Application of vesicles derived from xenogeneic mammalian cells in the preparation of vaccine vectors

Abstract

The present invention provides the use of vesicles derived from xenogeneic mammalian cells in the preparation of vaccine vectors, including tumor vaccines or infectious disease vaccines. The xenogeneic mammalian cell-derived vesicles can be made into xenogeneic cell vesicle nanovaccines, which can target dendritic cells through the body's pre-existing natural antibodies, improve antigen uptake efficiency, promote antigen cross-presentation, activate dendritic cells, promote antigen-specific immune responses, and enhance tumor suppression and COVID-19 prevention effects.

Description

Technical Field

[0001] The present invention belongs to the field of nanobiotechnology, and in particular relates to the application of vesicles derived from xenogeneic mammalian cells. Background Art

[0002] As a key technology industry, vaccines are currently a priority development target in clinical research and have good application value and prospects. However, existing research shows that vaccines still face the problems of weak antigen immune response, easy degradation, and uncontrollable biological behavior in the body. In order to solve the above problems, the current vaccine delivery systems developed by nanomaterials mainly adjust the nanosystem construction formula and active ligand modification to improve the physical and chemical properties of the nanomaterials themselves, promote antigen uptake, processing and enhance subsequent immune responses. Although these strategies improve the immune response effect of vaccines to a certain extent, the single theoretical mechanism and low antigen delivery efficiency still limit the application of vaccine delivery. Therefore, promoting the effective uptake of DC cells is an important direction for exploring efficient vaccine delivery systems.

[0003] The phenomenon of xenogeneic cells being recognized by natural antibodies during xenotransplantation can be exploited in the development of vaccine delivery systems. During organ transplantation, xenogeneic cells and tissues are recognized by pre-existing natural antibodies in the body. Antigen-presenting cells process the xenogeneic cells recognized by natural antibodies, subsequently activating antigen-related adaptive immune responses. Inspired by the xenogeneic rejection response triggered by natural antibodies against xenogeneic antigens, a novel xenogeneic cell vesicle nanovaccine has been constructed using cell membrane carrier technology. Nanovaccines constructed from xenogeneic cell vesicles can be recognized by pre-existing natural antibodies in the body, targeting DC cells, promoting the uptake, processing, and handling of antigens by DC cells, activating DC cells and antigen cross-presentation, and stimulating antigen-specific immune responses for tumor treatment and infectious disease prevention. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide applications of vesicles derived from xenogeneic mammalian cells.

[0005] In a first aspect, the present invention provides a use of vesicles derived from xenogeneic mammalian cells in the preparation of vaccines.

[0006] The present invention creatively discovered that xenogeneic mammalian cell-derived vesicles can be combined with natural antibodies based on the recognition phenomenon of natural antibodies on xenogeneic cells during xenotransplantation, and have potential application value.

[0007] Preferably, the xenogeneic mammalian cells are selected from any one of porcine endothelial cells, Chinese hamster ovary cells or African green monkey kidney cells.

[0008] The above-mentioned vesicles derived from xenogeneic mammals can avoid the risks of genomic contamination and viral infection during xenotransplantation, have good biocompatibility, and have the advantages of being phagocytosed by professional antigen-presenting cells such as dendritic cells, playing an important role in vaccine preparation.

[0009] Preferably, the particle size of the vesicles derived from xenogeneic mammalian cells is 50-200 nm.

[0010] The particle size of the vesicles derived from the xenogeneic mammalian cells can be 50 nm, 55 nm, 60 nm, 80 nm, 100 nm, 120 nm, 140 nm, 150 nm, 170 nm, 180 nm, 200 nm, etc. Specific point values ​​within the above range can be selected and will not be repeated here.

[0011] Preferably, the method for preparing vesicles derived from xenogeneic mammalian cells comprises the following steps:

[0012] The xenogeneic mammalian cells are resuspended and sonicated in a water bath; the sonicated solution is centrifuged for the first time to obtain a supernatant, and the precipitate is discarded. The supernatant is then centrifuged for a second time, and the precipitate is resuspended and extruded using a liposome extruder to obtain vesicles derived from the xenogeneic mammalian cells.

[0013] The water bath ultrasonic treatment lasts for 1-3 minutes at a temperature of 0-5°C;

[0014] The time of the water bath ultrasonic treatment can be 1 min, 1.2 min, 1.4 min, 2 min, 2.4 min, 2.7 min, 3 min, etc.

[0015] The temperature of the water bath ultrasonic treatment can be 0°C, 1°C, 2°C, 3°C, 4°C, 4.5°C, 5°C, etc.

[0016] The first centrifugation refers to centrifugation at 0-8°C and a centrifugal force of 8000-12000×g for 10-25 minutes;

[0017] The temperature of the first centrifugation can be 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 7°C, 8°C, etc.

[0018] The centrifugal force of the first centrifugation can be 8000×g, 8500×g, 9000×g, 9500×g, 10000×g, 10500×g, 11000×g, 12000×g, etc.

[0019] The time for the first centrifugation can be 10 min, 12 min, 15 min, 16 min, 17 min, 18 min, 20 min, 22 min, 24 min, 25 min, etc.

[0020] The second centrifugation refers to centrifugation at 0-8°C and a centrifugal force of 80,000-160,000×g for 30-45 minutes.

[0021] The temperature of the second centrifugation can be 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 7°C, 8°C, etc.

[0022] The centrifugal force of the second centrifugation can be 80,000×g, 85,000×g, 90,000×g, 95,000×g, 100,000×g, 105,000×g, 110,000×g, 120,000×g, 156,000×g, 160,000×g, etc.

[0023] The time for the second centrifugation can be 30 min, 32 min, 35 min, 36 min, 37 min, 38 min, 40 min, 42 min, 44 min, 45 min, etc.

[0024] Any specific point value within the above numerical range can be selected and will not be described in detail here.

[0025] In a second aspect, the present invention provides a xenogeneic cell vesicle nanovaccine, which comprises xenogeneic mammalian cell-derived vesicles and antigens encapsulated in the xenogeneic mammalian cell-derived vesicles.

[0026] Nanovaccines constructed from vesicles derived from xenogeneic mammalian cells can be recognized by the body's pre-stored natural antibodies, target dendritic cells, promote the uptake, processing and treatment of antigens by dendritic cells, activate dendritic cells and antigen cross-presentation, and activate antigen-specific immune responses for tumor treatment and infectious disease prevention.

[0027] The antigen is selected from any one of a polypeptide antigen, a protein antigen or an mRNA antigen.

[0028] When the antigen is a polypeptide antigen, the xenogeneic cell vesicle nanovaccine is prepared by a method comprising the following steps:

[0029] The polypeptide antigen is directly coated on vesicles derived from heterologous mammalian cells by electroporation or ultrasound, and then ultrafiltration and centrifugation are performed using an ultrafiltration tube to remove free polypeptide antigens.

[0030] Preferably, the ultrafiltration centrifugation refers to ultrafiltration centrifugation performed at 3000-5000 rpm using an 80-150 kDa ultrafiltration tube.

[0031] The molecular weight limit of the ultrafiltration tube can be 80kDa, 90kDa, 95kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, etc.

[0032] The rotation speed of the ultrafiltration centrifugation can be 3000 rpm, 3300 rpm, 3500 rpm, 4000 rpm, 4500 rpm, 4800 rpm, 5000 rpm, etc.

[0033] Preferably, when the antigen of the xenogeneic cell vesicle nanovaccine is mRNA, the xenogeneic cell vesicle nanovaccine is prepared by a method comprising the following steps:

[0034] Lipid nanoparticles encapsulating mRNA are prepared by microfluidic mixing or ethanol rapid injection, and unencapsulated mRNA is removed by ultrafiltration centrifugation. Vesicles derived from heterologous mammalian cells and lipid nanoparticles encapsulating mRNA are mixed and extruded using a liposome extruder.

[0035] Preferably, the ultrafiltration centrifugation refers to ultrafiltration centrifugation performed at 3000-5000 rpm using an 80-150 kDa ultrafiltration tube.

[0036] The molecular weight limit of the ultrafiltration tube can be 80kDa, 90kDa, 95kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, etc.

[0037] The rotation speed of the ultrafiltration centrifugation can be 3000 rpm, 3300 rpm, 3500 rpm, 4000 rpm, 4500 rpm, 4800 rpm, 5000 rpm, etc.

[0038] In a third aspect, the present invention provides a use of the xenogeneic cell vesicle nanovaccine as described in the second aspect in the preparation of tumor immunotherapy drugs or infectious disease prevention drugs.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention provides an application of vesicles derived from xenogeneic mammalian cells. The vesicles derived from xenogeneic mammalian cells can be made into xenogeneic cell vesicle nanovaccines. The xenogeneic cell vesicle nanovaccines can target dendritic cells through the body's pre-stored natural antibodies, improve the efficiency of antigen uptake, promote cross-presentation of antigens, activate dendritic cells, promote the body's antigen-specific immune response, and enhance the inhibitory effect on tumors and the prevention effect of new coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1This is a schematic diagram of the characterization of vesicles derived from xenogeneic mammalian cells under transmission electron microscopy;

[0042] Figure 2 Schematic diagram of the hydrated particle size and Zeta potential of vesicles derived from xenobiotic mammalian cells measured by Malvern laser particle size analyzer;

[0043] Figure 3 This is a statistical diagram of the targeting effect of XMV-Ag on DC cells;

[0044] Figure 4 DC cells secrete CD86 + Statistical graph of factor detection results;

[0045] Figure 5 DC cells secrete CD40 + Statistical graph of factor detection results;

[0046] Figure 6 This is a statistical chart of the detection results of TNF-α factor in DC cells;

[0047] Figure 7 This is a statistical chart of the detection results of IL-6 factor in DC cells;

[0048] Figure 8 This is a schematic diagram of the detection of DIR fluorescence targeting in mice through small animal in vivo imaging;

[0049] Figure 9 This is a schematic diagram of the fluorescence imaging of various organs after the mouse was dissected;

[0050] FIG10 is a statistical diagram of the relative fluorescence intensity of the main organs and lymph nodes after dissecting the mouse lymph nodes;

[0051] Figure 11 This is a schematic diagram of the tumor treatment effect of XMV-Ag;

[0052] Figure 12 This is a schematic diagram of the verification of the COVID-19 prevention effect of XMV-mRNA;

[0053] Figure 13 Schematic diagram of vaccinating mice with mRNA vaccine;

[0054] Figure 14 This is a statistical chart of the IgG antibody titers of mice against the spike protein antigen in the receptor binding region of the spike glycoprotein on the new coronavirus on different days. DETAILED DESCRIPTION

[0055] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0056] If specific techniques or conditions are not specified in the examples, the experiments were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments used without manufacturer specified are all conventional products commercially available through regular channels. The effects of the examples of the present invention will not be affected by the use of the same products from different manufacturers.

[0057] Example 1

[0058] Preparation of vesicles from xenogeneic mammalian cells:

[0059] (1) Cell collection: Culture porcine endothelial cells until they are in good growth condition and the amount of cells to be extracted is met, and then wash the cells three times with 0.01M PBS buffer. Use a cell scraper to treat the porcine endothelial cells, collect the cells, and wash them three times with 0.01M PBS buffer.

[0060] (2) The collected cells were washed three times with sterile PBS at a concentration of 0.01M, resuspended in 25mL of hypotonic lysis buffer containing a mixture of EDTA-free protease and phosphatase inhibitors, placed on ice for 20 minutes, and sonicated in a water bath for 2 minutes each time, for a total of 3 times. The sonicated solution was centrifuged at 4°C, 10,000×g for 20 minutes to obtain the supernatant, and the precipitate was discarded. The supernatant was then centrifuged at 4°C, 150,000×g for 35 minutes to collect the precipitate. The precipitate was resuspended in sterile PBS at a concentration of 0.01M, and the xenobiotic mammalian cell-derived vesicles were obtained using a liposome extruder with 400nm and 200nm filter membranes, hereinafter referred to as XMV.

[0061] Example 2

[0062] Characterization of Xenogeneic Mammalian Cell-Derived Vesicles:

[0063] The morphology, particle size and Zeta potential of the XMV prepared in Example 1 were characterized using a Malvern laser particle size analyzer and a transmission electron microscope. Figure 1 and Figure 2 As shown, Figure 1 The middle is an electron microscopic morphology of the prepared XMV vaccine universal carrier. The vesicles derived from heterologous mammalian cells have a spherical structure. Figure 2 The particle size of XMV measured by the Malvern laser particle size analyzer is about 100 nm and the Zeta potential is about -20 mV. When XMV is combined with immune serum, its particle size is about 125 nm and the Zeta potential is about -28 mV.

[0064] Example 3

[0065] This example provides a mouse autologous erythrocyte vesicle vaccine, hereinafter abbreviated as AUV-OVA 241-270 , mouse autologous erythrocyte vesicle vaccine (AUV-OVA 241-270 ) comprises the following steps: collecting fresh whole blood from mice, centrifuging at 4°C, 800×g for 5 minutes, and washing three times with 0.01M PBS. The obtained red blood cell pellet is resuspended with 0.25×0.01M PBS, ice-bathed for 20 minutes, centrifuged at 4°C, 8000×g for 5 minutes, and resuspended with 0.25×0.01M PBS. The pellet is then centrifuged and resuspended until the supernatant is free of red color, and the white red blood cell membrane pellet is collected and stored at -80°C. The multi-skin antigen is encapsulated by electroporation, and ultrafiltration and centrifugation are performed at 4000rpm using a 100kD ultrafiltration tube to remove the free multi-skin antigen to obtain AUV-OVA. 241-270 .

[0066] Example 4

[0067] This embodiment provides a xenogeneic mammalian cell-derived vesicle nanovaccine, hereinafter abbreviated as XMV-Ag), wherein Ag can be any one of a polypeptide or mRNA.

[0068] The preparation method of xenogeneic mammalian cell-derived vesicle nanovaccine (XMV-Ag) comprises the following steps:

[0069] (1) Xenogeneic mammalian cell-derived vesicle nanovaccine loaded with polypeptide antigens (hereinafter referred to as XMV-OVA) 241-270 ) preparation:

[0070] OVA 241-270 After physically mixing with xenogeneic mammalian cell-derived (porcine endothelial cell) cell vesicles at a mass ratio of 1:1, the peptide antigen was encapsulated by electroporation (voltage 400V, capacitance 125μF), and the excess antigen was removed by ultrafiltration centrifugation at 5000rpm using a 100kDa ultrafiltration tube to obtain XMV-OVA. 241-270 .

[0071] (2) Preparation of mammalian cell-derived vesicle nanovaccine (XMV-mRNA) for the novel coronavirus mRNA antigen:

[0072] First, according to the lipid nanoparticle process recipe, lipid nanoparticles encapsulating mRNA are prepared by microfluidic mixing or ethanol rapid injection method, and ultrafiltration centrifugation is performed at 4000 rpm for 15 minutes using a 100 kDa ultrafiltration tube. The unencapsulated mRNA is removed using the ultrafiltration tube to obtain lipid nanoparticles encapsulating mRNA.

[0073] Vesicles derived from heterologous mammalian cells and lipid nanoparticles encapsulating mRNA are mixed in a mass ratio of 1:1 and extruded using 400nm and 200nm liposome extruders.

[0074] Example 5

[0075] This embodiment provides a method of packaging OVA 241-270 Liposome vaccine, hereinafter referred to as Lip-OVA 241-270 .

[0076] Package OVA 241-270 Liposome vaccine (Lip-OVA 241-270 The preparation method of Lip-OVA comprises the following steps: using electroporation (parameters are voltage 400V, capacitance 125μF) to encapsulate polypeptide antigen, and ultrafiltration and centrifugation at 5000rpm using a 100kDa ultrafiltration tube to remove excess antigen, thereby obtaining Lip-OVA. 241-270 .

[0077] Example 6

[0078] This embodiment provides a liposome mRNA nanovaccine, hereinafter abbreviated as LNP-mRNA.

[0079] The preparation method of LNP-mRNA vaccine includes the following steps:

[0080] Prepare mRNA-encapsulated lipid nanoparticles according to Moderna's recipe. Dissolve DSPC, SM-102, DMG-PEG2000, and cholesterol in anhydrous ethanol at a molar ratio of 10%:50%:1.5%:38.5%. Dilute mRNA encoding SARS-CoV-2 in 0.5 mM citrate buffer (pH 4) at a lipid-to-mRNA mass ratio of 20:1 and an organic-to-aqueous phase volume ratio of 1:3. Rapidly mix and stir at room temperature for 3 minutes. Remove unencapsulated mRNA using a 100 kDa ultrafiltration tube to obtain LNP-mRNA.

[0081] Xenogeneic cell membrane protein and LNP-mRNA were mixed at a mass ratio of 1:1. Lipid nanoparticles (XMV-LNP-mRNA) coated with xenogeneic cell membranes were prepared using a liposome extruder with 400nm and 200nm filters. The mRNA encapsulation efficiency was measured using the Quant-iTRiboGreen RNA Assay Kit.

[0082] Test Example 1

[0083] This example verifies the XMV-OVA prepared in Example 4. 240-270 Vaccine targeting of dendritic cells.

[0084] Extraction of immunoglobulin G: Take whole blood from healthy C57BL / 6 mice, place the sample in a 1.5mL sterile, enzyme-free EP tube, and place it at room temperature for 2 hours. Then centrifuge at 2000rpm for 20 minutes to obtain normal mouse serum that is light yellow and non-hemolytic. At the same time, aliquot the serum for use to avoid repeated freezing and thawing. Before each use of the serum, centrifuge it at 4°C and 10,000×g for 30 minutes. Take the supernatant after centrifugation, separate and purify it using Protein A / G antibody magnetic beads, and quantify the extracted IgG using a BCA protein kit. The obtained IgG is aliquoted and stored at -20°C for subsequent experiments.

[0085] Subculture procedures for mouse bone marrow-derived dendritic cells (BMDCs): Mouse bone marrow-derived BMDCs were differentiated from bone marrow cells isolated from the tibia of male C57BL / 6 mice. The healthy mouse bone marrow cells were first cultured in RPMI 1640 complete medium supplemented with GM-CSF (20 ng / mL) and IL-4 (20 ng / mL). The medium was changed every other day, and on the sixth day, immature, fully differentiated mouse bone marrow-derived BMDCs were obtained.

[0086] This example provides four groups for flow cytometry testing, which are as follows:

[0087] (S1) blank control group (containing only PBS buffer);

[0088] (S2)XMV-OVA 240-270 group, which does not bind to IgG and FcR receptor blocking;

[0089] (S3)XMV-OVA 240-270 Group binds to IgG and FcR receptor blocks;

[0090] (S4)XMV-OVA 240-270 The group binds to IgG, but does not block FcR receptors;

[0091] XMV-OVA in the above groups 240-270 The binding method with IgG is as follows:

[0092] IgG and XMV-OVA 240-270 Incubate at 4°C for 30 min.

[0093] The FcR blocking method in the above group includes the following steps:

[0094] In XMV-OVA 241-270Before co-incubation with DC2.4 cells, DC cells were pre-treated with different concentrations of IgG or FcR inhibitors. After pre-treatment for 1 hour, fresh culture medium was replaced and XMV-OV A-loaded cells were added. 241-270 After 2 h, the cellular uptake was detected.

[0095] The test samples of the above groups were used to stimulate BMDCs and tested by flow cytometry. The results are as follows: Figure 3 As shown, the combination with IgG can effectively enhance the DC cell response to XMV-OVA 240-270 intake.

[0096] Test Example 2

[0097] This example verifies the activation effect of the nanovaccine prepared in Examples 3-5 on dendritic cells.

[0098] OVA 241-270 Free OVA with the same antigenic content 241-270 、Lip-OVA 241-270 、AUV-OVA 241-270 、XMV-OVA 241-270 Stimulate BMDC cells, set up blank control group, add only PBS solution to BMDC, and use ELISA kit to detect CD86 + 、CD40 + , IL-6, TNF-α and other related indicators were tested, and the results were as follows Figure 4-7 As shown, compared with other groups, XMV-OVA 241-270 It has a good activation effect on DC cells.

[0099] Test Example 3

[0100] This example verifies the targeting effect of the XMV-Ag vaccine prepared in Example 4 on lymph nodes

[0101] The XMV-Ag prepared in Example 4 was labeled with the fluorescent molecule DIR, and the DIR-labeled XMV-Ag was injected subcutaneously into the base of the mouse tail. The small animal imaging system was used to image the XMV-Ag targeting lymph nodes. Figure 8-1 As shown in Figure 0, the nanovaccine is clearly distributed in the mouse lymph nodes. At the same time, the relative fluorescence intensity of the lymph nodes is more obvious than that of other major organs.

[0102] Test Example 4

[0103] OVA 241-270 Free OVA with the same antigenic content 241-270 、Lip-OVA 241-270 、AUV-OVA 241-270、XMV-OVA 241-270 , verify the inhibitory effect of XMV-Ag on tumors.

[0104] Construction of melanoma model mice: On day 0, healthy female C57BL / 6 mice were subcutaneously inoculated with 2×10 5 B16F10-OVA mouse melanoma cells, such as Figure 11 shown.

[0105] The results are as follows Figure 12 As shown, on day 0, mice were inoculated with 2 × 10 5 B16-F10-OVA tumor cells were treated with the nanovaccine prepared in Example 2 on days 4, 11, and 18, respectively. The tumor size of the mice was measured every two days. It can be concluded that the nanovaccine has a significant inhibitory effect on mouse melanoma.

[0106] Test Example 5

[0107] The purpose of this example is to verify the new crown prevention effect of XMV-Ag

[0108] For the XMV-mRNA vaccine prepared in Example 4, the preventive effect of the nanovaccine platform against the new coronavirus was verified.

[0109] Healthy Balb / c mice were divided equally into XMV-mRNA, LNP-mRNA, and PBS groups, with 10 mice in each group. Each group of mice was housed individually in a clean, quiet environment with a temperature of 23-25°C and a humidity of 50-70%. Each group of mice had free access to food and water during the experiment. On day 0 and day 7, mice in the XMV-mRNA and LNP-mRNA groups were inoculated with the same amount of XMV-mRNA and LNP-mRNA, respectively. The PBS group was inoculated with 0.01M PBS buffer, as shown in Figure 2. Figure 13 As shown in the figure, the IgG antibody titer of the spike protein antigen on the receptor binding region of the spike glycoprotein of the new coronavirus in each group of mice was detected by ACRO Biosystems on the 7th, 14th, 21st and 42nd day. Figure 14 As shown in the results, both XMV-mRNA and LNP-mRNA can increase the binding antibody and neutralizing antibody titers in mice, and the XMV-mRNA nanovaccine can more significantly increase the binding antibody and neutralizing antibody titers in mice than the LNP-mRNA vaccine.

[0110] The applicant declares that the present invention uses the above-described embodiments to illustrate the detailed process flow of the present invention, but the present invention is not limited to the above-described detailed process flow, that is, it does not mean that the present invention must rely on the above-described detailed process flow to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. Application of porcine endothelial cell-derived vesicles in the preparation of vaccine vectors; The vaccine includes a tumor vaccine or an infectious disease vaccine; The porcine endothelial cell-derived vesicles are prepared by a preparation method comprising the following steps: The porcine endothelial cells were resuspended and sonicated in a water bath; the sonicated solution was centrifuged for the first time to obtain a supernatant, and the precipitate was discarded. The supernatant was then centrifuged for a second time, and the precipitate was resuspended and extruded using a liposome extruder to obtain porcine endothelial cell-derived vesicles.

2. The use according to claim 1, characterized in that The particle size of the porcine endothelial cell-derived vesicles is 50-200 nm.

3. The use according to claim 1, characterized in that The water bath ultrasonic treatment lasts for 1-10 minutes at a temperature of 0-8°C.

4. The use according to claim 1, characterized in that The first centrifugation refers to centrifugation at 0-8°C and a centrifugal force of 8000-12000×g for 10-25 minutes.

5. The use according to claim 1, characterized in that The second centrifugation refers to centrifugation at 0-8°C and a centrifugal force of 80,000-160,000×g for 30-45 min.

6. A porcine endothelial cell vesicle nanovaccine, characterized in that: The porcine endothelial cell vesicle nanovaccine comprises the porcine endothelial cell-derived vesicle according to claim 1 and an antigen encapsulated in the porcine endothelial cell-derived vesicle.

7. The porcine endothelial cell vesicle nanovaccine according to claim 6, characterized in that The antigen is selected from any one of a polypeptide antigen, a protein antigen or an mRNA antigen.

8. The porcine endothelial cell vesicle nanovaccine according to claim 7, characterized in that The antigen is a polypeptide antigen, and the porcine endothelial cell vesicle nanovaccine is prepared by a method comprising the following steps: The porcine endothelial cell-derived vesicles are directly coated with polypeptide antigens by electroporation or ultrasound, and ultrafiltration centrifugation is performed using an ultrafiltration tube to remove free polypeptide antigens.

9. The porcine endothelial cell vesicle nanovaccine according to claim 8, characterized in that The ultrafiltration centrifugation refers to ultrafiltration centrifugation performed at 3000-5000 rpm using an 80-150 kDa ultrafiltration tube.

10. The porcine endothelial cell vesicle nanovaccine according to claim 7, characterized in that The antigen is mRNA, and the porcine endothelial cell vesicle nanovaccine is prepared by a method comprising the following steps: Lipid nanoparticles encapsulating mRNA are prepared by a microfluidic mixing method or an ethanol rapid injection method, and unencapsulated mRNA is removed by ultrafiltration centrifugation. Vesicles derived from porcine endothelial cells and lipid nanoparticles encapsulating mRNA are mixed and extruded using a liposome extruder.

11. The porcine endothelial cell vesicle nanovaccine according to claim 10, characterized in that The ultrafiltration centrifugation refers to ultrafiltration centrifugation performed at 3000-5000 rpm using an 80-150 kDa ultrafiltration tube.

12. Use of the porcine endothelial cell vesicle nanovaccine according to any one of claims 6 to 11 in the preparation of tumor immunotherapy drugs or infectious disease prevention drugs.

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