A fish columnar disease subunit vaccine and its application

Abstract

The present invention discloses a fish columnar disease subunit vaccine and its application, wherein the subunit vaccine contains a recombinant protein with an amino acid sequence as shown in SEQ ID No.1 as an antigen component. The subunit vaccine has the highest protection rate of 88.9% for grass carp challenged with F. covae MU-04, and a protection rate of 100% for grass carp challenged with F. columnare JX-01. The specific IgM antibody titers induced by the vaccine in various tissues of grass carp are higher than those of the control group, and the vaccine can stimulate the grass carp to produce a strong antibody response. The present invention enriches the development of genetically engineered subunit vaccines for fish columnar disease and provides a new approach for the prevention and treatment of fish columnar disease.

Description

Technical Field

[0001] The invention belongs to the technical field of fish vaccines, and in particular relates to a fish columnar disease subunit vaccine and application thereof. Background Art

[0002] Columnar disease is an infectious disease caused by infection with Flavobacterium cloumnare. The researchers reclassified Flavobacterium cloumnare into four genetic groups through multilocus phylogenetic analysis (MLPA). The correspondence between MLPA and 16S-RFLP genotypes is as follows: Genetic group 1 corresponds to Genomovar I strains, Genetic group 2 and Genetic group 4 correspond to some strains in Genomovar II, and Genetic group 3 corresponds to Genomovar III strains. In 2022, LaFrentz et al. (2022) reclassified the strains of Genetic group 2, 3, and 4 into three new species of the genus Flavobacterium based on multiphase methods such as two-dimensional thin-layer chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), and multiple physiological and biochemical identifications, and named them Flavobacterium.covae, Flavobacterium.davisii, and Flavobacterium.oreochromis, respectively. The strains of Genetic group 1 still retained the original name—F.columnare.

[0003] Grass carp columnar disease is highly infectious and progresses rapidly, which can easily lead to large-scale deaths of farmed grass carp. It is one of the key reasons for the reduction in grass carp production and quality over the years, and has seriously restricted the development of the grass carp farming industry. At present, the prevention and treatment of grass carp columnar disease is still mainly based on chemical drugs and antibiotics, but this will affect the water environment and easily lead to bacterial resistance, which poses a great safety risk. Many research results show that it is feasible to develop vaccines for columnar disease. The vaccines reported so far mainly include inactivated vaccines, attenuated live vaccines and subunit vaccines, but only one AQUAVAC-COL attenuated live vaccine has been successfully launched in the United States. According to epidemiological surveys, the main pathogens of grass carp columnar disease in China are F. columnare and F. covae. F. covae grows slowly in artificial culture and is easy to aggregate, which increases the difficulty of preparing whole-bacterial vaccines; in addition, whole-bacterial vaccines will contain many invalid bacterial antigen components, inducing the host to produce a large number of invalid antibodies, thereby reducing the host's response intensity to protective antigens and the immune protection effect of the vaccine. Therefore, the development of genetically engineered subunit vaccines is very necessary. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a fish columnar disease subunit vaccine.

[0005] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0006] The present invention screens subunit vaccine antigens based on F. covae MU-04 strain, and obtains 5 candidate antigen targets based on the LC-MS / MS results of strain immune proteins, recombinantly expresses the candidate antigens through a prokaryotic expression system, and uses the candidate antigens to prepare vaccines for immunizing grass carp, and evaluates the protective effect of the vaccine on grass carp. The results show that the vaccine prepared by using a hypothetical protein (MU-04_GM002923) recombinant protein as an antigen has the highest protection rate of 88.9% for grass carp challenged with F. covae MU-04, and has a protection rate of 100% for grass carp challenged with F. columnare JX-01, and the titer of specific IgM antibodies induced by the vaccine in various tissues of the grass carp is higher than that of the control group.

[0007] Therefore, the present invention first provides the use of a hypothetical protein (MU-04_GM002923) as an antigen component in the preparation of a fish columnar disease subunit vaccine. Specifically, the hypothetical protein (MU-04_GM002923) is subjected to conventional in vitro recombinant expression to obtain a recombinant protein, and then the fish columnar disease subunit vaccine is prepared using the recombinant protein as an antigen.

[0008] The present invention further provides the use of the recombinant protein shown in SEQ ID No. 1 as an antigen component in the preparation of a fish columnar disease subunit vaccine.

[0009] Furthermore, the fish is grass carp.

[0010] Furthermore, the fish columnar disease is a fish columnar disease caused by Flavobacterium.covae and / or Flavobacterium.columnare.

[0011] The present invention also provides a fish columnar disease subunit vaccine, which contains a recombinant protein with an amino acid sequence as shown in SEQ ID No. 1 as an antigen component.

[0012] Furthermore, the nucleotide sequence of the gene encoding the recombinant protein is shown in SEQ ID No.2.

[0013] Furthermore, the fish columnar disease subunit vaccine also contains an adjuvant.

[0014] Furthermore, the adjuvant is Freund's adjuvant.

[0015] Preferably, the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant.

[0016] Furthermore, the volume ratio of the adjuvant to the recombinant protein is 1:1.

[0017] The present invention provides the use of any of the above-mentioned fish columnar disease subunit vaccines in the preparation of products for preventing and treating fish columnar disease.

[0018] Furthermore, the fish is grass carp.

[0019] Furthermore, the fish columnar disease is a fish columnar disease caused by Flavobacterium.covae and / or Flavobacterium.columnare.

[0020] Furthermore, the immunization dose of the fish columnar disease subunit vaccine is 80 to 100 μg of recombinant protein / fish.

[0021] Preferably, the immunization dose of the subunit vaccine is 80 μg of recombinant protein / fish.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a subunit vaccine for fish columnar disease, wherein the subunit vaccine contains a recombinant protein with an amino acid sequence as shown in SEQID No.1 as an antigen component. The subunit vaccine has the highest protection rate against grass carp challenged with F. covae, reaching 88.9%, and a protection rate against grass carp challenged with F. columnare of 100%. The specific IgM antibody titers induced by the vaccine in various tissues of grass carp are higher than those of the control group, and the vaccine can stimulate the grass carp to produce a strong antibody response. The present invention enriches the development of genetic engineering subunit vaccines for fish columnar disease, and provides a new approach for the prevention and treatment of fish columnar disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the sequence analysis of hypothetical protein; a is the nucleic acid sequence and amino acid sequence of the hypothetical protein gene, and the purple block is the signal peptide region; b is the Chou and Fasman β steering prediction; c is the Emini surface accessibility prediction; d is the Parker hydrophilicity prediction; and e is the linear antigen epitope prediction.

[0025] Figure 2 The figure shows the recombinant protein expression and purification of the hypothetical protein; a is the Western blot verification of protein expression; b is the result of washing the inclusion bodies with different concentrations of urea: 1: supernatant after bacterial lysis after ultrasonication, 2: 2M urea washing solution, 3: 4M urea washing solution, 4: 6M urea washing solution, 5: 6M precipitation; c is the result of protein purification by nickel column: 6: pET32a control, 7: protein induction, 8: purified protein. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0027] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0028] The Flavobacterium covae MU-04 and Flavobacterium columnare JX-01 strains were deposited in the Laboratory of Aquatic Animal Immunity and Disease Control, College of Oceanography, South China Agricultural University.

[0029] Example 1 Screening of subunit vaccine antigens

[0030] The present invention screens subunit vaccine antigens based on the F. covae MU-04 strain and screens based on the LC-MS / MS results of the strain immune protein. The specific method is as follows:

[0031] (1) Bacterial outer membrane protein extraction (Bacterial outer membrane protein extraction kit (BB-31512), purchased from Beibo Biotechnology Co., Ltd.)

[0032] a) Add 2 μL of protease inhibitor cocktail to every 500 μL of extract A;

[0033] b) 5 mL of bacterial solution was centrifuged at 6000 rpm for 5 min, the bacteria were collected and washed twice with PBS solution;

[0034] c) adding the solution in step a) to the bacterial cells in step b), mixing well and incubating at 4° C. with shaking for 1 h;

[0035] d) Centrifuge the solution in the previous step at 12,000 × g for 5 min at 4°C, remove the supernatant, and then incubate in a water bath at 37°C for 10 min;

[0036] e) Carefully discard the upper layer of liquid and keep 30 μL of liquid at the bottom of the tube;

[0037] f) Add 50 μL of pre-cooled membrane protein solubilization solution to the liquid in step e) and mix well, and store at -80°C for later use.

[0038] (2) LC-MS / MS sample preparation

[0039] a) 40 μL of outer membrane protein solution was mixed with 50 μL of grass carp serum immunized twice with MU-04 and incubated with shaking at room temperature for 1 h;

[0040] b) Add 60 μL of protein A equilibrated with 1×TBS to the liquid in step a) and place at 4°C overnight for shaking and binding;

[0041] c) After binding, centrifuge at 2000×g for 5 min and save the effluent. Wash the precipitate twice with 500 μL TBS solution (1000×g, 3 min) until A280 is 0;

[0042] d) Take 20 μL of the sample in step c), add 5× Loading Buffer, mix well, and boil in a 100°C water bath for 10 min;

[0043] e) The samples in the previous step were subjected to SDS-PAGE detection and then stained with Coomassie Brilliant Blue. After decolorization with a decolorizing solution, the gel bands were cut out and sent to Guangzhou Huijun Technology Biological Co., Ltd. for LC-MS / MS detection.

[0044] (3) LC-MS / MS result screening

[0045] The mass spectrometry results were compared with the MU-04 genome database (NCBI biosample number: PRJNA1078299) by Mascot search, and the predicted proteins were predicted by CELLO G — The subcellular localization program predicted the outer membrane proteins, and proteins with matching scores and high content were screened; then these proteins were compared with ATCC 49512, AH-01, and GX-01 proteins (NCBI: GCA_000240075.2, PRJNA841461, PRJNA841778), and finally the sequences with higher similarity in several bacteria were selected as candidate antigens.

[0046] The candidate antigens screened are shown in Table 1.

[0047] Table 1 Candidate antigens screened based on immune protein profiles

[0048]

[0049]

[0050] Note: The ID listed in the table is the gene number of this protein in the MU-04 genome.

[0051] Example 2 Subunit vaccine antigen expression and purification

[0052] (1) Antigen gene sequence analysis

[0053] The protein isoelectric point (pI) and molecular weight (Mw) were calculated using an online program ( http: / / web.expasy.org / compute_pi ), and the signal peptide ( https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ), transmembrane domain ( https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ) and antigen epitope ( http: / / tools.iedb.org / bcell / ) were also predicted.

[0054] (2) Prokaryotic expression of antigens

[0055] 1) Primer design

[0056] The recombinase primers were designed using CE Design software, and the primer sequences are shown in Table 2 (the primer names are short gene numbers).

[0057] 2) Gene amplification and purification

[0058] Phanta Max Super-Fidelity DNA Polymerase (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) was used to amplify the genes in Table 1 above. The PCR amplification system and program are shown in Tables 3 and 4.

[0059] Table 2 Antigen amplification primer sequences

[0060]

[0061]

[0062] Table 3 PCR amplification system

[0063]

[0064] Table 4 PCR reaction program

[0065]

[0066] The PCR amplified products were subjected to 1% agarose gel electrophoresis and then recovered by gel excision, and the concentration of the recovered products was determined.

[0067] 3) Expression vector construction

[0068] The pET32a strain was revived, and then a single colony was picked and placed in 5 mL LB liquid medium (containing Amp resistance) for overnight shaking. After collecting the bacteria in the logarithmic phase, plasmid extraction was performed. The obtained plasmid was double-digested with a fast-cutting enzyme (system is shown in Table 5) and incubated at 37°C for 20 minutes. The product after double-digestion was recovered by gel excision after 1% agarose gel electrophoresis, and the concentration of the gel-recovered product was determined.

[0069] The vector and target fragment were mixed according to Table 6, connected at 55°C for 20 min according to the instructions of the recombinase (ClonExpress Ultra OneStep Cloning Kit-C115, purchased from Nanjing Novozymes Biotechnology Co., Ltd.), and immediately placed on ice. At the same time, DH5α competent cells were taken out and transformed into Escherichia coli, and positive clones and sequencing were performed for identification. The strains with correct sequencing were amplified, the plasmids were extracted, and then transformed into Rosetta or plysS competent cells and stored at -80°C.

[0070] Table 5 pET32a double enzyme digestion reaction system

[0071]

[0072] Table 6 Connection system

[0073]

[0074] 4) Protein expression and purification

[0075] a) Resuscitate the strain preserved in step 3), pick a single colony and inoculate it into 5 mL of LB (Amp resistance) liquid culture medium, and culture at 37°C and 180 rpm for 12 to 16 hours;

[0076] b) Transfer 5 mL of overnight culture solution to 500 mL of liquid medium (with resistance) and culture at 37°C and 180 rpm for 4 h until OD600 reaches about 0.6;

[0077] c) Take 1 mL of the uninduced bacterial solution as a control, add IPTG to the remaining bacterial solution to a final concentration of 0.5 mM, continue culturing at 28°C for 4 h, take 1 mL of the induced bacterial solution as the product, and use SDS-PAGE to identify whether there is protein expression;

[0078] d) 500 mL of bacterial solution was centrifuged to collect bacterial precipitate, 20 mL of Lysis Buffer was added, and ultrasonic disruption was performed on ice at the following conditions: ultrasonic power 270 W, on for 9 seconds and off for 9 seconds, for 30 minutes, and the supernatant and precipitate were collected by centrifugation;

[0079] e) The precipitate obtained by centrifugation was washed with 2M, 4M, 6M, and 8M urea in sequence (the washing concentration was determined according to the dissolution), and then homogenized with a homogenizer for 5 minutes. Each component was collected and run on a gel together with the control to determine the component containing the target protein;

[0080] f) Purify the target protein using a nickel column, the steps are as follows: load the nickel filler into an affinity chromatography tube, add 1×TBS with a volume of 10 times the filler to pass through the column; turn off the bottom switch, add a protein sample containing the target protein, and bind overnight at 4°C; turn on the bottom switch and collect the effluent; add Wash buffer with a volume of 10 times the filler (add denatured or non-denatured as appropriate) to pass through the column, collect the effluent, and repeat this step once; add Elution buffer with a volume of 5 times the filler (add denatured or non-denatured as appropriate) to pass through the column, and collect the eluate; perform SDS-PAGE electrophoresis and staining on each collected component sample, and select the component where the target protein exists for subsequent renaturation;

[0081] g) Protein dialysis refolding: The protein solution was placed into a dialysis bag and placed in 6M, 4M, 2.5M, 1.5M, 0.5M, and 0M urea solutions in sequence (the next concentration was selected depending on the target protein's solubility), and finally transferred to a PBS solution for dialysis. Each gradient dialysis lasted for 6 hours. The entire refolding process was carried out at 4°C, and a magnetic stirrer was used for stirring.

[0082] Result analysis:

[0083] The predicted size of the recombinant protein of TonB-dependent receptor (MU-04_GM000527) is about 55kDa, and the actual expressed protein is at 70kDa with a single band. The predicted size of the recombinant protein of RND efflux transporter periplasmic adaptor subunit (MU-04_GM001036) is about 45kDa, and the actual expressed protein has a single band at 60kDa. The predicted size of the recombinant protein of electron transfer flavoprotein (MU-04_GM001325) is about 40kDa, and the actual expressed protein is at 55kDa. The predicted size of the recombinant protein of hypothetical protein (MU-04_GM002463) is about 21kDa, and the actual expressed protein has a single band at 35kDa.

[0084] The sequence analysis results of the gene of the hypothetical protein (MU-04_GM002923) are as follows Figure 1 As shown, the gene ORF is 621bp long, encoding 206 amino acids, the predicted protein molecular weight is 21.8kDa, the isoelectric point is 9.10, the first 20 amino acids are signal peptides, there is no transmembrane domain, and the comprehensive antigen epitope prediction positive result is 20-150aa. The protein expression and purification results of the recombinant protein of the hypothetical protein (MU-04_GM002923) are shown in Figure 2 As shown, the predicted size is about 25 kDa, and the actual expressed protein has a single band at 40 kDa.

[0085] The amino acid sequence of the hypothetical protein (MU-04_GM002923) is shown in SEQ ID No. 1:

[0086] MKKFLLTAAAVFAFGFANAQESFKPVKGLTLTTEVGVTGGILNTKFDAIGGAAKFRYFIKDDLAVRAGLGGSNKREENSIVNQNTPLEFTTEVARTTNRVITL GAEKHFAGTDRLSTYVGADIVFGFTGAEHKNTQSNNNNSTTVVGSVNALGVNGVGSSTFGVRATVGADYYFTKKLYLGVEVGLGYNSGTQDAVETTIRNLTP.

[0087] The nucleotide sequence of the gene of the hypothetical protein (MU-04_GM002923) is shown in SEQ ID No. 2:

[0088] .

[0089] Example 3: Grass carp immunization and sampling

[0090] (1) Vaccine preparation

[0091] The concentration of the protein expressed in Example 2 was determined according to the instructions of the BCA protein concentration determination kit (purchased from Shanghai Sangon Biotech Co., Ltd.). The protein solution was mixed with Freund's adjuvant (Freund's complete adjuvant (first immunization) or Freund's incomplete adjuvant (second immunization)) at a ratio of 1:1, then stirred and mixed with a homogenizer to emulsify it, and finally dropped on the water surface until it did not disperse, and sucked into a syringe to immunize grass carp.

[0092] (2) Grass carp immunization

[0093] Grass carp were immunized subcutaneously, and 50 grass carp were immunized in each vaccine group. Each fish (about 13-15 cm) was injected with 0.1 mL of vaccine. The first immunization was injected with Freund's complete adjuvant vaccine, and the booster immunization was performed two weeks after the first immunization, and the additional immunization was performed with Freund's incomplete adjuvant vaccine. A pure PBS solution group was set as the control group, and the grass carp was immunized in the same way. Since some protein may precipitate during protein renaturation, the absolute protein content of grass carp immunized with each vaccine group is as follows: TonB-dependent receptor recombinant protein (527) 100 μg / fish, RND efflux transporter periplasmic adaptor subunit recombinant protein (1036) 100 μg / fish, electron transfer flavoprotein (1325) 100 μg / fish, hypothetical protein 1 (2463) 100 μg / fish, hypothetical protein 2 (2923) 80 μg / fish.

[0094] (3) Grass carp poisoning

[0095] Two weeks after the booster immunization, grass carp were challenged subcutaneously with F. covae MU-04 and F. columnare JX-01, respectively. The challenge doses are shown in Tables 7 and 8. After the challenge, the symptoms of the diseased fish were observed and the mortality of the grass carp was recorded.

[0096] (4) Grass carp sampling and evaluation of grass carp IgM antibody titer

[0097] Four days after the challenge, grass carp were anesthetized with MS222, and skin mucus, serum, gills, skin and head kidney samples were collected. The specific method is as follows:

[0098] Skin mucus sample: Use a glass slide to gently scrape the mucus on the skin surface, let it stand at 4°C, centrifuge it at 13,000×g for 5 min, then aspirate the supernatant and place it in a new sterilized 1.5mL EP tube, and freeze it at -80°C for later use;

[0099] Serum sample: Use a 1mL syringe to draw blood from the caudal vein of grass carp, let it stand for 4 hours at room temperature, transfer it to 4℃ overnight, centrifuge it at 4℃ and 8000rpm, and take the supernatant to obtain the serum sample;

[0100] Skin samples: Use a sterile scalpel to cut the back skin samples and divide them into two parts in the same way as the gill samples, one for embedding and the other for tissue culture;

[0101] Head kidney samples: processed in the same way as gill and skin samples, and stored at -80℃ for later use.

[0102] The ELISA method was used to detect the antibody titer of grass carp after vaccine immunization.

[0103] Result analysis:

[0104] (1) Protective effect of the vaccine against F. covae MU-04

[0105] The detailed vaccine group protection information is listed in Table 7. The relative protection rate of the vaccine was calculated with the PBS group as the control group. The relative protection rates of the hypothetical protein 1 (2463) and hypothetical protein 2 (2923) subunit vaccines were relatively high, among which the hypothetical protein (2923) subunit vaccine had the highest relative protection rate of 88.9%.

[0106] Table 7 Immunoprotective effect of vaccines

[0107]

[0108] (2) Cross-protection effect of the vaccine against F. columnare JX-01

[0109] The hypothetical protein 1 (2463) subunit vaccine and hypothetical protein 2 (2923) subunit vaccine, which had good immune protection effects against the above-mentioned MU-04 bacteria, were subsequently challenged with F. columnare JX-01 strain, and their cross-protection effects were also tested. 2 to 3 days after the challenge, a large number of grass carp in the control group died, while only a small number of deaths occurred in the immunized group. Detailed protection information of the vaccine groups is listed in Table 8. Interestingly, compared with the protection effect against the own strain, the cross-protection effect of these vaccines was better, and the relative protection rate of the hypothetical protein (2923) subunit vaccine reached 100%.

[0110] Table 8 Cross-immunity protection effect of vaccines

[0111]

[0112] (3) Grass carp-specific IgM antibody titer test results

[0113] In the mucus and serum of grass carp in the immunized group, the production of specific IgM was detected, and the antibody titer was significantly higher than that of the control group. After two immunizations, not only can the antibody response of the local mucosal immune tissue of grass carp be activated, but the vaccine can also activate the systemic immunity of grass carp and produce specific IgM antibodies to fight bacterial infection. After immunization with hypothetical protein 1 (2463) and hypothetical protein 2 (2923) subunit vaccines, the specific IgM antibody titer test results of various tissues of grass carp are as follows:

[0114] 1) Serum specific IgM antibody titer

[0115] Hypothetical protein (2463) vaccine group: The average titer of specific IgM antibodies induced by this vaccine was 1:6w, which was about 1200 times higher than that of the PBS group and about 300 times higher than that of the PBS adjuvant group;

[0116] Hypothetical protein (2923) vaccine group: The average titer of specific IgM antibodies induced by this vaccine was 1:6w, which was about 3000 times higher than that of the two control groups;

[0117] 2) Mucus-specific IgM antibody titer

[0118] Hypothetical protein (2463) vaccine group: The average specific IgM antibody titer induced by this vaccine was about 1:600, which was about 48 times higher than that of the PBS group and about 24 times higher than that of the PBS adjuvant group;

[0119] Hypothetical protein (2923) vaccine group: The average titer of specific IgM antibodies induced by this vaccine was 1:600, which was about 600 times higher than that of the PBS group and about 24 times higher than that of the PBS adjuvant group;

[0120] 3) Specific IgM antibody titer in skin culture supernatant

[0121] Hypothetical protein (2463) vaccine group: The average titer of specific IgM antibodies induced by this vaccine was about 1:90, which was about 90 times higher than that of the control group;

[0122] Hypothetical protein (2923) vaccine group: The average titer of specific IgM antibodies induced by this vaccine was about 1:1000, which was about 1000 times higher than that of the PBS group and about 600 times higher than that of the PBS adjuvant group;

[0123] 4) Specific IgM antibody titer in head kidney culture supernatant

[0124] sprT family protein (2367) vaccine group: The average titer of grass carp-specific IgM antibody tested was about 1:64, which was about 64 times higher than that of the control group;

[0125] Hypothetical protein (2463) vaccine group: The average titer of specific IgM antibodies induced by this vaccine was 1:64, which was about 64 times higher than that of the control group;

[0126] Hypothetical protein (2923) vaccine group: The average titer of specific IgM antibodies induced by the vaccine in this group was about 1:300, which was about 300 times higher than that in the control group;

[0127] In summary, the hypothetical protein (2923) vaccine group has the best protective effect on grass carp after immunization, and the specific IgM antibody titers induced by it in various tissues of grass carp are higher than those in the control group.

Claims

1. Use of the recombinant protein shown in SEQ ID No.1 as an antigen component in the preparation of a fish columnar disease subunit vaccine, characterized in that: The fish columnar disease is Flavobacterium covae and / or Flavobacterium Columnare Caused by fish columnar disease.

2. A fish columnar disease subunit vaccine, characterized in that: The fish columnar disease subunit vaccine contains a recombinant protein with an amino acid sequence as shown in SEQ ID No. 1 as an antigen component; the fish columnar disease is Flavobacterium covae and / or Flavobacterium columnare Caused by fish columnar disease.

3. The fish columnar disease subunit vaccine according to claim 2, characterized in that: The nucleotide sequence of the gene encoding the recombinant protein is shown in SEQ ID No.

2.

4. The fish columnar disease subunit vaccine according to claim 2, characterized in that: The fish columnar disease subunit vaccine also contains an adjuvant.

5. The fish columnar disease subunit vaccine according to claim 4, characterized in that: The adjuvant is Freund's adjuvant.

6. The fish columnar disease subunit vaccine according to claim 5, characterized in that: The volume ratio of the adjuvant to the recombinant protein is 1:

1.

7. Use of the fish columnar disease subunit vaccine according to any one of claims 2 to 6 in the preparation of products for preventing and treating fish columnar disease, characterized in that: The fish columnar disease is Flavobacterium covae and / or Flavobacterium Columnare Caused by fish columnar disease.

8. The use according to claim 1 or 7, characterized in that: The fish is grass carp.

9. The use according to claim 8, characterized in that: The immunization dosage of the fish columnar disease subunit vaccine is 80-100 μg of recombinant protein / fish.

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