Aeromonas salmonicida OmpK subunit vaccine, and preparation method and application thereof

Abstract

The application discloses a kind of aeromonas veronii OmpK subunit vaccine and its preparation method and application, belong to genetic engineering and molecular immunology technical field.The technical scheme gist of the present application is as follows:one kind of aeromonas veronii OmpK subunit vaccine, the OmpK subunit vaccine protein is encoded by aeromonas veronii OmpK gene, its base sequence is shown in the sequence table SEQ ID No.1, the protein coded is aeromonas veronii OmpK subunit vaccine protein, its amino acid sequence is shown in the sequence table SEQ ID No.4.The subunit vaccine of the present application can significantly stimulate fish immune related genes (IL-1 beta, TNF-alpha, MHC-II, etc.) up-regulated expression and specific antibody (IgM) mass production after being injected into carp body, effectively improve the ability of carp to resist aeromonas veronii infection, and then be used to prevent bacterial septicemia of carp caused by aeromonas veronii in aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture animal vaccine technology, specifically relating to an OmpK subunit vaccine of Aeromonas cypriniformis, its preparation method, and its application. Background Technology

[0002] Carp is a core economic fish species in my country's freshwater aquaculture industry, characterized by large-scale farming and a complete industrial chain, playing a crucial role in stabilizing and increasing the efficiency of aquaculture production. Aeromonas veronii, a typical opportunistic pathogen, is the primary cause of bacterial septicemia in carp. It is characterized by high pathogenicity, rapid spread, and high mortality. After infection, fish exhibit symptoms such as surface hemorrhage, internal organ congestion, and ascites, with a mortality rate exceeding 80%, causing significant economic losses to the aquaculture industry. In recent years, with the increasing intensification and scale of aquaculture, the frequency and severity of bacterial diseases have continued to intensify, becoming a key bottleneck restricting the high-quality development of the aquaculture industry. Among them, Aeromonas veronii, as a typical opportunistic pathogen, is widely distributed in freshwater aquaculture environments and is the main pathogen causing bacterial septicemia in various economic fish species such as carp, crucian carp, and grass carp. The disease has a rapid onset, spreads quickly, and has a high mortality rate. It is particularly prone to outbreaks when the water temperature is 25-30℃, the water quality deteriorates, and the stocking density is too high, causing huge economic losses to the aquaculture industry.

[0003] Currently, the control of Aeromonas versicolor pathogens still heavily relies on antibiotics. Long-term, high-dose use not only easily induces multidrug resistance in bacteria but also causes drug residues and ecological imbalance in water bodies, seriously affecting the quality and safety of aquatic products and the sustainable development of the industry. Vaccination, as a green, safe, and efficient control method, has become the mainstream alternative to antibiotics. Existing Aeromonas versicolor vaccine research mainly focuses on inactivated vaccines. Inactivated vaccines have mature processes and good safety profiles, but their immunogenicity is weak and their protection rate is limited. Subunit vaccines, on the other hand, have high safety and clearly defined components, greatly reducing the possibility of harmful substances in the vaccine, providing a reliable basis for enhancing the effectiveness of green control in aquaculture.

[0004] Aeromonas versicolor outer membrane proteins (OMPs) are key virulence factors located on the bacterial surface, exhibiting high conservation and good immunogenicity, making them ideal candidate targets for subunit vaccine development. Among them, OmpK, as a major functional protein of the outer membrane, plays a crucial role in bacterial adhesion, invasion, and immune evasion. It also demonstrates strong conservation and abundant antigenic epitopes across different strains, possessing potential for vaccine development. Currently, there are no reports on constructing subunit vaccines using Aeromonas versicolor OmpK protein. Therefore, this invention constructs a subunit vaccine targeting the conserved outer membrane protein OmpK of Aeromonas versicolor, stimulating fish to produce anti-Aeromonas versicolor antibodies, thereby enhancing the fish's immunity and resistance to disease infection. Summary of the Invention

[0005] The purpose of this invention is to provide a Cyprinocystis ivermectin OmpK subunit vaccine for carp and its preparation method, which effectively solves the technical problems of low immunoprotection rate and insufficient safety of existing vaccines. The prepared subunit vaccine contains Cyprinocystis ivermectin outer membrane protein, which can simultaneously activate the specific and non-specific immunity of carp, significantly improve the ability to resist Cyprinocystis ivermectin infection, and can be used to prevent and treat bacterial septicemia in carp. It has a high immunoprotection rate, good safety, and can be produced on a large scale.

[0006] To achieve the above objectives, the present invention employs the following technical solution: a *Aeromonas vesiculosus* OmpK subunit vaccine, wherein the subunit vaccine protein is encoded by the *Aeromonas vesiculosus* OmpK gene, the base sequence of which is shown in SEQ ID No. 1 of the sequence listing, and the protein it encodes is the *Aeromonas vesiculosus* outer membrane protein OmpK subunit vaccine protein, the amino acid sequence of which is shown in SEQ ID No. 4 of the sequence listing.

[0007] A method for preparing an OmpK subunit vaccine from Aeromonas cypriniformes, the specific preparation steps of which are as follows:

[0008] Step S1, Construction of plasmid pET28a-OmpK: Using the isolated Aeromonas cyprinii genomic DNA as a template, the OmpK gene was amplified by PCR using specific primers. The upstream and downstream primer sequences of these specific primers are as follows:

[0009] OmpK-F: 5'-GCGGATCCATGAAAAAATTACTTCCTCTGATGATTGCCG-3';

[0010] OmpK-R: 5'-AAGCTTGAAGCGGTAGCCAACACC-3';

[0011] After purification, the amplified product was double-digested with BamHI and Hind III, and the digested gene fragments were recovered. At the same time, the plasmid pET28a(+) was double-digested with BamHI and Hind III, and the digested gene fragments were recovered. The two recovered gene fragments were ligated with T4 DNA ligase. The ligation product was transformed into competent Escherichia coli BL21(DE3) and cultured on LB solid medium containing kanamycin (Kan). Positive recombinant bacteria containing plasmid pET28a-OmpK were screened.

[0012] Step S2, induction and purification of vaccine protein: Positive recombinant bacteria containing plasmid pET28a-OmpK were cultured in LB liquid medium containing kanamycin at 37°C, and then the culture was transferred to fresh LB medium containing kanamycin and cultured at 37°C until OD2000. 600 The concentration was 0.6, and IPTG was added to a final concentration of 0.2 mM. The mixture was then induced and cultured at 25°C. The bacterial cells were collected by centrifugation and ultrasonically disrupted. The inclusion bodies were collected and purified by urea dissolution, Ni-IDA affinity chromatography, and gradient dialysis refolding to obtain high-purity Aeromonas villus outer membrane protein OmpK subunit vaccine protein.

[0013] The application of the Aeromonas cypriniforme OmpK subunit vaccine described in this invention in the preparation of aquaculture subunit vaccines.

[0014] The application of the Cypripedium vesiculosus OmpK subunit vaccine described in this invention in the preparation of a subunit vaccine against Cypripedium vesiculosus infection in carp involves injecting the Cypripedium vesiculosus OmpK subunit vaccine into the peritoneal cavity of carp at a dose of 2 μg subunit vaccine / g fish body weight. This stimulates the proliferation of carp immune cells, thereby enhancing the carp's ability to resist Cypripedium vesiculosus infection, with an immunoprotection rate of over 42%.

[0015] Further preferably, the carp immune cell-related genes are IL-1β, TNF-α, and MHC-II.

[0016] The application of the *Aeromonas versicolor* OmpK subunit vaccine of the present invention in the preparation of a subunit vaccine for the prevention and / or treatment of bacterial septicemia in carp caused by *Aeromonas versicolor* involves injecting the *Aeromonas versicolor* OmpK subunit vaccine into the peritoneal cavity of a carp at a dose of 2 μg subunit vaccine / g fish body weight. By inducing the fish to produce specific antibodies, the vaccine effectively enhances the carp's ability to resist *Aeromonas versicolor* infection, thereby effectively preventing and / or treating bacterial septicemia in carp caused by *Aeromonas versicolor* infection in aquaculture.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] 1. The OmpK protein used in the preparation of the Aeromonas versicolor OmpK subunit vaccine of carp in this invention is derived from the conserved outer membrane of Aeromonas versicolor, located on the surface of the bacteria, has good immunogenicity, and can effectively induce specific and non-specific immune responses in carp.

[0019] 2. The Aeromonas versicolor OmpK subunit vaccine prepared by this invention can induce carp to produce high-titer specific antibodies, significantly resisting Aeromonas versicolor infection, with an immunoprotective efficiency of 42.11%;

[0020] 3. The Aeromonas cyprini OmpK subunit vaccine prepared by this invention has high safety, contains no live bacteria, poses no risk of infection, has a single antigenic component, is easy to control in quality, has good stability, and is convenient for storage, transportation and large-scale application. Attached Figure Description

[0021] Figure 1 Electrophoresis diagram of PCR amplification of the OmpK gene in Aeromonas verrucosa (lane 1);

[0022] Figure 2 The image shows the electrophoresis pattern for double enzyme digestion verification of the OmpK recombinant plasmid. Lane 1 shows the plasmid double enzyme digestion, and lane 2 shows the OmpK gene PCR amplification band using the plasmid as a template.

[0023] Figure 3 The image shows an SDS-PAGE plot of the recombinant protein. The bands with a molecular weight of approximately 25 kDa in lanes 7 and 8 represent the purified recombinant protein.

[0024] Figure 4 A graph showing the changes in the expression of immune-related genes in the head kidney and spleen of carp after vaccination;

[0025] Figure 5 This graph shows the changes in serum-specific IgM antibody levels in carp after vaccination.

[0026] Figure 6 This is a comparison chart of the survival rates of carp in the experimental and control groups after viral challenge. Detailed Implementation

[0027] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0028] Example 1

[0029] Preparation of Aeromonas cypriniformis OmpK subunit vaccine:

[0030] 1. Construction of plasmid pET28a-OmpK

[0031] Aeromonas veronii strain XX237 was isolated from diseased carp in a pond in Xinxiang, Henan Province. It was identified as a pathogenic strain. Using the genomic DNA of this bacterium (which is basically consistent with the gene sequence of Aeromonas veronii strain 8C (GenBank: CABWLC010000004.1, reference: Jinghang Zhang, Dan Qiao, Haoyu Wang, Xianliang Zhao, Xinyu Jiang, Lei Zhu, Jie Zhang, Li Li, Xianghui Kong and Chao Pei. Mixed Infection in Common Carp (Cyprinus carpio) Caused by Aeromonas veronii, Aeromonas hydrophila, Plesiomonas shigelloides, and Citrobacterfreundii. Animals (Basel), 2025, 15(6): 805.) as a template, PCR amplification was performed using specific primers. The PCR amplification system was 2×Phusion Master Mix 25 μL of PCR product, 1 μL each of upstream / downstream primers (OmpK-F: 5'-GCGGATCCATGAAAAAATTACTTCCTCTGATGATTGCCG-3', OmpK-R: 5'-AAGCTTGAAGCGGTAGCCAACACC-3'), 2 μL of template DNA, and ddH2O to a final volume of 50 μL; amplification program: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 45 s, 60℃ annealing for 45 s, 72℃ for 1 min, 72℃ extension for 1 min, 30 cycles, and a final extension at 72℃ for 10 min. The purified PCR product was double-digested with BamHI and HindIII, and a gene fragment of approximately 850 bp was recovered. Simultaneously, plasmid pET28a(+) was double-digested with BamHI and HindIII, and a gene fragment of approximately 5.3 kb was recovered. Figure 2 The two recovered gene fragments were ligated using T4 DNA ligase. The ligation product was transformed into competent *E. coli* BL21(DE3) and cultured on LB agar containing ampicillin for 12 h. Single colonies that grew on the agar were picked and inoculated into liquid LB agar containing Kans for expansion. PCR was performed on each expanded colony, and positive bacteria were selected for plasmid DNA sequence analysis to verify the correct amplification of the target gene fragment and its correct ligation with the plasmid. The plasmid pET28a-OmpK was extracted from the expanded culture of the positive bacteria after sequencing verification.

[0032] 2. Induction and purification of subunit vaccine proteins

[0033] Transform the correctly identified pET28a-OmpK plasmid into BL21(DE3) strain, pick single colonies, and inoculate them into LB medium containing Kan (50 µg / mL), activating overnight at 37°C. The next day, transfer fresh LB medium containing Kan at a volume ratio of 1:100, and incubate at 37°C until OD200. 600 The value was approximately 0.6. Gradient induction was set up as follows: temperature 16℃ / 25℃, IPTG concentration 0.2~1.0 mM, time 4~10 h. 1 mL of bacterial culture from each group was collected by centrifugation, resuspended in PBS, boiled in 5× loading buffer, and subjected to 12% SDS-PAGE electrophoresis to compare band intensity. IPTG 0.2 mM induction at 25℃ for 10 h was selected as the optimal condition for large-scale expression. After small-scale induction under optimized conditions, 2 mL of bacterial culture was collected by centrifugation, resuspended in 300 μL PBS, and sonicated on ice (30% power, 3 s working / 3 s interval, total 2 min). The supernatant and precipitate were separated by centrifugation at 12000 g for 15 min at 4℃, and the precipitate was resuspended in an equal volume of PBS. 40 μL of supernatant / precipitate was added to 10 μL loading buffer and boiled. SDS-PAGE was used to detect solubility, showing that the protein was mainly expressed in inclusion body form. For large-scale induction, the overnight activated seed culture was transferred to 500 mL of LB medium containing Kans at a volume ratio of 1:100 and cultured at 37°C until OD500. 600 Approximately 0.6% of the optimal IPTG concentration was added, and the cells were induced and cultured at the optimized temperature for 10 h. After induction, the cells were collected by centrifugation at 8000 g for 30 min at 4 °C and washed twice with pre-cooled PBS. The cells were resuspended in 30 mL of binding buffer and sonicated on ice (35% power, 3 s working time / 3 s interval, total duration 30-40 min). The lysate was centrifuged at 12000 g for 20 min at 4 °C, and the supernatant was discarded. The precipitate was washed twice with binding buffer containing 1 M urea (centrifuged after 1 h of shaking each time) to remove some contaminating proteins. The final precipitate was dissolved overnight at 4 °C in 15 mL of denaturing binding buffer containing 8 M urea, centrifuged at 12000 g for 15 min, and the supernatant was collected as the column sample. Ni-IDA affinity chromatography purification was performed by packing 2 mL of Ni-IDA resin into a column, allowing it to settle naturally, and then equilibrating with 5 column volumes of binding buffer containing 8 M urea. The denaturing supernatant was slowly loaded onto the column, and the flow-through buffer could be repeated once. Unbound proteins were then washed away using 10 column volumes of 8 M urea-binding buffer.

[0034] During gradient elution, elution was performed sequentially with elution buffers containing 20 mM, 50 mM, 70 mM, 100 mM, 300 mM, and 500 mM imidazole (5 mL each), and the eluents were collected. 20 μL of each elution peak sample was added to 5 μL of 5× loading buffer, and SDS-PAGE was used to determine purity and concentration. The high-purity eluent fraction containing the target protein was combined. The purified eluent containing the target protein was placed in a pre-treated dialysis bag and dialyzed at 4°C with magnetic stirring in the following refolding buffer (20 mM PBS). After refolding, the dialysate was centrifuged at 12000 g for 10 min at 4°C to remove the precipitate. The protein concentration of the supernatant was determined using a NanoDrop spectrophotometer, and the purity and recovery were verified by SDS-PAGE. Figure 3 ).

[0035] Example 2

[0036] Application of Aeromonas cypriniformis OmpK protein subunit vaccine:

[0037] 1. Subunit vaccines can significantly increase the expression of disease-resistant genes in fish.

[0038] Following immunization with the OmpK subunit vaccine protein, TNF-α gene expression in the head kidneys rapidly initiated, significantly upregulating at 3 days (P<0.05) and reaching a peak, with high expression persisting until 28 days. High levels were reached at 7 and 14 days (P<0.01), before returning to control levels at 35 days. This indicates that the OmpK subunit vaccine protein provides strong but prolonged stimulation of TNF-α. Following immunization with the OmpK subunit vaccine protein, IL-1β in the carp head kidneys significantly increased at 3 days (P<0.05), peaked at 7 days, and rapidly decreased at 14 days. MHC-II in the head kidneys rapidly upregulated from 3 days (P<0.01), then rapidly decreased to control levels. Splenic IL-1β was significantly higher than the control group at 7 days (P<0.01), peaked at 14 days (P<0.01), and then slowly decreased. Splenic MHC-II peaked at 3 days (P<0.01) and then decreased. These results indicate that the OmpK subunit vaccine protein can effectively activate IL-1β responses and MHC-II-related adaptive immunity.

[0039] 2. Subunit vaccines can significantly stimulate fish to produce protective antibodies.

[0040] The changes in specific IgM antibody levels in carp serum after injection of OmpK subunit vaccine protein are as follows: Figure 5 As shown, the antibody response in the OmpK immunization group started rapidly, showing a significant increase on day 3 (P<0.01), peaking on day 28, and then gradually declining. On day 35, the difference from the control group was still extremely significant (P<0.01).

[0041] 3. Subunit vaccines have a significant protective effect against sepsis caused by Aeromonas verrucosa.

[0042] Eighty healthy carp (15±1.5g) were divided into two groups of 40 each, serving as the experimental group and the control group, respectively. The experimental group was injected with recombinant subunit vaccine protein (2 μg subunit vaccine protein / g fish body weight), while the control group was injected with PBS buffer. Twenty days after injection, each fish in both the experimental and control groups was injected with 0.1 mL of a 1×10⁻⁶ solution. 8 Fish were infected with CFU / mL Aeromonas vesiculosus bacterial suspension. After challenge, they were observed continuously for 15 days, and mortality was recorded. The number of deaths and survivors in both groups were counted, and the immunoprotective efficacy (RPS) of the subunit vaccine was calculated. RPS = (1 - mortality rate of immunized group / mortality rate of control group) × 100%. Based on the experimental results, the immunoprotective efficacy of the OmpK subunit vaccine against Aeromonas vesiculosus was 42.11%. Figure 6 This indicates that the OmpK subunit vaccine can significantly improve the ability of carp to resist Aeromonas verrucosa infection.

[0043] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

[0044] SEQUENCE LISTING <110> Henan Normal University <120> A Aeromonas cypriniformis OmpK subunit vaccine, its preparation method and application <130> 1 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 804 <212> DNA <213> Aeromonas veronii <400> 1 atggccaaat ttaccaagac tctgatcgct gccggtctgc tggctgctgc cgctaccccg 60 gccttcgctg ctgactacag cggtgatatc cacaagaatg actacaagtg gatgcagttc 120 aatatcatgc acaccatcga tcagaagccc catggtccgg atggttacaa cgatacttat 180 ttcgagatgg agtttggtgg tcgttcaggc atttttgacc tgtatggtta cgtcgattac 240 ttcgatattt ttgactccaa gcacagcgac aagcacaagc aagaaaacct cttcatgaag 300 tttgctccgc gtgtatctct ggatgcattg actggccaag acctctcttt tggtccggtg 360 caagaagttt acgttgctac cctgtttaac atcggtggtg tacaaggcct gaatgatgca 420 tttgtaggtc tgggtgctga cgttatggtg ccgtggtttg gcaaagttgg tatgaacctg 480 tacaagcgtc atgctaacga aaccttcagt ggtcaaggcg aaggctggaa cggccaacag 540 ttctccatga actggttcaa acctttctat accttctcca atggcagctt cgtctcttac 600 caaggttatc tggattacac ctggggcatg aaagaaacca gtcattctga tggtctggga 660 accttccacg gtatctactg gcattccgac cgttatgccg ttggctacgg tctgaagtac 720 ttcaacgaaa tctacggtat cgacaactct gacgcgttca agtctaccgg tttcggccac 780 tacttctctg ttacctacaa gttc 804 <210> 2 <211> 39 <212> DNA <213> Synthetic <400> 2 gcggatccat gaaaaaatta cttcctctga tgattgccg 39 <210> 3 <211> 24 <212> DNA <213> Synthetic <400> 3 aagcttgaag cggtagccaa cacc 24 <210> 4 <211> 268 <212> PRT <213> Aeromonas veronii <400> 4 Met Ala Lys Phe Thr Lys Thr Leu Ile Ala Ala Gly Leu Leu Ala Ala 1 5 10 15 Ala Ala Thr Pro Ala Phe Ala Ala Asp Tyr Ser Gly Asp Ile His Lys 20 25 30 Asn Asp Tyr Lys Trp Met Gln Phe Asn Ile Met His Thr Ile Asp Gln 35 40 45 Lys Pro His Gly Pro Asp Gly Tyr Asn Asp Thr Tyr Phe Glu Met Glu 50 55 60 Phe Gly Gly Arg Ser Gly Ile Phe Asp Leu Tyr Gly Tyr Val Asp Tyr 65 70 75 80 Phe Asp Ile Phe Asp Ser Lys His Ser Asp Lys His Lys Gln Glu Asn 85 90 95 Leu Phe Met Lys Phe Ala Pro Arg Val Ser Leu Asp Ala Leu Thr Gly 100 105 110 Gln Asp Leu Ser Phe Gly Pro Val Gln Glu Val Tyr Val Ala Thr Leu 115 120 125 Phe Asn Ile Gly Gly Val Gln Gly Leu Asn Asp Ala Phe Val Gly Leu 130 135 140 Gly Ala Asp Val Met Val Pro Trp Phe Gly Lys Val Gly Met Asn Leu 145 150 155 160 Tyr Lys Arg His Ala Asn Glu Thr Phe Ser Gly Gln Gly Glu Gly Trp 165 170 175 Asn Gly Gln Gln Phe Ser Met Asn Trp Phe Lys Pro Phe Tyr Thr Phe 180 185 190 Ser Asn Gly Ser Phe Val Ser Tyr Gln Gly Tyr Leu Asp Tyr Thr Trp 195 200 205 Gly Met Lys Glu Thr Ser His Ser Asp Gly Leu Gly Thr Phe His Gly 210 215 220 Ile Tyr Trp His Ser Asp Arg Tyr Ala Val Gly Tyr Gly Leu Lys Tyr 225 230 235 240 Phe Asn Glu Ile Tyr Gly Ile Asp Asn Ser Asp Ala Phe Lys Ser Thr 245 250 255 Gly Phe Gly His Tyr Phe Ser Val Thr Tyr Lys Phe 260 265

Claims

1. A Aeromonas cypriniformes OmpK subunit vaccine, characterized in that: The subunit vaccine protein is encoded by the Aeromonas villus ompK gene, the base sequence of which is shown in SEQ ID No. 1 of the sequence listing. The protein it encodes is the Aeromonas villus outer membrane protein OmpK subunit vaccine protein, the amino acid sequence of which is shown in SEQ ID No. 4 of the sequence listing.

2. A method for preparing the Aeromonas cypriniforme OmpK subunit vaccine according to claim 1, characterized in that... The specific preparation steps are as follows: Step S1, Construction of plasmid pET28a-OmpK: Using the isolated Aeromonas cyprinii genomic DNA as a template, the OmpK gene was amplified by PCR using specific primers. The upstream and downstream primer sequences of these specific primers are as follows: OmpK-F: 5'-GCGGATCCATGAAAAAATTACTTCCTCTGATGATTGCCG-3'; OmpK-R: 5'-AAGCTTGAAGCGGTAGCCAACACC-3'; After purification, the amplified product was double-digested with BamHI and Hind III, and the digested gene fragments were recovered. At the same time, the plasmid pET28a(+) was double-digested with BamHI and Hind III, and the digested gene fragments were recovered. The two recovered gene fragments were ligated with T4 DNA ligase. The ligation product was transformed into competent Escherichia coli BL21(DE3) and cultured on LB solid medium containing kanamycin. Positive recombinant bacteria containing plasmid pET28a-OmpK were screened. Step S2, induction and purification of vaccine protein: Positive recombinant bacteria containing plasmid pET28a-OmpK were cultured in LB liquid medium containing kanamycin at 37°C, and then the culture was transferred to fresh LB medium containing kanamycin and cultured at 37°C until OD2000. 600 The concentration was 0.6, and IPTG was added to a final concentration of 0.2 mM. The mixture was then induced and cultured at 25°C. The bacterial cells were collected by centrifugation and ultrasonically disrupted. The inclusion bodies were collected and purified by urea dissolution, Ni-IDA affinity chromatography, and gradient dialysis refolding to obtain high-purity Aeromonas villus outer membrane protein OmpK subunit vaccine protein.

3. The application of the Aeromonas cypriniforme OmpK subunit vaccine according to claim 1 in the preparation of aquaculture subunit vaccines.

4. The use of the Aeromonas versicolor OmpK subunit vaccine of claim 1 in the preparation of a subunit vaccine against Aeromonas versicolor infection in carp.

5. The application according to claim 4, characterized in that: The Aeromonas versicolor OmpK subunit vaccine was injected into the abdominal cavity of carp at a dose of 2 μg subunit vaccine / g fish body weight. This stimulated the proliferation of carp immune cells and enhanced the carp's ability to resist Aeromonas versicolor infection, with an immune protection rate of over 42%.

6. The application according to claim 5, characterized in that: The carp immune cell-related genes are IL-1β, TNF-α, and MHC-II.

7. The use of the Aeromonas versicolor OmpK subunit vaccine of claim 1 in the preparation of a subunit vaccine for the prevention and / or treatment of bacterial septicemia in carp caused by Aeromonas versicolor.

8. The application according to claim 7, characterized in that: The OmpK subunit vaccine of Aeromonas versicolor was injected into the abdominal cavity of carp at a dose of 2 μg subunit vaccine / g fish body weight. By inducing the fish to produce specific antibodies, the carp's ability to resist Aeromonas versicolor infection was effectively improved, thereby effectively preventing and / or treating bacterial septicemia of carp caused by Aeromonas versicolor infection in aquaculture.

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