A pcsk9 nanoparticle vaccine and preparation and application thereof

Abstract

The application relates to a PCSK9 nanoparticle vaccine and preparation and application thereof, the PCSK9 catalytic domain is directionally optimized by using an artificial intelligent protein design technology, the protein aggregation tendency is significantly reduced while the LDLR binding core epitope is reserved, the antigen stability and expression level are improved, and an engineered PCSK9 antigen is obtained. The engineered PCSK9 antigen is combined with an HPF nanoparticle carrier to realize high-density multivalent display, the body immune response is significantly enhanced, high-level functional anti-PCSK9 antibodies are induced, the interaction between PCSK9 and LDLR is blocked, the LDLR expression level is improved, plasma low-density lipoprotein cholesterol clearance is promoted, the purposes of reducing blood lipids and preventing cardiovascular diseases are achieved. The PCSK9 nanoparticle vaccine has the advantages of high immunogenicity, high safety, simple preparation process, suitability for large-scale production and the like, and has a good clinical application prospect.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a PCSK9 nanoparticle vaccine and its preparation and application. Background Technology

[0002] Cardiovascular disease (CVD) is currently the leading cause of death worldwide, and its high incidence rate has made it a serious public health problem. Clinical studies have shown that effectively controlling blood lipid levels is crucial for reducing the incidence and mortality of CVD. Currently, the traditional first-line lipid-lowering drugs are mainly statins, which effectively lower low-density lipoprotein cholesterol (LDL-C) levels in the blood, thereby reducing the occurrence of adverse cardiovascular events. However, some patients experience adverse reactions such as myalgia and elevated transaminases during long-term medication, exhibiting a significant phenomenon of "statin intolerance," which limits treatment adherence and target achievement rates.

[0003] Proprotein Convertase Subtilisin / Kexin type 9 (PCSK9) is a serine protease primarily synthesized and secreted by the liver. Its structure includes an N-terminal signal peptide, a prodomain, a catalytic domain, and a cysteine-rich C-terminal domain. The catalytic domain is the core functional region for PCSK9's binding to the low-density lipoprotein receptor (LDLR). PCSK9 is a crucial factor regulating low-density lipoprotein cholesterol (LDL-C) metabolism. Under physiological conditions, PCSK9 binds to the LDL-LR on the surface of hepatocytes, promoting LDLR internalization and targeting lysosomal degradation. This reduces the recycling of LDLR on the hepatocyte surface, decreases the liver's ability to clear circulating LDL-C, ultimately leading to elevated plasma LDL-C levels and increasing the risk of atherosclerotic cardiovascular disease. Studies have shown that serum LDL levels in individuals with PCSK9 dysfunction are reduced by about 40% compared to the general population, and the incidence of coronary heart disease is reduced by about 88%. Therefore, PCSK9 has become a highly attractive new target for lipid-lowering therapy.

[0004] Lipid-lowering drugs targeting PCSK9 work primarily on two levels: first, by blocking the binding of PCSK9 to LDLR (e.g., monoclonal antibodies); and second, by inhibiting the synthesis of the PCSK9 protein (e.g., small interfering RNA drugs). Currently, several PCSK9 inhibitors have been approved for marketing globally. Besides early drugs like evolocumab (Amgen), alirocumab (Sanofi), and the first siRNA drug, Inclisiran (Novartis), domestically developed fully human monoclonal antibodies (such as tolecithin) have also been approved for marketing. This fully demonstrates the feasibility and significant clinical application value of PCSK9 as a novel lipid-lowering target. However, existing drugs have certain clinical limitations: monoclonal antibodies typically require subcutaneous injections every two to four weeks, which not only limits long-term patient adherence but also easily stimulates the body to produce anti-drug antibodies (ADA), leading to efficacy tolerance; while Inclisiran extends the injection interval to six months, it still requires continuous passive injection, and the high treatment cost increases the economic burden on patients. Therefore, exploring new, more long-acting, economical, and accessible PCSK9 intervention methods remains of significant clinical importance.

[0005] Vaccines, as the most classic means of stimulating the body to produce active immunity, perfectly align with the demands of long-term chronic disease management. Therefore, leveraging the long-lasting advantage of vaccine-induced active immunity to overcome the disadvantages of existing targeted drugs—such as short half-life, frequent injections, and susceptibility to drug resistance—and combining this with the precise lipid-lowering target of PCSK9, the development of novel therapeutic vaccines for cardiovascular diseases has become a highly promising frontier direction. However, because PCSK9 is a highly conserved autoantigen within the body, inducing an immune response against it inevitably faces the body's natural and stringent immune tolerance barrier. Therefore, effectively breaking this immune tolerance and designing and constructing novel PCSK9 nanoparticle vaccines from the perspective of enhancing the immunogenicity of autoantigens has extremely important scientific research significance and broad clinical translation prospects. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies, such as the large molecular weight of full-length PCSK9 protein, the large number of non-functional epitopes, insufficient immune focusing, and the tendency of truncated antigens to aggregate. This invention provides a PCSK9 antigen and its nanoparticle vaccine based on artificial intelligence protein design, and provides a method for preparing the nanoparticle vaccine and its application.

[0007] The PCSK9 nanoparticles of this invention, used as the active ingredient in a vaccine, retain the core functional epitope of PCSK9 binding to the low-density lipoprotein receptor (LDLR). Computational protein design techniques are used to optimize protein stability and solubility, and a multivalent nanoparticle vaccine is further constructed, thereby significantly improving the immune response against PCSK9. This nanoparticle vaccine exhibits highly effective lipid-lowering properties, providing a novel and effective option and technical solution for the prevention and treatment of hyperlipidemia, atherosclerosis, and other PCSK9-related cardiovascular diseases.

[0008] The first objective of this invention is to provide a recombinant protein.

[0009] A second objective of this invention is to provide the use of the recombinant protein in the preparation of lipid-lowering drugs or vaccines.

[0010] A third objective of this invention is to provide a nanoparticle.

[0011] A fourth object of the present invention is to provide a composition.

[0012] The fifth objective of this invention is to provide a biomaterial.

[0013] The sixth object of the present invention is to provide a composition.

[0014] A seventh object of the present invention is to provide the use of any of the nanoparticles, the composition, the biomaterial, or the composition in the preparation of lipid-lowering drugs or vaccines.

[0015] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention claims protection for a recombinant protein having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 6.

[0016] It also claims protection for the use of the recombinant protein in the preparation of lipid-lowering drugs or vaccines.

[0017] This invention claims protection for a nanoparticle comprising a nanoparticle carrier, an engineered PCSK9 antigen, and a self-assembly system, wherein the engineered PCSK9 antigen and the nanoparticle carrier are respectively connected to the self-assembly system via linking peptides, and form multivalent antigen nanoparticles through self-assembly. The amino acid sequence of the engineered PCSK9 antigen is shown in SEQ ID NO: 1.

[0018] Preferably, the self-assembly system is a peptide chain or fusion protein capable of spontaneous bonding.

[0019] Preferably, the self-assembly system is a self-assembly system based on isopeptide bonds; the self-assembly system based on isopeptide bonds is a GvTagOpti / SdCatcher (GvO / Sd) system or a SpyTag / SpyCatcher system. The nanoparticle carrier is: Helicobacter pylori ferritin tetratrimole nanoparticles, I53-50 nanoparticles, mi3 nanoparticles, or Gag-Like protein nanoparticles.

[0020] The Gag-Like protein nanoparticles are protein nanoparticles that are beneficial for synthesizing HIV-1 p24 and human endogenous retrovirus K capsid protein CA. For details, see CN119841901A A short peptide composition that inhibits inclusion body formation and promotes the self-assembly of Gag-Like protein nanoparticles.

[0021] More preferably, the self-assembly system is connected to the N-terminus or C-terminus of the nanoparticle carrier and the engineered PCSK9 antigen via GSG peptides, respectively.

[0022] As a specific implementation method, the recombinant protein with the amino acid sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6 is self-assembled.

[0023] This invention claims a composition containing a recombinant protein with amino acid sequences as shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0024] This invention claims protection for a biological material, which is any one of the following: (1) A nucleic acid molecule encoding a recombinant protein with an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 6; (2) Nucleic acid molecules encoding each component of the composition; (3) An expression cassette containing the nucleic acid molecule described in (1) or (2); (4) A recombinant vector containing the nucleic acid molecule described in (1) or (2) or the expression cassette described in (3); (5) A recombinant microorganism containing the nucleic acid molecule described in (1) or (2), the expression cassette described in (3), or the recombinant vector described in (4); (6) A cell line containing the nucleic acid molecule described in (1) or (2), the expression cassette described in (3), or the recombinant vector described in (4).

[0025] This invention claims protection for a composition comprising the composition and an adjuvant; or comprising any of the nanoparticles and an adjuvant.

[0026] The present invention also claims protection for the use of any of the nanoparticles, the composition, the biomaterial, or the composition in the preparation of lipid-lowering drugs or vaccines.

[0027] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to employ artificial intelligence protein design technology to target and optimize the catalytic domain of PCSK9, significantly reducing protein aggregation tendency and improving antigen stability and expression levels while retaining the LDLR-binding core epitope. Furthermore, by combining it with 24-mer HPF nanoparticle carriers, high-density multivalent display of engineered PCSK9 antigens is achieved, significantly enhancing the body's immune response to PCSK9, inducing high levels of functional anti-PCSK9 antibodies, thereby blocking the interaction between PCSK9 and LDLR, increasing LDLR expression levels, and promoting the clearance of low-density lipoprotein cholesterol (LDL-C) in plasma, thus achieving the goal of lowering blood lipids and preventing cardiovascular diseases. The PCSK9 nanoparticle vaccine described in this invention has advantages such as strong immunogenicity, high safety, simple preparation process, and suitability for large-scale production, and has good prospects for clinical application. Attached Figure Description

[0028] Figure 1 The design process for the PCSK9 catalytic domain; cyan: LDLR, green: PCSK9. The catalytic domain was extracted, and an optimized interface was defined. Partial diffusion was used for interface redesign. After experimental screening, the optimal design was selected and assembled into a nanoparticle vaccine.

[0029] Figure 2 Below is the designed PCSK9 mutant library, with four candidate designs shown in red.

[0030] Figure 3 The image shows the purification results of PCSK9 nanoparticles by pre-packed Siperose6 Increase 10 / 300 GL chromatography; the curve shows the absorption peak at 100 nm; the elution peak of PCSK9 nanoparticles is around 17 mL.

[0031] Figure 4 The image shows PCSK9 nanoparticles as captured by a transmission electron microscope. The PCSK9 nanoparticles are uniform in size and have a stable structure and morphology, with a diameter of about 200 nm.

[0032] Figure 5 A schematic diagram of a hyperlipidemia animal model using PCSK9 nanoparticles as a vaccine.

[0033] Figure 6The results show the dynamic detection of T-CHO and LDL levels in the serum of hyperlipidemic mice under the action of PCSK9 nanoparticle vaccine; HPF is GvO-HPF protein.

[0034] Figure 7 The graph shows the dynamic changes in the level of PCSK9-specific IgG antibodies in the serum of hyperlipidemic mice under the influence of PCSK9 nanoparticle vaccine; HPF refers to GvO-HPF protein.

[0035] Figure 8 The graph shows the affinity of Sd-PCSK9 to LDLR; its equilibrium dissociation constant KD is 2.36E-07 M. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0037] The GvTagOpti / Sdcatcher (Gv / Sd) system based on isopeptide bonds is described in Chinese Patent CN113621031A, which describes a combination of peptide linkers that utilize spontaneous isopeptide bonds for protein covalent self-assembly.

[0038] Example 1: PCSK9 engineered antigen designed based on artificial intelligence proteins I. Experimental Methods Specific design methods include Figure 1 As shown.

[0039] Using the PCSK9 catalytic domain as a design template, the propeptide and CHRD domains were deleted, retaining only the core functional regions related to LDLR binding to improve antigen focusing and reduce interference from non-functional epitopes on the immune response. However, directly truncating the catalytic domain exposes some hydrophobic amino acids that were originally embedded within the native protein to the protein surface, easily leading to protein aggregation; simultaneously, some cysteine ​​residues, after losing their native structural constraints, may form incorrect disulfide bonds, thus affecting proper protein folding and expression.

[0040] Therefore, the extracted catalytic domain was further optimized in its structure design: First, protein structure analysis was used to identify hydrophobic residues exposed on the protein surface due to domain truncation, as well as potential aggregation hotspots. A systematic analysis of cysteine ​​residues in the catalytic domain was then performed to assess their impact on protein stability and aggregation tendency. Based on the analysis results, key sites that may lead to protein aggregation, misfolding, or decreased expression levels were screened as redesign regions.

[0041] Subsequently, the partial diffusion design strategy in the RF Diffusion protein design platform was used for structure optimization. The PCSK9-LDLR binding interface was defined as a fixed region, preserving its native conformation and key functional residues to maintain the integrity of the LDLR binding-related epitopes. At the same time, the potential aggregation regions, exposed hydrophobic regions, and non-critical surface regions obtained from the previous screening were defined as designable regions, and the local sequence and structure were regenerated using a diffusion model.

[0042] Using the above design approach, while maintaining the basic structure of the LDLR binding interface, the amino acid residues that may lead to aggregation are targeted for optimization. This includes reducing surface hydrophobicity, eliminating unnecessary free cysteine ​​residues, and improving surface charge distribution, thereby enhancing the protein's solubility, stability, and expression level. After multiple rounds of iterative design, several candidate engineered PCSK9 sequences were obtained.

[0043] Furthermore, the AlphaFold structure prediction platform was used to screen and evaluate candidate designs. The overall structural reliability was evaluated using the predicted local distance difference test (pLDDT), and the predicted interface alignment error (iPAE) was used to evaluate the structural rationality between the redesigned and retained regions, as well as the retention of the LDLR binding interface. Candidate molecules with high pLDDT scores, low iPAE scores, and intact LDLR binding epitopes were preferred for subsequent experimental validation.

[0044] II. Experimental Results See results Figure 2 A total of 64 designs were obtained, a PCSK9 mutation library was constructed, and four candidate PCSK9 engineered antigens with high pLDDT scores, low RMSD scores and intact LDLR binding epitopes were selected for subsequent experiments.

[0045] Example 2: Characterization of PCSK9 engineered antigen I. Expression and purification of PCSK9 engineered antigen 1. Experimental Methods The four candidate PCSK9 engineered antigens obtained in Example 1 were expressed and purified using the following methods: For PCSK9, an Sp signal peptide is linked to the N-terminus via a linker (GSG), resulting in Sp-GSG-PCSK9. The 3' end of the Sp-GSG-PCSK9 coding gene also contains a stop codon, TAATAA, which is linked between the XholI and XbaI restriction sites of the pcDNA3.1-GFP vector by placing XholI and XbaI restriction sites at the 5' and 3' ends, respectively, to obtain the recombinant expression vector pcDNA3.1-PCSK9.

[0046] The recombinant expression vector pcDNA3.1-PCSK9, which was verified to be correct by sequencing, was transformed into E. coli BL21(DE3) competent cells to obtain the corresponding engineered strain. Positive single clones were picked and inoculated into 5 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C and 220 rpm with shaking.

[0047] The overnight culture was transferred to 500 mL of LB liquid medium containing kanamycin and cultured at 37°C and 220 rpm. When the absorbance (OD) of the culture reached approximately 0.6 (about 4–5 hours), IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 1 mM. The temperature was then lowered to 16°C, and protein expression was induced at 220 rpm for 18 hours.

[0048] After induction, the bacterial cell pellet was collected by centrifugation. The cells were then fully resuspended in pre-cooled buffer (containing 20 mM Tris-HCl, 50 mM NaCl, pH 7.5). The bacterial suspension was then disrupted using an autoclave, and the supernatant was collected by centrifugation after complete disruption to obtain the crude extract of PCSK9.

[0049] Preliminary purification was performed using DEAE anion exchange chromatography. The crude sample was then incubated and finally purified using a Superose6 Increase 10 / 300 GL molecular sieve column (GE Healthcare) to obtain high-purity PCSK9 engineered antigen obtained in Example 1.

[0050] 2. Experimental Results The results showed that of the four candidate PCSK9 engineered antigens obtained in Example 1, three could not be expressed, and only one was expressed well. Its amino acid sequence is shown in SEQ ID NO: 1.

[0051] II. Size Exclusion Chromatography (SEC) 1. Experimental Methods The high-purity amino acid sequence of the PCSK9 engineered antigen sample, as shown in SEQ ID NO: 1, was incubated in 20 mM Tris-50 mM NaCl pH 7.5 buffer at 4°C and 37°C for 12–16 hours. After incubation, size exclusion chromatography (SEC) was performed with an elution flow rate of 0.5 mL / min, and the target protein eluted in the range of 10–12 mL elution volumes. Each component produced a corresponding chromatographic peak under a UV detector (typically 280 nm), and the content of each component was calculated using peak area normalization. The structural and storage stability of the candidate antigens were evaluated by changes in monomer retention and aggregation levels.

[0052] 2. Experimental Results Size exclusion chromatography (SEC) analysis showed that the peak range was concentrated in 10–12 ml (spanning approximately 2 ml), indicating that the target substance was relatively concentrated during the elution process.

[0053] III. Dynamic Light Scattering (DLS) 1. Experimental Methods Take a high-purity PCSK9 engineered antigen sample with the amino acid sequence shown in SEQ ID NO: 1, at a concentration of 100–500 μg / mL, and place it in a micro-sample cell (100 μl volume). Cover the sample cell and insert it into the BeNano 180Zeae nanoparticle size and Zata potential analyzer. The specific method is as follows: create a new test project on the instrument's computer, input the test SOP parameters, select "particle size test" as the test type, enter the sample name, set the temperature to 25℃, and perform the test after a constant temperature of 60–120 seconds.

[0054] 2. Experimental Results Dynamic light scattering (DLS) analysis showed that the particle size of the samples did not fluctuate significantly under different storage conditions, and the particle size distribution was uniform and remained stable, indicating that the protein particles were in good condition and there was no obvious aggregation.

[0055] IV. Thermal Stability Analysis 1. Experimental Methods High-purity PCSK9 engineered antigen samples with amino acid sequences as shown in SEQ ID NO: 1 were stored at 4℃, 37℃ and room temperature, respectively, and samples were taken at regular intervals. Physicochemical indicators were detected by electrophoresis, liquid chromatography and dynamic light scattering, respectively.

[0056] 2. Experimental Results The results showed that the high-purity PCSK9 engineered antigen sample obtained in Example 1 had a uniform and stable particle size, no significant decrease in purity, was not prone to degradation and aggregation, and had good temperature tolerance and storage stability.

[0057] In summary, this invention, through AI-guided design and a series of rigorous physicochemical characterization processes, successfully screened a structurally stable, non-aggregating, highly soluble, and highly homogeneous PCSK9 engineered antigen (amino acid sequence shown in SEQ ID NO: 1) from numerous candidates. This antigen not only perfectly overcomes the technical bottleneck of easy aggregation of traditional PCSK9 truncated proteins, but also provides the most ideal core active ingredient for the subsequent construction of efficient and stable multivalent nanoparticle vaccines. Example 3: Design and preparation of a self-contained PCSK9 nanoparticle vaccine I. Experimental Methods 1. Design of an independently developed PCSK9 nanoparticle vaccine PCSK9 nanoparticle vaccines were prepared using Helicobacter pylori nonheme ferritin (HPF, SEQ ID NO: 2) as a nanoparticle carrier. The engineered PCSK9 antigen, with an amino acid sequence as shown in SEQ ID NO: 1, was regularly, densely, and multivalently displayed on the surface of the nanoparticles using the GvTagOpti / SdCatcher (GvO / Sd) system, thereby enhancing the B cell receptor cross-linking effect and improving antigen presentation efficiency and immunogenicity.

[0058] The amino acid sequence of GvTagOpti (GvO) is shown in SEQ ID NO: 3; The amino acid sequence of SdCatcher (SD) is shown in SEQ ID NO: 4; The amino acid sequence of the first fusion protein GvO-HPF is shown in SEQ ID NO: 5; The amino acid sequence of the second fusion protein SD-PCSK9 is shown in SEQ ID NO: 6.

[0059] 2. Preparation of PCSK9 nanoparticle vaccine using self-contained technology (1) Construction of recombinant expression vector pcDNA3.1-SD-PCSK9 For SD-PCSK9, the Sp signal peptide is linked to the N-terminus of the SD peptide via a linker (GSG), resulting in Sp-GSG-SD-PCSK9. The 3' end of the gene encoding Sp-GSG-SD-PCSK9 also contains a stop codon, TAATAA, which is linked between the XholI and XbaI restriction sites of the pcDNA3.1-GFP vector by placing them at the 5' and 3' ends, respectively, to obtain the recombinant expression vector pcDNA3.1-SD-PCSK9.

[0060] (2) Construction of the fusion protein GVO-HPF The 3' end of the gene encoding the fusion protein GVO-HPF also contains a 6×his gene, which is then linked to the NcoⅠ and XholⅠ restriction sites of pET28a by designing NcoⅠ and XholⅠ restriction sites at the 5' end and 3' end, to obtain the recombinant expression vector pET28a-GVO-HPF.

[0061] 3. Induced expression and crude extraction of proteins (1) Plasmid transformation and strain activation The recombinant expression vectors pET28a-GVO-HPF and pcDNA3.1-SD-PCSK9, which were verified to be correct by sequencing, were transformed into Escherichia coli BL21(DE3) competent cells to obtain the corresponding engineered strains.

[0062] Positive single clones were picked and inoculated into 5 mL of LB liquid medium containing kanamycin, and cultured overnight with shaking at 37°C and 220 rpm.

[0063] (2) Expanded culture and protein induction The overnight culture was transferred to 500 mL of LB liquid medium containing kanamycin and cultured at 37°C and 220 rpm. When the absorbance (OD) of the culture reached approximately 0.6 (about 4–5 hours), IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 1 mM. The temperature was then lowered to 16°C, and protein expression was induced at 220 rpm for 18 hours.

[0064] (3) Collection and lysis of bacterial cells After induction, the bacterial cell pellet was collected by centrifugation. The cells were then fully resuspended in pre-cooled buffer (containing 20 mM Tris-HCl, 50 mM NaCl, pH 7.5). The bacterial suspension was then disrupted using a high-pressure cell disruptor. After complete disruption, the supernatant was collected by centrifugation, ultimately yielding crude extracts of the first fusion protein GvO-HPF and the second fusion protein SD-PCSK9.

[0065] 2. Protein purification (1) The crude extract of the first fusion protein GvO-HPF was heated in a 70°C water bath for 15 minutes to denature and precipitate most of the heat-sensitive impurities. The precipitate was then removed by centrifugation at 12,000 rpm for 10 minutes, and the supernatant was filtered through a 0.45 μm microporous membrane (filter head) to remove small suspended particles. Finally, the filtered supernatant was transferred to an ultrafiltration concentrator with a molecular weight cutoff of 100 kDa for centrifugation and concentration.

[0066] (2) The crude extract of the second fusion protein SD-PCSK9 was first centrifuged through an ultrafiltration concentrator with a molecular weight cutoff of 100 kDa, and the filtrate was collected (to remove impurity proteins with a molecular weight greater than 100 kDa); then the filtrate was transferred to an ultrafiltration concentrator with a molecular weight cutoff of 10 kDa, and the target protein was concentrated by centrifugation.

[0067] (3) Molecular sieve chromatography purification The concentrated first fusion protein GvO-HPF and the second fusion protein SD-PCSK9 were respectively loaded onto a Superose 6 Increase 10 / 300 GL molecular sieve chromatography column (GE Healthcare) for purification. The corresponding elution peaks were collected, and the protein purity was analyzed by reducing SDS-PAGE electrophoresis. The concentration of the purified protein was determined by the BCA method.

[0068] 3. Assembly and Identification of PCSK9 Nanoparticles (1) Protein incubation and assembly The first fusion protein GvO-HPF and the second fusion protein SD-PCSK9 were mixed at a molar ratio of 1:1 and placed in a buffer solution (containing 20 mM Tris-HCl, 50 mM NaCl, pH 7.5) and incubated overnight at room temperature. This allowed them to fully bind and self-assemble to form PCSK9 nanoparticles (PCSK9-NP).

[0069] (2) Purification and identification of nanoparticles The incubated and assembled reaction solution was re-loaded onto a Superose 6 Increase 10 / 300 GL molecular sieve column for purification, and the target elution peak was collected. The microstructure of the PCSK9 nanoparticles was observed using transmission electron microscopy (TEM).

[0070] II. Experimental Results The purification chromatogram of PCSK9 nanoparticles by pre-packed Siperose6 Increase 10 / 300 GL chromatography is shown below. Figure 3 As shown in the figure, the curve represents the absorption peak at 100 nm. The elution peak of PCSK9 nanoparticles is around 17 ml.

[0071] The purified first fusion protein GvO-HPF, the second fusion protein SD-PCSK9, and the PCSK9 nanoparticles after incubation were photographed under a transmission electron microscope as shown in the following figure. Figure 4As shown, the PCSK9 nanoparticles are uniform in size and have a stable structure and morphology, with a diameter of approximately 200 nm. The results indicate that the PCSK9 engineered antigen designed in Example 1 did not disrupt the original self-assembly properties of the HPF nanocarrier, and successfully prepared PCSK9 nanoparticles with regular morphology and complete structure, fully meeting the expected engineering design requirements.

[0072] Example 4: Effect test of PCSK9 nanoparticles as a vaccine on blood lipids in mice and detection of antibodies. I. Immunization of hyperlipidemic mice with PCSK9 nanoparticle vaccine A mouse model of hyperlipidemia was constructed to test the effect of the PCSK9 nanoparticles prepared in Example 3 as a vaccine on the blood lipid levels of mice. The control protein was the GvO-HPF protein (HPF) purified in Example 3.

[0073] Six- to eight-week-old B6-hPCSK9 (Ki / Ki) humanized mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were immunized three times with PCSK9 nanoparticle vaccine at week 0, week 3 and week 6 (dosage: 10 μg / mouse).

[0074] Meanwhile, starting from week 0 (W0), a high-fat diet was administered to establish an animal model of hyperlipidemia, as illustrated in the diagram below. Figure 5 .

[0075] II. Detection of serum total cholesterol (T-CHO) and low-density lipoprotein (LDL) 1. Experimental Methods Sample collection began at week 0 prior to immunization. Blood was collected from the orbital venous plexus of mice after fasting, every two weeks. Serum was separated for dynamic detection of serum total cholesterol (T-CHO) and low-density lipoprotein (LDL) using reagent kits from Nanjing Jiancheng Technology Co., Ltd., as follows: T-CHO assay: First, take 2.5 μL each of serum from different groups, physiological saline (negative control), and standard (positive control). Then add 250 μL of test solution, incubate at 37℃ for 10 minutes, and use an ELISA reader to detect the absorbance at 500 nm. Calculate the final result based on the OD value and the concentration of the standard.

[0076] LDL assay: Take 2.5 μL each of serum from different groups, physiological saline (negative control), and standard (positive control). Add 180 μL of reagent one, incubate at 37°C for 5 minutes, and then measure the absorbance (OD1) at 550 nm using a microplate reader. Add 60 μL of reagent two, incubate at 37°C for 10 minutes, and then measure the absorbance (OD2) at 550 nm again. Calculate the final result based on the two OD values ​​and the standard concentration.

[0077] 2. Experimental Results The dynamic detection results of serum T-CHO and LDL levels in hyperlipidemic mice under PCSK9 nanoparticle vaccine treatment are as follows: Figure 6 The study demonstrated that, compared with the control GvO-HPF protein, the PCSK9 nanoparticle vaccine effectively reduced the levels of T-CHO and LDL in the serum of humanized mice under high-fat feeding conditions.

[0078] III. ELISA detection of PCSK9-specific antibodies in serum 1. Experimental Methods The level of PCSK9-specific antibodies in the serum of high-fat model mice was determined using enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: The concentration of the purified second fusion protein SD-PCSK9 from Example 3 was adjusted to 5 μg / mL using ELISA coating buffer (purchased from Beijing Solarbio Science & Technology Co., Ltd.), and 50 μL was plated into 96-well plates at 4°C overnight. The plates were then incubated overnight at room temperature. The next day, the microplates were blocked with 5% skim milk at room temperature for 1 hour.

[0079] The serum samples to be tested were serially diluted with PBS solution, starting at a ratio of 1:30, and then 7 serially diluted 3-fold before being added to the well plates and incubated at 37°C for 2 hours.

[0080] Next, wash three times with PBST, 200 μL / well each time. Then add 100 μL of HRP-conjugated goat anti-mouse secondary antibody IgG diluted with PBS at a ratio of 1:4000 to each well, and incubate at 37°C for 1 hour.

[0081] Wash the plate four times with 200 μL / well PBST solution, then add 50 μL / well TMB substrate solution and incubate at room temperature for color development. After 5 min of substrate development, add 50 μL / well stop solution to stop the reaction, and measure the absorbance of each well at 450 nm using a microplate reader.

[0082] 2. Experimental Results Dynamic changes in PCSK9-specific IgG antibody levels, such as Figure 7As shown in the figure. The results indicate that, compared with the control group inoculated with GvO-HPF protein, the PCSK9 nanoparticle vaccine can effectively activate the body's immune response and produce specific IgG antibodies against the PCSK9 catalytic domain.

[0083] Example 5: Surface Plasmon Resonance (SPR) Detection of Affinity between Sd-PCSK9 and LDLR I. Experimental Methods 1. Surface activation and ligand immobilization of sensor chips Using the CM5 chip as a solid-phase carrier, LDLR was immobilized on the active channel of the chip surface using the standard amino coupling method: (1) Surface activation: EDC (0.4 M) and NHS (0.1 M) were mixed at a volume ratio of 1:1 and injected into the experimental channel and the reference channel at a flow rate of 10 μL / min for 7 minutes to activate the carboxyl groups on the chip surface.

[0084] (2) Ligand immobilization: LDLR was diluted to 50 μg / mL with 10 mM sodium acetate buffer (pH 4.5). The diluted LDLR was injected into the experimental channel at a flow rate of 10 μL / min to set the target coupling amount. The reference channel was not injected with protein, but only buffer was flowed through it as a control.

[0085] (3) Surface blocking: 1M ethanolamine hydrochloride (1 M pH 8.5) was injected into the two channels at a flow rate of 10 μL / min for 7 minutes to block the remaining unreacted active sites on the chip surface and wash away non-specifically adsorbed proteins.

[0086] 2. Analysis of binding and dissociation kinetics The second fusion protein SD-PCSK9 purified in Example 3 was serially diluted using running buffer to prepare a series of test samples at different concentrations (starting from 2 μM, two serial dilutions were performed, a total of 7 concentration gradients were set, and a buffer blank control with a concentration of 0 was also provided). The system flow rate was set to 60 μL / min, and the operating temperature was set to 25 °C. Sd-PCSK9 solutions of different concentration gradients were sequentially flowed through the reference channel and the experimental channel from low to high concentration. The association phase time was set to 120 seconds; subsequently, elution was performed using the run buffer (1× HBS-EP+ buffer: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P2O, pH 7.4), and the dissociation phase time was set to 600 seconds.

[0087] According to formula K D = k off / k on The equilibrium dissociation constant K between the second fusion protein SD-PCSK9 and LDLR obtained in Example 3 was calculated. D .

[0088] Figure 8 The results showed that Sd-PCSK9 exhibited a high binding affinity to LDLR, K D 2.36 x 10 -7 M.

[0089] These results clearly demonstrate that despite the significant truncation and engineering modifications to the PCSK9 protein in this invention, the engineered antigen Sd-PCSK9 can still effectively and specifically bind to the target receptor LDLR. This confirms that the design strategy of this invention successfully preserved the key binding surface (core functional epitope) for the PCSK9-LDLR interaction while removing non-core domains, demonstrating the scientific validity and feasibility of this engineered antigen as a highly effective vaccine active ingredient.

Claims

1. A recombinant protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO:

6.

2. The use of the recombinant protein according to claim 1 in the preparation of lipid-lowering drugs.

3. A nanoparticle, characterized in that, The nanoparticles include a nanoparticle carrier, an engineered PCSK9 antigen, and a self-assembly system. The engineered PCSK9 antigen and the nanoparticle carrier are respectively connected to the self-assembly system via linking peptides, and form multivalent antigen nanoparticles through self-assembly. The amino acid sequence of the engineered PCSK9 antigen is shown in SEQ ID NO:

1.

4. The nanoparticles according to claim 3, characterized in that, The self-assembly system is a self-assembly system based on isopeptide bonds; the self-assembly system based on isopeptide bonds is a GvTagOpti / SdCatcher system or a SpyTag / SpyCatcher system; the nanoparticle carrier is: Helicobacter pylori ferritin tetratrimole nanoparticles, I53-50 nanoparticles or mi3 nanoparticles.

5. The nanoparticles according to claim 3, characterized in that, The self-assembly system is connected to the N-terminus or C-terminus of the nanoparticle carrier and the engineered PCSK9 antigen via GSG peptides, respectively.

6. The nanoparticles according to claim 3, characterized in that, The recombinant protein, with amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, was obtained through self-assembly.

7. A composition, characterized in that, Recombinant proteins containing amino acid sequences as shown in SEQ ID NO: 5 and SEQ ID NO:

6.

8. A biomaterial, characterized in that, It is any one of the following: (1) A nucleic acid molecule encoding a recombinant protein with an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 6; (2) Nucleic acid molecules encoding each recombinant protein in the composition of claim 7; (3) An expression cassette containing the nucleic acid molecule described in (1) or (2); (4) A recombinant vector containing the nucleic acid molecule described in (1) or (2) or the expression cassette described in (3); (5) A recombinant microorganism containing the nucleic acid molecule described in (1) or (2), the expression cassette described in (3), or the recombinant vector described in (4); (6) A cell line containing the nucleic acid molecule described in (1) or (2), the expression cassette described in (3), or the recombinant vector described in (4).

9. A composition, characterized in that, It contains the composition and adjuvant of claim 7; or contains the nanoparticles and adjuvant of any one of claims 3 to 6.

10. The use of any of the nanoparticles of claims 3 to 6, the composition of claim 7, the biomaterial of claim 8, or the composition of claim 9 in the preparation of a lipid-lowering drug.

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