PDADMAC modified silicon dioxide mineralized foot-and-mouth disease virus-like particle as well as preparation method and application thereof

Abstract

The invention discloses PDADMAC modified silicon dioxide mineralized foot-and-mouth disease virus-like particles as well as a preparation method and application thereof. The PDADMAC modified silicon dioxide mineralized foot-and-mouth disease virus-like particle (VLPs-SiO2) is a mineralized particle obtained by forming a silicon dioxide mineralized foot-and-mouth disease virus-like particle under the action of PDADMAC and then further modifying the silicon dioxide mineralized foot-and-mouth disease virus-like particle with PDADMAC, and the SiO2 mineralization rate of FMDV VLPs reaches 90% or above. The observation of a transmission electron microscope and a scanning electron microscope shows that the VLPS-SiO2 is spherical-like nano-particles with the particle size of 80-100 nm. According to the present invention, the level of FMDVVLPs ingestion by DC2.4 cells is increased by SiO2 mineralization, after the mice are immunized by nasal dripping, the specific antibody level in the serum of the VLPS-SiO2 immune group and the secretory IgA level in the lung, the cecum and the excrement are higher than the level of the FMDVVLPs immune group, and the levels of Tfh, Th1, Th2, memory B cells and memory T cells are increased. The invention provides an effective technical means for enhancing mucosal immune protection of the VLPs vaccine.

Description

Technical Field

[0001] This invention relates to a foot-and-mouth disease virus-like particle, its preparation method, and its application, particularly to a PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particle, its preparation method, and its application. This invention belongs to the field of biotechnology. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, and highly contagious disease caused by the foot-and-mouth disease virus (FMDV). FMDV can infect cloven-hoofed animals such as pigs, cattle, and sheep. Clinical symptoms mainly include fever and vesicles on the oral mucosa, coronary band, and udder. FMD has a wide range of susceptible animals and spreads rapidly, causing significant economic losses due to numerous outbreaks worldwide. In my country, FMD is listed as a Class A infectious disease, and the World Organisation for Animal Health (OIE) designates it as a disease requiring mandatory reporting. FMDV can infect susceptible animals on a large scale via aerosol transmission. Therefore, vaccines that can induce good mucosal immune protection are of significant economic and social value in the prevention and control of FMDV.

[0003] Mucosa serves as the gateway between the body and the external environment, acting as the body's first line of defense and playing a crucial role in protecting animals from invading pathogens. Compared to intramuscular or subcutaneous immunization, mucosal immunization offers advantages such as ease of administration, suitability for large-scale intensive farming, and minimal immune stress. Developing vaccines that induce favorable mucosal immune responses is of paramount importance for effectively controlling FMD and achieving ultimate eradication. Currently, inactivated FMDV vaccines are primarily administered via intramuscular injection. However, inactivated FMD vaccines require production in high-biosafety-level facilities, and intramuscular injection increases stress on immunized animals. VLPs (viral protein chains) vaccines, composed of FMDV structural proteins (VP0, VP1, and VP3), lack viral genetic material, exhibiting high safety. Furthermore, their morphology and structure are similar to natural viruses, inducing favorable immune responses. Therefore, FMDV VLPs are a promising candidate to replace inactivated vaccines. However, mucosal immunization requires antigens to cross the protease-rich mucosal barrier; therefore, the development of novel adjuvants that assist VLPs in crossing the mucosal barrier and enhance their immune efficacy is necessary.

[0004] In nature, minerals composed of different elements with well-structured and functional compounds protect organisms from adverse environmental influences. The SiO2 shell protects various plants and animals from damage caused by adverse factors, such as enhancing the heat resistance of archaea and hot spring bacteria. Therefore, SiO2 mineralization has been extensively studied and used to improve the stability of biomolecules such as enzymes, viruses, and yeast. However, some biomolecules cannot spontaneously catalyze SiO2 deposition and require modification to catalyze SiO2 formation and binding. Polydimethyldiallyl ammonium chloride (PDADMAC) can catalyze the hydrolysis of metasilicic acid to form SiO2, and amino groups can serve as SiO2 binding sites to form a mineralized layer. Silicon mineralization of yeast modified with PDADMAC resulted in the formation of a silicon mineralized layer on the yeast cell surface.

[0005] This invention focuses on the scientific problem of improving the mucosal immune efficacy of FMDM VLPs vaccines. It utilizes electrostatic interactions combined with PDADMAC to form a SiO2 mineralization layer under PDADMAC catalysis, which is further modified using PDADMAC. Cellular uptake and immunization experiments show that SiO2 mineralization enhances the uptake of FMDV VLPs by DC2.4 cells and the VLPs-induced mucosal immune effect. This invention provides a technical means for developing FMDV VLPs vaccines with good mucosal immune efficacy. Summary of the Invention

[0006] The purpose of this invention is to provide PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particles, their preparation method, and their applications.

[0007] To achieve the above objectives, the present invention employs the following technical means:

[0008] The present invention discloses a PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particle, wherein the PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particle is formed by forming silica-mineralized foot-and-mouth disease virus-like particles under the action of polydimethyldiallyl ammonium chloride (PDADMAC), and then further modified with PDADMAC.

[0009] Preferably, the morphology of the PDADMAC-modified silica mineralized foot-and-mouth disease virus-like particles is spherical with a size of 80-100 nm and a mineralization level of over 90%.

[0010] Furthermore, the present invention also proposes a method for preparing the PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particles, comprising the following steps:

[0011] First, PDADMAC solution was stirred together with foot-and-mouth disease virus-like particles to form VLPs-PDADMAC complex; then, sodium metasilicate mineralization solution was added and stirred, allowed to stand, and the mineralized particles were collected by centrifugation to obtain silica-mineralized foot-and-mouth disease virus-like particles. Then, the silica-mineralized foot-and-mouth disease virus-like particles were mixed with PDADMAC to form PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particles.

[0012] Preferably, the concentration of the PDADMAC solution is 0.2–3 mg / mL; and the concentration of the sodium metasilicate mineralization solution is 10–30 mM.

[0013] Preferably, the PDADMAC solution is stirred with foot-and-mouth disease virus-like particles at 4°C and 1500 rpm for 30 min to form a VLPs-PDADMAC complex.

[0014] Preferably, the sodium metasilicate mineralization solution is added and stirred at 1500 rpm for 2 hours at 4°C, and then allowed to stand at 4°C for 8 hours; the centrifugation is carried out at 8000 rpm for 10 minutes.

[0015] Preferably, the mineralized particle precipitate obtained by centrifugation is stirred with PDADMAC solution at 4°C and 1500 rpm for 30 min to form PDADMAC-modified silica mineralized foot-and-mouth disease virus-like particles.

[0016] Furthermore, the present invention also proposes the application of the PDADMAC-modified silica mineralized foot-and-mouth disease virus-like particles in the preparation of foot-and-mouth disease virus-like particle vaccines.

[0017] Preferably, the silica mineralization layer enhances the mucosal immune response induced by foot-and-mouth disease virus-like particles, increases the level of specific antibodies, secretory IgA in the lungs, cecum and feces, as well as the levels of Tfh, Th1, Th2, memory B cells and memory T cells.

[0018] Finally, the present invention also proposes a silica-mineralized foot-and-mouth disease virus-like particle vaccine, wherein the vaccine contains the PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particles.

[0019] In this invention, the foot-and-mouth disease virus-like particles can be prepared using existing methods in the art. In one specific embodiment of this invention, the virus-like particles are foot-and-mouth disease virus-like particles, prepared using the method disclosed in patent application CN106479986B, entitled "An O-type foot-and-mouth disease virus-like particle and its preparation method and use". Alternatively, they can be prepared according to the method disclosed in patent application CN101914501B, entitled "Foot-and-mouth disease virus-like particles and their preparation method and use".

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention forms a SiO2 mineralization layer on the surface of FMDVVLPs under the action of PDADMAC, and further modifies it with PDADMAC. The resulting mineralized particles are spherical with a size of 80-100 nm and a mineralization level of over 90%. The chemically modified SiO2 particles also promote antigen uptake by APCs. The SiO2-mineralized VLP particles, after PDADMAC modification, have a positively charged surface, making them more easily adsorbed onto negatively charged cell membranes via electrostatic interactions. Western blot and laser confocal microscopy analysis of cell uptake levels showed that SiO2 mineralization increased the uptake of FMDVVLPs by DC2.4 cells. SiO2 also plays a role in assisting antigen-induced immune responses. After being phagocytosed by APCs, SiO2 dissolves to produce silicate ions, which in turn activate the NLRP3 inflammasome, promote the secretion of cytokines such as IL-1β and IL-18, and enhance humoral immune responses. Under acidic lysosomal conditions, the SiO2 shell triggers a proton sponge effect, promoting the release of encapsulated antigens into the cytoplasm and providing conditions for inducing a favorable cellular immune response. Mouse immunization experiments showed that SiO2 mineralization increased the levels of specific antibodies and Th1 cells induced by VLPs, revealing the effect of SiO2 mineralization on enhancing both cellular and humoral immune responses induced by FMDVVLPs. The "penetration-delivery-activation" properties of SiO2 mineralized particles can penetrate the mucosal barrier, significantly enhancing sIgA levels in mucosal sites. ELISA detection of sIgA levels in the lungs, cecum, and feces of immunized mice showed that SiO2 mineralization enhanced the mucosal immune effect after intranasal immunization with FMDVVLPs. One of the main goals of vaccine immunization is to induce a favorable memory immune response, which generates a rapid immune response when the body is threatened by pathogen infection, protecting the body from pathogen infection. Flow cytometry results indicated that SiO2 mineralization enhanced the levels of memory B cells and memory T cells induced by the FMDVVLPs vaccine. Therefore, SiO2 mineralization provides a technical means for constructing vaccines that can induce good mucosal immunity. Attached Figure Description

[0022] Figure 1 Preparation for FMDVVLPs;

[0023] Among them, (A) the purified and enzyme-digested FMDV structural proteins were identified by SDS-PAGE; (B) the antigen specificity before and after enzyme digestion was analyzed by Western blot; and (C) the morphology of assembled FMDVVLPs was observed by TEM.

[0024] Figure 2 Preparation and characterization of VLPs-SiO2;

[0025] Among them, (A) TEM was used to observe the morphology and size of VLPs-SiO2; (B) SEM was used to observe the three-dimensional structure of VLPs-SiO2; (C) DLS was used to determine the Zeta potential of different substances during mineralization; (D) FTIR was used to detect the absorption peaks of functional groups in FMDVVLPs and VLPs-SiO2 particles; (E) Western blot was used to detect the VLP levels in the mineralized precipitate and supernatant; (F) Dot blot was used to detect the VLP levels on the surface of the mineralized supernatant and precipitate particles; and (G) the mineralization level was determined by a protein concentration assay kit.

[0026] Figure 3 This refers to cellular uptake levels.

[0027] Among them, (A) Western blot was used to determine the level of VLPs uptake in DC2.4 cells at different times (0h, 1h, 2h, 4h and 6h) after incubation with FMDV VLPs and VLPs-SiO2; (B) laser confocal microscopy was used to observe the level of intracellular FMDV VLPs and VLPs-SiO2 in DC2.4 cells after incubation for 6h.

[0028] Figure 4 The levels of specific antibodies and IgA in immunized mice;

[0029] Among them, (A) the specific antibody levels of mice immunized with FMDVVLPs and VLPs-SiO2 at different time points (3d, 7d, 14d, 21d and 28d) after final immunization; and the IgA levels in (B) the lungs, (C) the cecum and (D) the feces of mice immunized with FMDVVLPs and VLPs-SiO2 at different time points (3d, 7d, 14d and 28d) after final immunization.

[0030] Figure 5 Three days after final immunization, flow cytometry was used to analyze the levels of (A) Tfh cells, (B) Th1 cells and (C) Th2 cells in spleen lymphocytes of FMDVVLPs and VLPs-SiO2 immunized mice.

[0031] Figure 6To detect memory B cells and central memory CD4 in the FMDVVLPs group and VLPs-SiO2 group 28 days after final immunization using flow cytometry + T (CD4T TCM) cells, effector memory CD4 + T (CD4T TEM) cells, central memory CD8 + T (CD8T TCM) cells and effector memory CD8 + The level of T(CD8T TEM) cells. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0033] Example 1: Preparation of PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particles

[0034] 1. Materials and Methods

[0035] 1.1 Cells and main reagents

[0036] The recombinant fusion protein strains expressing His-SUMO and type O FMDV structural proteins VP0, VP1, and VP3 (described in patent application CN106479986B, entitled "A type O foot-and-mouth disease virus-like particle and its preparation method and uses") and the strains expressing SUMO protease are preserved by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

[0037] 1.2 Purification and Silicate Biomimetic Mineralization of FMDVVLPs

[0038] 1.2.1 Purification and Identification of FMDVVLPs

[0039] Following the method established in patent application CN106479986B, entitled "An O-type Foot-and-Mouth Disease Virus-like Particle and Its Preparation Method and Use," E. coli expressing FMDV structural proteins VP0, VP1, and VP3 were proliferated, induced to express, and purified. The His-Sumo tag was removed using Sumo enzyme, and the particles were assembled in an assembly buffer to obtain O-type foot-and-mouth disease virus-like particles (FMDVVLPs). The assembled FMDVVLPs were analyzed using SDS-PAGE.

[0040] 1.2.2 Preparation and Identification of VLPs-SiO2

[0041] First, 0.5 mg / mL PDADMAC and FMDVVLPs were stirred at 4℃ and 1500 rpm for 30 min to form a VLPs-PDADMAC complex. Then, 15 mM sodium metasilicate mineralization solution was added and stirred (4℃, 1500 rpm) for 2 h, followed by standing at 4℃ for 8 h. The mineralized particles were collected by centrifugation (8000 rpm, 10 min) and the supernatant was collected. Then, 0.5 mg / mL PDADMAC was added to the mineralized particle precipitate and stirred at 1500 rpm for 30 min to form PDADMAC-modified VLPs-SiO2. The morphology of the mineralized VLPs-SiO2 particles was observed using transmission electron microscopy (TEM), the three-dimensional structure of the mineralized VLPs-SiO2 particles was analyzed using scanning electron microscopy (SEM), and the change in zeta potential of the mineralized VLPs-SiO2 particles during mineralization was analyzed using dynamic light scattering (DLS). Fourier transform infrared spectroscopy (FTIR) was used to analyze the functional groups contained in the mineralized VLPs-SiO2 particles. The levels of FMDV VLPs in the mineralized precipitate and supernatant were analyzed by Western blot and Dot blot. The content of added FMDV VLPs and protein in the supernatant was determined using a BCA protein concentration kit, and the mineralization rate was calculated using the formula: Mineralization rate = (Total protein content - Protein content in supernatant) / Total protein content × 100%.

[0042] 1.3 FMDVVLPs-SiO2 uptake level

[0043] DC2.4 cells were incubated with the same dose of FMDVVLPs (5 μg) in FMDVVLPs-SiO2 and FMDVVLPs for different time periods (1 h, 2 h, 4 h, and 6 h). Western blot analysis was used to analyze the level of FMDVVLPs uptake in DC2. DC2.4 cells incubated with FMDVVLPs and VLPs-SiO2 for 6 h underwent indirect immunofluorescence assays. Incubation was performed with anti-FMDV porcine serum, followed by incubation with FITC-labeled goat anti-porcine antibody, and cell nuclei were stained with DAPI. Each step involved washing four times with PBST solution. Intracellular FITC and DAPI fluorescence images in DC2.4 cells were analyzed by laser confocal microscopy and merged. The level of FMDVVLPs uptake in DC2.4 cells was determined based on the intracellular FITC fluorescence level.

[0044] 1.4 Animal Experiments

[0045] 1.4.1 Mouse Immunization

[0046] Thirty-six 6-8 week old female BALB / c mice were randomly divided into three groups and immunized intranasally with PBS, FMDV VLPs, and FMDVVLPs-SiO2, respectively. Immunization was administered three times on days 0, 7, and 14. Serum was collected from mice on days 3, 7, 14, 21, and 28 post-immunization. Three mice were sacrificed on days 3, 7, 14, and 28 post-immunization, and bronchoalveolar lavage fluid, cecal fragment extract, and fecal extract were prepared. Spleens were isolated from mice on days 3 and 28 post-immunization, and splenic lymphocyte typing was analyzed using flow cytometry.

[0047] 1.4.2 Specific antibody levels

[0048] The levels of specific antibodies in serum were measured by liquid-phase blocking ELISA at 3, 7, 14 and 28 days post-immunization.

[0049] 1.4.3 IgA Levels

[0050] The levels of IgA in lung lavage fluid, feces, and cecal extract of mice were determined by indirect ELISA at 3, 7, 14, and 28 days post-immunization.

[0051] 1.4.4 Splenic lymphocyte classification

[0052] Splenic lymphocytes were obtained from mice 3 days and 28 days after final immunization using mouse lymphocyte separation medium and counted. The splenic lymphocytes were diluted to 1×10⁻⁶. 7 Cells / mL, according to 100 μL / well (1×10⁻⁶ cells / mL). 6 (Number of cells) were added to a serum dilution plate, centrifuged to collect the cells, and washed with flow cytometry washing buffer.

[0053] Expiratory cell staining: First, mouse spleen lymphocytes were blocked with CD16 / 32, then live and dead cells were labeled with FixableViability Stain 700. Subsequently, Tfh cells (FITC-CD3, BV421-CD4, Y763-PC7-CXCR-5, BV-650-PD-1) and memory B cells (FITC-CD3-, BV500-B220, PE-Cy) were labeled with fluorescently tagged antibodies. TM 7-IgM and PE-IgD), CD4 + T memory cells (FITC-CD3, BV421-CD4, APC-CD44, PerCP-Cy) TM 5.5-CD62L) and CD8 + T memory cells (FITC-CD3, APC-Cy) Tm7- CD8, APC-CD44, PerCP-Cy TM5.5-CD62L). Each operation involved washing with flow cytometry. Finally, cells were fixed with 0.5% paraformaldehyde.

[0054] Intracellular flow cytometry staining: Splenic lymphocytes from mice in different immunization groups were stimulated with FMDVVLPs. After 2 hours of stimulation, brefiltrant A (BFA) was added to inhibit the secretion of cytokines into the extracellular space. After 8 hours of continued stimulation, the splenic lymphocytes were blocked, followed by live / dead labeling. Splenic lymphocytes were labeled with fluorescently tagged CD3, CD4, and CD8, then fixed with paraformaldehyde solution and ruptured with cell membrane lysis solution. Finally, splenic lymphocytes were labeled with fluorescently tagged IL-4 and IFN-γ, and Th1 cells (FITC-CD3, BV421-CD4, PerCP-Cy) were analyzed. Tm Levels of 5.5-IFN-γ and Th2 cells (FITC-CD3, BV421-CD4, PE-IL-4). Washing with flow cytometry was performed after each step.

[0055] 2 Results

[0056] 2.1 Preparation of FMD VLPs

[0057] FMDVVLPs are assembled from structural proteins. After expression using laboratory-stored expression bacteria, the FMDVVLPs structural proteins with a Sumo-His tag were purified by nickel column chromatography. SDS-PAGE analysis showed that the purified protein size matched the expected protein size. Figure 1 A) The molecular weight of the structural protein obtained after Sumo enzyme digestion is consistent with the theoretical calculation value. Figure 1 A). Western blot analysis showed that both the protein before and after enzyme digestion specifically reacted with the anti-FMDV antibody, indicating that the FMDV structural protein was successfully prepared. Figure 1 B). Transmission electron microscopy (TEM) revealed empty capsids of approximately 30 nm in size in the assembled sample, indicating successful preparation of FMDV VLPs. Figure 1 C).

[0058] 2.2 Preparation and Characterization of FMDVVLPs-SiO2

[0059] FMDVVLPs were modified with PDADMAC and then subjected to silicon mineralization. TEM observation showed that the mineralized sample consisted of nanoparticles with poor light transmittance of 80–100 nm. Figure 2 A). SEM results show that the mineralized sample is an irregular spherical shape. Figure 2B). DLS analysis showed that the Zeta potentials of the FMDV VLPs, PDADMAC, VLPs-PDADMAC, VLPs-PDADMAC-SiO2, and VLPs-PDADMAC-SiO2-PDADMAC in the mineralization process were approximately -5.27 mV, 15.05 mV, 10.11 mV, -13 mV, and 8.3 mV, respectively. Figure 2 C). FTIR analysis showed that the mineralized particles were at 1095 cm⁻¹. -1 It exhibits a broad and strong absorption peak at 798 cm⁻¹, which is the antisymmetric stretching vibration peak of the Si-O-Si bond; -1 The absorption peak at 466 cm⁻¹ is a symmetric stretching vibration peak of Si-O-Si. -1 The absorption peaks appearing nearby are due to the stretching vibrations of Si-O-Si; additionally, at 1648 cm⁻¹... -1 The detection of a characteristic absorption peak of amide I bond indicates that the mineralized particles contain SiO2 and VLPs ( Figure 2 D). Western blot and Dot blot analyses showed that the mineralized supernatant and the surface of the precipitated particles contained only a small amount of protein, while a large number of VLPs were encapsulated inside the particles. Figure 2 E and 2F). The protein concentration kit determined the level of SiO2-mineralized FMDVVLPs to be approximately 94.9% (E and 2F). Figure 2 G).

[0060] 2.3 Cellular uptake level

[0061] The level of antigen uptake by antigen-presenting cells (APCs) is closely related to the antigen-induced immune response. When DC2.4 cells were incubated with the same amount of VLPs from FMDVVLPs and VLPs-SiO2, Western blot analysis showed that the level of VLP uptake gradually increased over time, and the VLP uptake level in the VLPs-SiO2 incubation group was significantly higher than that in the FMDVVLPs group. Figure 3 A). Further observation using laser confocal microscopy showed that the intensity of green fluorescence (FMDVVLPs) in DC2.4 cells in the VLPs-SiO2 incubation group was significantly higher than that in the FMDVVLPs incubation group (A). Figure 3 B). The above results indicate that SiO2 mineralization facilitates the uptake of FMDV VLPs by APCs, which may be due to the positive charge of PDADMAC-modified mineralized particles, making them more likely to bind to the negatively charged cell membrane.

[0062] 2.4 Mouse Immune-Specific Antibody and IgA Levels

[0063] Specific antibody levels are a key indicator for measuring the systemic immune response induced by a vaccine. ELISA analysis showed that the specific antibody levels in mice immunized with VLPs-SiO2 were higher than those in mice immunized with FMDVVLPs, especially on days 3 and 7 after final immunization, where there were significant differences in specific antibody levels between the two groups. Figure 4 A) indicates that SiO2 mineralization significantly enhances the immune response induced by FMDVVLPs. The level of secreted IgA (sIgA) is an important indicator for evaluating vaccine-induced mucosal immunity and plays a crucial role in blocking pathogen invasion of the mucosa. The level of sIgA in the lungs of immunized mice was detected by ELISA. Figure 4 B) Cecum ( Figure 4 C) and feces ( Figure 4 The sIgA level results in D) showed that the sIgA level in the VLPs-SiO2 immunization group was higher than that in the FMDVVLPs immunization group in all tested samples. Specifically, the sIgA level in the VLPs-SiO2 group was significantly higher than that in the FMDVVLPs group in lung samples 7 days after immunization and intestinal samples 3, 7, and 14 days after immunization. These results indicate that VLPs-SiO2 induced a higher level of specific immune response and mucosal immunity, demonstrating a good immune-enhancing effect.

[0064] 2.6 Splenic lymphocyte typing in immunized mice

[0065] After vaccination, the vaccine is taken up by APCs and presented to different immune cells, which are activated by the combined action of surface molecules and cytokines, thereby activating an adaptive immune response. Analyzing the levels of various immune cells in the spleen lymphocytes of immunized mice can clarify the effectiveness of the vaccine-induced immune response. Flow cytometry was used to detect Tfh(α-cells) in spleen lymphocytes. Figure 5 A) Th1( Figure 5 B) and Th2 Figure 5 C) The levels of each cell in the VLPs-SiO2 immunization group were higher than those in the FMDVVLPs immunization group, indicating that SiO2 mineralization effectively enhanced FMDVVLPs-induced cellular and humoral immunity.

[0066] 2.6 Levels of memory immune cells in immunized mice

[0067] Memory cells are an important component of the adaptive immune response, playing a crucial role in long-term defense and rapid response, and are one of the important indicators for evaluating vaccine immunization. Therefore, the levels of various memory cells in the spleen lymphocytes of mice 28 days after intranasal immunization were measured. Flow cytometry results showed that the levels of memory B cells, central memory T cells, and effector memory T cells in the VLPs-SiO2 immunization group were higher than those in the FMDVVLPs immunization group, with effector memory CD4 cells being the most abundant. + T cells showed significant differences ( Figure 6The above results indicate that SiO2 mineralization can enhance the level of memory immune response induced by FMDVVLPs vaccine, which helps the body to quickly initiate an immune response when infected by pathogens.

Claims

1. A PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particle, characterized in that, The PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particles are obtained by forming silica-mineralized foot-and-mouth disease virus-like particles under the action of polydimethyldiallylammonium chloride (PDADMAC), and then further modifying them with PDADMAC.

2. The PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particles as described in claim 1, characterized in that, The morphology of the PDADMAC-modified silica mineralized foot-and-mouth disease virus-like particles is that of spherical particles with a size of 80-100 nm and a mineralization level of over 90%.

3. A method for preparing PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particles as described in claim 1 or 2, characterized in that, Includes the following steps: First, PDADMAC solution was stirred together with foot-and-mouth disease virus-like particles to form VLPs-PDADMAC complex; then, sodium metasilicate mineralization solution was added and stirred, allowed to stand, and the mineralized particles were collected by centrifugation to obtain silica-mineralized foot-and-mouth disease virus-like particles. Then, the silica-mineralized foot-and-mouth disease virus-like particles were mixed with PDADMAC to form PDADMAC-modified silica-mineralized foot-and-mouth disease virus-like particles.

4. The method as described in claim 3, characterized in that, The concentration of the PDADMAC solution is 0.2–3 mg / mL; the concentration of the sodium metasilicate mineralization solution is 10–30 mM.

5. The method as described in claim 3, characterized in that, The PDADMAC solution was mixed with foot-and-mouth disease virus-like particles at 4°C and 1500 rpm for 30 min to form VLPs-PDADMAC complex.

6. The method as described in claim 3, characterized in that, Add sodium metasilicate mineralization solution, stir at 1500 rpm for 2 hours at 4°C, and let stand at 4°C for 8 hours; the centrifugation is carried out at 8000 rpm for 10 minutes.

7. The method as described in claim 3, characterized in that, The mineralized particle precipitate obtained by centrifugation was mixed with PDADMAC solution at 4℃ and 1500 rpm for 30 min to form PDADMAC-modified silica mineralized foot-and-mouth disease virus-like particles.

8. The use of the PDADMAC-modified silica mineralized foot-and-mouth disease virus-like particles as described in claim 1 or 2 in the preparation of foot-and-mouth disease virus-like particle vaccines.

9. The application as described in claim 8, characterized in that, The silica mineralization layer enhanced the mucosal immune response induced by foot-and-mouth disease virus-like particles, increased the level of specific antibodies, secretory IgA levels in the lungs, cecum and feces, and the levels of Tfh, Th1, Th2, memory B cells and memory T cells in the spleen.

10. A silica-mineralized foot-and-mouth disease virus-like particle vaccine, characterized in that, The vaccine contains silica-mineralized foot-and-mouth disease virus-like particles modified with PDADMAC as described in claim 1 or 2.

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