A subunit nanovaccine of Aeromonas hydrophila, its preparation method and application

Abstract

This invention discloses a subunit nanovaccine of Aeromonas hydrophila, its preparation method, and its application, belonging to the field of biological immunology. The invention uses cationic lipids, hydrophobic stabilizers, and surfactants to prepare a cationic nanovaccine by co-loading Aeromonas hydrophila OMPP5 recombinant protein antigen and an immune adjuvant through a double emulsification process. Immersion administration promotes the uptake of the nanovaccine by fish gills, thereby improving drug utilization. Its application in largemouth bass farming shows strong immunoprotective effects. This nanovaccine is convenient to prepare, simple to use, and exhibits stable immunogenicity and high protection rate.

Description

Technical Field

[0001] This invention relates to a subunit nanovaccine of Aeromonas hydrophila, its preparation method and application, belonging to the field of biological immunology. Background Art

[0002] Aquaculture meets nearly half of the world's fish and shellfish consumption needs. The green and healthy development of aquaculture is a crucial guarantee for food security and is closely related to human health. Disease is one of the major factors restricting the development of aquaculture. Existing research shows that disease losses in aquatic animals account for 15% of total fish production in my country. Aeromonas hydrophila (… Aeromonas hydrophila Aeromonas hydrophila is one of the important pathogenic microorganisms causing diseases in aquatic animals, and it is widely distributed in various aquatic environments. Fish affected by Aeromonas hydrophila mainly include largemouth bass, carp, and tilapia. The colonization and infection of the host by Aeromonas hydrophila are influenced by its structural components, toxins, and extracellular products, either individually or collectively, and are also affected by the host's immune system. Symptoms of fish diseases caused by Aeromonas hydrophila infection include septicemia, red fin disease, and enteritis.

[0003] For decades, vaccines have played a crucial role in controlling infectious diseases in fish. In aquaculture, they protect fish from infectious diseases, effectively reduce antibiotic use, and avoid the risk of antibiotic-induced bacterial resistance. Currently, methods of vaccinating fish include oral administration, immersion administration, and injection. Immersion administration involves applying the vaccine to the surface of the fish. The fish ingests the antigen through its gills, skin, and lateral line, stimulating mucosal immunity and inducing a local mucosal immune response. This leads to the local induction and production of mucosal IgT and systemic IgM, thereby achieving an immunization effect. It has been proven effective in preventing diseases caused by various fish viruses and bacteria. Immersion vaccines can be administered by briefly immersing the fish in a concentrated vaccine solution or by spraying the vaccine solution onto the fish. Immersion administration is suitable for large-scale vaccination of young fish that cannot be vaccinated in high-throughput injections, and it causes minimal stress to the fish. However, the development of immersion vaccines faces several challenges, resulting in relatively slow research progress. First, due to the low uptake efficiency of antigens on mucous membranes, immersion immunization requires a large amount of antigen protein. Second, antigens are unstable in water and easily decomposed, making immersion vaccines less effective than intraperitoneal vaccines. Existing research indicates that subunit antigens have advantages such as convenient production, large-scale preparation through fermentation expression, and higher safety, making them suitable for immersion vaccine development. Currently, research on Aeromonas hydrophila subunit antigens mainly focuses on outer membrane proteins (OMPs). OMPs are epitopes exposed on the cell surface, making them more likely to interact with the host immune system and thus good candidates for vaccine development. OMP P5, one of the OMPs of Aeromonas hydrophila, has high immunogenicity, can induce a strong immune response, and exhibits good immunoprotective effects. However, subunit antigens have low immunogenicity and are unstable, requiring adjuvants and effective delivery systems to enhance their drug-likeness. Nanoparticle vaccines formed by co-loading subunit antigens and adjuvants using nanodelivery technology offer advantages such as protecting the antigen from enzymatic degradation in water, enhancing the drug-likeness of the adjuvant, and promoting uptake and presentation by antigen-presenting cells. Summary of the Invention

[0004] This invention combines the characteristics of subunit antigens, nanodelivery technology, and immersion inoculation. Based on the principle that biological mucosal tissues are mainly composed of negatively charged proteins and other biological macromolecules, and that preparing positively charged nanovaccines can promote the uptake of vaccines by fish and thus improve vaccine utilization, a *Aeromonas hydrophila* subunit nanovaccines, their preparation method, and applications have been developed.

[0005] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures described herein are all standard procedures widely used in the relevant fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0006] As used herein, the term “particle” refers to a geometric shape within a certain size range, characterized by the presence of discrete particles, pellets, beads, or clumps, regardless of their size, shape, or morphology.

[0007] As used in this article, the term "nanoparticle" refers to a particle with a size (i.e., the diameter in the longest dimension of the particle) of less than 100 nanometers.

[0008] As used in this article, the term "particle size" is equivalent to "equivalent particle size," which means that when a certain physical property or physical behavior of the particle being tested is most similar to that of a homogeneous sphere (or combination) of a certain diameter, the diameter of that sphere (or combination) is taken as the equivalent particle size (or particle size distribution) of the particle being tested.

[0009] As used herein, the term "average particle size" refers to the diameter of an actual particle swarm consisting of particles of different sizes and shapes, compared to an imaginary particle swarm consisting of uniform spherical particles, if the total length of the particles is the same. Methods for measuring average particle size are known to those skilled in the art, such as light scattering; instruments for measuring average particle size include, but are not limited to, Malvern particle size analyzers.

[0010] As used in this article, the term "room temperature" refers to 25 ± 5 °C.

[0011] As used herein, the term "immune adjuvant" refers to a substance that, when administered to the body together with or before an antigen, enhances immunogenicity or alters the type of immune response. Immunoadjuvants may or may not be immunogenic.

[0012] As used herein, the term "antigen" or "immunogen" refers to a substance capable of inducing a specific immune response in a host. Antigens can include whole organisms (e.g., inactivated, attenuated, or live organisms); subunits or portions of an organism; recombinant vectors containing immunogenic inserts; portions or fragments of DNA that can induce an immune response upon presentation to a host; proteins, glycoproteins, lipoproteins, polypeptides, peptides, antigenic epitopes, haptens, toxins, antitoxins, or any combination thereof.

[0013] This invention selects biocompatible cationic lipid materials, PLGA and the electrically neutral surfactant DSPE-PEG as modifying materials, uses recombinant protein OMP P5 as the antigen, and common molecular adjuvants as immune agonists to prepare three novel nanovaccines suitable for immersion immunization. The protective efficacy of the novel nanovaccines was evaluated by statistically analyzing the relative survival rate of largemouth bass after immersion immunization. The data indicate that the OMP P5 recombinant protein nanovaccines can serve as candidate vaccines for preventing Aeromonas hydrophila infection in largemouth bass.

[0014] The purpose of this invention is to overcome the shortcomings and defects of existing fish subunit vaccines and to provide a novel fish subunit nano-vaccine.

[0015] Another object of the present invention is to provide a method for preparing the novel nanovaccine.

[0016] Another object of the present invention is to provide the application of the novel nanovaccine.

[0017] The above-mentioned objective of this invention is achieved through the following technical solution:

[0018] This invention provides a nano-vaccine comprising OMP P5 protein, an immune adjuvant, a cationic polymer, a surfactant, and a hydrophobic stabilizer.

[0019] In one embodiment, the amino acid sequence of the OMP P5 protein is GenBank ABK37458.1.

[0020] In one embodiment, the immune adjuvant is selected from one or more of poly(I:C), CpG, MPLA, and squalene.

[0021] In one embodiment, the immune adjuvant is CpG and / or MPLA.

[0022] OMP P5 is an antigen exposed on the cell surface that can interact with the host immune system. It possesses high immunogenicity and can induce a strong immune response, thus producing a high level of immune protection. Studies have shown that OMP P5 has a good preventive effect against Aeromonas hydrophila infection in largemouth bass. This invention utilizes electrostatic interactions to load OMPP5 protein and anionic hydrophilic adjuvants onto cationic phospholipids, stabilizing the particles with a hydrophobic stabilizer. The hydrophobic adjuvant is loaded simultaneously with the hydrophobic stabilizer and modified using a surfactant to form a nanovaccine containing OMP P5 protein and a specific immune adjuvant. Compared to OMP P5 protein alone, the nanovaccine of this invention produces a superior immune effect.

[0023] The term "encapsulation" is not limited to completely placing the OMP P5 protein and immune adjuvant inside the nanovaccine. In the nanovaccine of the present invention, the OMP P5 protein and immune adjuvant may be entirely located inside the nanovaccine or partially located on the surface of the nanovaccine.

[0024] In one embodiment, the cationic polymer is selected from one or more of the following: trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), trimethyl-2,3-diolenoyloxypropylammonium bromide (DOTMA), dimethyl-2,3-diolenoyloxypropyl-2-(2-spermineformylamino)ethylammonium trifluoroacetate (DOSPA), dimethyl bis(octadecyl)ammonium bromide (DDAB), trimethyl dodecylammonium bromide (DTAB), trimethyl tetradecylammonium bromide (TTAB), trimethyl hexadecylammonium bromide (CTAB), 1,2-dioleoyl-3-succinyl-sn-glycerolcholine ester (DOSC), 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl]cholesterol (DC-Chol), and stearylamine (SA).

[0025] In one embodiment, the cationic polymer is DOTAP.

[0026] In one embodiment, the hydrophobic stabilizer is selected from one or more of polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), polyethylene oxide (PEO), and polylactic acid (PLA).

[0027] In one embodiment, the hydrophobic stabilizer is PLGA.

[0028] In one embodiment, the surfactant is selected from one or more of distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG-2000), PF68, and PF127.

[0029] In one embodiment, the surfactant is DSPE-PEG-2000.

[0030] In one embodiment, the nanovaccine is approximately spherical.

[0031] In one embodiment, the particle size of the nanovaccine is 50-200 nm.

[0032] In one embodiment, the mass ratio of OMP P5 protein: immune adjuvant: cationic lipid: hydrophobic stabilizer: surfactant is 10~30:5~20:3~12:20~50:40~80.

[0033] In one embodiment, when the adjuvant is Poly(I:C) and CpG, the mass ratio of OMP P5 protein: immune adjuvant: cationic lipid: hydrophobic stabilizer: surfactant is 20:10:9:45:60; when the adjuvant is MPLA and squalene, the mass ratio of OMP P5 protein: immune adjuvant: cationic lipid: hydrophobic stabilizer: surfactant is 20:1:7:40:60.

[0034] The present invention also provides a method for preparing the above-mentioned nano-vaccine, the method comprising the following steps:

[0035] (1) Mix one volume of OMP P5 protein with anionic hydrophilic adjuvant, stir magnetically at 600 rpm, add dropwise to 10-25 volumes of cationic lipid solution, mix for 10 minutes, and crosslink to obtain a mixture; or, mix one volume of OMP P5 protein with hydrophobic adjuvant, stir magnetically at 600 rpm, add dropwise to 15-25 volumes of cationic lipid solution, mix for 10 minutes, and crosslink to obtain a mixture.

[0036] (2) Add 12-25 volumes of hydrophobic stabilizer or a hydrophobic stabilizer solution containing hydrophobic adjuvant to the mixture formed in step 1 while stirring to obtain nanoparticles.

[0037] (3) Add the nanoparticles obtained in step (2) to 50-150 volumes of surfactant, stir for 5 minutes, and then dialyze to remove the organic solvent.

[0038] In one embodiment, OMP P5 protein is dissolved in a buffer solution to obtain a 10-30 mg / mL OMP P5 protein solution; cationic lipids are dissolved in an organic solvent to obtain a 3-12 mg / mL cationic lipid solution; a hydrophobic adjuvant is dissolved in an organic solvent to obtain a 5-20 mg / mL hydrophobic adjuvant; a hydrophobic stabilizer is dissolved in an organic solvent to obtain a 20-50 mg / mL hydrophobic stabilizer solution; and a surfactant is dissolved in water to obtain a 20-50 mg / mL surfactant solution.

[0039] In one embodiment, the buffer solution is a PBS salt solution with pH=8.0.

[0040] In one embodiment, the hydrophobic stabilizer is dissolved in DMSO.

[0041] In one embodiment, the cationic lipid is soluble in ethanol.

[0042] In one embodiment, the method involves adding one volume of OMP P5 protein and an anionic hydrophilic adjuvant to 18 volumes of a cationic lipid solution.

[0043] Beneficial effects:

[0044] (1) The present invention uses OMP P5 recombinant protein as antigen to prepare nano-vaccines. The prepared nano-vaccines have regular morphology, round shape, smooth surface, good dispersibility, and no obvious adhesion, damage, collapse or other phenomena.

[0045] (2) In the nano-vaccine of the present invention, the OMP P5 recombinant protein and the immune adjuvant have a high encapsulation rate;

[0046] (3) When the nano-vaccine of the present invention is applied to test fish, it can produce a strong immune response and the immune effect is better than that of free subunit vaccines;

[0047] (4) The nano-vaccine of the present invention can protect the antigen and prolong the stability and effectiveness of the antigen;

[0048] (5) The nano-vaccine of the present invention can be immunized by immersion, and enhances fish absorption by efficiently binding to the gills through cationic nanoparticles, while improving drug utilization and reducing the amount of antigen and adjuvant used. Attached Figure Description

[0049] Figure 1 The images show the morphology of the nanovaccine in Example 4. A shows the morphology of the nanovaccine encapsulating OMP P5 recombinant protein and CpG adjuvant under a transmission electron microscope; B shows the morphology of the nanovaccine encapsulating OMP P5 recombinant protein and MPLA adjuvant under a transmission electron microscope; C shows the morphology of the nanovaccine encapsulating OMP P5 recombinant protein, CpG, and MPLA dual adjuvants under a transmission electron microscope.

[0050] Figure 2 The particle size distribution of the nanovaccine in Example 4 is shown in Figure A. Figure A shows the particle size distribution of the nanovaccine encapsulating OMP P5 recombinant protein and CpG adjuvant; Figure B shows the particle size distribution of the nanovaccine encapsulating OMP P5 recombinant protein and MPLA adjuvant; Figure C shows the particle size distribution of the nanovaccine encapsulating OMP P5 recombinant protein, CpG, and MPLA dual adjuvants.

[0051] Figure 3 The survival rate of largemouth bass challenged with Aeromonas hydrophila 20 days after treatment in Example 6 is shown.

[0052] Figure 4 The relative expression levels of TNF-α, IL-1β, IgM and IgT genes in the gills of largemouth bass after being vaccinated with nanoviruses in Example 7. Detailed Implementation

[0053] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0054] Example 1. Preparation of NP-OC nanovaccine

[0055] 1. Reagents: The OMP P5 recombinant protein was produced by expression in an *E. coli* expression system, followed by dissolution and renaturation. Its amino acid sequence was obtained from GenBank (ABK37458.1). First, the target fragment was amplified using specific primers: the upstream primer sequence was 5ʹ-CGCGGATCCATGAATAAAACACTGATTACCTTGC-3ʹ; the downstream primer sequence was 5ʹ-CCCAAGCTTCTGCTGAACTTCCGAGATCC-3ʹ. Then, the amplified product was purified and ligated into the pET-28a vector to construct the recombinant expression vector. Subsequently, the recombinant expression vector was transformed into *E. coli* (DE3), and positive transformants were screened by colony PCR and sequencing. The positive transformants were then cultured in LB medium and induced with IPTG. Finally, the expression product was purified and renatured to obtain the OMP P5 recombinant protein. Detailed operating procedures can be found in *Molecular Cloning: A Laboratory Manual*, 4th edition.

[0056] All other reagents were purchased commercially.

[0057] 2. Preparation process:

[0058] (1) The cationic polymer DOTAP was dissolved in anhydrous ethanol and dissolved under magnetic stirring to obtain a DOTAP concentration of 1 mg / mL. The surfactant DSPE-PEG-2000 was dissolved in water to obtain a DSPE-PEG-2000 concentration of 1 mg / mL. The OMP P5 recombinant protein and the anionic hydrophilic adjuvant CpG were dissolved in 1×PBS buffer to obtain OMP P5 solution with a concentration of 40 mg / mL and CpG solution with a concentration of 20 mg / mL, respectively. The hydrophobic stabilizer PLGA was dissolved in DMSO to a concentration of 5 mg / mL.

[0059] (2) Mix one volume of OMP P5 solution and one volume of CpG solution, stir magnetically at 600 rpm, and add dropwise to 18 volumes of DOTAP solution. Mix DOTAP with CpG and OMP P5 protein for 10 minutes, and crosslink the mixture through electrostatic interaction.

[0060] (3) Then, 20 volumes of PLGA solution are added dropwise to the mixture formed in (2) while stirring, forming nanoparticles coated with hydrophobic stabilizer.

[0061] (4) The obtained nanoparticles were added dropwise to 120 volumes of DSPE-PEG-2000, stirred for 5 min, and then dialyzed to remove the organic solvent to form a nano-vaccine (named NP-OC nano-vaccine solution).

[0062] Example 2. Preparation of NP-OM nanovaccine

[0063] (1) The cationic polymer DOTAP was dissolved in anhydrous ethanol and dissolved under magnetic stirring to obtain a DOTAP concentration of 1 mg / mL. The surfactant DSPE-PEG-2000 was dissolved in water to obtain a DSPE-PEG-2000 concentration of 1 mg / mL. OMP P5 was dissolved in 1×PBS to obtain OMP P5 solutions with a concentration of 20 mg / mL. The hydrophobic adjuvant MPLA and the hydrophobic stabilizer PLGA were dissolved in DMSO to a concentration of 1 mg / mL and 5 mg / mL, respectively.

[0064] (2) Two volumes of OMP P5 protein were magnetically stirred at 600 rpm and added dropwise to 18 volumes of DOTAP solution. The mixture containing DOTAP and OMP P5 protein was thoroughly mixed for 10 minutes and cross-linked by electrostatic action to obtain the mixture.

[0065] (3) Then, 20 volumes of MPLA and PLGA solution were added dropwise to the mixture formed in (2) while stirring, forming nanoparticles coated with hydrophobic stabilizer.

[0066] (4) The obtained nanoparticles were added dropwise to 120 volumes of DSPE-PEG-2000, stirred for 5 min, and then dialyzed to remove the organic solvent to obtain the nano-vaccine (named NP-OM nano-vaccine solution).

[0067] Example 3. Preparation of NP-OCM nanovaccine

[0068] (1) The cationic polymer DOTAP was dissolved in anhydrous ethanol and dissolved under magnetic stirring to obtain a DOTAP concentration of 1 mg / mL. The surfactant DSPE-PEG-2000 was dissolved in water to obtain a DSPE-PEG-2000 concentration of 1 mg / mL. OMP P5 and CpG were dissolved in 1×PBS to obtain OMP P5 solution with a concentration of 20 mg / mL and CpG solution with a concentration of 10 mg / mL, respectively. The hydrophobic adjuvant MPLA and the hydrophobic stabilizer PLGA were dissolved in DMSO to obtain concentrations of 1 mg / mL and 5 mg / mL, respectively.

[0069] (2) Two volumes of OMP P5 protein and CpG mixed solution were added dropwise to 18 volumes of DOTAP solution, and DOTAP and OMP P5 protein were thoroughly mixed for 10 minutes to obtain a mixture through electrostatic cross-linking.

[0070] (3) Then, 20 volumes of MPLA and PLGA solution were added dropwise to the mixture formed in (2) while stirring, forming nanoparticles coated with hydrophobic stabilizer.

[0071] (4) The obtained nanoparticles were added dropwise to 120 volumes of DSPE-PEG-2000, stirred for 5 min, and then dialyzed to remove the organic solvent to obtain the nano-vaccine (named NP-OCM nano-vaccine solution).

[0072] Example 4. Morphological characterization, particle size analysis, and potential analysis of nanovaccines

[0073] 1. Morphological characterization

[0074] NP-OC, NP-OM, and NP-OCM nanovaccines were observed using transmission electron microscopy. The morphologies of the three nanovaccines are as follows: Figure 1 As shown in A, 1B, and 1C. Figure 1 A shows the morphology of the nanovaccine encapsulating OMP P5 recombinant protein and CpG immune adjuvant under a transmission electron microscope. Figure 1 B shows the morphology of the nanovaccine encapsulating OMP P5 recombinant protein and MPLA immune adjuvant under a transmission electron microscope. Figure 1 C represents the morphology of the nanovaccine encapsulating the OMP P5 recombinant protein CpG and MPLA dual adjuvants under a transmission electron microscope.

[0075] like Figure 1 As shown, the three types of nano-vaccines have regular morphology, round shape, smooth surface, and good dispersibility, with no obvious adhesion, damage, or collapse.

[0076] 2. Particle size testing and zeta potential testing:

[0077] The average particle size and zeta potential of NP-OC, NP-OM, and NP-OCM nanovaccines were tested using a Malvern particle size analyzer (with a dynamic light scattering detector), and the results are shown in Table 1.

[0078] Table 1. Average particle size and Zeta potential of nanovaccines

[0079]

[0080] Figure 2 A, 2B, and 2C are particle size distribution diagrams of NP-OC, NP-OM, and NP-OCM nanovaccines, respectively. Figure 2A is the particle size distribution diagram of the nanovaccine encapsulating OMP P5 recombinant protein and CpG adjuvant; Figure 2 B is the particle size distribution diagram of the nanovaccine encapsulating OMP P5 recombinant protein and MPLA immune adjuvant; Figure 2 C represents the particle size distribution of the nanovaccine encapsulating the OMP P5 recombinant protein CpG and MPLA dual adjuvants. (See diagram for reference.) Figure 2 As shown, all four nanovaccines have a narrow particle size distribution.

[0081] Example 5. Calculation of encapsulation efficiency of OMP P5 recombinant protein in nano-vaccines

[0082] 1. Take 5 mL of NP-OC nanovaccine solution into a 300 kDa ultrafiltration tube, centrifuge at 4 ℃ and 3000 rpm for 30 min, take the filtrate, and use the Bradford protein assay kit to detect the content of free OMP P5 recombinant protein in the filtrate. Calculate the encapsulation efficiency of OMP P5 recombinant protein in the nanovaccine according to the following formula.

[0083] Encapsulation efficiency of OMP P5 recombinant protein = w0 - w1 / w0 × 100%, where w0 is the total amount of added OMP P5 recombinant protein; w1 is the total amount of free OMP P5 recombinant protein in the filtrate below.

[0084] 2. Determine the encapsulation efficiency of OMP P5 recombinant protein in NP-OM and NP-OCM nanovaccines respectively, following the method in step 1.

[0085] The results of the encapsulation efficiency determination are shown in Table 2.

[0086] Table 2 Encapsulation efficiency of OMP P5 recombinant protein

[0087]

[0088] As can be seen from the results in Table 2, among the three nano-vaccines of the present invention, the OMP P5 recombinant protein has a higher encapsulation rate, which is beneficial for the nano-vaccines to induce a strong immune effect.

[0089] Example 6. Evaluation of the immunoprotective effect of nano-vaccines against Aeromonas hydrophila infection in largemouth bass.

[0090] Largemouth bass were vaccinated with OMP P5 subunit nanovaccine: One hundred healthy largemouth bass (3±0.3g) of uniform size were randomly divided into 5 groups of 20 fish each. Immunization was performed using an immersion method: the control group (PBS), the free protein antigen group (FO), and the OMP P5 subunit nanovaccine groups (NP-OC, NP-OM, NP-OCM) were immersed in water containing PBS, FO, NP-OC, NP-OM, and NP-OCM, respectively, for 5 minutes. The antigen dosage for the FO, NP-OC, NP-OM, and NP-OCM groups was 50 μg / ml. After vaccination, the experimental fish were transferred to new tanks for further rearing.

[0091] Post-inoculation bacterial challenge: 21 days after immunization, the experimental fish were transferred to an environment containing Aeromonas hydrophila at a final concentration of 1×10⁻⁶. 7 The fish were immersed in water containing CFU / mL for 1 hour. After challenge, the experimental fish were transferred to new tanks for further rearing and observed for 20 consecutive days, with daily mortality recorded. The immune protection rate (RPS) was calculated using the following formula: RPS = [1 - (mortality rate in the immunized group / mortality rate in the control group)] × 100%.

[0092] The results are as follows Figure 3 As shown, the cumulative mortality rates of the experimental fish in the PBS group, FO group, NP-OC group, NP-OM group, and NP-OCM group were 90%, 85%, 20%, 25%, and 15%, respectively. Calculations showed that, compared with the PBS control, the immunoprotective rates of FO, NP-OC, NP-OM, and NP-OCM vaccines against largemouth bass were 5%, 78%, 72%, and 83%, respectively. Therefore, the three nano-vaccines, NP-OC, NP-OM, and NP-OCM, can effectively provide immunoprotection against largemouth bass to varying degrees, thereby enhancing their resistance to Aeromonas hydrophila infection.

[0093] Example 7. Nano-vaccines enhance the immune response of largemouth bass to Aeromonas hydrophila.

[0094] RNA extraction and reverse transcription: On days 1, 3, and 14 after recombinant OMP P5 nanovaccination, gill tissue from three fish in each group was collected for RNA extraction. Total RNA was extracted using the Trizol method, and DNase I was added and digested at 37°C for 1 h to remove genomic DNA contamination. Reverse transcription was then performed, and the reverse transcription product was used as a template for quantitative fluorescence detection.

[0095] qRT-PCR was used to detect the expression levels of immune-related genes in fish: TNF-α, IL-1β, IgM, and IgT genes in the gills of largemouth bass were detected using quantitative real-time PCR, with β-actin as an internal control. Specific primers and sequences are shown in Table 3.

[0096] Table 3. Specific primers and sequences for real-time PCR

[0097]

[0098] The results are as follows Figure 4 As shown, compared with the FO group, the expression levels of TNF-α, IL-1β, IgM and IgT genes in the gills of fish treated with NP-OC, NP-OM and NP-OCM were significantly increased. This indicates that soaking in OMP P5 subunit nano-vaccines can enhance the immune response of largemouth bass and improve the host's resistance to Aeromonas hydrophila infection.

[0099] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A nano-vaccine for fish that has been inoculated by immersion, characterized in that, The nanovaccine has a particle size of 50-200 nm and contains OMP P5 protein, an immune adjuvant, cationic lipids, a surfactant, and a hydrophobic stabilizer; the amino acid sequence of the OMP P5 protein is GenBank ABK37458.1; the immune adjuvant includes anionic hydrophilic adjuvants and / or hydrophobic adjuvants; the hydrophobic stabilizer is polylactic acid-glycolic acid copolymer; The mass ratio of OMP P5 protein: immune adjuvant: cationic lipid: hydrophobic stabilizer: surfactant is 10-30:5-20:3-12:20-50:40-80. The immune adjuvant is selected from one or more of poly(I:C), CpG, MPLA, and squalene; The cationic lipid is selected from one or more of the following: trimethyl-2,3-dioleoyloxypropylammonium bromide, trimethyl-2,3-dioleenooxypropylammonium bromide, dimethyl-2,3-dioleenooxypropyl-2-(2-spermineformylamino)ethylammonium bromide, dimethyl dioctadecylammonium bromide, trimethyl dodecylammonium bromide, trimethyl tetradecylammonium bromide, trimethyl hexadecylammonium bromide, 1,2-dioleoyl-3-succinyl-sn-glycerolcholine ester, 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl]cholesterol, and stearylamine. The surfactant is distearate phosphatidylethanolamine-polyethylene glycol 2000; The preparation method includes the following steps: S1. Mix one volume of OMP P5 protein solution and anionic hydrophilic adjuvant solution, stir, and add dropwise to 10-25 volumes of cationic lipid solution, mix, and crosslink to obtain a mixture; S2. Add 12-25 volumes of hydrophobic stabilizer solution or a solution containing both hydrophobic stabilizer and hydrophobic adjuvant to the mixture formed by S1 while stirring, to form nanoparticles coated with hydrophobic stabilizer. S3. Add the nanoparticles obtained in S2 dropwise to a surfactant solution of 50-150 volumes, stir, and then dialyze to remove the organic solvent; or, S1. Take one volume of OMP P5 protein solution, stir, and add it dropwise to 15-25 volumes of cationic lipid solution. Mix and crosslink to obtain a mixture. S2. While stirring, 12-25 volumes of a solution containing both a hydrophobic stabilizer and a hydrophobic adjuvant are added dropwise to the mixture formed by S1 to form nanoparticles coated with the hydrophobic stabilizer. S3. Add the nanoparticles obtained in S2 dropwise to a surfactant solution of 50-150 volumes, stir, and then dialyze to remove the organic solvent.

2. The method for preparing the fish nano-vaccine after immersion inoculation as described in claim 1, characterized in that, Includes the following steps: S1. Mix one volume of OMP P5 protein solution and anionic hydrophilic adjuvant solution, stir, and add dropwise to 10-25 volumes of cationic lipid solution, mix, and crosslink to obtain a mixture; S2. Add 12-25 volumes of hydrophobic stabilizer solution or a solution containing both hydrophobic stabilizer and hydrophobic adjuvant to the mixture formed by S1 while stirring, to form nanoparticles coated with hydrophobic stabilizer. S3. Add the nanoparticles obtained in S2 dropwise to a surfactant solution of 50-150 volumes, stir, and then dialyze to remove the organic solvent; or, S1. Take one volume of OMP P5 protein solution, stir, and add it dropwise to 15-25 volumes of cationic lipid solution. Mix and crosslink to obtain a mixture. S2. While stirring, 12-25 volumes of a solution containing both a hydrophobic stabilizer and a hydrophobic adjuvant are added dropwise to the mixture formed by S1 to form nanoparticles coated with the hydrophobic stabilizer. S3. Add the nanoparticles obtained in S2 dropwise to a surfactant solution of 50-150 volumes, stir, and then dialyze to remove the organic solvent.

3. The method according to claim 2, characterized in that, The OMP P5 protein solution is a 10-30 mg / mL OMP P5 protein solution obtained by dissolving OMP P5 protein in a buffer solution; the cationic lipid solution is a 3-12 mg / mL cationic lipid solution obtained by dissolving cationic lipids in an organic solvent; the hydrophobic adjuvant solution is a 5-20 mg / mL hydrophobic adjuvant solution obtained by dissolving a hydrophobic adjuvant in an organic solvent; the hydrophobic stabilizer solution is a 20-50 mg / mL hydrophobic stabilizer solution obtained by dissolving a hydrophobic stabilizer in an organic solvent; and the surfactant solution is a 20-50 mg / mL surfactant solution obtained by dissolving a surfactant in water.

4. The method according to claim 3, characterized in that, The buffer solution is a PBS salt solution; the hydrophobic stabilizer solution uses DMSO as the organic solvent; and the cationic lipid solution uses ethanol as the organic solvent.

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