Ferritin nanoparticles, their preparation method and applications

Abstract

This application relates to the technical fields of bioengineering, nanomaterials, and vaccine carriers, specifically to a ferritin nanoparticle, its preparation method, and its applications. This application involves designing a mutant of *Vorticoid fibrillosa* ferritin to obtain a ferritin variant with the amino acid sequence shown in SEQ ID NO:2. A linker is then used to connect a Spycatcher variant with the amino acid sequence shown in SEQ ID NO:4 to the N-terminus of this ferritin variant, thus constructing ferritin. This ferritin, after recombinant expression and purification in *E. coli*, can autonomously assemble into ferritin nanoparticles. The ferritin nanoparticles provided by this application exhibit high expression levels, excellent stability, and uniform particle size. Through the SpyTag-SpyCatcher covalent coupling system, multiple antigens can be efficiently displayed on the nanoparticle surface, providing an efficient and stable antigen display carrier for vaccine development.

Description

Technical Field

[0001] This application relates to the technical fields of bioengineering, nanomaterials and vaccine carriers, and in particular to a ferritin nanoparticle, its preparation method and application. Background Technology

[0002] Ferritin is a class of naturally occurring iron storage proteins widely found in organisms. It consists of 24 subunits that self-assemble into a hollow spherical nanocage structure with an outer diameter of approximately 12 nm and an inner lumen of approximately 8 nm. It possesses excellent biocompatibility, high structural stability, and strong modifiability, making it an ideal carrier for nanovaccines, drug delivery, and bioimaging. Its naturally hollow structure can load metal ions, drug molecules, or antigenic proteins, and the exposed amino acid residues on its surface can be targeted and functionalized through genetic engineering or chemical modification, demonstrating great potential in the development of vaccines for infectious diseases such as influenza, malaria, and rabies.

[0003] In recent years, significant progress has been made in the research of ferritin nanoparticles as vaccine carriers. For example, the team led by Guo Yu at Nankai University developed a self-assembled nanovaccine based on human ferritin heavy chain (FTH1), which, through non-covalent coupling with rabies virus G protein domain III (GDIII), provided complete protection against rabies virus intracerebral challenge in mice with a single immunization (Self-assembling nanoparticle engineered from the ferritinophagy complex as arabies virus vaccine candidate, 2024). The team led by Yan Xiyun at the Chinese Academy of Sciences successfully loaded 400 doxorubicin molecules (10 times the drug loading capacity of human ferritin) using the high stability of Pyrococcus furiosus ferritin (PfFtn), and achieved precise killing of liver cancer cells through the surface targeting peptide SP94 (GRP78-targetedferritin nanocaged ultra-high dose of doxorubicin for hepatocellular carcinoma therapy, 2019).

[0004] Pyrococcus furiosus, a hyperthermophilic archaea, possesses ferritin (PfFtn) with extreme thermostability (half-life of 48 hours at 100°C and 85 minutes at 120°C) and a unique ferrooxidase center (FC) structure, exhibiting special advantages in the field of nanocarriers (A highly thermostable ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus, 2006). Studies have shown that all 24 subunits of PfFtn contain diferric binding sites, its iron oxidation rate is 3-5 times that of mammalian ferritin, and its lumen can efficiently load metal ions or drug molecules.

[0005] Despite the aforementioned advantages of PfFtn, significant gaps remain in current technologies: 1. Lack of systematic mutant design: The self-assembly interface of natural PfFtn depends on conserved amino acid residues (such as Asp and Glu), and the fusion of exogenous proteins easily disrupts its 24-mer structure. There are no reports on optimizing assembly efficiency through site-directed mutagenesis; 2. Fuse expression with SpyCatcher has not been achieved: Existing SpyCatcher-ferritin fusion proteins are mostly based on human or E. coli ferritin, failing to utilize the thermal stability of PfFtn to address protein denaturation issues during high-temperature purification; 3. Unoptimized preparation process: Traditional PfFtn purification requires multiple chromatographic steps, with a recovery rate of less than 50%, and lacks efficient separation methods for fusion proteins. Summary of the Invention

[0006] This application provides ferritin nanoparticles, their preparation method, and applications. The aim of this application is to obtain ferritin nanoparticles with high expression levels, high self-assembly efficiency, high stability, and high antigen display effectiveness, providing an efficient and stable antigen display carrier for vaccine development.

[0007] This application constructs ferritin by designing a mutant of *Porcine flavonoids* ferritin and linking the Spycatcher variant (SEQ ID NO:4) to the N-terminus of the ferritin variant (SEQ ID NO:2) via a linker (amino acid sequence shown in SEQ ID NO:5). This ferritin can autonomously assemble into ferritin nanoparticles, which exhibit excellent stability, high recovery rate, and uniform particle size. Furthermore, the ferritin nanoparticles of this application can covalently link with SpyTag-expressing antigen proteins through SpyCatcher-SpyTag specific interactions, forming a stable antigen-nanoparticle protein complex.

[0008] In a first aspect, this application provides a ferritin nanoparticle, which adopts the following technical solution:

[0009] A ferritin nanoparticle is constructed by designing a mutation of ferritin from *Ficus viridans* to obtain a ferritin variant with the amino acid sequence shown in SEQ ID NO:2, and then using a linker to connect the Spycatcher variant with the amino acid sequence shown in SEQ ID NO:4 to the N-terminus of the ferritin variant to obtain ferritin; the ferritin is then recombinantly expressed and purified in *Escherichia coli* and autonomously assembled into ferritin nanoparticles.

[0010] Optionally, the amino acid sequence of the linker is shown in SEQ ID No. 5.

[0011] Optionally, the amino acid sequence of the ferritin nanoparticles is as shown in SEQ ID No. 6.

[0012] Secondly, this application provides a method for preparing ferritin nanoparticles, employing the following technical solution:

[0013] A method for preparing ferritin nanoparticles includes the following steps: gene synthesis and vector construction, plasmid transformation, induced expression, cell disruption, thermal precipitation, and molecular sieve chromatography.

[0014] Optionally, the preparation method includes: collecting bacterial cell precipitate after inducing expression, adding lysate and preparing bacterial cell lysate under high pressure homogenization conditions, centrifuging and filtering to obtain bacterial cell lysate supernatant filtrate; subjecting the bacterial cell lysate supernatant filtrate to heat precipitation treatment to remove heat-denatured impurity protein precipitate, filtering, concentrating, and performing analytical sieve chromatography on the supernatant to obtain ferritin nanoparticles.

[0015] Secondly, this application provides a gene. This gene encodes the aforementioned ferritin nanoparticles.

[0016] Thirdly, this application provides an expression vector. The expression vector includes the aforementioned gene.

[0017] Fourthly, this application provides a host cell. This host cell expresses the aforementioned gene or the aforementioned expression vector.

[0018] Fifthly, this application provides the use of the above-mentioned ferritin nanoparticles in the preparation of vaccines or compositions for drug delivery.

[0019] Sixthly, this application provides a vaccine, which adopts the following technical solution:

[0020] A vaccine comprising the aforementioned ferritin nanoparticles, an antigen, and a pharmaceutically acceptable carrier or excipient.

[0021] In summary, this application includes at least one of the following beneficial technical effects:

[0022] This application describes the construction of a ferritin protein by designing a mutant of Pyrococcus furiosus ferritin and expressing the ferritin variant in conjunction with the SpyCatcher variant. This ferritin protein can be efficiently recombinantly expressed in Escherichia coli at high levels, meeting the requirements for large-scale production.

[0023] This application modifies the N-terminus of *Vorticoides fibrillosa* ferritin by truncating it and introducing site-directed mutations of E121A and E130A to optimize the interfacial interactions between ferritin subunits, obtaining a ferritin variant with the amino acid sequence shown in SEQ ID NO:2. Then, using a linker with the amino acid sequence shown in SEQ ID NO:5, a SpyCatcher variant with the amino acid sequence shown in SEQ ID NO:4 is ligated to the N-terminus of this ferritin variant to construct ferritin. After recombinant expression and purification in *E. coli*, the ferritin autonomously assembles into ferritin nanoparticles, resulting in ferritin nanoparticles with the amino acid sequence shown in SEQ ID NO:6. Stability testing showed that these ferritin nanoparticles maintained their dispersed nanoparticle morphology after being placed at 25°C for two weeks, exhibiting excellent stability without significant aggregation or degradation.

[0024] The method for preparing ferritin nanoparticles provided in this application includes core steps such as heat treatment, ultrafiltration concentration, and molecular sieve chromatography: the induced bacterial cells are harvested and lysed; heat treatment efficiently removes heat-labile impurities; and further purification is achieved through molecular sieve chromatography to obtain high-purity ferritin nanoparticles. This process is simple to operate, highly reproducible, and suitable for large-scale production.

[0025] The ferritin nanoparticles prepared using the process described in this application are mainly spherical with an average particle size of about 12 nm. The particles are uniform in size and well dispersed, providing a stable carrier basis for subsequent efficient conjugation of antigens.

[0026] The ferritin nanoparticles of this application can be efficiently covalently linked to antigen proteins expressing SpyTag through the SpyCatcher-SpyTag covalent coupling system, with a coupling efficiency of up to 95%. This allows for the efficient display of multiple antigens, providing an efficient and stable antigen display vector for vaccine development. Attached Figure Description

[0027] Figure 1 This is the map of the ferritin expression vector pET-28a-SpyC∆N-pfuF03 of this application.

[0028] Figure 2 This is the single colony morphology of Escherichia coli transformed by the ferritin expression vector of this application.

[0029] Figure 3This is the SDS-PAGE result of ferritin expression in this application.

[0030] Figure 4 This is the SDS-PAGE result of the purified ferritin in this application.

[0031] Figure 5 This is a negative-stained electron microscope image of the ferritin nanoparticles in this application (including samples placed at 25°C for 2 weeks).

[0032] Figure 6 The results are obtained by SDS-PAGE and molecular sieve chromatography of the ferritin nanoparticles covalently coupled with the SpyTag-expressing antigen protein in this application. Detailed Implementation

[0033] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0034] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0035] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0036] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0037] This application provides a ferritin nanoparticle. The ferritin nanoparticle utilizes a linker to connect a ferritin variant and a Spycatcher variant; the amino acid sequence of the ferritin nanoparticle is shown in SEQ ID No. 6. Specifically, the amino acid sequence of the ferritin variant is shown in SEQ ID No. 2, and the amino acid sequence of the Spycatcher variant is shown in SEQ ID No. 4.

[0038] This application also provides a method for preparing the above-mentioned ferritin nanoparticles. The preparation method specifically includes the following steps:

[0039] (1) Gene synthesis and vector construction

[0040] The full-length codon sequence of ferritin (SpyC∆N-pfuF03) was optimized and synthesized, then cloned into the pET-28a(+) vector to construct the recombinant plasmid pET-28a-SpyC∆N-pfuF03. After the recombinant plasmid was verified by sequencing, it was amplified and purified to obtain a high-purity plasmid. The plasmid powder was then concentrated by vacuum centrifugation for subsequent transformation.

[0041] (2) Plasmid transformation

[0042] Take the plasmid powder, centrifuge to allow the plasmid powder to settle at the bottom of the tube, and add sterile water to dissolve it completely; add the plasmid solution to competent cells and gently pipette to mix; incubate on ice for 30 min, heat shock at 42℃ for 45 s, and then immediately incubate on ice for 2 min; add antibiotic-free LB liquid medium and culture at a constant temperature with shaking; spread the bacterial solution on LB plates (containing kanamycin) and incubate upside down overnight.

[0043] (3) Induced expression

[0044] Pick a single positive colony from an LB agar plate and inoculate it into LB liquid medium (containing kanamycin), shake and incubate overnight to obtain a seed culture; inoculate the seed culture into fresh LB liquid medium (containing kanamycin) and incubate until OD reaches [value missing]. 600 Add IPTG to a concentration of approximately 0.6-0.8 to induce expression; centrifuge to collect bacterial cells, discard the supernatant, and store the bacterial cell pellet for later use.

[0045] (4) Cell disruption

[0046] Add the lysis buffer to the wet bacterial cells at a ratio of 1g wet bacterial cells to 10-30mL of lysis buffer, resuspend, and stir until homogeneous. Under high pressure homogenization, circulate and lyse until the bacterial solution becomes clear, obtaining the lysate. Centrifuge to collect the supernatant, filter through a membrane, and obtain the lysate supernatant for later use.

[0047] (5) Thermal precipitation

[0048] The supernatant filtrate of the lysed bacterial cells was incubated in a metal bath, then quickly transferred to ice to cool, centrifuged to collect the supernatant, and the heat-denatured protein precipitate was removed.

[0049] (6) Molecular sieve chromatography

[0050] The supernatant after thermal precipitation was filtered again through a filter membrane and concentrated to obtain a concentrate. After equilibration of the molecular sieve chromatography column, the concentrate was loaded, eluted, and ferritin nanoparticles were obtained.

[0051] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0052] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0053] The present application will be further described in detail below with reference to the embodiments and test results.

[0054] Preparation Example 1

[0055] This preparation example provides a ferritin variant.

[0056] The engineering process of this ferritin variant is as follows: Pyrococcus furiosus ferritin (PfFtn, amino acid sequence as shown in SEQ ID NO:1) was engineered by removing the "MLSE" part at the N-terminus and simultaneously performing E121A and E130A mutations to obtain the ferritin variant (pfuF03), whose amino acid sequence is shown in SEQ ID NO:2.

[0057] Preparation Example 2

[0058] This preparation example provides a Spycatcher variant.

[0059] The engineering process of the Spycatcher variant is as follows: Spycatcher (amino acid sequence as shown in SEQ ID NO:3) is engineered by removing 21 amino acids from the N-terminus to obtain the Spycatcher variant (SpyC∆N), whose amino acid sequence is shown in SEQ ID NO:4.

[0060] Preparation Example 3

[0061] This preparation example provides a ferritin.

[0062] The engineering process of this ferritin is as follows: the Spycatcher variant (SEQ ID NO:4) is linked to the N-terminus of the ferritin variant (SEQ ID NO:2) via a linker (amino acid sequence as shown in SEQ ID NO:5) to obtain ferritin (SpyC∆N-pfuF03), whose amino acid sequence is shown in SEQ ID NO:6.

[0063] Example 1

[0064] This embodiment provides a method for preparing ferritin.

[0065] The preparation method of this ferritin specifically includes the following steps:

[0066] (1) Gene synthesis and vector construction

[0067] The full-length codons of ferritin (SpyC∆N-pfuF03) were optimized and synthesized, then cloned into the NcoI-BamHI site of the pET-28a(+) vector to construct the recombinant plasmid pET-28a-SpyC∆N-pfuF03 (plasmid map shown). Figure 1 (As shown). After the recombinant plasmid was verified by sequencing, it was amplified and purified to obtain a high-purity plasmid. The plasmid powder was then concentrated by vacuum centrifugation for subsequent transformation.

[0068] (2) Plasmid transformation

[0069] Take the preserved plasmid powder, centrifuge at 12000 rpm for 1 min to allow the powder to settle at the bottom of the tube, and add 100 μl of sterile water to dissolve it completely. Add 2 μl of the plasmid solution to 100 μl of E. coli BL21(DE3) competent cells and gently pipette to mix. Incubate the mixture on ice for 30 min, then heat shock at 42℃ for 45 s, followed immediately by incubation on ice for 2 min. Add 1 mL of antibiotic-free LB liquid medium and incubate at 37℃ with shaking at 200 rpm for 1 h. Spread 200 μl of the above bacterial solution evenly onto LB solid medium plates containing 50 μg / mL kanamycin and incubate in an inverted incubator at 37℃ overnight. The morphology of single colonies after plasmid transformation is as follows: Figure 2 As shown.

[0070] (3) Induction of expression and expression detection

[0071] Pick a single positive colony from the above LB solid medium plate and inoculate it into 20 mL of LB liquid medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C with shaking at 200 rpm to prepare a seed culture. Take the seed culture and inoculate it at a 1:100 ratio into 1000 mL of fresh LB liquid medium containing 50 μg / mL kanamycin. Incubate at 37°C with shaking at 200 rpm until OD reaches 1000. 600 ≈0.6-0.8 (culture time approximately 3.5h), add IPTG to the system and adjust its final concentration to 0.5mM, and set two sets of induction conditions: 30℃, 200rpm isothermal oscillation for 4.5h induction; 25℃, 200rpm isothermal oscillation for 20h induction.

[0072] The bacterial culture samples before and after induction were diluted to the same OD600 value, and 10 μl of each sample was taken for SDS-PAGE electrophoresis to analyze the protein expression induction. The results of protein expression detection before and after induction are as follows: Figure 3 As shown.

[0073] Depend on Figure 3 It can be seen that after IPTG induction, a large number of target protein (SpyC∆N-pfuF03) expression bands appeared in the sample, and the molecular weight of the target protein was consistent with the expectation, about 30kDa.

[0074] (4) Cell disruption

[0075] Centrifuge the induced bacterial culture at 4℃ and 8000 rpm for 10 min to collect the bacterial cells. Discard the supernatant and store the bacterial precipitate at -80℃ for later use. Take 6.4 g of wet bacterial cells and add 128 mL of lysis buffer (40 mM Tris, 150 mM NaCl, 0.5% Triton-X100, pH 7.5-8.5) at a ratio of 1 g of wet bacterial cells to 20 mL of lysis buffer. Resuspend and stir well. Use a high-pressure homogenizer to circulate and homogenize 3-5 times at 500-900 psi until the bacterial culture becomes clear to obtain the bacterial cell lysate. Centrifuge the bacterial cell lysate at 4℃ and 12000 rpm for 30 min. Collect 126 mL of the supernatant and filter it through a 0.22 μm filter membrane to obtain the bacterial cell lysate supernatant filtrate for later use.

[0076] (5) Thermal precipitation

[0077] Incubate the supernatant filtrate of the lysed bacterial cells in a metal bath at 80-90℃ for 10-20 min, then quickly transfer it to ice to cool for 10 min. Centrifuge at 4℃ and 12000 r / min for 15 min, and take the supernatant (115 ml) to remove heat-denatured protein precipitates.

[0078] (6) Molecular sieve chromatography

[0079] The supernatant after heat precipitation was filtered again through a 0.22 μm filter membrane and then concentrated using a 30 kD ultrafiltration centrifuge tube at 4 °C and 3500 r / min for 30 min, to approximately 10 mL, to obtain the concentrated solution. The molecular sieve chromatography column was equilibrated using molecular sieve equilibration buffer (20 mM PB, 150 mM NaCl, 5% trehalose, pH 6.5-7.5). After equilibration, 5 mL of the concentrated solution was loaded at a single flow rate of 0.5 mL / min, and the target protein peak was collected by SDS-PAGE. The collected peaks were then stored at 4 °C for later use, yielding ferritin (SpyC∆N-pfuFO3) nanoparticles.

[0080] Example 2

[0081] This embodiment verifies the structure of the ferritin (SpyC∆N-pfuF03) nanoparticles prepared in Example 1.

[0082] (1) SDS-PAGE analysis

[0083] The purity of ferritin nanoparticles was determined by 12% SDS-PAGE gel electrophoresis. The results are as follows: Figure 4 As shown.

[0084] Depend on Figure 4 It can be seen that after Coomassie brilliant blue staining, ferritin nanoparticles show a single band with a molecular weight of approximately 30 kDa.

[0085] (2) Negative staining electron microscopy analysis

[0086] The grid (Cu, 300 mesh, continuous carbon film) was treated with a PELCO easiGlow Glow Discharge instrument under the following conditions: 90 seconds, 15 mA. The sample was diluted to 0.06 mg / mL with phosphate buffer, and 3 μL of the sample was added to the glow discharge-treated grid. After incubation for 60 seconds, excess liquid was aspirated with filter paper. 3 μL of 0.75% uranium formate staining solution was added to the grid and stained for 20 seconds. Excess liquid was aspirated with filter paper. The grid was dried for 2 minutes before loading the sample.

[0087] Insert the sample holder at room temperature into the Talos L120C 120kV transmission electron microscope. Once the microscope vacuum stabilizes, turn on the light path, adjust the microscope, and observe the sample. Locate suitable staining areas at low magnification and use a Ceta camera at 73,000x magnification to capture images. The detection results are as follows: Figure 5 As shown.

[0088] Depend on Figure 5 Electron microscopy revealed that the ferritin nanoparticles were predominantly spherical with an average particle size of approximately 12 nm, exhibiting uniform structure and good dispersion. After being placed at 25°C for two weeks, the nanoparticles maintained their dispersed morphology without significant aggregation or degradation, indicating good stability.

[0089] Example 3

[0090] This embodiment verifies the function of the ferritin (SpyC∆N-pfuF03) nanoparticles prepared in the previous embodiments, focusing on verifying their covalent coupling ability with antigen proteins fused to express the SpyTag tag. The coupling scheme is shown in Table 1, and the specific scheme is as follows:

[0091] (1) Sample pretreatment: The high-purity ferritin (SpyC∆N-pfuF03) nanoparticles purified in the example and the antigen protein fused to express the SpyTag tag (such as preF3-SpyT protein) were dialyzed twice with the specified buffer (40mM PB, 100mM sodium chloride, 2mM magnesium chloride, 10% trehalose, 0.1% Tween-80, pH 7.0), with each dialyze lasting 1 hour to remove residual purification reagents and impurities from the sample and ensure a stable coupling reaction environment. After dialysis, the concentrations of the two proteins were determined by the BCA method and adjusted to the target concentrations: ferritin nanoparticles 8 μM (corresponding to a mass concentration of 1.24 mg / ml) and antigen protein fused to express the SpyTag tag 8.8 μM (corresponding to a mass concentration of 1.91 mg / ml) for later use.

[0092] (2) Preparation of coupling reaction system: Take 3 ml of the pretreated ferritin nanoparticles (8 μM, 1.24 mg / ml) and 3.74 ml of the antigen protein fused to express the SpyTag tag (8.8 μM, 1.91 mg / ml) and add them to a sterile centrifuge tube. Add the specified buffer (40 mM PB, 100 mM sodium chloride, 2 mM magnesium chloride, 10% trehalose, 0.1% Tween-80, pH 7.0) to adjust the total volume of the reaction system to 10 ml. Gently pipette to mix, avoiding vigorous shaking that could cause nanoparticle aggregation or protein conformational changes.

[0093] (3) Coupling reaction conditions control: The prepared reaction system was placed in a constant temperature environment of 25°C and incubated for 5 hours by rotating mixing to ensure that the two proteins were in full contact and to promote the specific covalent binding of the SpyCatcher variant to the SpyTag tag (pH 7.0).

[0094] (4) Verification of coupling effect: Take the above-mentioned coupling reaction samples, uncoupled ferritin nanoparticle samples, and uncoupled SpyTag antigen protein samples, and perform SDS-PAGE electrophoresis and molecular sieve chromatography detection respectively. By comparing the band position and elution peak changes, the success of the coupling reaction and the coupling efficiency are verified.

[0095] Table 1. Antigen-protein conjugation schemes between ferritin nanoparticles and SpyTag fusion expression

[0096]

[0097] The results of SDS-PAGE electrophoresis and molecular sieve chromatography are as follows: Figure 6As shown in the figure. The left figure is an SDS-PAGE electrophoresis image, in which: "Fusion Ferritin Lane" corresponds to the SpyC∆N-pfuF03 ferritin nanoparticle carrier of this application, and the band position is consistent with the expected molecular weight; "Antigen Protein Lane" corresponds to the preF3-SpyT antigen protein, with a clear band, which is one of the substrates for the coupling reaction; "Conjugation Product Lane" is the complex after covalent coupling of SpyC∆N-pfuF03 and preF3-SpyT, with a significantly increased molecular weight and the band position is higher than the substrate protein; "A1-A6 Lanes" are the elution fractions collected during protein purification, corresponding to the collection tube numbers of the UV280 absorption peak area in the chromatogram, used to verify the presence and purity of the target coupling product in each fraction; "Marker Lane" is the molecular weight standard, used to estimate the molecular weight of each protein band. The right figure is a chromatogram of protein purification. The "UV280 curve (blue)" represents the protein concentration in the eluent. A single, sharp absorption peak appears at about 30-40 mL, indicating that the target conjugate product is concentratedly eluted at this stage. The "conductivity curve (green)" reflects the change in the salt concentration of the eluent. A sudden change occurs at about 100 mL, corresponding to the switch of the elution program. The "component markers (red)" indicate the collection locations of FcWaste (flowthrough / waste liquid) and components A1-A6, which correspond perfectly to the UV280 absorption peak.

[0098] Depend on Figure 6 The SDS-PAGE results showed that bands with significantly increased molecular weight appeared in both the lanes of the coupling product and in the A1–A6 fractions, with very few impurities. This indicates that SpyC∆N-pfuF03 and preF3-SpyT underwent efficient covalent coupling, forming a stable coupling complex with high product purity. The UV280 curve in the chromatogram showed a single, symmetrical main peak with no obvious impurities, indicating good homogeneity of the coupling reaction, minimal formation of byproducts, and effective separation of the target coupling product by the purification process.

[0099] In summary, the ferritin nanoparticles (SpyC∆N-pfuF03) of this application can be efficiently conjugated to the antigen protein (preF3-SpyT) through the SpyTag / SpyCatcher system. The conjugation efficiency is high and the product is stable. It has the core functional characteristics of a vaccine antigen display vector and can be used for the construction and evaluation of subsequent vaccine candidate molecules.

[0100] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A ferritin nanoparticle characterized in that, By designing a mutation in the ferritin of *Ficus viridans*, a ferritin variant with the amino acid sequence shown in SEQ ID NO:2 was obtained. The Spycatcher variant with the amino acid sequence shown in SEQ ID NO:4 was then linked to the N-terminus of the ferritin variant using a linker to construct ferritin. The ferritin was then recombinantly expressed and purified in *E. coli* and autonomously assembled into ferritin nanoparticles.

2. The ferritin nanoparticle of claim 1, wherein, The amino acid sequence of the linker is shown in SEQ ID No.

5.

3. The ferritin nanoparticle of claim 1, wherein, The amino acid sequence of the ferritin nanoparticles is shown in SEQ ID No.

6.

4. A method of producing the ferritin nanoparticle according to any one of claims 1 to 3, characterized in that, The preparation method specifically includes the following steps: gene synthesis and vector construction, plasmid transformation, induced expression, cell disruption, thermal precipitation, and molecular sieve chromatography.

5. The preparation method according to claim 4, characterized in that, The preparation method includes: collecting bacterial cell precipitate after inducing expression, adding bacterial lysis solution and preparing bacterial cell lysate under high pressure homogenization conditions, centrifuging and filtering to obtain bacterial cell lysate supernatant filtrate; subjecting the bacterial cell lysate supernatant filtrate to heat precipitation treatment to remove heat-denatured impurity protein precipitate, filtering, concentrating, and performing analytical sieve chromatography on the supernatant to obtain ferritin nanoparticles.

6. A gene characterized in that, The gene encodes the ferritin nanoparticles of claim 1.

7. An expression carrier, characterized in that, The expression vector comprises the gene as described in claim 6.

8. A host cell, characterized in that, The host cell expresses the gene of claim 6 or the expression vector of claim 7.

9. The use of any one of the ferritin nanoparticles according to claims 1-3 in the preparation of vaccines or compositions for drug delivery.

10. A vaccine, characterized in that, The vaccine comprises any one of the ferritin nanoparticles, antigens, and pharmaceutically acceptable carriers or excipients as described in any one of claims 1-3.

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