A vaccine based on an Angelica polysaccharide derivative delivery vector, its preparation method and application

Abstract

This invention provides a vaccine based on an Angelica sinensis polysaccharide derivative delivery carrier, its preparation method, and its application, belonging to the field of biomedical technology. The invention provides a method for preparing the vaccine, using an amphiphilic Angelica sinensis polysaccharide derivative as a delivery carrier to obtain a W / O / W type Pickering emulsion, which is the vaccine. The vaccine of this invention improves safety and reduces antigen degradation in the digestive tract through the delivery of the Angelica sinensis polysaccharide derivative. This invention further provides the application of the vaccine in the preparation of fish farming-related products and a fish feed, wherein the feed pellets contain the vaccine. These vaccine feed pellets stimulate higher levels of innate and adaptive immunity in fish, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a vaccine based on a delivery carrier of Angelica sinensis polysaccharide derivatives, its preparation method, and its application. Background Technology

[0002] Aquaculture is a crucial part of the agricultural economy. In recent years, with the development of ecological transformation in fisheries, intensive aquaculture models such as indoor and factory farming have become increasingly mainstream. However, the accompanying aquatic disease problems have become increasingly prominent, posing a serious challenge to the aquaculture industry due to frequent disease outbreaks. Currently, the prevention and control of fish diseases still largely rely on chemical drugs. The overuse of chemical drugs may lead to food and environmental problems, as well as the potential dangers of antibiotic abuse, and does not align with the goals of modern aquaculture that are "efficient, safe, and environmentally friendly." Immunization is one of the effective means of preventing and controlling fish diseases. Oral immunization, as an immunization route, is convenient to use, causes minimal stress to fish populations, and is suitable for large-scale and repeated immunization, making it particularly suitable for the immunization of aquatic animals.

[0003] Current research indicates that the pinocytosis capacity of fish posterior intestinal cells enables the absorption and transport of soluble or particulate antigens, and allows for the rapid release of antigens into the intercellular space. These antigens can be presented by intraepithelial macrophages, making them a key effector site for intestinal mucosal immunity in fish. However, due to the disruptive environment of the fish digestive tract and the presence of various digestive enzymes, vaccine antigens are largely degraded before reaching the posterior intestinal mucosa. Even the small amount of antigen that reaches the mucosa is difficult for the immune system to efficiently recognize and capture, thus hindering the induction of a high-level mucosal immune response. To protect antigens from the disruptive digestive environment, combining vaccines with specific oral delivery systems to maintain stable antigen presence in the digestive tract, while simultaneously enhancing the immune system's response and improving antigen utilization efficiency, is essential.

[0004] Angelica sinensis polysaccharide is the main active ingredient in the traditional Chinese medicine Angelica sinensis, possessing biological activities such as immune enhancement, anti-oxidation, anti-tumor activity, and promotion of hematopoietic function. However, there are currently no successfully developed and applied Angelica sinensis polysaccharide-based vaccines for aquatic animals, and the efficacy of Angelica sinensis polysaccharide as a vaccine carrier in fish remains unknown. Summary of the Invention

[0005] To address the problems existing in the prior art, the first objective of this invention is to provide a vaccine based on an Angelica polysaccharide derivative delivery carrier and a method for preparing the same.

[0006] A second objective of the present invention is to provide the application of the vaccine in the preparation of fish farming-related products.

[0007] A third objective of this invention is to provide a fish feed in which the feed pellets contain the vaccine.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a vaccine based on an Angelica sinensis polysaccharide derivative delivery vector, comprising the following steps: (1) Stearic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in DMSO in a molar ratio of 1:1:1.2 and activated to obtain a reaction solution. The reaction solution was added dropwise to the DMSO solution of Angelica polysaccharide and reacted at room temperature. After the reaction was completed, it was added dropwise to anhydrous ethanol under ultrasonic conditions and the precipitate was collected by centrifugation. The precipitate was dispersed in water and dialyzed. After dialysis, it was freeze-dried to obtain the Angelica polysaccharide derivative. (2) Angelica polysaccharide derivatives were dispersed in sodium alginate solution to obtain an external aqueous phase; (3) The antigen was dissolved in PBS to obtain the inner aqueous phase; (4) Peanut oil and polyglycerol polyricinoleate are mixed to obtain the oil phase; (5) The aqueous and oil phases are mixed and sheared to obtain the primary emulsion; (6) Mix and stir the external aqueous phase and the colostrum to obtain a W / O / W type Pickering emulsion.

[0009] Preferably, in step (1), the final concentration of stearic acid in DMSO is 50 mmol / L; and the activation reaction time is 30 min.

[0010] Preferably, in step (1), the final concentration of Angelica polysaccharide in the DMSO solution is 10 mg / mL to 50 mg / mL; and the reaction time at room temperature is 36 h.

[0011] Preferably, in step (1), the ultrasonic power is 100~200W, the centrifugation conditions are 8000rpm for 10min; the molecular weight cutoff for dialysis is 8000~14000, and the time is 48h; the freeze-drying temperature is -50℃~-80℃, the vacuum degree is 2~10Pa, and the time is 24~48h.

[0012] Preferably, the concentration of sodium alginate solution in the external aqueous phase is 0.5%, and the concentration of Angelica polysaccharide derivative is 1 g / 100 mL; the concentration of antigen in the internal aqueous phase is 50 mg / mL; and the volume ratio of peanut oil to polyglycerol polyricinoleate in the oil phase is 10:1.

[0013] Preferably, the volume ratio of the internal aqueous phase to the oil phase is 2:8, 3:7, 4:6 or 5:5, the shearing speed is 1000 rpm, and the time is 2 min.

[0014] Preferably, the volume ratio of the promulgated emulsion to the external aqueous phase is 4:6, 5:5, 6:4 or 8:2, the stirring speed is 8000 rpm, and the stirring time is 1 min.

[0015] The present invention also provides a vaccine prepared by the above preparation method.

[0016] This invention also provides the application of the above-mentioned vaccine in the preparation of fish farming-related products.

[0017] The present invention also provides a fish feed, wherein the feed pellets contain the above-mentioned vaccine, and the vaccine content is 0.1% to 0.5% of the feed pellet mass.

[0018] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention uses Angelica sinensis polysaccharide derivatives as a delivery carrier, which has many advantages such as low cost, convenient extraction, good biocompatibility and tolerability, and ease of modification. The final W / O / W type Pickering emulsion has high encapsulation efficiency, good formulation stability, and no cytotoxicity. This invention improves vaccine safety and reduces antigen degradation in the digestive tract.

[0019] The present invention further provides the application of the vaccine in the preparation of fish farming-related products and a fish feed, wherein the feed pellets contain the vaccine, and the vaccine feed pellets stimulate higher levels of innate and adaptive immunity in fish, providing higher protection and showing good application prospects. Attached Figure Description

[0020] Figure 1 Example 1: Fourier transform infrared characterization results of ASP-SA powder and ASP powder.

[0021] Figure 2 Example 1: Morphological characterization results of ASP-SA.

[0022] Figure 3 Example 1: Measurement results of ASP-SA particle size and zeta potential.

[0023] Figure 4 Example 1: Morphology of the vaccine under an optical microscope.

[0024] Figure 5 Serum antibody levels of each group at different time points.

[0025] Figure 6 Results of immune-related enzyme activity assays in each group after immunization.

[0026] Figure 7 Expression levels of immune-related genes in each group.

[0027] Figure 8 : Display of contact angle measurement results for each group.

[0028] Figure 9 Statistical analysis of contact angle measurement results for each group.

[0029] Figure 10 Verification of results with different mixing ratios of internal water phase and oil phase.

[0030] Figure 11 : Validation of results with different ASP-SA concentrations.

[0031] Figure 12 : Validation of results with different mixing ratios of external aqueous phase and colostrum.

[0032] Figure 13 Example 4: Observation results of vaccine morphology.

[0033] Figure 14 Survival rate of each group of leopard gill spiny perch after being challenged with Streptococcus dolphinus. Detailed Implementation

[0034] This invention provides a method for preparing a vaccine based on an Angelica sinensis polysaccharide derivative delivery vector, comprising the following steps: (1) Stearic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in DMSO and activated to obtain a reaction solution. The reaction solution was added dropwise to the DMSO solution of Angelica polysaccharide and reacted at room temperature. After the reaction was completed, it was added dropwise to anhydrous ethanol under ultrasonic conditions and the precipitate was collected by centrifugation. The precipitate was dispersed in water and dialyzed. After the dialyzation was completed, it was freeze-dried to obtain the Angelica polysaccharide derivative.

[0035] In this invention, the molar ratio of stearic acid (SA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and 4-dimethylaminopyridine (DMAP) is 1:1:1.2; the final concentration of stearic acid in the DMSO is 50 mmol / L, the final concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 50 mmol / L, and the final concentration of 4-dimethylaminopyridine is 60 mmol / L; stearic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine are dissolved in dimethyl sulfoxide and activated at room temperature for 30 min, and this is referred to as the reaction solution.

[0036] In this invention, Angelica polysaccharide is dissolved in dimethyl sulfoxide (DMSO), and the final concentration of Angelica polysaccharide in the DMSO solution is 10 mg / mL to 50 mg / mL, preferably 20 mg / mL, 30 mg / mL or 40 mg / mL; the reaction solution is added dropwise to the DMSO solution of Angelica polysaccharide, and the reaction time at room temperature is preferably 36 h.

[0037] The ultrasonic power described in this invention is 100~200W, preferably 150W; the centrifugation conditions are 8000 rpm for 10 min. As an optional embodiment, the precipitate (white precipitate) collected by centrifugation is washed three times with anhydrous ethanol and then collected by centrifugation.

[0038] The precipitate collected in this invention is preferably dispersed in double-distilled water and transferred to a dialysis bag with a molecular weight cutoff of 8000~14000 for dialysis. The dialysis solution is preferably ultrapure water, and the dialysis time is preferably 48 hours. After dialysis, the product is freeze-dried to obtain white granular product, which is the Angelica polysaccharide derivative (which can be stored at 4°C for later use). The freeze-drying temperature in this invention is -50°C to -80°C, the vacuum degree is 2~10 Pa, and the time is 24~48 hours, preferably 36 hours.

[0039] This invention couples stearic acid to the sugar chain of Angelica sinensis polysaccharide to form an Angelica sinensis polysaccharide derivative (ASP-SA) that has both hydrophilic and hydrophobic properties.

[0040] (2) Angelica polysaccharide derivatives were dispersed in sodium alginate solution to obtain an external aqueous phase; In this invention, the concentration (mass-volume ratio, g / 100ml) of sodium alginate solution in the external aqueous phase is 0.5%, and the concentration of Angelica polysaccharide derivative is 1g / 100mL. As an optional embodiment, an appropriate amount of Angelica polysaccharide derivative is weighed and dispersed in a 0.5% sodium alginate solution, and ultrasonicated for 60s to ensure that the particles are fully dispersed in the aqueous phase. The ultrasonic power is preferably 100~200W.

[0041] (3) The antigen was dissolved in PBS to obtain the inner aqueous phase; The concentration of the antigen in the aqueous phase of this invention is 50 mg / mL. The antigen is a water-soluble antigen. As an optional embodiment, the antigen includes ovalbumin (OVA) and / or α-enolase antigen (ENO). The ovalbumin antigen may be purchased from Solarbio; the α-enolase antigen may be obtained by expression based on the α-enolase nucleic acid sequence (KF460454.1) of *Streptococcus dolphinus*, or by having Beijing Qingke Biotechnology Co., Ltd. synthesize plasmid pET32a-ENO (plasmid restriction site is...) based on the *Streptococcus dolphinus* α-enolase nucleic acid sequence (KF460454.1). Bam HI and XhoI), expressed using conventional methods in the field.

[0042] (4) Peanut oil and polyglycerol polyricinoleate are mixed to obtain the oil phase; In this invention, the volume ratio of peanut oil to polyglycerol polyricinoleate in the oil phase is 10:1. As an optional embodiment, 1 ml of polyglycerol ricinoleate (PGPR) is dissolved in 10 ml of peanut oil and thoroughly shaken to obtain the oil phase for later use.

[0043] (5) The aqueous and oil phases are mixed and sheared to obtain the primary emulsion; The volume ratio of the aqueous phase to the oil phase in this invention is 2:8, 3:7, 4:6, or 5:5. Preferably, the aqueous and oil phases are mixed and then subjected to stirring, shearing, and emulsification in a high-speed mixer to obtain a W / O type emulsion, which is the primary emulsion. The shearing speed in this invention is 1000 rpm, and the time is 2 minutes.

[0044] (6) Mix and stir the external aqueous phase and the colostrum to obtain a W / O / W type Pickering emulsion.

[0045] The volume ratio of the colostrum to the external aqueous phase in this invention is 4:6, 5:5, 6:4, or 8:2. After mixing, the external aqueous phase and colostrum are placed in a high-speed mixer and stirred until fully emulsified to obtain a W / O / W type Pickering emulsion, i.e., the finished vaccine. The stirring speed in this invention is 8000 rpm, and the stirring time is 1 minute.

[0046] The present invention also provides a vaccine prepared by the above preparation method.

[0047] This invention also provides the application of vaccines in the preparation of fish farming-related products, which preferably include feed and immunizing agents. As an optional implementation, this invention involves feeding the feed or immunizing agent prepared from the vaccine to the fish in small, frequent batches until the corresponding immunization dose is reached, ensuring that the immunization dose received by each fish is approximately consistent.

[0048] This invention also provides a fish feed, wherein the feed pellets contain the vaccine, and the vaccine content is 0.1% to 0.5% of the feed pellet mass. In this invention, the vaccine, carrying an antigen, is injected into the feed pellets, wherein the antigen concentration of the vaccine is not less than 10 mg / ml, and the volume of vaccine injected into each feed pellet is not more than 5 µl. Preferably, each feed pellet contains 40 mg of the vaccine and is a spherical pellet with a diameter of 3.0 to 4.5 mm. As an optional embodiment, this invention selects a general-purpose fish feed with a pellet size of 3.0 to 4.5 mm, hollows out the pellets with a needle, and injects the vaccine into the feed pellets at a specific dose through a syringe.

[0049] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0050] In a specific embodiment of the present invention, the α-enolase antigen is expressed based on the nucleic acid sequence information of Streptococcus dolphinii α-enolase (KF460454.1): Beijing Qingke Biotechnology Co., Ltd. was commissioned to design and synthesize plasmid pET32a-ENO (plasmid restriction site is KF460454.1) based on the nucleic acid sequence of Streptococcus dolphinus α-enolase. Bam HI and Xho I) After sequencing verification, the plasmid was heat-transformed (heat shock at 42℃ for 90 seconds) into E. coli BL21(DE3) chemically competent cells. The transformed E. coli were plated on LB agar containing ampicillin and incubated overnight at 37℃. Single clones were then picked for sequencing identification. Strains with correct sequencing were inoculated into LB liquid medium, with ampicillin added to a final concentration of 50 μg / mL. The medium was then cultured at 37℃ on a shaker at 160 rpm for expansion until the bacterial concentration reached OD500. 600 When the value is 0.8~1.0, add isopropyl thiogalactoside (IPTG) to a final concentration of 0.001 mol / L, and induce expression at 37℃ for 6 h.

[0051] Cell disruption and isolation of ENO antigen: The fermentation mixture was centrifuged at 8000 r / min at room temperature to collect the cells. The cells were washed three times with a equilibration buffer (20 mM phosphate, 500 mM NaCl, 10 mM imidazole, pH 7.4). The collected moist cells were resuspended with 10 mL of equilibration buffer per 1 g of moist cells and sonicated under ice bath conditions. The sonicated bacterial solution was placed at 4°C and centrifuged at 12000 r / min for 5 min to collect the supernatant, which yielded a solution of contaminating proteins containing ENO antigen.

[0052] Purification of ENO antigen: Purification was performed using a Ni-NTA gravity column (Shanghai Sangon Biotech Co., Ltd.). First, the column was equilibrated with equilibration buffer at a flow rate of 1 ml / min, allowing the buffer to slowly drain from the resin. Then, two column volumes of contaminating protein solution containing ENO antigen were added to the column, followed by washing with two column volumes of equilibration buffer. Elution was then performed with two column volumes of elution buffer (20 mM phosphate, 500 mM NaCl, 250 mM imidazole, pH 7.4), and the protein solution was collected. 30 mL of purified protein solution was placed in a dialysis bag with a molecular weight cutoff of 8000–14000 and dialyzed in 5 L of PBS buffer at 4°C for 24 h, with the buffer changed every 6 h. The final purified ENO antigen solution was obtained. The obtained ENO antigen was detected by SDS-PAGE. When the result showed a protein size of approximately 66 kDa and the purified antigen solution was free of contaminating proteins, it was ready for use.

[0053] Unless otherwise specified, the following embodiments are all conventional methods.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0055] Example 1 A method for preparing a vaccine based on an Angelica polysaccharide derivative delivery vector: (1) Weigh 1 mmol stearic acid, 1 mmol 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.2 mmol 4-dimethylaminopyridine and dissolve them in 20 mL The reaction solution was activated in DMSO at room temperature for 30 min to obtain a reaction solution. 500 mg of Angelica polysaccharide was dissolved in 20 mL of dimethyl sulfoxide. The reaction solution was added dropwise to the DMSO solution of Angelica polysaccharide and reacted at room temperature for 36 h. After the reaction was completed, anhydrous ethanol was added dropwise under ultrasonic conditions (150 W). The mixture was centrifuged at 8000 rpm for 10 min to collect the white precipitate. The precipitate was washed three times with anhydrous ethanol and centrifuged at 8000 rpm for 10 min to collect the precipitate. The precipitate was dispersed in double-distilled water and transferred to a dialysis bag with a molecular weight cutoff of 8000~14000 for dialysis for 48 h. After dialysis, the precipitate was freeze-dried (-80℃, vacuum degree of 6 Pa, time for 48 h) to obtain a white granular product, which is the Angelica polysaccharide derivative (ASP-SA), and stored at 4℃ for later use.

[0056] (2) Weigh an appropriate amount of Angelica polysaccharide derivative and disperse it in sodium alginate solution. Sonicate for 60s to fully disperse the particles and obtain an external aqueous phase. The ultrasonic power is 150W, the concentration of sodium alginate solution is 0.5%, and the concentration of Angelica polysaccharide derivative is 1g / 100mL.

[0057] (3) Weigh an appropriate amount of ovalbumin antigen (OVA, purchased from Solarbio) and dissolve it in PBS to obtain an inner aqueous phase; the ovalbumin concentration is 50 mg / mL.

[0058] (4) Dissolve 1 ml of polyglycerol ricinoleate (PGPR) in 10 ml of peanut oil and shake well to form the oil phase.

[0059] (5) The aqueous phase and oil phase are mixed at a volume ratio of 3:7 and then placed in a high-speed mixer for stirring and shearing emulsification to obtain a W / O type emulsion, which is the primary emulsion. The shearing speed is 1000 rpm and the time is 2 min.

[0060] (6) The colostrum and external aqueous phase were mixed at a volume ratio of 6:4 and then placed in a high-speed mixer for stirring to fully emulsify and obtain a W / O / W type Pickering emulsion, which is the finished vaccine. The stirring speed was 8000 rpm and the time was 1 min.

[0061] Experimental Example 1 1. Characterization of the Angelica polysaccharide derivative (ASP-SA) prepared in Example 1: (1) Fourier transform infrared characterization of ASP-SA ASP-SA powder and ASP (Angelica polysaccharide) powder were compressed into tablets, and their analysis was performed using Fourier transform infrared spectroscopy. The results are as follows: Figure 1 As shown, compared with ASP, ASP-SA retains the OH stretching vibration peak on the ASP sugar chain, and also exhibits a peak at 2851 cm⁻¹. -1 Nearby, a methylene peak representing the carbon chain of stearic acid also appeared at 1744 cm⁻¹. -1 Nearby, an ester bond peak appeared, indicating that stearic acid was successfully grafted onto the inulin backbone molecule. This series of peak changes indicates that the stearic acid modification of Angelica polysaccharide was successful.

[0062] (2) Morphological characterization of ASP-SA The microstructure of ASP-SA was observed using a desktop scanning electron microscope. ASP-SA powder was adhered to a conductive adhesive, and the sample was sputter-coated with gold. The morphology of the ASP-SA particles was then observed at ideal magnification under an accelerating voltage of 20 kV. The results are as follows: Figure 2 As shown in the figure, the scale bar is 1000 nm. Based on the microstructure of ASP-SA, it can be seen that the ASP-SA particles are spherical and their size is between 300 and 4000 nm.

[0063] (3) Measurement of ASP-SA particle size and zeta potential ASP-SA was dispersed in deionized water, and the particle size and zeta potential of the sample were determined using a nanoparticle size analyzer and a zeta potential analyzer. The results are as follows: Figure 3As shown in the figure, A represents the nanoparticle size and B represents the zeta potential. The figure shows that the average particle size of ASP-SA particles is between 536.87 ± 29.92 nm, and the zeta potential is approximately -29.90 ± 1.14 mV.

[0064] 2. The vaccine prepared in Example 1 was observed and tested: (1) Morphological observation of ASP-SA stable Pickering emulsion oral vaccine The prepared oral vaccine was diluted 50 times in pure water. An appropriate amount was placed on a glass slide and observed under an inverted microscope. The results were as follows: Figure 4 As shown in the figure, with a scale bar of 10 μm, the emulsion morphology consists of microspheres ranging from 1 to 10 micrometers in size. The solid particles adsorbed on the surface of the microspheres are tightly packed to form a dense interfacial film. An aqueous phase structure containing antigens can be seen inside the microspheres. Overall, the emulsion microspheres are uniformly distributed without aggregation, indicating good overall stability.

[0065] (2) Vaccine antigen encapsulation rate test Dilute the above vaccine solution in PBS at a ratio of 1:100 and mix thoroughly. Let stand for 5 hours. Carefully aspirate 200 μL of the aqueous phase (W1) from the above mixture, being careful not to aspirate the vaccine microspheres. Freeze the remaining mixture at -20°C to demulsify the vaccine microspheres. After thawing, let stand for 5 hours and carefully aspirate the aqueous phase (W2). Measure the protein concentration in W1 and W2 using a BCA protein assay kit, and calculate the antigen encapsulation efficiency (n=3). The results are shown in Table 1. The results show that the average encapsulation efficiency is 44.37%.

[0066] The formula for calculating antigen encapsulation efficiency is: EE% = ((W2 - W1) / W2) × 100% Table 1. Results of vaccine antigen encapsulation rate test

[0067] Example 2 A fish feed, wherein the feed pellets contain the vaccine prepared in Example 1.

[0068] Preparation method: Select general-purpose fish feed (expanded marine fish feed, 40mg per pellet, purchased from Fuzhou Seahorse Feed Co., Ltd.) with a pellet size of 3.0~4.5mm, and hollow out the inside of each pellet with a needle. Inject the vaccine into the feed pellets at a certain dose through a syringe, with each pellet containing a vaccine dose equivalent to 25µg OVA.

[0069] Example 3 A fish feed, wherein the feed pellets contain the vaccine prepared in Example 1.

[0070] Preparation method: Select general-purpose fish feed (expanded marine fish feed, 40mg per pellet, purchased from Fuzhou Seahorse Feed Co., Ltd.) with a pellet size of 3.0~4.5mm, and hollow out the inside of each pellet with a needle. Inject the vaccine into the feed pellets at a certain dose through a syringe, with each pellet containing a vaccine dose equivalent to 50µg OVA.

[0071] Experimental Example 2 1. Immunization with ASP-SA stabilized Pickering emulsion vaccine (using feed prepared in Examples 2-3): 280 healthy grouper weighing 9-10g were taken and divided into 7 groups of 40 each. One group served as the control group, and the rest as the experimental groups. The grouper were fed vaccine-containing feed pellets in batches. The feeding methods for each group were as follows: PBS control group (or PBS): fed with a diet containing an equal volume of PBS. The volume is calculated based on the volume used in the highest dose immunization group.

[0072] ASP-SA Empty Carrier Group (ASP-SA PE): Feed containing an equal volume of antigen-free empty emulsion carrier. Volume is calculated based on the volume used in the maximum dose immunization group.

[0073] OVA Immunization Group (OVA): Feed containing pure OVA antigen. Each pellet contains 50µg of OVA antigen, given in 8 divided doses, for a total immunization dose of 40µg / g.

[0074] ASP-SA@OVA 5µg / g Immunization Group (ASP-SA@OVA 5µg / g): Feed containing ASP-SA@OVA emulsion vaccine was administered. Each feed pellet contained 25µg of OVA, and the feed was given in two doses. The total immunization dose was 5µg / g.

[0075] ASP-SA@OVA 10µg / g Immunization Group (ASP-SA@OVA 10µg / g): Feed containing ASP-SA@OVA emulsion vaccine was administered. Each feed pellet contained 25µg of OVA, and the feed was administered in 4 divided doses. The total immunization dose was 10µg / g.

[0076] ASP-SA@OVA 20µg / g Immunization Group (ASP-SA@OVA 20µg / g): Feed containing ASP-SA@OVA emulsion vaccine was administered. Each feed pellet contained 50µg of OVA, and the feed was given in 4 divided doses. The total immunization dose was 20µg / g.

[0077] ASP-SA@OVA 40µg / g Immunization Group (ASP-SA@OVA 40µg / g): Feed containing ASP-SA@OVA emulsion vaccine was administered. Each feed pellet contained 50µg of OVA, and the feed was given in 8 divided doses. The total immunization dose was 40µg / g.

[0078] 2. Evaluation of vaccine immunization efficacy (1) Serum antibody titer detection Blood was collected from each group of spotted lemurs on days 7, 14, 21, 28, and 35 post-immunization and incubated overnight at 4°C. The next day, the serum supernatant was collected by centrifugation at 5000g for 10 minutes at 4°C. The serum was diluted 1:500 using coating buffer (Solepro ELISA coating buffer) and 100µL was added to each well of a 96-well microplate, incubated overnight at 4°C. After incubation, the liquid in the wells was discarded, and 5% fetal bovine serum was added for blocking at 37°C for 1 hour. After blocking, the plates were washed three times with TBST for 3 minutes each. Horseradish peroxidase-labeled ovalbumin (OVA) was diluted according to the instructions of the Horseradish Peroxidase-Labeled OVA Kit (Solepro) and 100µL was added to each well of a 96-well microplate, incubated at 37°C for 50 minutes, and then washed three times with TBST for 3 minutes each. Add 100 µL of TMB colorimetric solution to each well and react at 37 °C for 15 min. Then add 50 µL of stop solution to each well to terminate the reaction and measure the absorbance at 450 nm.

[0079] The results are as follows Figure 5 As shown in the figure, the serum antibody levels in each emulsion vaccine immunization group gradually increased over time, exhibiting a dose-gradient effect. The levels peaked on day 28 post-immunization, began to decline on day 35, but remained at a relatively high level. Furthermore, under the same dosage, the antibody levels in the emulsion vaccine immunization group were higher than those in the pure antigen immunization group.

[0080] (2) Detection of immune-related physiological indicators Blood samples were collected from each group of *Stellaria media* on days 3, 7, and 14 post-immunization. The collected blood was incubated overnight at 4°C. The following day, the serum supernatant was collected by centrifugation at 5000g for 10 minutes at 4°C. Superoxide dismutase (SOD), acid phosphatase (ACP), and alkaline phosphatase (AKP) activities were measured using a superoxide dismutase activity assay kit (Nanjing Jiancheng Engineering Research Institute), an acid phosphatase activity assay kit (Nanjing Jiancheng Engineering Research Institute), and an alkaline phosphatase activity assay kit (Nanjing Jiancheng Engineering Research Institute). The procedures were performed according to the kit instructions.

[0081] The results are as follows Figure 6 As shown in the figure, on day 3 post-immunization, the enzyme activity levels in each emulsion vaccine immunization group were significantly higher than those in the control group, exhibiting a dose-gradient effect. At the same concentration, the enzyme activity levels in the emulsion vaccine immunization group were higher than those in the pure antigen group.

[0082] (3) Detection of expression levels of immune-related genes Spleen tissues from *Symplocos edulis* var. *mongolica* were collected on days 3, 14, and 28 post-immunization to extract RNA, which was then reverse transcribed into cDNA. Real-time quantitative PCR was performed according to the kit (ChamQ SYBR Color qPCR Master Mix, Nanjing Novizan). The relevant genes and primers used for detection are shown in the table below.

[0083] Table 2 Primer sequences for quantitative PCR detection

[0084] The results are as follows Figure 7 As shown in the figure, A represents the relative gene expression level on day 3 after immunization, B represents the relative gene expression level on day 14 after immunization, and C represents the relative gene expression level on day 28 after immunization. On day 3 post-immunization, all emulsion vaccine immunization groups showed varying degrees of increase in immune genes related to innate immunity, reflecting the rapid response of the immune system after vaccination and the initiation of the corresponding immunization program. On day 14 after immunization, all emulsion vaccine immunization groups showed varying degrees of increase in immune-related genes compared to the control group, reflecting that both innate and adaptive immunity were activated to varying degrees and interacted with each other. On day 28 after immunization, all emulsion vaccine immunization groups continued to show varying degrees of change compared to the control group, especially with a significant increase in immunoglobulin genes reflecting adaptive immunity, exhibiting a clear dose gradient effect.

[0085] Experimental Example 3 1. An investigation into the dosage of stearic acid (SA) The amounts of stearic acid were 0.5, 1, 2, 4, and 6 mmol, respectively, and all other steps were the same as in Example 1.

[0086] The contact angle of the prepared Angelica polysaccharide derivative (ASP-SA) was measured, and the results are as follows: Figures 8-9 As shown. Figure 8 The contact angle measurement results for each group are displayed. Figure 9 The contact angle measurements for each group were statistically analyzed. The results showed that as the amount of stearic acid increased, the contact angle first increased, then decreased, and then increased again, with 1 mmol of stearic acid being the most suitable.

[0087] 2. To investigate the mixing ratio of the internal aqueous phase and oil phase. The volume ratios of the internal aqueous phase and oil phase were set to 2:8, 3:7, 4:6, and 5:5, respectively, and all other steps were the same as in Example 1.

[0088] The morphology of the stable Pickering emulsion oral vaccine for ASP-SA was observed using the method described in Example 1. The results are as follows: Figure 10 As shown, the scale bar is 10µm. In the figure, 0d represents the morphology of the vaccine immediately after preparation, 1d represents the morphology of the vaccine after one day, and 3d represents the morphology of the vaccine after three days. The results show that as the proportion of oil phase increases, the droplet surface is covered by more oil phase, resulting in an increase in the number and size of multiple droplets. Conversely, as the proportion of water phase increases, the droplet fluidity increases, making it less susceptible to being encapsulated by the oil phase, thus reducing the number of emulsion droplets. Based on this, observation of microscopic images on days 0, 1, and 3 after emulsion preparation revealed that when the water-oil ratio was 3:7, the droplet size and number showed little difference and were relatively stable.

[0089] 3. Investigate the concentration of the external aqueous phase. The concentrations of Angelica polysaccharide derivatives in the external aqueous phase were set to 1 g / 100 mL (1%), 2 g / 100 mL (2%), 4 g / 100 mL (4%), and 8 g / 100 mL (8%), respectively, and the other steps were the same as in Example 1.

[0090] The morphology of the stable Pickering emulsion oral vaccine for ASP-SA was observed using the method described in Example 1. The results are as follows: Figure 11 As shown, the scale bar is 20 µm. The results indicate that the aggregation of polysaccharides in the emulsion gradually increases with increasing particle concentration. At particle concentrations of 4% and 8%, clumps of polysaccharide particles were observed in the emulsion, resulting in poor particle dispersion. In contrast, a particle concentration of 2% achieved sufficient droplet coverage while preventing the aggregation of excess polysaccharides, demonstrating better emulsion stability.

[0091] 4. To investigate the mixing ratio of the external aqueous phase and colostrum. The volume ratios of the promulgated emulsion and the external aqueous phase were set to 4:6, 5:5, 6:4, and 8:2, respectively, and all other steps were the same as in Example 1.

[0092] The morphology of the stable Pickering emulsion oral vaccine for ASP-SA was observed using the method described in Example 1. The results are as follows: Figure 12 As shown, the scale bar is 10µm. The 0d group represents the morphology of the vaccine immediately after preparation, the 1d group represents the morphology of the vaccine after one day, and the 3d group represents the morphology of the vaccine after three days. The results show that as the aqueous phase increases, emulsion droplets tend to aggregate and fuse, leading to demulsification; increasing the emulsion phase results in larger emulsion droplets with insufficient solid particle coverage. Overall, when the ratio of initial emulsion to external aqueous phase is 6:4, the emulsion droplet size and stability are relatively good.

[0093] Example 4 A method for preparing a vaccine based on an Angelica polysaccharide derivative delivery vector: (1) Same as Example 1.

[0094] (2) Weigh an appropriate amount of Angelica polysaccharide derivative and disperse it in sodium alginate solution. Sonicate for 60s to fully disperse the particles and obtain an external aqueous phase. The ultrasonic power is 150W, the concentration of sodium alginate solution is 0.5%, and the concentration of Angelica polysaccharide derivative is 2g / 100mL.

[0095] (3) Weigh an appropriate amount of α-enolase antigen and dissolve it in PBS to obtain an inner aqueous phase; the antigen concentration is 20 mg / mL.

[0096] (4) Dissolve 1 ml of polyglycerol ricinoleate (PGPR) in 10 ml of peanut oil and shake well to form the oil phase.

[0097] (5) The aqueous phase and oil phase are mixed at a volume ratio of 3:7 and then placed in a high-speed mixer for stirring and shearing emulsification to obtain a W / O type emulsion, which is the primary emulsion. The shearing speed is 1000 rpm and the time is 2 min.

[0098] (6) The external aqueous phase and colostrum were mixed at a volume ratio of 4:6 and then placed in a high-speed mixer for stirring to fully emulsify and obtain a W / O / W type Pickering emulsion, which is the finished vaccine. The stirring speed was 8000 rpm and the time was 1 min.

[0099] The morphology of the vaccine loaded with α-enolase (ENO) antigen was observed using the method described in Example 1. The results are as follows: Figure 13 As shown, the scale bar is 10µm. The results show that the emulsion droplets are mainly spherical or nearly spherical in shape, exhibiting a typical three-phase two-film structure. The solid particles adsorbed on the droplet surface are clearly visible, and these particles are closely arranged at the oil-water interface, forming a dense covering film. The emulsion system is stable and in good overall condition.

[0100] Example 5 A fish feed, wherein the feed pellets contain the vaccine prepared in Example 4.

[0101] Preparation method: Select general-purpose fish feed (expanded marine fish feed, 40mg per pellet, purchased from Fuzhou Seahorse Feed Co., Ltd.) with a pellet size of 3.0~4.5mm, and hollow out the inside of each pellet with a needle. Inject the vaccine into the feed pellets in a certain dose through a syringe, with each pellet containing 50µg of ENO equivalent vaccine.

[0102] Test Example 4 ASP-SA stable Pickering emulsion oral vaccine (encapsulated with α-enolase antigen) immunization: Ninety healthy leopard-gill spiny perch weighing 9-10g were collected and divided into three groups of 30 each. One group served as the control group, and the rest as the experimental groups. Vaccine-containing feed pellets were fed to each group in batches. The feeding methods for each group were as follows: PBS (control group): Feed containing an equal volume of PBS was administered. The volume is calculated based on the volume used in the highest dose immunization group.

[0103] ENO (experimental group): Feed containing pure OVA antigen was administered. Each pellet contained 50µg of ENO antigen, administered in 8 divided doses, with a total immunization dose of 40µg / g.

[0104] ASP-SA@ENO (Experimental Group): Feed containing ASP-SA@OVA emulsion vaccine was administered. Each feed pellet contained 50µg of ENO, and the feed was given in 8 divided doses. The total immunization dose was 40µg / g.

[0105] On day 14 post-immunization, the leopard-gill sea bass in each group were challenged with *Streptococcus dolphinus*. The *Streptococcus dolphinus* suspension was administered intraperitoneally at a concentration of 1×10⁻⁶. 5 CFU / ml, dose 100µL / fish. Morbidity and survival of the leopard-gill spiny perch were recorded over 14 days. Survival rate was calculated, and relative percentage survival (RPS) was determined. The formula was: RPS = (1 - mortality rate in the immunized group / mortality rate in the control group) × 100%. Survival rate results for each group are shown below. Figure 14 As shown, the relative protection rate (RPS) of the ENO group was 38.46%, and the relative protection rate (RPS) of the ASP-SA@ENO group was 69.24%. The results indicate that the ASP-SA@ENO group had a better immune response.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a vaccine based on an Angelica sinensis polysaccharide derivative delivery carrier, characterized in that, Includes the following steps: (1) Stearic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in DMSO in a molar ratio of 1:1:1.2 and activated to obtain a reaction solution. The reaction solution was added dropwise to the DMSO solution of Angelica polysaccharide and reacted at room temperature. After the reaction was completed, it was added dropwise to anhydrous ethanol under ultrasonic conditions and the precipitate was collected by centrifugation. The precipitate was dispersed in water and dialyzed. After dialysis, it was freeze-dried to obtain the Angelica polysaccharide derivative. The final concentration of stearic acid in the DMSO is 50 mmol / L; the final concentration of Angelica polysaccharide in the DMSO solution is 10 mg / mL to 50 mg / mL. (2) Angelica polysaccharide derivatives were dispersed in sodium alginate solution to obtain an external aqueous phase; (3) The antigen was dissolved in PBS to obtain the inner aqueous phase; (4) Peanut oil and polyglycerol polyricinoleate are mixed to obtain the oil phase; (5) The aqueous and oil phases are mixed and sheared to obtain the primary emulsion; (6) Mix and stir the external aqueous phase and the colostrum to obtain a W / O / W type Pickering emulsion; The concentration of sodium alginate solution in the external aqueous phase is 0.5%, and the concentration of Angelica polysaccharide derivative is 1 g / 100 mL; the concentration of antigen in the internal aqueous phase is 50 mg / mL; the volume ratio of peanut oil and polyglycerol polyricinoleate in the oil phase is 10:

1. The antigen is ovalbumin or α-enolase antigen.

2. The preparation method according to claim 1, characterized in that, In step (1), the activation reaction takes 30 minutes.

3. The preparation method according to claim 1, characterized in that, In step (1), the room temperature reaction time is 36 hours.

4. The preparation method according to claim 1, characterized in that, In step (1), the ultrasonic power is 100~200W, the centrifugation conditions are 8000rpm for 10min; the molecular weight cutoff for dialysis is 8000~14000, and the time is 48h; the freeze-drying temperature is -50℃~-80℃, the vacuum degree is 2~10Pa, and the time is 24~48h.

5. The preparation method according to claim 1, characterized in that, The volume ratio of the internal aqueous phase to the oil phase is 2:8, 3:7, 4:6, or 5:5, the shearing speed is 1000 rpm, and the time is 2 min.

6. The preparation method according to claim 1, characterized in that, The volume ratio of the primary emulsion to the external aqueous phase is 4:6, 5:5, 6:4, or 8:2, the stirring speed is 8000 rpm, and the stirring time is 1 min.

7. The vaccine prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the vaccine according to claim 7 in the preparation of related products that improve the immune level of fish, characterized in that, The relevant products are feed or immune preparations.

9. A fish feed, characterized in that, The feed pellets contain the vaccine as described in claim 7, wherein the vaccine content is 0.1% to 0.5% of the feed pellet mass.

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