Four weeks after the model was established (i.e., week 7), blood was collected from the tails of the mice to perform the third titer detection. The results of the vaccine titers are shown in B of Figure 2. As can be seen from this figure, high antibody titers were generated in all the mice immunized with the four types of vaccines 3 weeks and 7 weeks later.

[0042] 2. Inhibition of neointima formation Four weeks after creating the model, the fixation was carried out and the materials were collected. That is, after fixation overnight with 4% paraformaldehyde, it was embedded in OCT (purchased from Sakura Finetek USA, product number 4583) gel and stored at -80 °C until frozen sections were obtained. For the frozen sections, six sections at 200 μm, 350 μm, 500 μm, 1 mm, 1.5 mm, and 2 mm from the ligated site were left, with each section being 7 μm thick. Subsequently, HE staining was performed, and the staining results for each group of mice are shown in C of Figure 2. Four indicators, namely the neointimal area, the ratio of the neointimal area to the medial area, the external elastic lamina perimeter, and the medial area, were calculated. As shown in D, E, F, and G of Figure 2, the results of statistics using Image Pro Plus software showed that in total carotid artery ligated mice, after injection of vaccine DP-9 (represented as "DP-9" in the figure) and CD-9 (represented as "CD-9" in the figure), compared with the mice in the control group (represented as "KLH" in the figure), the neointimal area and the ratio of the neointimal area to the medial area were significantly decreased. Also, in mice injected with CP-9 (represented as "CP-9" in the figure) and CD-8 (represented as "CD-8" in the figure), there was no statistically significant difference in the ratio of the neointimal area to the medial area compared with the control group (represented as "KLH" in the figure). Therefore, vaccine DP-9 and vaccine CD-9 had a significant effect in suppressing neointimal formation in the ligation model.

[0043] Example 3, Suppression of neointimal formation by vaccine DP-9 and vaccine CD-9 in the guide wire scratching model 1. Creation of the guide wire scratching model for immunized mice A total of 30 six-week-old C57BL / 6 male mice (purchased from the Animal Department of Peking University Health Science Center) weighing 21 - 23 g were randomly divided into five groups: experimental group 1, experimental group 2, experimental group 3, experimental group 4, and the control group, with 6 mice in each group.

[0044] The four types of conjugate dry powders (KLH-CP9, KLH-DP9, KLH-CD9, KLH-CD8) obtained in Step 2 of Example 1 were each dissolved in sterile physiological saline to obtain four types of conjugate solutions with a concentration of 1 mg / mL. Each of the four types of conjugate solutions was mixed with an aluminum hydroxide adjuvant (purchased from HEART, product number BF040) at a volume ratio of 10:1 to obtain four types of vaccines, namely Vaccine CP-9, Vaccine DP-9, Vaccine CD-9, and Vaccine CD-8. These were injected into the mice in Experimental Groups 1, 2, 3, and 4 by subcutaneous injection at 3 - 4 sites respectively. KLH was dissolved in sterile physiological saline to obtain a KLH solution with a concentration of 1 mg / mL. This KLH solution and an aluminum hydroxide adjuvant (purchased from HEART, product number BF040) were mixed at a volume ratio of 10:1 to obtain a KLH mixture, which was injected into the mice in the control group by subcutaneous injection at 3 - 4 sites. The injection dose per mouse in Experimental Groups 1 - 4 was 50 μg in terms of conjugate, and the injection dose per mouse in the control group was 50 μg in terms of KLH. As shown in Figure 3A, the first vaccine injection (week 0, denoted as 0W) was performed at 6 weeks of age. Blood was collected from the mouse tail to measure the first titer and body weight. The second vaccine injection was performed at week 2, and blood was collected from the mouse tail at week 3 to detect the second titer. Here, the titer detection method was the same as in Example 1. As shown in Figure 3B, after the mouse had an immune response, it was ready for a left common carotid artery guide wire scratching experiment (i.e., model creation). That is, before the experiment, 150 μl of 1% sodium pentobarbital was injected intraperitoneally. After disinfecting the neck skin, a median anterior incision of the neck was made, the external carotid artery was ligated, and the blood flow of the internal carotid artery and the common carotid artery was temporarily blocked with a vascular clip. The microvascular scissors were used to obliquely cut the ligation site near the distal end of the external carotid artery. A metal guide wire with a diameter of 0.38 mm was inserted, advanced and retracted 5 times while rotating to rub the blood vessel wall, causing damage to the common carotid artery. The guide wire was withdrawn, the proximal end of the arteriotomy was ligated, and the skin was sutured. Four weeks after the model was created (i.e., week 7), blood was collected from the mouse tail to perform the third titer detection.The results of the vaccine titers are shown in Figure 3B. As can be seen from this figure, vaccines CP-9 and DP-9 were able to produce high antibody titers after immunizing mice.

[0045] 2. Inhibition of neointima formation Four weeks after the model was created, the animals were fixed and samples were collected. That is, after fixation overnight with 4% paraformaldehyde, they were embedded in OCT (purchased from Sakura Finetek USA, product number 4583) gel and stored at -80°C until frozen sections were obtained. For the frozen sections, six sections at 200 μm, 350 μm, 500 μm, 1 mm, 1.5 mm, and 2 mm from the ligated site were retained, and each section was 7 μm thick. Subsequently, HE staining was performed, and the staining results for each group of mice are shown in Figure 3C. Four indicators, namely the neointima area, the ratio of the neointima area to the media area, the external elastic lamina perimeter, and the media area, were calculated. As shown in Figures 3D, E, F, and G, the results of statistical analysis using Image Pro Plus software showed that in total carotid artery wire-scratching mice, after injection of vaccine DP-9 (represented as "DP-9" in the figure) and CD-9 (represented as "CD-9" in the figure), the neointima area and the ratio of the neointima area to the media area were significantly decreased compared with those of the mice in the control group (represented as "KLH" in the figure). Therefore, vaccines DP-9 and CD-9 had a significant effect in suppressing neointima formation in the wire-scratching model.

[0046] Example 4. Suppression of atherosclerotic lesion formation in LDLR- / - mice fed a high-fat diet by vaccine DP-9 1. Establishment of an atherosclerotic model A total of 18 eight-week-old male LDLR- / - mice (purchased from the Animal Department of Peking University Health Science Center) weighing 23 - 25 g were randomly divided into three groups: experimental group 1, experimental group 2, and a control group, with 6 mice in each group.

[0047] The two types of conjugate dry powders (KLH-DP9, KLH-CD9) obtained in step 2 of Example 1 were each dissolved in sterile physiological saline to obtain two types of conjugate solutions with a concentration of 1 mg / mL. The two types of conjugate solutions and aluminum hydroxide adjuvant (purchased from HEART, product number BF040) were mixed at a volume ratio of 10:1 to obtain two types of vaccines (vaccine DP-9, vaccine CD-9), which were injected into the mice in experimental group 1 and experimental group 2 by subcutaneous injection at 3-4 points respectively. KLH was dissolved in sterile physiological saline to obtain a KLH solution with a concentration of 1 mg / mL. This KLH solution and aluminum hydroxide adjuvant (purchased from HEART, product number BF040) were mixed at a volume ratio of 10:1 to obtain a KLH mixture, which was administered to the mice in the control group by subcutaneous injection at 3-4 points. The injection dose per mouse in experimental group 1 and experimental group 2 was 50 μg in terms of conjugate, and the injection dose per mouse in the control group was 50 μg in terms of KLH. As shown in A of Figure 4, the first vaccine injection was performed at 8 weeks of age (week 0, denoted as 0W), and vaccine injections were performed at 2, 4, 12, and 15 weeks respectively. From week 4 to week 18, an atherosclerotic diet (high-fat diet, the feed was purchased from Research Diets, product number: D12108C, the content of the main components was cholesterol 1.25%, fat 40 kcal%) was continued. Then, vaccine titer detection and body weight detection were performed at 3, 9, 13, and 18 weeks. Here, the method for detecting the titer was the same as that in Example 1. As shown in B and C of Figure 4, it was shown that vaccines CD-9 and DP-9 could produce high antibody titers after immunizing the mice, and high antibody titers could also be detected at 18 weeks.

[0048] 2. Inhibition of the formation of atherosclerotic lesions Under a microscope, the entire blood vessels of the mouse were dissected using a microscopic instrument, then fixed with 4% paraformaldehyde for 6 h, next immersed in 60% isopropyl alcohol for 5 min, and then stained with Oil Red O for 30 min under light-shielding conditions. After that, the skin color was washed away with 60% isopropanol solution. Subsequently, under the microscope, the adventitial fat was dissected. After being dissected cleanly, the blood vessels were longitudinally incised with micro scissors. Then, the same steps were repeated, immersed in 60% isopropanol for 5 min, and then stained with Oil Red O for 30 min under light-shielding conditions. After that, the skin color was washed away with 60% isopropanol solution. Finally, the stained blood vessels were fixed on a plate to obtain a schematic diagram. As shown in D of Figure 4, atherosclerotic plaques were formed in all of the control group, experimental group 1, and experimental group 2. As a result of statistically analyzing the plaque area using Image Pro Plus software, when the plaque area of the control group (represented by "KLH" in the figure) was set to 1, the relative plaque areas of experimental group 2 (represented by "CD-9" in the figure) and experimental group 1 (represented by "DP-9" in the figure) were calculated. The results showed that compared with the control group (represented by "KLH" in the figure), the relative plaque area of experimental group 1 (represented by "DP-9" in the figure) was significantly decreased (p < 0.001), and there was no difference in the relative plaque area of experimental group 2 (represented by "CD-9" in the figure) (p > 0.05). From this, it can be seen that the vaccine DP-9 significantly inhibited the formation of atherosclerotic plaques, and there was no significant difference in the vaccine CD-9 compared with the control group KLH.

[0049] In the present invention, the following statistical methods were used for analysis. (1) A continuous random variable with a normal distribution is represented as Mean ± Standard Error of the Mean (Mean ± SEM). (2) For paired data, all corresponding t-tests (two-sided tests) were used. (3) For unpaired data, Student's t-test (two-sided test) was used. (4) When comparing the results of a single factor among multiple groups, one-way analysis of variance was used. When further comparing two groups at a time, the Student-Newman-Keuls test was used. (5) When comparing the results of two factors among multiple groups, two-way analysis of variance was used. When further comparing two groups at a time, the Bonferroni test was used.

[0050] The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range with the same parameters, concentrations, and conditions without departing from the gist and scope of the present invention and without performing unnecessary experiments. Although the present invention shows specific embodiments, it should be understood that the present invention may be further improved. In short, according to the principle of the present invention, the present application intends to include any modifications, uses, and improvements of the present invention that depart from the scope disclosed in the present application but include changes by the prior art known in the art. According to the following appended claims, the application of some basic features can be carried out.

Industrial Applicability

[0051] 1. The short peptide of metalloprotease ADAMTS-7 screened by the present invention can cause a specific immune response in wild-type C57BL / 6 mice and ApoE- / - mice, specifically bind to the catalytic domain of metalloprotease ADAMTS-7, and can produce specific antibodies against the short peptide that can block the activity of its hydrolase. 2. The vaccine produced by the present invention can significantly suppress the intimal hyperplasia in a vascular restenosis mouse model (i.e., a ligation model and a guide wire scratching model) and the occurrence of atherosclerosis in high-fat diet-fed mice, and is useful for the prevention or treatment of atherosclerosis and / or vascular restenosis. 3. The hetero-bifunctional cross-linking agent used in the production process of the vaccine of the present invention has the characteristic of being able to simultaneously link two types of groups, and moreover, different groups can be linked in sequence in two steps, and the operation is simple and efficient.

Claims

1. A short peptide, characterized in that it is any one of the following A1) to A2): A1) A short peptide whose amino acid sequence is shown in SEQ ID NO: 1 in the Sequence Listing; A2) A short peptide whose amino acid sequence is shown in SEQ ID NO: 2 in the Sequence Listing.

2. A conjugate of the short peptide according to Claim 1, which is a complete antigen obtained by conjugating the short peptide according to Claim 1 with a carrier protein.

3. The conjugate according to Claim 2, wherein the carrier protein is keyhole limpet hemocyanin, recombinant Qβ phage particle protein, recombinant bovine papillomavirus protein, recombinant hepatitis B virus protein, or tetanus toxoid.

4. Use, characterized in that it is any one of the following: P1) Use of the short peptide according to Claim 1 in the manufacture of a product for preventing or treating atherosclerosis; P2) Use of the conjugate according to Claim 2 in the manufacture of a product for preventing or treating atherosclerosis; P3) Use of the conjugate according to Claim 3 in the manufacture of a product for preventing or treating atherosclerosis; P4) Use of the short peptide according to Claim 1 in the manufacture of a product for preventing or treating vascular restenosis; P5) Use of the conjugate according to Claim 2 in the manufacture of a product for preventing or treating vascular restenosis; P6) Use of the conjugate according to Claim 3 in the manufacture of a product for preventing or treating vascular restenosis; P7) Use of the short peptide according to Claim 1 in the manufacture of a product for preventing or treating atherosclerosis; P8) Use of the conjugate according to Claim 2 in the manufacture of a product for preventing or treating atherosclerosis; P9) Use of the conjugate according to Claim 3 in the manufacture of a product for preventing or treating atherosclerosis; P10) Use of the short peptide according to Claim 1 in the manufacture of a product for reducing the neointimal area of an injured artery; P11) Use of the conjugate according to Claim 2 in the manufacture of a product for reducing the neointimal area of an injured artery; P12) Use of the conjugate according to Claim 3 in the manufacture of a product for reducing the neointimal area of an injured artery; P13) Use of the short peptide according to Claim 1 in the manufacture of a product for reducing the ratio of the neointimal area to the medial area of an injured artery; Use of the conjugate according to claim 2 in the manufacture of a product for reducing the ratio of intimal area to medial area of an injured artery. Use of the conjugate according to claim 3 in the manufacture of a product for reducing the ratio of intimal area to medial area of an injured artery. **Claim 5** A vaccine for preventing or treating atherosclerosis and / or preventing or treating vascular restenosis and / or reducing the neointimal area of an injured artery and / or reducing the ratio of intimal area to medial area of an injured artery, characterized in that it comprises the conjugate according to claim 2 or 3. **Claim 6** The vaccine according to claim 5, characterized in that it consists of the conjugate according to claim 2 or 3 and an immunoadjuvant. **Claim 7** Use of the vaccine according to claim 5 or 6 in the manufacture of a product for preventing or treating atherosclerosis and / or preventing or treating vascular restenosis and / or reducing the neointimal area of an injured artery and / or reducing the ratio of intimal area to medial area of an injured artery. **Claim 8** A composition for preventing or treating atherosclerosis and / or preventing or treating vascular restenosis and / or reducing the neointimal area of an injured artery and / or reducing the ratio of intimal area to medial area of an injured artery, characterized in that it comprises the conjugate according to claim 2 or 3 or the vaccine according to claim 5 or 6.

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