Fusion protein, pedv rbd nanoparticle antigen and preparation method and application thereof

Abstract

This invention discloses a fusion protein, PEDV RBD nanoparticle antigen, its preparation method, and its applications. The fusion protein is obtained by fusing the receptor-binding domain of the porcine epidemic diarrhea virus (PEDV) Spike protein to the N-terminus of a ferritin monomer via a linker peptide. It can self-assemble into PEDV RBD nanoparticle antigens with an average diameter of approximately 15 nm. The nanoparticle antigens exhibit structural integrity, enabling high-density multivalent display of the RBD, and possess excellent antigenic activity, specificity, thermal stability, and biocompatibility, making them suitable for large-scale production. This invention provides a safe, infection-free, and easily industrialized vaccine strategy for addressing PEDV infection. It can be further expanded for applications in veterinary vaccine formulation optimization and the research and development of diagnostic antigens and reagents, demonstrating good practicality and industrialization prospects.

Description

Technical Field

[0001] This invention relates to a nanoparticle antigen, its preparation method and application, and more particularly to a fusion protein, a PEDV RBD nanoparticle antigen, its preparation method and application. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). It primarily affects the epithelial cells of the small intestine in pigs, clinically manifesting as watery diarrhea, vomiting, dehydration, and rapid weight loss. It is particularly prevalent in suckling piglets, leading to high morbidity and mortality rates and significant economic losses. Since PEDV is mainly transmitted via the fecal-oral route, it replicates efficiently and sheds large amounts of virus locally in the intestine, forming a highly shedding-high transmission chain. Therefore, there is an urgent need to develop a novel immunization strategy that can both induce systemic immunity and effectively establish an intestinal mucosal immune barrier.

[0003] Currently, the immunization and control of PEDV mainly relies on traditional vaccines, including inactivated vaccines, live attenuated vaccines, and their combined immunization regimens, supplemented by comprehensive measures such as biosafety and feeding management. However, existing vaccines still have significant limitations in practical applications: (1) PEDV has high genetic variability, and there are significant differences in antigens between different circulating strains, which can easily lead to insufficient cross-protection, especially when new variant strains are prevalent or there are large differences in regional strains, the risk of immunization failure increases significantly; (2) Traditional inactivated vaccines have weak immunogenicity and usually require strong adjuvants or multiple immunizations to obtain a certain level of neutralizing antibodies, and the induction effect on intestinal mucosal immunity (such as secretory IgA) is unstable; (3) Although live attenuated vaccines can stimulate mucosal immunity to a certain extent, there are potential safety hazards, such as pathogenicity due to vaccine virus residues, immunosuppression, risk of co-infection with other pathogens, and difficulty in controlling genetic stability and quality consistency during the production process. The above problems seriously restrict the effective control of PED, and there is an urgent need to develop a new antigen platform that is safer, more efficient, broad-spectrum, and can take into account both systemic and mucosal immunity.

[0004] In recent years, nanoparticle antigens have attracted widespread attention as an emerging antigen delivery and display strategy. The core concept is to present key neutralizing epitopes in a regular, multi-copy, and high-density manner on the surface of nanoscale particles, thereby mimicking the spatial conformation characteristics of natural viruses. This enhances antigen retention and uptake efficiency in lymphoid tissues, improves antigen presentation capabilities, and strongly activates B cell responses through multivalent cross-linking effects. Compared to soluble monomeric antigens, nanoparticle antigens can typically induce higher titers of neutralizing antibodies at lower doses, promote germinal center formation and antibody affinity maturation, and possess superior physical stability, storage tolerance, and cold chain transport adaptability, which is beneficial for the industrial production and field application of vaccines.

[0005] Ferritin (FR) is a class of iron storage proteins widely found in organisms, possessing the natural ability to self-assemble into highly symmetrical nanocage-like structures. Typical ferritin consists of 24 identical subunits that self-assemble into spherical particles approximately 12 nm in diameter, exhibiting stable, uniform structure that is easily engineered. By linking viral antigens to the N- or C-terminus of ferritin subunits through gene fusion technology, the antigens can be displayed in a high-density, orderly manner on the nanoparticle surface during the self-assembly process, achieving multivalent antigen presentation. Compared to methods such as chemical coupling or non-covalent adsorption, gene fusion strategies offer advantages such as well-defined construction, good batch-to-batch consistency, scalable expression and purification, and easier quality control. Furthermore, ferritin nanoparticles exhibit good stability under varying environmental conditions such as temperature and pH, and have been successfully applied in the development of various viral vaccines.

[0006] Research and development of nanoparticle antigens targeting PEDV is also underway. PEDV belongs to the genus Alphacoronavirus of the family Coronaviridae. It is an enveloped, single-stranded, positive-sense RNA virus whose genome encodes various structural proteins. Among them, the spike glycoprotein (S protein) located on the viral surface is a key factor mediating viral adsorption and host cell invasion, and is also the main target for inducing neutralizing antibodies. Invention patent CN 120004990 A provides a porcine epidemic diarrhea virus antigen fusion protein, its encoding gene, and a porcine epidemic diarrhea vaccine prepared therefrom. The antigen fusion protein is obtained by fusing the homosensitive sequence of the screened PEDV S protein with a monomeric porcine ferritin heavy chain subunit. The provided vaccine induces broadly neutralizing anti-PEDV antibodies by displaying the PEDV S protein on the surface of the porcine ferritin cage structure. Patent application CN 117264075 A discloses a multi-epitope fusion protein of porcine epidemic diarrhea virus, its preparation method and application, which connects multiple B lymphocyte epitopes and T lymphocyte epitopes of porcine epidemic diarrhea virus spike protein, and uses fusion expression technology to embed them on ferritin nanoparticles to construct a multi-epitope recombinant protein of porcine epidemic diarrhea virus.

[0007] However, existing PEDV nanoparticle antigens still have many shortcomings: (1) fusion proteins contain non-essential sequences, which can cause immune-irrelevant reactions and result in a low level of specific immune response; (2) fusion proteins constructed by peptide fusion may lead to conformation loss or weakened immunogenicity; (3) the glycosylation and other modifications of proteins expressed by insect or prokaryotic systems are quite different from those expressed by mammals, affecting the immunogenicity of the antigen. Summary of the Invention

[0008] Purpose of the invention: The purpose of this invention is to provide a fusion protein composed of the receptor-binding domain (RBD) of porcine epidemic diarrhea virus spike protein and ferritin monomers. The fusion protein can self-assemble to form PEDV RBD nanoparticle antigens with high immunogenicity and stability. This invention also provides a method for preparing the nanoparticle antigens and their applications.

[0009] Technical solution: The fusion protein is obtained by linking the spike protein receptor-binding domain of porcine epidemic diarrhea virus with ferritin monomers via a linker peptide.

[0010] Based on the analysis of ferritin structure, this invention fuses the RBD of the Spike protein of PEDV with the N-terminus of a ferritin monomer using a linker peptide to obtain the fusion protein. After self-assembly into particles, this ensures that the RBD is fully exposed on the particle surface and maintains its conformation. The reason for using the S-RBD as the core for designing antigens and developing PEDV vaccines in this invention is that the S protein of porcine epidemic diarrhea virus consists of two functional subunits, S1 and S2. The S1 subunit is responsible for receptor recognition and dominates antigenicity differentiation. Its receptor-binding domain is rich in multiple key neutralizing epitopes, which are closely related to viral infectivity and immune protection. The S-RBD is the core region of the S protein that mediates viral invasion and induces neutralizing antibodies. Compared to the full S protein sequence, the S-RBD reduces non-essential sequences, thereby reducing immune-irrelevant responses while retaining key conformational epitopes and improving specific immune responses. Compared to multi-epitope peptide clusters, this invention uses the complete S-RBD domain, which better preserves the three-dimensional structure and native epitopes, avoiding conformational loss or weakened immunogenicity that may result from peptide fusion.

[0011] Preferably, the amino acid sequence of the receptor-binding domain of the porcine epidemic diarrhea virus spike protein is shown in SEQ ID NO.2, and the amino acid sequence of the ferritin monomer is shown in SEQ ID NO.4.

[0012] Preferably, the fusion protein includes a sterically hindered peptide. The self-assembly of the fusion protein to form PEDV RBD nanoparticle antigen can be regulated by cleaving the sterically hindered peptide. This cleavage can be performed using the TEV protease, which recognizes the amino acid sequence ENLYFQG at the cleavage site.

[0013] Preferably, the fusion protein further includes a signal peptide sequence, a His tag sequence, and a Kozak sequence located at the N-terminus. These sequences optimize protein expression and purification in eukaryotic cells. The signal peptide sequence is shown in SEQ ID NO.1, the His tag sequence is HHHHHHHHHH, and the Kozak sequence is GCCACCATGG.

[0014] Preferably, the linker peptide is a Foldon tag capable of spontaneously forming trimers and a flexible linker peptide to ensure the correct folding and spatial exposure of the RBD antigen, thereby enhancing the stability of the nanoparticles. The Foldon tag sequence is shown in SEQ ID NO.3. The Foldon tag, by forming a stable β-helix structure, can spontaneously trimerize and is widely used in vaccine design, enzyme engineering, and the construction of protein nanostructures. Optional flexible linker peptide sequences are shown in Table 1, with the SGG linker peptide being the most preferred.

[0015] Table 1 Flexible Connecting Peptides

[0016] Serial Number sequence 1 SGG 2 GGSG 3 GGSGGGSG 4 GGSGGGSGGGGSG 5 GGSGGGSGGGSGGGSG 6 GGGGS 7 GGGGSGGGGS 8 GGGGSGGGGSGGGGS

[0017] The PEDV RBD nanoparticle antigen is composed of the fusion protein.

[0018] The PEDV RBD nanoparticle antigen is composed of 24 fusion proteins. Based on the ferritin monomers in the fusion proteins, it self-assembles into spherical nanoparticles with a diameter of 10-30 nanometers and an average diameter of 15 nanometers. The hollow cage-like structure formed by the spontaneous assembly of ferritin exhibits excellent thermal stability, chemical stability, and biocompatibility. The PEDV RBD nanoparticle antigen can achieve regular high-density display of RBD antigens, mimicking the natural multivalent conformation on the viral surface, effectively activating B cell responses and inducing the production of highly efficient neutralizing antibodies.

[0019] This invention also provides a vector encoding the nanoparticle antigen, wherein the vector is a eukaryotic expression vector pCAGGS-PEDV-RBD-FR or a lentiviral vector pLVX-PEDV-RBD-FR. The lentiviral vector enables stable expression and facilitates large-scale production. Preferably, the vector has an EcoRI restriction enzyme site at the 5' end and a HindIII restriction enzyme site at the 3' end, facilitating vector construction and identification.

[0020] The present invention also provides recombinant cells expressing the nanoparticle antigen, said recombinant cells being HEK293T cells or ExpiCHO cells. Both HEK293T and ExpiCHO cells are mammalian cells, and nanoparticle antigens expressed using mammalian systems exhibit higher immunogenicity because these systems provide post-translational modifications, such as glycosylation, that are closer to natural. Glycosylation affects epitope exposure and immune recognition; therefore, this choice is crucial for coronavirus S proteins (such as PEDV S-RBD).

[0021] The present invention also provides a method for preparing the nanoparticle antigen, comprising the following steps: transfecting or transducing a vector encoding the nanoparticle antigen into eukaryotic cells; culturing for 48 hours; collecting cell culture supernatant and cell pellet; affinity purification to obtain the target protein; and purifying the protein to self-assemble into nanoparticles.

[0022] The preferred steps of the preparation method are as follows:

[0023] (1) Construction of recombinant expression vector: The gene encoding PEDV RBD-ferritin fusion protein is cloned into a eukaryotic expression vector or lentiviral vector;

[0024] (2) Cell transfection and expression: The recombinant vector was transfected into HEK293T cells or ExpiCHO cells and cultured at 37°C and 5% CO2 for 48 hours;

[0025] (3) Protein collection and lysis: Collect cell culture supernatant and cell pellet, treat the cell pellet with RIPA lysis buffer and sonicate;

[0026] (4) Protein purification: Purify the target protein using His-tagged affinity chromatography (HisSep Ni-NTA magnetic beads);

[0027] (5) Dialysis refolding: The purified protein was dialyzed in refolding buffer (50 mM Tris-HCl, 150 mM NaCl, 400 mM L-Arg, 5% glycerol) to promote the self-assembly of nanoparticles;

[0028] (6) Quality inspection: The formation, uniformity and size distribution of nanoparticles were verified by negative staining transmission electron microscopy and dynamic light scattering technology.

[0029] Preferably, the ExpiCHO suspension cell expression system is used for large-scale protein production, and a stable expression cell line is established through lentiviral transduction to achieve efficient expression of the target protein.

[0030] Preferably, the expression of the fusion protein is verified using SDS-PAGE electrophoresis and Western blot techniques, with an expected molecular weight of approximately 70 kDa.

[0031] This invention also provides the use of the nanoparticle antigen in the preparation of PEDV vaccines or anti-PEDV neutralizing antibodies, or in the preparation of PEDV diagnostic reagents. This antigen can be used to immunize animals to prepare highly specific antibodies, or directly as a coating antigen for ELISA detection kits.

[0032] The present invention also provides a vaccine composition comprising the above-described nanoparticle antigen, the composition further comprising a pharmaceutically acceptable carrier, adjuvant or preservative, suitable for immunization via intramuscular injection or oral administration.

[0033] This invention also provides an antigen display platform composed of the aforementioned nanoparticle antigens, with 24 PEDV RBD antigen units regularly displayed on the surface of each particle, achieving high-density multivalent antigen presentation. This platform possesses characteristics such as enhanced immunogenicity potential, modular production process with strong scalability, and easily standardized quality control, making it more suitable for large-scale industrial transformation.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention solves the problems of limited immunogenicity of traditional subunit vaccines and low antigen-specific immune response level and weak immunogenicity of PEDV nanoparticles.

[0035] In vitro validation showed that the PEDV RBD nanoparticle antigen provided by this invention can form uniform and regular spherical particles, achieving high-density multivalent display of RBD (24 copies / particle), exhibiting good structural integrity and antigen display potential; and can achieve efficient delivery and presentation of antigen without introducing the risk of replicating pathogens, with high biosafety; control experiments showed that the PEDV RBD nanoparticle antigen has good antigen activity and specificity; it also has excellent thermal stability and biocompatibility, making it suitable for large-scale production.

[0036] This invention provides a safe, infection-free, and easily industrialized vaccine strategy for addressing PEDV infection. It offers a new strategic framework for antigen design and delivery of PEDV and related coronavirus candidate vaccines, and can be further expanded for the optimization of veterinary vaccine formulations and the research and development of diagnostic antigens and reagents. It has good practicality and industrialization prospects. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the fusion protein gene construction strategy of the present invention;

[0038] Figure 2 Map of the recombinant plasmid pCAGGS-PEDV-RBD-FR encoding the fusion protein of this invention;

[0039] Figure 3Map of the recombinant plasmid PLVX-PEDV-RBD-FR encoding the fusion protein of this invention;

[0040] Figure 4 This is a Western Blot analysis result of the expression product of the fusion protein of the present invention;

[0041] Figure 5 These are representative images of the IFAT detection results of the fusion protein expression product in HEK293T cells of this invention (a is the positive fluorescence signal of the cells transfected with the fusion protein expression plasmid reacting with the specific antibody, and b is the negative control of the cells transfected with the empty vector control plasmid reacting with the antibody).

[0042] Figure 6 This is a Western Blot analysis result of the expression product of the fusion protein of the present invention;

[0043] Figure 7 These are representative images of the immunofluorescence observation results of the fusion protein expression product in ExpiCHO cells of this invention (a is the green fluorescence signal of cells transfected with the fusion protein expression plasmid, and b is the negative fluorescence signal of untransfected control cells).

[0044] Figure 8 The figure shows the analytical results of the purified PEDV-S-RBD-FR recombinant protein;

[0045] Figure 9 These are transmission electron microscope (TEM) images of the nanoparticle antigen of this invention (a and b are electron microscope images of nanoparticles under different fields of view).

[0046] Figure 10 The results are from a crossover control ELISA trial. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0048] This invention provides a fusion protein and a PEDVRBD nanoparticle antigen based on the self-assembly of Helicobacter pylori ferritin therein. Its core lies in achieving the controllable depolymerization and repolymerization of ferritin nanoparticles in vitro through rational molecular design, thereby displaying the porcine epidemic diarrhea virus spike protein receptor binding domain on the surface of the nanoparticles in a high-density and high conformational fidelity manner.

[0049] Example 1

[0050] This implementation describes the construction of the pCAGGS-PEDV-RBD-FR eukaryotic expression vector.

[0051] Based on the spike protein sequence of the classic PEDV strain CV777, a gene encoding the PEDV RBD region (amino acid positions 498-638) was designed. A segment of the C-terminus of the RBD was retained, resulting in the sequence shown in SEQ ID NO.2. A Foldon tag (sequence shown in SEQ ID NO.3) capable of spontaneously forming a trimer and a flexible linker peptide SGG were used to link the PEDV RBD to the ferritin gene. A Kozak sequence (GCCACCATGG), a signal peptide GTP sequence (MGWSCIILFLVATATGVHS), and a His tag sequence (HHHHHHHHHHH) were sequentially introduced at the N-terminus of the PEDV RBD gene. An EcoRI restriction enzyme site (GAATTC) was introduced at the 5' end of the target gene, and a HindIII restriction enzyme site (AAGCTT) was introduced at the 3' end. The construction strategy is as follows: Figure 1 As shown, this includes the splicing method of the coding sequences for RBD, Foldon tag, flexible linker peptide, and ferritin.

[0052] The empty pCAGGS vector (preserved in the laboratory) was double-digested with EcoRI and HindIII. The vector fragment and target gene fragment were recovered separately using a Tiangen gel recovery kit (DP219, Tiangen Biotech (Beijing) Co., Ltd.). The vector and target fragment were mixed at a 1:3 molar ratio, and T4 DNA ligase was added. Ligation was performed overnight at 16°C. The ligation product was transformed into DH5α competent cells and plated on LB agar plates containing ampicillin resistance. The cells were incubated at 37°C for 12 hours. Single clones were picked for colony PCR verification. Plasmids were extracted from positive clones and sent for sequencing verification (Suzhou Genewiz Biotechnology Co., Ltd.). The correctly sequenced recombinant vector was named pCAGGS-PEDV-RBD-FR, and the chromatogram is shown below. Figure 2 As shown, the multiple cloning site, promoter, resistance gene, and insertion fragment location are marked.

[0053] Example 2

[0054] This example demonstrates the expression of recombinant protein in HEK293T cells.

[0055] HEK293T cells were revived and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin until the logarithmic growth phase. One day before transfection, cells were cultured at 5 × 10⁻⁶ cells / day. 5 The cells were seeded at a density of 1 cell per well in 6-well plates and cultured until the cell density reached 80-90% confluence.

[0056] Following the instructions for the X-treme GENE HP DNA transfection reagent (Cat# 6366236001, Sigma), 2 μg pCAGGS-PEDV-RBD-FR plasmid was mixed with 4 μL of transfection reagent in 100 μL of serum-free DMEM. After incubation at room temperature for 20 minutes, the mixture was added dropwise to the cells. The cell culture supernatant and cell pellet were collected 48 hours after transfection.

[0057] Cell pellets were treated with RIPA lysis buffer and sonicated, then 6×SDS-PAGE loading buffer was added, and the pellets were boiled at 100°C for 10 minutes. Protein electrophoresis was performed on a 10% SDS-PAGE gel, followed by Western blot analysis. Results are shown below. Figure 4 The results showed a specific protein band at approximately 70 kDa, consistent with the expected molecular weight, confirming successful expression of the PEDV-RBD-FR fusion protein in HEK293T cells. IFAT analysis results are shown below. Figure 5 .

[0058] Example 3

[0059] This embodiment describes the construction of lentiviral packaging vectors and the preparation of recombinant lentiviruses.

[0060] EcoRI and BamHI restriction enzyme sites were introduced at both ends of the PEDV-RBD-FR target gene, and the lentiviral vector pLVX-IRES-ZsGreen1 (Cat# LM1460, LMAI Bio, China) was double-digested with the corresponding enzymes. The target gene was cloned into the vector using T4 DNA ligase, transformed, screened, and sequenced for verification, yielding the pLVX-PEDV-RBD-FR lentiviral packaging vector. (See diagram below.) Figure 3 As shown.

[0061] Recombinant lentiviruses were prepared using a three-plasmid packaging system. Well-grown HEK293T cells were seeded in 10 cm culture dishes and transfected when the cell density reached 80-90%. A transfection system was prepared using the packaging plasmid pLVX-PEDV-RBD-FR, helper plasmid psPAX2, and envelope plasmid pMD2.G (Addgene, Watertown, MA, USA) in a 3:2:1 ratio (6 μg, 4 μg, and 2 μg, respectively). The plasmids were mixed with PolyJet transfection reagent at a 1:2 ratio, incubated at room temperature for 20 minutes, and then added to the cell culture medium.

[0062] Six hours after transfection, the culture medium was replaced with fresh medium, and the cells were cultured for another 48 hours. A clear green fluorescent signal was observed under a fluorescence microscope, indicating successful lentivirus packaging. The cell culture supernatant was collected, centrifuged at 6000 rpm for 15 minutes to remove cell debris, filtered through a 0.45 μm filter for sterilization, and stored at 4°C for later use.

[0063] Example 4

[0064] This example demonstrates lentiviral transduction and large-scale protein expression in ExpiCHO cells (A29127, Thermo Fisher Scientific, Waltham, MA, USA).

[0065] ExpiCHO cells were cultured to the logarithmic growth phase, with cell viability maintained above 95%. Lentiviral transduction was performed using an MOI of 20, with the addition of 8 μg / mL polybrene (Polybrene Infection / Transfection Reagent, TR-1003-G, MilliporeSigma) to enhance transduction efficiency. Forty-eight hours after transduction, positive cells exhibited significant green fluorescence under a fluorescence microscope. IFAT analysis results are shown below. Figure 7 .

[0066] Green fluorescent positive cells were sorted by flow cytometry to establish a stable ExpiCHO cell line expressing PEDV-RBD-FR. The stable cell line was cultured in suspension in CHO Grow CD serum-free medium (Cat# MED-CHO-K1-001, SAIOS, Wuhan SAIOS Biotechnology Co., Ltd.) at 37°C, 8% CO2, and 125 rpm with shaking.

[0067] For large-scale culture, use 2 L shake flasks or bioreactors to achieve a cell density of 2 × 10⁻⁶. 6 Cell collection began when the cell count reached 100 cells / mL. Cells were cultured continuously for 7 days, with supernatant collected every 24 hours. Western blot results are shown below. Figure 6 The results showed that the target protein was highly expressed in the culture supernatant, with an expression level of approximately 50-80 mg / L.

[0068] Example 5

[0069] This example demonstrates the affinity purification of recombinant proteins and the self-assembly of nanoparticles.

[0070] The collected ExpiCHO cell culture supernatant was centrifuged to clarify the supernatant at 2000 rpm for 10 minutes to remove cell debris. Pre-equilibrated HisSep Ni-NTA magnetic beads were added to the clarified supernatant (at a ratio of 1 mL of magnetic beads to 1 L of supernatant), and incubated overnight at 4°C.

[0071] The magnetic beads were separated from the supernatant using a magnetic separator. The beads were washed three times with washing buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 20 mM imidazole), each wash lasting 10 minutes. The target protein was then eluted with elution buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 250 mM imidazole). The purification results are shown below. Figure 8 .

[0072] The eluted protein solution was placed in a dialysis bag and dialyzed overnight at 4°C in refolding buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 400 mM L-arginine, 5% glycerol) to promote proper protein folding and nanoparticle self-assembly. The dialyzed protein solution was sterilized through a 0.22 μm filter and aliquoted for storage at -80°C.

[0073] Example 6

[0074] This embodiment describes the characterization of the nanoparticle structure and the verification of its bioactivity.

[0075] The particle size distribution of the purified protein was detected using dynamic light scattering (DLS). The hydrated particle size of the protein samples was mainly distributed in the range of 10-30 nm, with an average diameter of 15 nm and a polydispersity index of less than 0.2, indicating that the particle size was uniform.

[0076] The morphology of the nanoparticles was further verified by negative staining transmission electron microscopy. The protein sample was diluted to 50 μg / mL, and 4 μL was added to a copper grid treated with glow discharge. After standing for 2 minutes, excess liquid was blotted off with filter paper. Then, 4 μL of 3% uranium acetate staining solution was added, stained for 2 minutes, blotted dry, and allowed to air dry at room temperature before observation under a transmission electron microscope. The electron micrographs are shown below. Figure 9 The results showed that the sample contained a large number of uniformly sized spherical nanoparticles with an average diameter of about 15 nanometers, which is consistent with the dynamic light scattering results.

[0077] The immunoreactivity of nanoparticle antigens was detected using an ELISA method. Purified PEDV-RBD nanoparticles were coated onto an ELISA plate at a concentration of 1 μg / mL and incubated overnight at 4°C. The plate was blocked with PBS containing 5% skim milk for 2 hours, and then incubated with anti-PEDV positive serum at different dilutions for 1 hour at 37°C. After washing, HRP-labeled goat anti-pig IgG secondary antibody was added, and colorimetric detection was performed. Results are shown below. Figure 10This indicates that the nanoparticle antigen exhibits a strong specific reaction with PEDV-positive serum, confirming that it maintains good antigenic activity.

Claims

1. A fusion protein, characterized in that, It is obtained by linking the spike protein receptor-binding domain of porcine epidemic diarrhea virus with ferritin monomers via a linker peptide.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the spike protein receptor-binding domain of the porcine epidemic diarrhea virus is shown in SEQ ID NO.2, and the amino acid sequence of the ferritin monomer is shown in SEQ ID NO.

4.

3. The fusion protein according to claim 1, characterized in that, The fusion protein includes a sterically hindered peptide.

4. The fusion protein according to claim 1, characterized in that, The fusion protein also includes a signal peptide sequence, a His tag sequence, and a Kozak sequence located at the N-terminus.

5. The fusion protein according to claim 1, characterized in that, The linker peptide is a Foldon tag capable of spontaneously forming trimers and a flexible linker peptide.

6. A PEDV RBD nanoparticle antigen composed of the fusion protein according to any one of claims 1-5.

7. A carrier encoding the nanoparticle antigen of claim 6, characterized in that, The vector is either the eukaryotic expression vector pCAGGS-PEDV-RBD-FR or the lentiviral vector pLVX-PEDV-RBD-FR.

8. A recombinant cell expressing the nanoparticle antigen of claim 6, characterized in that, The recombinant cells are HEK293T cells or ExpiCHO cells.

9. A method for preparing the nanoparticle antigen according to claim 6, characterized in that, Includes the following steps: The vector encoding the nanoparticle antigen was transfected or transduced into eukaryotic cells; cultured for 48 hours; cell culture supernatant and cell pellet were collected; the target protein was obtained through affinity purification; and the purified protein was self-assembled into nanoparticles.

10. The use of the nanoparticle antigen of claim 6 in the preparation of PEDV vaccines, anti-PEDV neutralizing antibodies, or PEDV diagnostic reagents.

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