A tanguus virus ediii self-assembled nanoparticle and preparation method and application thereof

Abstract

The application discloses a kind of tanbusu virus EDIII self-assembly nanoparticles and preparation method and application thereof.The application realizes efficient coupling of duck tanbusu virus EDIII and Ferritin NP by "molecular glue" SpyCatcher / SpyTag, and prepares EDIII-Ferritin NP.EDIII-Ferritin NP can stimulate strong specific humoral immune response and cellular immune response after immunizing duckling, provides stronger immune protection efficacy, can effectively reduce the tissue damage caused by viral infection and effectively inhibit the replication of virus in duckling, and its production process is simple, easy to operate, and coupling product is stable.Therefore, the EDIII-Ferritin NP prepared by the application can be used in the preparation of tanbusu virus live vaccine, and industrial production application is realized.

Description

Technical Field

[0001] This invention relates to the field of molecular immunology, specifically to a Tembusu virus EDIII self-assembled nanoparticle, its preparation method, and its application. Background Technology

[0002] Duck Tembusu virus (DTMUV) is a major pathogen affecting waterfowl farming, and vaccination is currently the primary control strategy. The E protein of DTMUV is the main target for inducing neutralizing antibodies in the host. However, conserved regions of the full-length E protein, such as the fusion loop, easily induce cross-reactive weak neutralizing and non-neutralizing antibodies, potentially leading to antibody-dependent enhancement (ADE) and increasing the risk of viral infection. Therefore, in the design of DTMUV subunit vaccines, ensuring antigen neutralization activity while minimizing the risk of ADE is a key consideration during antigen screening. EDIII, located at the C-terminus of the E protein, exhibits a typical immunoglobulin-like structure and is rich in specific neutralizing epitopes, primarily inducing highly efficient and specific neutralizing antibodies. Therefore, EDIII is considered a candidate antigen that can help reduce the risk of ADE.

[0003] Despite its high safety profile, EDIII suffers from insufficient immunogenicity. On one hand, monomeric proteins generally have drawbacks such as small molecular weight, low epitope density, and lack of spatial repeatability, making it difficult to achieve regular and dense antigen display. Therefore, they are typically less efficient than nanoparticles in promoting B cell receptor cross-linking and germinal center responses. On the other hand, EDIII readily enters the bloodstream and is rapidly cleared, with relatively low efficiency in drainage to lymph nodes, local accumulation, and uptake by dendritic cells, thus failing to generate a strong immune stimulus. Summary of the Invention

[0004] The purpose of this invention is to solve the above-mentioned problems existing in the prior art, and to provide a Tembusu virus EDIII self-assembled nanoparticle, its preparation method and application.

[0005] The technical solution adopted in this invention is as follows:

[0006] A method for preparing Tembusu virus EDIII self-assembled nanoparticles, characterized by comprising the following steps:

[0007] S1. Ferritin was fused to SpyCatcher at the N-terminus via (GGS)4linker, and the resulting SpyCatcher-Ferritin amino acid sequence is shown in SEQ ID NO.1; EDIII was fused to SpyTag at the C-terminus via (GSG)3linker, and the resulting EDIII-SpyTag amino acid sequence is shown in SEQ ID NO.2; codon optimization was performed on SpyCatcher-Ferritin and EDIII-SpyTag, and the codon-optimized SpyCatcher-Ferritin and EDIII-SpyTag genes were cloned into pET28a(+) and pET32a(+) vectors, respectively, to construct recombinant expression plasmids;

[0008] S2. The recombinant expression plasmid was transformed and induced to express, then purified to obtain purified EDIII-SpyTag and SpyCatcher-Ferritin.

[0009] S3. The purified EDIII-SpyTag was coupled with SpyCatcher-Ferritin to prepare Tembusu virus EDIII self-assembled nanoparticles.

[0010] Preferably, in S3, SpyCatcher-Ferritin and EDIII-SpyTag are coupled in a molar ratio of 1:2.

[0011] The present invention also provides Tembusu virus EDIII self-assembled nanoparticles prepared by the above preparation method.

[0012] Preferably, the Tembusu virus EDIII self-assembled nanoparticles are spherical with a radius of 22.0±3.7 nm.

[0013] This invention also provides the application of the above-mentioned Tembusu virus EDIII self-assembled nanoparticles in the preparation of Tembusu virus live vaccines.

[0014] The beneficial effects of this invention are as follows:

[0015] (1) This invention uses duck Tembusu virus EDIII as the target antigen and prepares EDIII-Ferritin NP with Ferritin NP through the SpyCatcher / SpyTag system. Compared with existing live attenuated vaccines and inactivated vaccines, it has a clear target and no risk of strong virulence.

[0016] (2) The preparation method in this invention is simple and easy to operate, the coupling product is stable, and it has the feasibility of scale-up and can be applied to clinical trials relatively quickly.

[0017] (3) The EDIII-Ferritin NP prepared in this invention can elicit strong specific humoral and cellular immune responses in ducklings after immunization. Regarding humoral immunity, EDIII-Ferritin NP induces durable and high levels of specific IgY antibodies. In contrast, the IgY antibody levels throughout the detection period are significantly higher than those of monomeric protein EDIII and commercially available inactivated vaccines (InV-HB). Furthermore, the neutralizing antibody titers induced by EDIII-Ferritin NP are significantly higher than those of InV-HB, and the decrease in neutralizing antibody levels over time is significantly less than that of monomeric protein EDIII. Regarding cellular immunity, EDIII-Ferritin NP stimulates more intense PBMC proliferation than monomeric protein EDIII. The specific immune response induced by EDIII-Ferritin NP provides stronger immunoprotective efficacy, effectively reducing tissue damage caused by viral infection and strongly inhibiting viral replication in ducklings. Therefore, it can be applied to the preparation of Tembusu virus live vaccines. Attached Figure Description

[0018] Figure 1 A schematic diagram of the coupling between EDIII-SpyTag and Ferritin NP;

[0019] Figure 2 This is a schematic diagram of the immunization and virus challenge process;

[0020] Figure 3 This is a schematic diagram showing the purification results of the target protein;

[0021] Figure 4 This is a schematic diagram of Western blot identification of the target protein.

[0022] Figure 5 A schematic diagram of SDS-PAGE analysis of the coupling results;

[0023] Figure 6 This is a schematic diagram of particle size analysis of Ferritin NP and EDIII-Ferritin NP.

[0024] Figure 7 A schematic diagram of EDIII-Ferritin NP under TEM observation;

[0025] Figure 8 This is a schematic diagram illustrating the detection of serum-specific IgY antibodies from week 1 to week 8 after the initial immunization.

[0026] Figure 9 This is a schematic diagram showing the determination of serum neutralizing antibody titers at 4 and 8 weeks after the initial immunization.

[0027] Figure 10A schematic diagram of the stimulation index of PBMCs at 5 weeks post-primary immunization against DTMUV infection.

[0028] Figure 11 Survival rate of ducklings after viral challenge;

[0029] Figure 12 This is a gross lesion image of ducklings after being challenged with the virus;

[0030] Figure 13 This is a diagram showing the pathological changes in ducklings after viral challenge.

[0031] Figure 14 This diagram illustrates the viral load in the brain, liver, and spleen tissues of ducklings after challenge with the virus. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0033] The experimental materials used in this invention are shown below:

[0034] Tembusu virus (CQW1 strain) was deposited by the Poultry Disease Control and Prevention Research Center of Sichuan Agricultural University, NCBI accession number GenBank: KM233707.1; cells (DH5α, BL21(DE3), Shuffle T7-K12 and BHK-21) and plasmids (pET28a(+) and pET32a(+)), 1-day-old ducklings, mouse anti-duck IgY monoclonal antibody and other materials were all purchased commercially.

[0035] Example 1

[0036] 1. Preparation of Tembusu virus EDIII self-assembled nanoparticles EDIII-Ferritin NP

[0037] 1.1 Design and codon optimization of Ferritin and EDIII sequences

[0038] Based on the codon usage preferences of *E. coli*, the existing gene sequences of Ferritin and EDIII were optimized using codons. The optimized gene sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. His6-SpyCatcher is linked to the N-terminus of Ferritin via (GGS)4 and inserted into the MCS region of pET28a(+) to construct the recombinant expression plasmid pET28a-SpyCatcher-Ferritin. The C-terminus of EDIII is linked to SpyTag via (GSG)3, and EDIII-SpyTag is inserted between TrxA and His6 in pET32a(+) to construct the recombinant expression plasmid pET32a-EDIII-SpyTag.

[0039] 1.2 Expression and purification of the target protein

[0040] pET32a-EDIII-SpyTag and pET28a-SpyCatcher-Ferritin were transformed into BL21(DE3) competent cells, respectively. Positive clones of pET32a-EDIII-SpyTag and pET28a-SpyCatcher-Ferritin were picked and inoculated into LB broth containing 50 μg / mL kanamycin. After overnight incubation at 37°C and 220 rpm on a shaker, the cells were then inoculated at a 1:10 ratio into 200 mL of LB broth containing the corresponding antibiotic. The cells were cultured with shaking until OD... 600 When the concentration of the bacterial cell was 0.6–0.8, IPTG was added to a final concentration of 0.5 mmol / L, and the mixture was induced at 25°C and 150 rpm in a constant-temperature shaker for 16–18 h. After induction, the bacterial cell pellet was collected by centrifugation, resuspended in 10 mL of lysis buffer (25 mM Tris-HCl, pH 8.0, 300 mmol / L NaCl, 0.5 mg / mL lysozyme, 50 μg / mL DNase I), and subjected to repeated freeze-thaw cycles. The pellet was then sonicated on ice for 5 min: 3 s on, 5 s off. After sonication, the pellet was centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant and pellet were collected separately. The pellet was dissolved in 8 mol / L urea, centrifuged, and the supernatant was collected and labeled as inclusion bodies. 40 μL of each of the supernatant and inclusion bodies were added to 10 μL of 5× Loading Buffer for SDS-PAGE analysis.

[0041] EDIII-SpyTag was purified by affinity chromatography using a nickel column: The harvested EDIII-SpyTag supernatant was added to a chromatography column containing 4 mL of Ni-NTA and incubated at 4°C for 2 h. The column was washed with 5 column volumes of Wash Buffer (25 mmol / L Tris-HCl, pH=8.0, 500 mmol / L NaCl, 30 mmol / L imidazole), followed by elution with 8 mL of Elution Buffer (25 mmol / L Tris-HCl, pH=8.0, 500 mmol / L NaCl, 300 mmol / L imidazole). The eluent was then transferred to a 7000 Da dialysis bag and dialyzed against 1000 times Tris-HCl buffer (25 mmol / L Tris-HCl, pH=8.0, 100 mmol / L NaCl) at 4°C for 24 h to remove imidazole. SpyCatcher-Ferritin was purified using ammonium sulfate precipitation: 170 mg / mL ammonium sulfate was added to the supernatant of SpyCatcher-Ferritin and incubated at 4°C for 1 h. Then, it was centrifuged at 3000 rpm for 10 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in 8 mL of Tris-HCl buffer (25 mmol / L Tris-HCl, pH=8.0, 100 mmol / L NaCl). Subsequently, it was dialyzed 1000 times in the same buffer for 24 h to remove residual ammonium sulfate. After dialyzing, it was centrifuged at 12000 rpm for 30 min at 4°C to remove any insoluble substances. Finally, the purified EDIII-SpyTag and SpyCatcher-Ferritin were analyzed by SDS-PAGE and Western blotting.

[0042] The SpyCatcher-Ferritin (Ferritin NP) protein sequence is shown in SEQ ID NO.1; the EDIII-SpyTag protein sequence is shown in SEQ ID NO.2.

[0043] 1.3 Coupling of EDIII with Nanoparticles

[0044] Couple EDIII-SpyTag with SpyCatcher-Ferritin (e.g.) Figure 1The specific procedure is as follows: The concentration of the purified target protein is determined using a BCA protein concentration assay kit. The measured mass concentration is then converted into molar concentration. Based on the calculated molar concentration, 8 μmol / L SpyCatcher-Ferritin and EDIII-SpyTag are mixed at different molar ratios (1:1, 1:1.5, 1:2, 1:2.5, 1:3) and incubated overnight at 4°C. The coupled nanoparticles are named EDIII-Ferritin NP.

[0045] Based on the above results, SpyCatcher-Ferritin:EDIII-SpyTag was coupled at a molar ratio of 1:2. After the reaction, the reaction solution was transferred to a 100 kDa dialysis bag, and then dialyzed at 4°C for 24 h with 1000-fold Tris-HCl buffer (25 mmol / L Tris-HCl, pH=8.0, 100 mmol / L NaCl) to remove free EDIII-SpyTag. The resulting EDIII-Ferritin NP was used for subsequent analysis of coupling results and construction of EDIII nanoparticle vaccines.

[0046] 1.4 Analysis of Coupling Results

[0047] The consumption of nanoparticles and the formation of coupling products under different molar ratios were analyzed by SDS-PAGE. The particle size, dispersion and morphology of nanoparticles before and after coupling were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0048] DLS detection was performed using a Zetasizer Nano S90 instrument. The specific steps were as follows: purified SpyCatcher-Ferritin was diluted to 0.5 mg / mL and placed in a microplate. At 25°C, the intensity of the scattered light from the sample was measured using an avalanche photodiode detector placed at a measurement angle of 173° to determine the distribution of nanoparticles, and the average values ​​of the hydrodynamic particle size and polydispersity index of the sample were recorded.

[0049] TEM observation was performed using a JEOL-JEM-2100Plus electron microscope. The specific steps were as follows: The concentrations of SpyCatcher-Ferritin and EDIII-Ferritin were both adjusted to 0.2 mg / mL. 5 μL–10 μL of each solution was dropped onto a copper grid, allowed to absorb for 2 min, and excess liquid was absorbed with filter paper. 5 μL of 1% phosphotungstic acid was dropped onto the copper grid containing the sample, negatively stained for 2 min, excess stain was absorbed with filter paper, and the grid was air-dried at room temperature. The grid was then imaged using an electron microscope at a working voltage of 120 kV.

[0050] 2. Data Analysis

[0051] 2.1 Expression and purification of the target protein

[0052] Analyze the purification effect of the target protein. For example... Figure 3 As shown in (A), a distinct and single band appears between 25 kDa and 30 kDa, consistent with the theoretical value of 27.6 kDa for EDIII-SpyTag. (See figure below.) Figure 3 As shown in (B), a distinct and single band appeared between 30 kDa and 40 kDa, consistent with the theoretical value of SpyCatcher-Ferritin at 31.5 kDa. Subsequently, the concentrations of EDIII-SpyTag and SpyCatcher-Ferritin were determined by the BCA method to be 1.8 mg / mL and 1.7 mg / mL, respectively, which translates to molar concentrations of 65.2 μmol / L and 54 μmol / L.

[0053] 2.2 Western blot identification of the target protein

[0054] The purified protein was identified by Western blot. For example... Figure 4 As shown in (A), a specific band appears between 25 kDa and 30 kDa, consistent with the theoretical value of EDIII-SpyTag. Figure 4 As shown in (B), specific bands appeared in the range of 30kDa to 40kDa, which is consistent with the theoretical value of SpyCatcher-Ferritin.

[0055] 2.3 SDS-PAGE analysis of coupling results

[0056] SpyCatcher-Ferritin and EDIII-SpyTag were coupled at different molar ratios, and the coupling results were analyzed by SDS-PAGE. Figure 5As shown, all successfully conjugated EDIII-Ferritin bands exhibited specific bands between 50 kDa and 72 kDa, consistent with the theoretical value of 59.1 kDa. Grayscale analysis of the protein bands using ImageJ revealed that SpyCatcher-Ferritin was almost depleted when the molar ratio reached 1:2, indicating that the conjugation reaction had reached saturation.

[0057] DLS detection of 2.4 nanoparticles

[0058] The size of nanoparticles was detected using DLS. Figure 6 As shown, the radius of SpyCatcher-Ferritin is 19.3±6.7 nm, while the radius of EDIII-Ferritin is 22.0±3.7 nm. These results suggest that EDIII-SpyTag was successfully displayed on the surface of the nanoparticles.

[0059] TEM observation of 2.5 nanoparticles

[0060] The microstructure of the prepared nanoparticles was observed using TEM. Figure 7 As shown, low-magnification TEM images (scale bar 100 nm) reveal a tendency for EDIII-Ferritin NPs to aggregate in a chain-like manner. High-resolution images (scale bar 20 nm) show that EDIII-Ferritin NPs are spherical. The TEM results further confirm the successful preparation of EDIII-Ferritin NPs.

[0061] Example 2

[0062] 1. Application of EDIII-Ferritin NP—Immunity and Immunological Evaluation in Ducklings

[0063] 1.1 Immunization and DTMUV Challenge Experiment in Ducklings

[0064] An adjuvant-emulsified immunomodulatory group (Tris-HCl, EDIII, and EDIII-Ferritin NP) and InV-HB were prepared. The specific emulsification steps were as follows: The oil phase (adjuvant) and aqueous phase were heated to 32°C and mixed at a 1:1 volume ratio. Then, the mixture was rapidly and repeatedly pushed and pulled with a syringe until a homogeneous emulsion was formed. After thorough emulsification, the emulsion was stored at 4°C and centrifuged at 3000 rpm for 15 min to evaluate its stability by observing whether stratification occurred.

[0065] Forty-eight one-day-old ducklings were randomly divided into four groups of 12 each. Figure 2As shown, ducks were initially immunized at 7 days of age, with a booster immunization at 2 weeks, using the same dose for both immunizations. Each group of ducks was immunized with 0.5 mL / bird Tris-HCl, 0.5 mL / bird EDIII (EDIII 0.2 mg / mL), 0.5 mL / bird EDIII-Ferritin NP (EDIII 0.2 mg / mL), and 0.5 mL / bird InV-HB, respectively. One week after the booster immunization, 6 ducks from each group were randomly selected and 10... 6 TCID 50 Challenge the virus with a dose of / animal.

[0066] 1.2 Detection of serum-specific IgY antibodies

[0067] EDIII protein diluted with coating buffer was coated onto microplates (4 μg / well) and incubated at 4°C for 12 h. After coating, the plates were washed three times with PBST, then 200 μL of 5% skim milk powder was added, and the plates were blocked at 37°C for 2 h. The blocking buffer was discarded, the plates were washed three times with PBST, and serum from each group of immunized ducks diluted 1:800 was added, and the plates were incubated at 37°C for 1.5 h (100 μL / well). The primary antibody was discarded, the plates were washed three times with PBST, and mouse anti-duck IgY monoclonal antibody diluted 1:5000 was added, and the plates were incubated at 37°C for 1 h (100 μL / well). The secondary antibody was discarded, the plates were washed three times with PBST, and TMB chromogenic solution (100 μL / well) was added, and the plates were incubated at 37°C in the dark for 15 min. The reaction was then terminated by adding 100 μL of 2 mol / L H2SO4, and the OD values ​​were read using a microplate reader. 450 Numerical value.

[0068] 1.3 Detection of serum neutralizing antibodies

[0069] One day before the first immunization, and at weeks 1, 2 (before booster immunization), 3, 4, 5, 6, 7, and 8 after the first immunization, six ducks were randomly selected from each group for jugular vein blood collection and serum separation. Serum neutralizing antibodies were then detected using a micro-neutralization test (Micro-NT). The detection procedure was as follows: the serum was inactivated at 56°C for 30 min, and then serially diluted 2-fold (2... -1 ~2 -8 Subsequently, 50 μL of serum at different dilutions was taken and mixed with 50 μL of 200 TCID50 solution. 50Mix DTMUV thoroughly and incubate at 37°C for 1 hour. After incubation, add the mixture to BHK-21 cells (96-well plate) with 80% confluence, with 8 replicates for each dilution. After incubation at 37°C for 2 hours, discard the mixture and add DMEM medium containing 1% fetal bovine serum. Culture for 5–7 days, observe and record cytopathic effect (CPE), and calculate the 50% neutralization titer (NT) using the Reed-Muench method. 50 ).

[0070] 1.4 PBMC Proliferation Assay

[0071] Peripheral blood mononuclear cells (PBMCs) were extracted at week 5 post-primary immunization according to the instructions of the peripheral blood mononuclear cell isolation kit. They were then diluted to 1×10⁻⁶. 7 Cells / mL, and seeded at 80 μL per well into a 96-well plate, then 20 μL of DTMUV (10 6 TCID 50 The cells were cultured in 37°C at 5% CO2 for 48 hours. PBMCs from the Tris-HCL group were used as a negative control, and RPMI 1640 medium without cells was used as a blank control. After incubation at 37°C in a 5% CO2 incubator for 4 hours, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the dark at 37°C in a 5% CO2 incubator for another 4 hours. After incubation, the OD values ​​were measured using a microplate reader. 450 The value of the value is calculated, and the stimulation index SI (SI = DTMUV stimulation hole - blank control) / (negative control - blank control) is calculated.

[0072] 1.5 Observation of clinical symptoms and pathological changes

[0073] Following viral challenge, the clinical symptoms and mortality of ducklings were observed for 9 consecutive days. On days 5 and 9 post-challenge, three ducks from each group were randomly euthanized, and gross lesions of the liver, spleen, and brain were photographed and recorded. Liver, spleen, and brain tissues were collected on day 9 post-challenge. The collected tissues were fixed with 4% paraformaldehyde, stained with hematoxylin and eosin (HE), and then subjected to histopathological examination.

[0074] 1.6 Detection of viral load in tissues

[0075] Liver, spleen, and brain tissues were collected on days 5 and 9 post-infection. RNA was extracted from each tissue using Trizol, and 1 μg of RNA was then reverse transcribed into cDNA. Subsequently, DTMUV NS3-specific primers were used: forward primer, ...

[0076] 5'-TAAAGAGGGAGCATACTGG-3'; reverse primer, 5'-GCAGGGTCTGTGAAGTGA-3'. RNA levels were detected by real-time quantitative PCR (RT-qPCR). Viral copy number in each tissue was calculated based on the RT-qPCR results.

[0077] 2. Experimental Results and Data Analysis

[0078] 2.1 Detection of serum-specific IgY antibodies

[0079] EDIII protein was used as the coating antigen, and serum-specific IgY antibodies were detected by indirect ELISA. Results are as follows: Figure 8 As shown, throughout the immunization period, the IgY antibody level in the EDIII-Ferritin NP group gradually increased, and rose sharply after booster immunization, reaching a peak at week 3. Thereafter, it plateaued at a high level, showing only a slight decrease over time, and at each time point was significantly higher than that in the InV-HB group (P<0.0001) and the EDIII group (P<0.0001). These results indicate that EDIII-Ferritin NP can induce durable and high levels of specific IgY antibodies.

[0080] 2.2 Determination of serum neutralizing antibody titers

[0081] 200TCID 50 After co-incubation with serum at different dilutions, the cells were seeded into BHK-21 cells, and the titers of neutralizing antibodies in the serum at week 4 and week 8 post-primary immunization were measured using Micro-NT assay. Results are as follows: Figure 9 As shown, the neutralizing antibody titers (NT) in each group from week 4 to week 8 post-immunization were... 50 The levels of neutralizing antibodies showed a downward trend. Specifically, the EDIII-Ferritin NP group exhibited the highest neutralizing antibody levels at both time points, with a relatively small decrease (from 60 to 44). The InV-HB group (from 46 to 37) showed a decrease in neutralizing antibody titers, but still maintained a relatively high level. These results indicate that, compared to monomeric EDIII, EDIII-Ferritin NP can further increase neutralizing antibody levels and enhance the persistence of humoral immune responses.

[0082] 2.3 Proliferative response of PBMCs to DTMUV infection

[0083] To assess whether DTUMV can effectively activate cellular immunity, the stimulation index of PBMCs was measured at 5 weeks post-primary immunization. Figure 10As shown, the stimulation index of each immune group was significantly higher than that of the negative control group (Tris-HCl). Specifically, the EDIII-Ferritin NP group exhibited stronger PBMC proliferation stimulation than the EDIII group. These results indicate that the Ferritin NP platform can effectively enhance cellular immune responses.

[0084] 2.4 Clinical symptoms and pathological changes after DTMUV infection

[0085] To assess the protective effect of each immunization group, patients were observed continuously for 9 days after challenge. The Tris-HCl group showed decreased feed intake and green, watery stools on day 3 post-challenge. Survival rates for each group are as follows: Figure 11 As shown, one animal in the Tris-HCl group died on day 5 and day 8, with a survival rate of 66.7%.

[0086] To observe gross lesions in the brain, liver, and spleen, autopsies were performed on days 5 and 9 post-infection. Figure 12 As shown in Figure A, on day 5 post-DTMUV infection, varying degrees of cerebral congestion were observed in all groups. The Tris-HCl group showed the most severe congestion, with highly distended meningeal vessels. In contrast, the EDIII and InV-HB groups exhibited reduced vascular congestion, displaying a dendritic pattern, while the EDIII-Ferritin NP group showed a return to a more normal physiological state. Congestion and enlargement were observed in the liver in all groups. Splenic lesions were most prominent in the Tris-HCl group, with splenomegaly exhibiting a marbled appearance. Gross lesions on day 9 post-infection are shown below. Figure 12 As shown in Figure B, dendritic congestion was observed in the brain of the Tris-HCl, EDIII, and InV-HB groups, while no obvious lesions were seen in the brain of the EDIII-Ferritin NP group. Liver lesions were most pronounced in the Tris-HCl group, with congestion, enlargement, and hemorrhagic plaques on the surface. Only a few hemorrhagic plaques were observed on the surface of the liver in the other groups. Splenomegaly was observed to varying degrees in all groups, with more significant enlargement in the Tris-HCl and EDIII groups.

[0087] Brain, liver, and spleen tissues were collected on day 9 post-infection and prepared for HE staining. Microscopic observation results are as follows: Figure 13As shown, the brain tissue in the Tris-HCl and EDIII groups showed edema, loose parenchyma separation, and vacuolar degeneration (black arrow 1); the meningeal edema in the InV-HB group showed separation of the membrane from the parenchyma (black arrow 1); the brain tissue in the EDIII-Ferritin NP group showed perivascular inflammatory cell infiltration, exhibiting a cuffing phenomenon (black arrow 1). Hepatocytes in all groups showed varying degrees of swelling and vacuolar appearance (blue arrow 1 in each group); in addition, the liver tissue in the Tris-HCl group showed sinusoidal congestion (blue arrow 2); the liver tissue in the EDIII and InV-HB groups showed focal inflammatory cell infiltration (blue arrow 2) and sinusoidal congestion (blue arrow 3). The boundary between the red and white pulp in the spleen tissue of the Tris-HCl group was indistinct (red arrow 1); the splenic sinusoids in the EDIII group were dilated and congested (red arrow 1).

[0088] 2.5 RT-qPCR detection of viral load in tissues

[0089] To further evaluate the effectiveness of immune protection, brain, liver, and spleen tissues were collected on days 5 and 9 post-challenge, and viral copy number was detected by RT-qPCR. Figure 14 As shown, the viral copy number in the liver (day 5 post-challenge) and brain (day 9 post-challenge) of the EDIII-Ferritin NP group was significantly lower than that of the EDIII group.

[0090] In summary, the EDIII-Ferritin NP prepared by this invention can provide stronger immune protection, effectively inhibit viral replication, and effectively reduce tissue damage caused by viral infection.

[0091] The specification and drawings of this invention are intended to be illustrative rather than restrictive. Based on this invention, those skilled in the art can make substitutions and modifications to some of the technical features without creative effort, and all such modifications are within the scope of protection of this invention.

Claims

1. A method for preparing Tembusu virus EDIII self-assembled nanoparticles, characterized in that, Includes the following steps: S1. Ferritin was fused to SpyCatcher at the N-terminus via (GGS)4linker, and the resulting SpyCatcher-Ferritin amino acid sequence is shown in SEQ ID NO.1; EDIII was fused to SpyTag at the C-terminus via (GSG)3linker, and the resulting EDIII-SpyTag amino acid sequence is shown in SEQ ID NO.2; codon optimization was performed on SpyCatcher-Ferritin and EDIII-SpyTag, and the codon-optimized SpyCatcher-Ferritin and EDIII-SpyTag genes were cloned into pET28a(+) and pET32a(+) vectors, respectively, to construct recombinant expression plasmids; S2. The recombinant expression plasmid was transformed and induced to express, then purified to obtain purified EDIII-SpyTag and SpyCatcher-Ferritin. S3. The purified EDIII-SpyTag was coupled with SpyCatcher-Ferritin to prepare Tembusu virus EDIII self-assembled nanoparticles.

2. The preparation method according to claim 1, characterized in that, In S3, SpyCatcher-Ferritin and EDIII-SpyTag are coupled in a molar ratio of 1:

2.

3. The Tembusu virus EDIII self-assembled nanoparticles prepared by the method described in claim 1.

4. The Tembusu virus EDIII self-assembled nanoparticles according to claim 3, characterized in that, The Tembusu virus EDIII self-assembled nanoparticles are spherical with a radius of 22.0±3.7 nm.

5. The application of the Tembusu virus EDIII self-assembled nanoparticles as described in claim 3 or 4 in the preparation of a Tembusu virus live vaccine.

Images