A large mouth bass ferritin self-assembled nano vaccine and a preparation method thereof

Abstract

The application discloses a large-mouth bass ferritin self-assembled nano vaccine and a preparation method thereof, and relates to the technical field of biological medicines. The large-mouth bass ferritin self-assembled nano vaccine comprises a polypeptide LMWP, a multi-epitope antigen MEV and a bass ferritin MFH connected in series. The amino acid sequences of the polypeptide LMWP, the multi-epitope antigen MEV and the bass ferritin MFH are shown in SEQ ID NO. 3-5. The cell-penetrating peptide, the multi-epitope antigen and the self-assembled ferritin carrier are organically combined, the delivery efficiency, the immunological activity and the use convenience are considered, the application short board of the traditional aquatic vaccine is overcome, an efficient new scheme is provided for the prevention and control of the iridovirus disease, meanwhile, a referable technical paradigm is provided for the research and development, optimization and industrial popularization of the self-assembled nano bath vaccine for aquatic animals, and the technical paradigm has important practical values for promoting the green and healthy development of the large-mouth bass breeding industry.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a largemouth bass ferritin self-assembled nanovaccine and its preparation method. Background Technology

[0002] Largemouth bass ( Micropterus salmoides Largemouth bass, commonly known as California bass, is a high-quality freshwater economic fish species cultivated on a large scale. However, with the popularization of intensive and high-density farming models, disease problems in largemouth bass farming have become increasingly prominent. Among them, viral diseases caused by largemouth bass ranavirus (LMBV) have a mortality rate of over 70%, seriously restricting the healthy and sustainable development of the industry and becoming a major bottleneck in current aquaculture production.

[0003] LMBV belongs to the family Iridoviridae ( Iridoviridae Frog virus genus ( Ranavirus LMBV is a double-stranded DNA virus with an icosahedral symmetry structure, a diameter of 150-170 nm, and a genome size of 100-210 kbp. Its encoded main capsid protein (MCP) is the core structural protein of the virus, with a molecular weight of 48-55 kDa. It not only maintains the stability of the viral particle structure and participates in viral assembly, but also possesses extremely strong immunogenicity, inducing a specific immune response in the body. It is the core antigen target for LMBV vaccine development.

[0004] The applicant previously used phage display and bioinformatics analysis to screen for dominant antigenic epitopes of multiple MCP proteins. Based on this, a multi-epitope vaccine was constructed that induced strong specific antibody levels in largemouth bass after intraperitoneal injection. However, intraperitoneal injection suffers from a series of problems, including low operational efficiency, high labor costs, significant fish damage, and strong stress responses, making it unsuitable for the large-scale, intensive farming of largemouth bass and extremely difficult to commercialize. Immersion immunization, due to its ability to achieve batch immunization of the entire pond, simple operation, and minimal harm to the fish, has become the mainstream method for promoting and applying fish vaccines. However, traditional direct antigen immersion suffers from problems such as easy antigen dilution and degradation, poor skin penetration, and unstable immunization effects, making it difficult to realize the immunization potential of multi-epitope vaccines. Therefore, this invention aims to develop a new LMBV vaccine to overcome the shortcomings of traditional vaccines while ensuring both safety and immunoprotective efficacy. Summary of the Invention

[0005] The purpose of this invention is to provide a largemouth bass ferritin self-assembled nanovaccine and its preparation method, thereby solving the problems existing in the prior art. This invention constructs a largemouth bass ferritin self-assembled nanovaccine composed of the cell-penetrating peptide LMWP, the multi-epitope antigen MEV, and largemouth bass ferritin MFH in tandem. This nanovaccine balances delivery efficiency, immunogenicity, and ease of use, overcoming the shortcomings of traditional aquatic vaccines and providing a highly efficient new solution for the prevention and control of iridovirus diseases.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a largemouth bass ferritin self-assembled nanovaccine for the prevention of LMBV virus, comprising a polypeptide LMWP, a multi-epitope antigen MEV, and bass ferritin MFH in sequence. The amino acid sequence of the polypeptide LMWP is shown in SEQ ID NO.3; The amino acid sequence of the multi-epitope antigen MEV is shown in SEQ ID NO.4; The amino acid sequence of the perch ferritin MFH is shown in SEQ ID NO.5.

[0007] Furthermore, the polypeptide LMWP and the multiepitope antigen MEV are linked by a first linker peptide; the multiepitope antigen MEV and the sea bass ferritin MFH are linked by a second linker peptide.

[0008] Furthermore, the amino acid sequence of the first linker peptide is KK; the amino acid sequence of the second linker peptide is shown in SEQ ID NO.6.

[0009] The present invention also provides a gene encoding the above-mentioned largemouth bass ferritin self-assembled nanovaccine.

[0010] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0011] The invention also provides a gene expression cassette containing the aforementioned encoding gene.

[0012] The invention also provides a recombinant plasmid comprising the gene expression cassette described above.

[0013] The invention also provides a recombinant microbial strain comprising the aforementioned recombinant plasmid.

[0014] The invention also provides the application of the above-mentioned coding gene, gene expression cassette, recombinant plasmid or recombinant microbial strain in the preparation of the above-mentioned largemouth bass ferritin self-assembled nanovaccine.

[0015] The invention also provides a method for preparing a largemouth bass ferritin self-assembled nanovaccine, comprising the following steps: The recombinant microbial strains were induced and cultured to obtain bacterial cells; After the bacterial cells were broken down, they were denatured and purified to obtain the largemouth bass ferritin self-assembled nanovaccine.

[0016] The present invention discloses the following technical effects: This invention constructs a largemouth bass ferritin self-assembled nanovaccine composed of cell-penetrating peptide LMWP, multi-epitope antigen MEV, and largemouth bass ferritin MFH in series. Relying on the natural 24-tetrammer cage-like self-assembly characteristics of ferritin, spherical nanovaccine particles with a particle size of about 24 nm were successfully prepared. The particles have uniform morphology and stable structure, effectively solving the pain points of traditional fish vaccines such as easy degradation of antigens, low delivery efficiency, and unstable immunization effect.

[0017] This invention's vaccine uses endogenous ferritin from largemouth bass as its backbone carrier, exhibiting excellent biocompatibility, containing no exogenous harmful components, and avoiding immune rejection or adverse reactions in fish, thus demonstrating outstanding safety. Simultaneously, it incorporates linker peptides with distinct functions at both ends of the multi-epitope antigens. These linker peptides ensure the independent spatial conformation of each functional segment, preventing abnormal protein folding and maximizing the preservation of the immunogenicity of the multi-epitope antigens and the self-assembly ability of ferritin. This also guarantees the bioactivity of the cell-penetrating peptide LMWP. LMWP possesses excellent cell membrane penetration capabilities, significantly enhancing the antigen uptake efficiency of largemouth bass mucosal epithelial cells. This overcomes the technical challenge of insufficient antigen penetration barrier in fish immersion immunization, making the immersion immunization mode truly suitable for large-scale largemouth bass farming. Compared to traditional intraperitoneal injection vaccines, it significantly reduces labor costs and minimizes fish damage and stress responses.

[0018] After prokaryotic expression, denaturation purification, and self-assembly, this nanovaccine can remain stable in the aquatic environment and is not easily diluted or degraded. Immersion immunization experiments showed that different concentrations of the vaccine consistently activated a specific immune response in largemouth bass. Serum antibody titers gradually increased with immunization time, reaching a peak on day 28. The 40 mg / L dose group exhibited the best antibody levels, demonstrating a sustained and strong immune response.

[0019] In the challenge protection test, the vaccine of this invention showed good antiviral protection effect: the relative immune protection rates against the largemouth bass iris virus in the three bath dose groups of 10 mg / L, 20 mg / L and 40 mg / L reached 60.53%, 68.42% and 73.68% respectively, which were much higher than those in the blank control group, and could effectively reduce the mortality rate of fish after viral infection.

[0020] In summary, this invention organically combines cell-penetrating peptides, multi-epitope antigens, and self-assembled ferritin carriers, balancing delivery efficiency, immunomodulatory activity, and ease of use. It not only overcomes the shortcomings of traditional aquatic vaccines but also provides a highly efficient new solution for the prevention and control of iridovirus diseases. Furthermore, it offers a referable technical paradigm for the research, optimization, and industrialization of self-assembled nano-immersion vaccines for aquatic animals, and has significant practical value for promoting the green and healthy development of the largemouth bass farming industry. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 A schematic diagram illustrating the construction of the largemouth bass ferritin self-assembled nanovaccine MEV-MFH; Figure 2 Electrophoresis image of PCR amplification of MEV-MFH; Figure 3 This is a graph showing the sequencing results of pET28a-MEV-MFH. Figure 4 Image showing the SDS-PAGE identification results of MEV-MFH; Figure 5 The image shows the Western Blot identification results of MEV-MFH. Figure 6 Transmission electron microscopy analysis of a ferritin-based self-assembled nanovaccine from largemouth bass; Figure 7 Particle size analysis diagram of ferritin-based self-assembled nanovaccine from largemouth bass; Figure 8 The image shows the serum antibody titer test results for the ferritin-based self-assembled nanovaccine from largemouth bass. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] The experimental materials used in the following examples are as follows: Largemouth bass (1.9±0.3g, 4.8±0.6 cm) were bred at the Wan-Yue Fish Fry Breeding Base in Guangzhou, Guangdong Province. After two weeks of temporary rearing in a laboratory recirculating aquaculture system, the experiment was conducted at a water temperature of 22±0.5℃. The largemouth bass iridovirus used in this example was the LMBV-FS001 strain (GenBank accession No. ON936874), preserved in our laboratory. Escherichia coli (E. coli) BL21 and DH5α were obtained from Beijing Qingke Biotechnology Co., Ltd.

[0029] The reagent information used in the following examples is as follows: Trizol, Thermo Fisher Scientific (China) Co., Ltd.; Mouse anti-bass IgM monoclonal antibody, Fointech Technology (Wuhan) Co., Ltd.; Mouse His-tagged monoclonal antibody and goat anti-mouse IgG (HRP) monoclonal antibody, Sangon Biotech (Shanghai) Co., Ltd.; His-tagged protein purification kit, Sangon Biotech (Shanghai) Co., Ltd.; Single-component TMB chromogenic solution and ELISA stop solution, Beijing Solarbio Science & Technology Co., Ltd.; Kanamycin (KAN), Sigma-Aldrich; Reverse transcription kit, Jiangsu Bristol-Myers Squibb Biotechnology Co., Ltd.; Universal gel extraction kit, Tiangen Biotech (Beijing) Co., Ltd.; Plasmid miniprep kit, Jiangnan Aikerui Biotechnology Co., Ltd.

[0030] The experimental instrument information used in the following examples is as follows: ALC-1100.2 electronic balance, Beijing Sartorius Instrument Systems Co., Ltd.; HH-4 digital display constant temperature water bath, Shanghai Techeng Machinery Equipment Co., Ltd.; H1650-W benchtop high-speed microcentrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; 1-15K high-speed refrigerated centrifuge, Sigma-Aldrich, USA; Universal Hood gel imaging system, Bio-Rad, USA; DYCZ-24DN vertical electrophoresis system, Beijing Liuyi Instrument Factory; Thermo Multiskan MK3 microplate reader, Thermo Fisher Scientific, USA; Ultrasonic cell disruptor, Ningbo Xinzhi Biotechnology Co., Ltd.

[0031] Example 1 This embodiment describes the design of a largemouth bass ferritin self-assembled nanovaccine.

[0032] The cell-penetrating peptide LMWP, the multi-epitope antigen MEV, and the sea bass ferritin MFH were tandemly linked using overlap extension PCR technology to construct the pET28a-MEV-MFH recombinant expression plasmid. The constructed recombinant expression plasmid was then transformed into Escherichia coli. E. coli The following steps were taken from BL21(DE3) competent cells and single colonies were selected for prokaryotic expression: The primers used in this embodiment are shown in Table 1. The primers for ferritin gene amplification were designed based on the reference sequence for the largemouth bass ferritin gene (GenBank: XM_038701750.1) in NCBI. Total RNA was extracted from the spleen tissue of largemouth bass (n=3) using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. Using reverse-transcribed cDNA and the preserved pET32a-MEV plasmid (purchased from Suzhou Genewiz Biotechnology Co., Ltd.) as templates, the MFH and MEV gene sequences were amplified, respectively. The LMWP gene, Linker 1 coding sequence, MEV gene, Linker 2 coding sequence, and MFH gene were then tandemly constructed to obtain the recombinant fusion protein coding gene, named MEV-MFH gene, whose nucleotide sequence is shown in SEQ ID NO.1. In the recombinant fusion protein MEV-MFH (amino acid sequence shown in SEQ ID NO.2), LMWP (amino acid sequence of LMWP shown in SEQ ID NO.3) and MEV (amino acid sequence of MEV shown in SEQ ID NO.4) are linked using Linker 1 (KK), and MEV and MFH (amino acid sequence of MFH shown in SEQ ID NO.5) are linked using Linker 2 (amino acid sequence shown in SEQ ID NO.6). Figure 1 ).

[0033] SEQ ID NO. 1: ATGGGCAGCGTGAGCCGCCGCCGCCGCCGCGGCGGCCGCCGCCGCAAAAAAACCGGCAGCGGCATTACGAGCGGCTTTATTGATGGCCCGGGCCCGGGCGCGACCACCTATTTTGTGAAAGAACATGGTCCGGGTCCGGGCGTGAAGGAACACTATCCGGTGGGCTGGTTTACCAAACTGGGTCCGGGCCCGGGCACCATTAGCCTGCGCAGCATTCAAGATCTGGGTCCGGGCCCGGGCCCGGATATGAGCGTGGAATATTATAGCCTGGTGCAGCCGTGGTATTATGGCCCGGGTCCGGGCATTCCGATTAGCACCGGCCATCATCTGTATAGCTATGCGCTGAGCCTGAACGATCCGCATCCGAGCGGCAGCGGCGGCGGCGGCAGCGGCGGTGGTGGTAGCAGTTCCCAGGTGAGACAGAACTTCCACCAAGATTGCGAGGCTGCAATTAACAGGCAGATCAACATGGAGCTGTATGCCTCCTACGTCTACCTGTCTATGTCATACTACTTTGACCGGGATGACCAGGCATTGCACAACTTTGCCAAGTTCTTCCGTAGTCAGTCACACGAGGAGCGTGAGCACGCTGAGAAACTAATGAAACTGCAGAACCAGAGGGGAGGAAGGATCTTCCTACAAGATGTCAAGAAGCCAGAGAGGGATGAGTGGGGCAGTGGTATCGAGGCCCTTGAATGCGCCCTGCAGCTCGAGAAGACTGTGAACCAGTCCCTGCTGGACTTGCACAAAGTCAGCTCTGATCACAATGACCCACATCTGTGTGACTTCATTGAGACACACTACCTGGACGAGCAGGTGAAATCCATCAAAGAGCTGTCAGACTGGGTAACCAACCTGCGCCGCATGGGAGCTCCTCAGAATGGCCTGGCCGAGTACCTGTTTGACAAGCATACCCTGGGCAAAGAAAGCAGC; wherein, bases 1-45 are the LMWP gene; bases 47-511 are the coding sequence of Linker 1Bases 52-375 represent the MEV gene; bases 376-405 represent the Linker 2 coding sequence; bases 406-933 represent the MFH gene.

[0034] SEQ ID NO.2:MGSVSRRRRRGRRKKTGSGITSGFIDGPGPGATTYFVKEHGPGPGVKEHYPVGWFTKLGPGPGTISLRSIQDLGPGPDMSVEYYSLVQPWYYGPGPGIPISTGHHLYSYALSLNDPHPSGSGGGGSGGGGSSSQVRQNFHQDCEAAINR QINMELYASYVYLSMSYYFDRDDQALHNFAKFFRSQSHEEREHAEKLMKLQNQRGGRIFLQDVKKPERDEWGSGIEALECALQLEKTVNQSLLDLHKVSSDHNDPHLCDFIETHYLDEQVKSIKELSDWVTNLRRMGAPQNGLAEYLFDKHTLGKESS.

[0035] SEQ ID NO. 3: MGSSVSRRRRGGRRR.

[0036] SEQ ID NO. 4: TGSGITSGFIDGPGPGATTYFVKEHGPGPGVKEHYPVGWFTKLGPGPGTISLRSIQDLGPGPGPDMSVEYYSLVQPWYYGPGPGIPISTGHHLYSYALSLNDPHPSGS.

[0037] SEQ ID NO.5: SSQVRQNFHQDCEAAINRQINMELYASYVYLSMSYYFDDRDDQALHNFAKFFRSQSHEEREHAEKLMKLQNQRGGRIFLQDVKKPERDEWGSGIEALECALQLEKTVNQSLLDLHKVSSDHNDPHLCDFIETHYLDEQVKSIKELSDWVTNLRRMGAPQNGLAEYLFDKHTLGKESS.

[0038] SEQ ID NO.6: GGGGSGGGGS.

[0039] PCR results as follows Figure 2 As shown, the amplified MEV-MFH gene is approximately 933 bp in length, consistent with the expected result.

[0040] The MEV-MFH gene was cloned into the pET28a expression vector using seamless cloning technology to obtain the recombinant plasmid pET28a-MEV-MFH, which was then transformed into Escherichia coli. E. coli Recombinant *E. coli* was obtained from BL21(DE3) competent cells. The recombinant plasmid pET28a-MEV-MFH was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequencing results ( Figure 3 The results showed that the sequencing results of pET28a-MEV-MFH were consistent with the designed sequence, indicating that the recombinant plasmid pET28a-MEV-MFH was successfully constructed.

[0041] Table 1 Primers for MEV-MFH gene amplification

[0042] Recombinant *E. coli* with correct sequencing were inoculated into LB liquid medium containing KAN resistance and cultured overnight at 37°C and 120 rpm. The next day, isopropyl-β-D-thiogalactopyranoside (IPTG) inducer was added to the bacterial culture at a ratio of 1:1000, and induction was performed at 37°C and 150 rpm for 8 h. The induced bacterial culture was centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the bacterial cells were collected, resuspended in deionized water, and disrupted using an ultrasonic homogenizer. After SDS-PAGE analysis, Western blotting was performed using a mouse His-tagged monoclonal antibody as the primary antibody and a goat anti-mouse IgG (HRP) monoclonal antibody as the secondary antibody.

[0043] SDS-PAGE results are as follows Figure 4 As shown, the molecular weight of MEV-MFH proteins is approximately 39.6 kDa, which is consistent with the expected results, and the expressed MEV-MFH mainly exists in the form of inclusion bodies.

[0044] Western Blot results are as follows Figure 5 As shown, the expressed protein can recognize a mouse-derived His-tagged monoclonal antibody. This result indicates that the MEV-MFH protein was successfully expressed in Escherichia coli.

[0045] Example 2 Preparation method of ferritin-based self-assembled nanovaccine for largemouth bass: The recombinant *E. coli* constructed in Example 1 was inoculated into LB liquid medium containing KAN resistance and cultured overnight at 37°C and 120 rpm. The next day, isopropyl-β-D-thiogalactopyranoside (IPTG) inducer was added to the bacterial culture at a ratio of 1:1000, and induction was performed at 37°C and 150 rpm for 8 h. The induced bacterial culture was centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the bacterial cells were collected, resuspended in deionized water, and disrupted using an ultrasonic homogenizer. The disrupted bacterial cells were denatured by dissolution with 8 M urea and then filtered through a 0.22 μm filter membrane. The protein filtrate was purified by nickel column chromatography. The purified protein was dialyzed and refolded sequentially using 6 M, 4 M, and 2 M urea, with each dialysis and refolding step lasting 24 h. Finally, it was refolded with pure water, which was changed at least 3 times to remove as much excess urea and other impurities as possible. The purified recombinant fusion protein MEV-MFH was lyophilized and stored at -80 °C for later use.

[0046] Example 3 This embodiment describes the characterization and analysis of a largemouth bass ferritin-based self-assembled nanovaccine.

[0047] The self-assembly process of ferritin nanocages is reversible; at neutral pH (pH = 7.2), ferritin subunits can assemble into spherical nanocage structures. The protein concentration of the recombinant fusion protein MEV-MFH expressed in Example 2 was determined, and a protein concentration range of 0.2–0.3 mg / mL was selected for further analysis. The morphological characteristics of the samples were observed using a Hitachi HT7800 transmission electron microscope (TEM). 2 μL of sample was dropped onto the surface of a copper mesh, air-dried at room temperature, and then negatively stained with 2% phosphotungstic acid. After the copper mesh dried, the particle morphology was observed under a TEM. The particle size of the nanoparticles was determined using a Nano ZS90 Zetasizer (Malvern Panalytical, UK).

[0048] Figure 6 Transmission electron microscopy results showed that MEV-MFH consisted of single, uniformly distributed spherical nanoparticles. Figure 7 The results showed that the average particle size of MEV-DFH was approximately 24 nanometers. These results collectively confirm the successful construction of the self-assembled nanovaccine.

[0049] Example 4 This embodiment describes the evaluation of the immunogenicity of a largemouth bass ferritin-based self-assembled nanovaccine.

[0050] Largemouth bass immunization: The largemouth bass ferritin self-assembled nanovaccine prepared in Example 2 was used to immunize largemouth bass by bathing at different doses. Three concentrations were set: 10 mg / L, 20 mg / L, and 40 mg / L. Six hours after immunization, the largemouth bass in different immunization groups were transferred to different recirculating aquaculture systems. The immunization period lasted for four weeks, during which the fish were fed regularly, and their immune status was monitored and recorded daily. The immunization experiment was conducted in four groups: a PBS blank control group, a MEV-MFH (10 mg / L) immunization group, a MEV-MFH (20 mg / L) immunization group, and a MEV-MFH (40 mg / L) immunization group, with 80 fish in each group and three parallel treatments.

[0051] Serum-specific antibody level detection: Blood samples were collected on days 7, 4, 21, and 28 after immersion immunization. Serum antibody titers were detected by indirect ELISA. Results are as follows: Figure 8 As shown in the figure. The results showed that after immersion immunization, the serum antibody titer of the immunized group was significantly higher than that of the PBS blank control group. Except for the PBS blank control group, the serum antibody titers of the largemouth bass in the other groups gradually increased after injection immunization, and reached a peak on day 28 of immunization. Among the groups, the serum antibody titer of the MEV-MFH (40 mg / L) immunization group was the highest.

[0052] Example 5 This embodiment describes the relative immunoprotective effect of a largemouth bass ferritin-based self-assembled nanovaccine after LMBV infection.

[0053] In Example 4, 28 days after immunization, 40 largemouth bass from each experimental group were randomly selected for LMBV infection testing. LMBV virus solution (20 μL / fish, 10 μL / tail) was injected intraperitoneally. 5 TCID 50 / mL), recording the disease incidence and survival of largemouth bass within 14 days. During the experiment, the water temperature was controlled at 25±0.5℃, and other culture conditions remained unchanged. The relative percentage survival (RPS) was calculated. RPS calculation method: RPS = (1 - mortality rate of the immunized group / mortality rate of the control group) × 100.

[0054] Table 2 Relative immune protection rate

[0055] The experimental results are shown in Table 2. 14 days after LMBV infection, the mortality rate in the PBS blank control group was 95.00%, while the relative immunoprotection rate in the MEV-MFH (40 mg / L) immunization group was 73.68%, significantly improved compared to the 10 mg / L immersion dose. These results indicate that the MEV-MFH self-assembled nano-immersion vaccine can provide good immunoprotection for largemouth bass.

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A ferritin-based self-assembled nanovaccine for preventing LMBV virus in largemouth bass, characterized in that, It includes the polypeptide LMWP, the multi-epitope antigen MEV, and the perch ferritin MFH, which are linked in sequence. The amino acid sequence of the polypeptide LMWP is shown in SEQ ID NO.3; The amino acid sequence of the multi-epitope antigen MEV is shown in SEQ ID NO.4; The amino acid sequence of the perch ferritin MFH is shown in SEQ ID NO.

5.

2. The largemouth bass ferritin self-assembled nanovaccine according to claim 1, characterized in that, The polypeptide LMWP and the multi-epitope antigen MEV are linked by a first linker peptide; the multi-epitope antigen MEV and the perch ferritin MFH are linked by a second linker peptide.

3. The largemouth bass ferritin self-assembled nanovaccine according to claim 2, characterized in that, The amino acid sequence of the first linker peptide is KK; the amino acid sequence of the second linker peptide is shown in SEQ ID NO.

6.

4. The encoding gene of a largemouth bass ferritin self-assembled nanovaccine as described in any one of claims 1-3.

5. The encoding gene according to claim 4, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

6. A gene expression cassette, characterized in that, It includes the coding gene as described in claim 4 or 5.

7. A recombinant plasmid, characterized in that, It includes the gene expression cassette as described in claim 6.

8. A recombinant microbial strain, characterized in that, It includes the recombinant plasmid as described in claim 7.

9. The use of the encoding gene as described in claim 4 or 5, the gene expression cassette as described in claim 6, the recombinant plasmid as described in claim 7, or the recombinant microbial strain as described in claim 8 in the preparation of a largemouth bass ferritin self-assembled nanovaccine as described in any one of claims 1-3.

10. A method for preparing a largemouth bass ferritin self-assembled nanovaccine as described in any one of claims 1-3, characterized in that, Includes the following steps: The recombinant microbial strain of claim 8 was induced and cultured to obtain bacterial cells; After the bacterial cells were broken down, they were denatured and purified to obtain the largemouth bass ferritin self-assembled nanovaccine.

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