Protein subunit vaccine adjuvant for pathogenic bacteria of pelteobagrus fulvidraco as well as preparation method and application of protein subunit vaccine adjuvant

Abstract

The invention discloses a protein subunit vaccine adjuvant aiming at pathogenic bacteria of pelteobagrus fulvidraco, a preparation method and application, the protein subunit vaccine adjuvant is coded by a nucleotide sequence, the nucleotide sequence is shown as SEQ ID NO.1, the protein coded by the nucleotide sequence is the protein subunit vaccine adjuvant, and the protein subunit vaccine adjuvant is a protein subunit vaccine adjuvant. The protein subunit vaccine adjuvant comprises an amino acid as shown in SEQ ID NO. 2. The protein subunit vaccine adjuvant is pelteobagrus fulvidraco IL6 protein and can be used as a vaccine adjuvant, the immune effect of a pelteobagrus fulvidraco vaccine is remarkably improved, specifically, the activity of lysozyme in serum and the content of complement C3 are improved, the expression of immune-related genes (such as IL-1beta and TNF-alpha) is enhanced, and the protection rate of the vaccine on edwardsiella ictaluri after toxin attacking is remarkably increased. The invention further discloses a combined immune composition composed of the IL6 protein and the edwardsiella ictaluri OmpC protein, and application of the combined immune composition in preparation of edwardsiella ictaluri subunit vaccines.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of vaccine prevention and control, and particularly relates to a protein subunit vaccine adjuvant for pathogenic bacteria of Pelteobagrus fulvidraco, a preparation method and application. BACKGROUND

[0002] Pelteobagrus fulvidraco (Richardson), also known as yellow hot pepper and on fish, is an important freshwater aquaculture fish, which is delicious, has no muscle and is deeply loved by breeders and consumers. However, with the development of intensive farming, diseases frequently occur in Pelteobagrus fulvidraco seedlings and long-term breeding, and Edwardsiella tarda and Aeromonas veronii are two main bacterial pathogens, which cause a large amount of economic losses to Pelteobagrus fulvidraco breeding industry. Tachysurus fulvidraco In order to effectively improve the immune effect of seedlings on pathogenic bacteria, a large amount of antibiotics are used in the industry, which has caused the attention of the society to the field of aquatic food safety, and the development and application of aquatic vaccines are urgently needed. In the application process of aquatic vaccines, there are some difficulties. For example, the application of vaccine in seedling period can realize the resistance to specific pathogens at minimum cost, but how to further improve the immune effect and duration is an important content which needs to be further developed and researched in the field of aquatic vaccines.

[0003] SUMMARY In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a protein subunit vaccine adjuvant for pathogenic bacteria of Pelteobagrus fulvidraco, a preparation method and application. The IL6 protein of the present application can be used as an adjuvant for other specific bacterial vaccines to improve the immune effect and protection efficiency of the vaccine.

[0004] In order to achieve the above purpose, the present application provides the following scheme:

[0005] In the first aspect, the present application provides a protein subunit vaccine adjuvant for pathogenic bacteria of Pelteobagrus fulvidraco, which is encoded by a nucleic acid sequence, the nucleic acid sequence comprises SEQ ID NO. 1, the encoded protein of which is the protein subunit vaccine adjuvant, and the protein subunit vaccine adjuvant comprises the amino acid of SEQ ID NO. 2. Preferably, the pathogenic bacteria is Edwardsiella tarda.

[0006] It should be noted that the above protein subunit vaccine adjuvant is actually interleukin 6 protein (abbreviated as IL6 protein).

[0007] In the second aspect, the present application provides a preparation method of the above protein subunit vaccine adjuvant, which comprises the following steps:

[0008] ​Constructing expression plasmid: the cDNA library of Pelteobagrus vachelli is used to obtain the Pelteobagrus vachelli IL-6 homologous gene fragment by primer amplification, and the expression plasmid is obtained by connecting the expression vector. Protein expression and purification are used to obtain the required protein subunit vaccine adjuvant: the obtained expression plasmid is transformed into an expression strain to obtain a positive recombinant bacteria, the positive recombinant bacteria is induced to express, and the required protein is obtained by extraction and purification.

[0009] Preferably, the expression plasmid is pET-21d.

[0010] Preferably, the expression strain is Escherichia coli Rosetta.

[0011] Preferably, the protein expression and purification step specifically includes the following operations: The positive clone bacteria containing the plasmid pET-21d-IL6 are cultured in LB medium containing Amp at 37°C overnight, then inoculated into LB medium containing Amp, and cultured at 37°C until the OD 600 is 0.4-0.8, 1 mM IPTG is added, and the culture is continued, then the bacteria are centrifuged, the bacteria are collected by centrifugation, resuspended in buffer, lysed buffer is added, ultrasonic broken, and the precipitate is collected by centrifugation. The precipitate is denatured by urea method, then renatured by using a dry dialysis bag, purified by Ni-NTA affinity chromatography, and then concentrated to obtain the required protein, i.e., the protein subunit vaccine adjuvant.

[0012] In a third aspect, the present application provides a combined immunization composition comprising the above-mentioned protein subunit vaccine adjuvant and the OmpC protein of Edwardsiellaictaluri.

[0013] Preferably, the OmpC protein of Edwardsiellaictaluri comprises the amino acid shown as SEQ ID NO. 4, and the coding sequence of the OmpC protein gene is shown as SEQ ID No. 3.

[0014] Preferably, the mass ratio of the protein subunit vaccine adjuvant to the OmpC protein of Edwardsiellaictaluri is 1:1.

[0015] In a fourth aspect, the present application provides one of the following applications of the above-mentioned protein subunit vaccine adjuvant or the above-mentioned combined immunization composition: In the preparation, screening or prevention of diseases caused by bacterial pathogenic bacteria of fish; In the preparation, screening or prevention of diseases caused by bacterial pathogenic bacteria of fish; In the preparation, screening or prevention of detection kits related to bacterial pathogenic bacteria of fish.

[0016] In the technical scheme described in the present application, the method for preparing the IL6 protein of Pelteobagrus fulvidraco by using a prokaryotic expression system comprises the following steps: constructing a prokaryotic expression plasmid vector, inducing expression in vitro, and purifying and separating the IL6 protein.

[0017] Preferably, the prokaryotic expression plasmid is pET-21d, and the expression strain is Rosetta.

[0018] When the prokaryotic expression system is expressed, the OD 600 is 0.4-0.8, IPTG is added to a final concentration of 1 mM, induction is performed overnight at 37 DEG C and 180 rpm, and the recombinant protein is obtained by the inclusion body renaturation method.

[0019] Compared with the prior art, the present application has the following technical effects: (1) The present application provides an IL6 protein of Pelteobagrus fulvidraco, which can be used as a vaccine adjuvant for the vaccine of Pelteobagrus fulvidraco. By improving the lysozyme and complement C3 activity of the fry, the expression of IL-1beta and TNF alpha is improved, and the immune effect of the vaccine is improved, and the protection efficiency of the vaccine is improved.

[0020] (2) The present application simultaneously provides an OmpC protein derived from Edwardsiellaictaluri, which can be used as a subunit vaccine for immunizing Edwardsiellaictaluri. The expression system is further optimized in the present application, and the content of the obtained recombinant protein is 2.1 mg / L of bacterial liquid. DETAILED DESCRIPTION

[0021] Figure 1 The figure shows the cloning and plasmid construction verification results of the IL6 of Pelteobagrus fulvidraco, wherein A shows the IL6 gene cloning results; B shows the single colony PCR verification results, and M represents Marker.

[0022] Figure 2 The figure shows the SDS-PAGE verification results of the IL6 protein purification of Pelteobagrus fulvidraco, wherein M represents Marker, and IL6 shows the SDS-PAGE electrophoresis results after IL6 purification.

[0023] Figure 3 The figure shows the Western blot verification results of the IL6 protein purification of Pelteobagrus fulvidraco, wherein M represents Marker, and IL6 represents the WB results after IL6 purification.

[0024] Figure 4 The figure shows the cloning and plasmid construction verification results of the OmpC gene of Edwardsiellaictaluri, wherein A is the OmpC gene cloning result, B shows the single colony PCR verification result, and M represents Marker.

[0025] Figure 5SDS-PAGE verification result of the purified OmpC protein of Edwardsiella tarda.

[0026] Figure 6 Western blot verification result of the purified OmpC protein of Edwardsiella tarda.

[0027] Figure 7 Figure of the lysozyme activity and complement C3 content of the serum of the immunized Pelteobagrus fulvidraco.

[0028] Figure 8 Figure of the expression levels of immune-related genes in the liver, head kidney and spleen of the immunized Pelteobagrus fulvidraco.

[0029] Figure 9 Figure of the survival rate of Edwardsiella tarda challenge of the immunized Pelteobagrus fulvidraco. DETAILED DESCRIPTION

[0030] The following examples are only used to further illustrate the content of the present application, but should not be understood as limiting the present application. Modifications or replacements of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application. The experimental methods not specified in the examples and the reagents and materials not specified in the formula are all according to the conventional conditions in the art, and the reagents used can be commercially available.

[0031] The formula of the LB culture medium is: yeast extract 5 g, sodium chloride 10 g, tryptone 10 g, dissolved in 1 L of deionized water, 121℃ high pressure sterilization for 15 min.

[0032] Example 1: Construction of recombinant expression plasmid (1) Construction of pET-21d-IL6 expression plasmid The total RNA of the liver tissue of Pelteobagrus fulvidraco was extracted by TriZol method, and the cDNA library of the liver tissue of Pelteobagrus fulvidraco was synthesized by M-MuLV Reverse Transcriptase. The homologous gene of IL6 of Pelteobagrus fulvidraco was cloned by using PfIL6-F: 5'-ATGGATTTCTATGAAACATCTGG-3' (as shown in SEQ ID NO. 5) and PfIL6-R: 5'-TTGCTGGTGTTTTGAGATC-3' (as shown in SEQ ID NO. 6) as primers. Pelteobagrus fulvidraco Pf- IL6 ), as shown in SEQ ID NO. 1; and the coding sequence thereof, as shown in SEQ ID NO. 2.

[0033] SEQ ID NO. 1: ​In the diagram, ATG (in bold) represents the start codon, and TGA (in bold) represents the stop codon.

[0034] SEQ ID NO.2: MDFYETSGSELQNEAHAPDQKWVDVAKQLRNEVKNARDEQFHSAIGGTQNTTIYAYKGIKMPILNDSDGCLRSDFNAQKCLRLIYTGLRVYQVNMPHLE QDSQFITNVLNIKAGTYRLLHLIKETVKVNDEKVPQVDLSEFPDTAWNQNIIAHSILHSFTDFMTEASRAINYMKNKKLMRQHLKEDKKVDKWISKHQQ.

[0035] The cloned fragment was ligated into the pET-21d vector to obtain the pET-21d-IL6 expression plasmid.

[0036] For relevant experimental results, please refer to Figure 1 . Figure 1 The results of IL6 cloning and plasmid construction verification for yellow catfish are shown. A shows the IL6 gene cloning results; B shows the single colony PCR verification results; and M represents the marker.

[0037] (2) Construction of pET-21d-OmpC expression plasmid Edwardsiella tarda was extracted using a high-temperature lysis method. Edwardsiella ictaluri The genome of Edwardsiella tarda was cloned using EiOmpC-F:5'-ATGAAACGCAATATTCTTGCAG-3' (as shown in SEQ ID NO. 7) and EiOmpC-R:5'-ATAGAACTGGTAGATCATACCAACG-3' (as shown in SEQ ID NO. 8) as primers, and the encoding amino acid sequence of Edwardsiella tarda was obtained as shown in SEQ ID NO. 4.

[0038] SEQ ID NO.3: In the diagram, ATG (in bold) represents the start codon, and TAA (in bold) represents the stop codon.

[0039] SEQ ID NO.4 is shown below: MKRNILAVVIPALLVAGAANAAELYNKDGNKLSLYGKVDARYKFTNDKSDDGDHTYARFGFKGETQINSELTGYGQWEAEAKAKKPESDSGNFKTRLGFAGLKFADYGSLDYGRNYGVVYDVEAWTDVLPVFGGDTYASPDNYMTYRTNNVLTYRNNGFFGLVDGLNVALQYQGKNGAVG ESNNGRGLAEQNGDGFGMSASYDLGWGVSAAAAYSNSNRTVAQKELDGANKAEVWTAGLKYDANNVYIATMYAETRNMTWVGGDQGGIAPKTKNFEAVAQYQFDFGLRPSIAYLQSRAEGYGDKVDVVKYVDLGATYYFNKNMSTYVDYKINLLDNNDFTNAAGISTDNIVGVGMIYQF.

[0040] For relevant experimental results, please refer to Figure 4 . Figure 4 The results of OmpC gene cloning and plasmid construction verification of Edwardsiella tarda are shown. In the figure, A shows the OmpC gene cloning result, B shows the single colony PCR verification result, and M represents the marker.

[0041] Example 2: Expression and purification of IL6 in yellow catfish and OmpC in Edwardsiella tarda. The obtained pET-21d-IL6 and pET-21d-OmpC were transformed into the Escherichia coli expression strain Rosetta by heat shock method.

[0042] Single clones were selected and verified by PCR and sequencing. 100 μL of the positive clone was added to 20 mL of LB medium containing Amp (adenosine monophosphate) and incubated overnight (6-8 h) at 37°C and 180 rpm to obtain the stock solution. The stock solution was then inoculated into 1 L of LB medium containing Amp and incubated at 37°C and 180 rpm for 3-4 h until the bacterial growth rate reached OD. 600Add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 1 mM, and induce overnight (6-8 h) at 37 ℃ and 180 rpm. Centrifuge the 1 L LRosetta strain induced overnight at 4000 rpm for 10 min, resuspend and concentrate in a 50 mL centrifuge tube. Add 20 mL of Lysis Buffer to the 50 mL centrifuge tube, place the centrifuge tube in a 100 mL beaker filled with ice, and place it in an ultrasonic homogenizer with the probe inserted to about 50 mm from the bottom of the tube. Set the ultrasonic amplitude bar power to 50%, sonicate for 3 s, stop for 6 s, for a total of 30 min. Centrifuge at 12000 rpm for 10 min; the precipitate at this point is the inclusion body.

[0043] The recombinant protein was denatured and refolded using the urea method. The specific steps were as follows: (1) Wash the insoluble inclusion bodies with 2% Triton X-100, 50 mM Tris-HCl (pH should be more than 2.0 different from the isoelectric point of the protein), and 1 / 1000 DTT. Stir with a magnetic stir bar at RT for 1 h, then centrifuge at 12000 rpm for 10 min and discard the supernatant. (2) Wash the insoluble inclusion bodies with 2% Triton X-100 and 50 mM Tris-HCl. Stir with a magnetic stir bar at RT for 1 h, then centrifuge at 12000 rpm for 10 min and discard the supernatant. (3) Wash the insoluble inclusion bodies with 50 mM Tris-HCl. Stir with a magnetic stir bar at RT for 1 h, then centrifuge at 12000 rpm for 10 min and discard the supernatant. (4) The inclusion bodies were collected and dissolved using 10 mM Tris-HCl containing 8 M urea and 100 mM NaH2PO4. The mixture was stirred with a magnetic stir bar at RT for 1 h, followed by centrifugation at 12000 rpm for 10 min, and the supernatant was collected.

[0044] The inclusion bodies were refolded by dialysis using a dry dialysis bag. The specific operation was as follows: the dialysis bag was boiled in a MiliQ containing 2% NaHCO3 and 1mM EDTA for 10 min, washed in MiliQ, boiled in a MiliQ containing 1mM EDTA for 10 min, and then placed in MiliQ for later use. (1) The dissolved inclusion bodies were put into the dialysis bag, and the dialysis bag was placed in 500 mL of dialysis solution containing 4 M urea, 0.1 mM oxidized glutathione, 0.9 mM reduced glutathione and 20 mM Tris-HCl. The solution was magnetically stirred and placed in an ice bath for 12 h. (2) The urea concentration of the dialysis solution was reduced to 2 M and dialysis was performed for 12 h. (3) The urea concentration of the dialysis solution was reduced to 0 M, and NaCl was added to the dialysis solution to a final concentration of 1 mM. The solution was then dialyzed for 12 h. (4) Change the dialysis solvent to PBS, add NaCl and glycerol, and dialyze for 12 h. After dialysis, the refolding is completed and the refolding solution in the dialysis bag is collected.

[0045] Purification was performed using Ni-NTA affinity chromatography. The specific procedures were as follows: (1) Equilibrate the column with two column volumes of protein buffer, and then pass the supernatant through the column for adsorption. (2) Next, equilibrate with two column volumes of 8M urea and 100mM NaH2PO4 in 10mM Tris-HCl, pass the supernatant through the column for adsorption, and wash twice with 20mM imidazole, 8M urea and 100mM NaH2PO4 in 10mM Tris-HCl, with each elution volume being twice the column volume. (3) Perform gradient elution with 8M urea, 250mM imidazole and 100mM NaH2PO4 in 10mM Tris-HCl (each concentration being twice the column volume).

[0046] The refolded protein was concentrated as follows: (1) Select a MiliQ filter membrane to wet the filter. (2) Take the eluent containing the target protein after verification and put it into the ultrafiltration membrane, 4000 rpm, 10 min. (3) Discard the liquid in the ultrafiltration tube, add 10 mL of 1×PBS containing 5% glycerol to the ultrafiltration membrane, 4000 rpm, 10 min. (4) Repeat step 3, changing the solvent of the protein eluent. (5) After the last centrifugation, collect the protein into a new sterile EP tube with a pipette tip. This is the desired protein. It can be stored at 4℃ for short-term storage and at -80℃ for long-term storage.

[0047] SDS-PAGE analysis confirmed that the protein size was consistent with the predicted Pf-IL6 23 kDa ( Figure 2 ) and OmpC 38 kDa ( Figure 5 This is consistent with the results. Western blotting further validated the recombinant protein Pf-IL6 (Pf-IL6) using an anti-his antibody. Figure 3 ) and OmpC ( Figure 6The presence of the protein at the expected molecular weight was observed. SDS-PAGE showed that the purified recombinant yellow catfish IL6 and Edwardsiella edodes OmpC protein had a purity exceeding 80%.

[0048] Example 3: Application of IL6 from yellow catfish as an adjuvant for yellow catfish vaccines Yellow catfish were divided into small and large groups for injection. Ninety healthy yellow catfish (10g ± 2.5g) were randomly divided into three groups of 30 each. Sixty healthy yellow catfish (100g ± 5g) were randomly divided into three groups of 20 each. Two immunization groups received OmpC and OmpC+IL6, respectively, while the control group received PBS. The subunit vaccine dose was 1μg / g, and the subunit vaccine was mixed with adjuvant at a 1:1 ratio, maintaining the same subunit vaccine antigen level. A booster immunization was administered two weeks after the primary immunization, according to the system shown in Table 1 below. Table 1. Samples were collected 7 days after booster immunization, and 3 yellow catfish were randomly selected from each group and anesthetized with MS-222.

[0049] Blood samples were collected via the tail vein, approximately 500 μL per tail, and the collected blood was allowed to stand at room temperature for 2 hours. Afterwards, centrifuge at 1000 rpm for 30 min, collect the serum, and store at -20℃ for later use.

[0050] Dissect the yellow catfish and remove its liver, head, kidneys, and spleen. After rinsing with DEPC H2O, transfer the organs to enzyme-free EP tubes and store at -80℃ for later use.

[0051] Example 4: Serum lysozyme activity detection results Serum lysozyme activity was measured using a lysozyme assay kit (lysozyme (LZM) activity assay kit (Nanjing Jiancheng Institute of Biotechnology). The experiment used a blank control method to determine lysozyme activity. The experimental procedures and calculation methods were performed according to the kit instructions.

[0052] This experiment measured the lysozyme activity in the serum of yellow catfish after two immunizations (Figure 7). The results showed that lysozyme activity increased after subunit vaccine administration, and the adjuvant vaccine group was significantly higher than the PBS group (p<0.05). However, there was no significant difference between the OmpC subunit vaccine group and the PBS group or the adjuvant vaccine group (p<0.05). This indicates that OmpC subunit vaccine alone cannot significantly increase lysozyme activity in fish serum, while IL6, as an adjuvant, can enhance lysozyme activity in serum to achieve an immune-enhancing effect.

[0053] (2) Detection of serum complement C3 content Serum complement C3 levels were determined using a complement C3 assay kit (Suzhou Grease Biotechnology Co., Ltd.).

[0054] The results of serum complement C3 activity assay (Figure 7) showed that C3 activity increased in the serum of fish in the OmpC subunit vaccine group and the OmpC+IL6 co-immunization group. The co-immunization group showed a significant increase in serum C3 activity compared to the PBS group (p<0.05). While the OmpC subunit vaccine group showed an increasing trend compared to the PBS group, the difference was not statistically significant. This indicates that IL6, as an adjuvant, can also enhance the body's ability to produce C3, thereby strengthening the immune response.

[0055] (3) Expression of immune-related genes To further evaluate the immunogenicity and mechanism of the subunit vaccine, the inventors performed qRT-PCR on the liver, head kidney, and spleen of yellow catfish 7 days after the second immunization to assess the gene expression levels of key cytokines interleukin-1β (IL-1β), interleukin-6 (IL6), tumor necrosis factor (TNF-α), and interleukin-6 receptor (IL6R). The results showed (Figure 8): subunit vaccine administration increased IL-1β expression in the liver and head kidney. Co-injection of IL6 as an adjuvant significantly increased IL-1β expression in both the liver and head kidney, while there was no significant difference in IL-1β expression in the spleen. Figure 8 (Figure A) Subunit vaccination leads to high IL6 expression in the liver, while the addition of an IL6 adjuvant inhibits this result, presumably because the active addition of IL6 antagonizes the production of IL6 in the body. There is no significant difference in IL6 expression between the head kidney and spleen (Figure B). After immunization, TNF-α expression in the liver increases, while TNF-α expression in the spleen decreases. Injection of IL6 as an adjuvant significantly amplifies the trend in both cases. There is no significant difference in TNF-α expression in the head kidney (Figure C). Vaccination significantly reduces IL6R expression in the liver, while the changes in IL6R expression in the head kidney and spleen are not significant. Figure 8 (D).

[0056] Example 5: Immunoprotective effect of yellow catfish IL6 and Edwardsiella tarda OmpC against Edwardsiella tarda infection Ninety juvenile yellow catfish (10g±2.5g) were randomly divided into three groups of 30 fish each. Immunization was performed according to the prescribed schedule, with an antigen injection dose of 1 μg / g and an injection volume of 10 μL. Seven days after booster immunization, due to the limited tolerance of the juvenile fish, the inventors challenged them with immersion in a solution of undiluted Edwardsiella catfish. The three groups of fish were immersed in a solution of uninactivated Edwardsiella catfish at a concentration of 1.0×10⁻⁶. 7 The cfu / mL solution was soaked for 30 min, then rinsed three times in clean water for 10 min each time. During the challenge process, the water was aerated with an air pump and the water temperature was maintained at 28 ℃. The fish were observed continuously for 30 days, and the mortality rate of each group of yellow catfish was recorded.

[0057] The results of the Edwardsiella tarda challenge experiment (Figure 9) showed that the experimental fish began to die successively on the fourth day after challenge, reaching a peak mortality rate around the tenth day, and then leveling off on the twenty-fourth day. The survival rate of the PBS group after 30 days of continuous observation was 48.65%, while the survival rates of the OmpC subunit vaccine injection group and the group co-injected with adjuvant and vaccine were 57.15% and 63.12%, respectively. These results indicate that the OmpC subunit vaccine has a certain protective efficacy, and IL6, as a vaccine adjuvant, can enhance the vaccine's protective rate. Furthermore, all the dead yellow catfish exhibited head cracking symptoms, suggesting that the deaths were likely caused by Edwardsiella tarda.

Claims

1. A protein subunit vaccine adjuvant against pathogenic bacteria of Pelteobagrus, characterized in that, The protein subunit vaccine adjuvant is encoded by a nucleic acid sequence comprising SEQ ID NO. 1, and the encoded protein is the protein subunit vaccine adjuvant comprising the amino acid of SEQ ID NO.

2.

2. The protein subunit vaccine adjuvant according to claim 1, characterized in that, The pathogenic bacteria are Edwardsiellaictaluri.

3. A process for the preparation of a protein subunit vaccine adjuvant as claimed in claim 1 or 2, characterized in that, The preparation method comprises the following steps: Constructing an expression plasmid: a cDNA library of Pelteobagrus fulvidraco liver tissue is used to obtain a Pelteobagrus fulvidraco interleukin 6 homologous gene fragment by primer amplification, which is connected to an expression vector to obtain the required expression plasmid; Protein expression and purification to obtain the required protein subunit vaccine adjuvant: the obtained expression plasmid is transformed into an expression strain to obtain positive recombinant bacteria, the positive recombinant bacteria are induced to express, and extraction and purification are performed to obtain the required protein.

4. The production method according to claim 3, characterized by, The expression plasmid is pET-21d.

5. The preparation method according to claim 3, characterized in that, The expression strain is Escherichia coli Rosetta.

6. The method of any one of claims 3-5, wherein, In the protein expression and purification step, the following operations are specifically included: The positive clone bacteria containing plasmid pET-21d-IL6 were cultured in LB medium containing Amp at 37°C overnight, then inoculated into LB medium containing Amp, and cultured at 37°C until OD 600 was 0.4-0.8, IPTG was added to a final concentration of 1 mM, and the culture was continued, then the bacteria solution was centrifuged, the bacteria were collected by centrifugation, resuspended in buffer, lysed buffer was added, and the precipitate was collected by ultrasonic crushing and centrifugation; The urea method is used to denature and overlap the precipitate, then dialysis renaturation is performed by using a dry dialysis bag, Ni-NTA affinity chromatography purification is performed, and then concentration is performed to obtain the required protein, i.e., the protein subunit vaccine adjuvant.

7. A combination immunization composition comprising, in combination, a first immunization composition comprising a first antigen and a second immunization composition comprising a second antigen. The combined immunization composition comprises the protein subunit vaccine adjuvant of claim 1 or 2 and the Edwardsiellaictaluri OmpC protein.

8. The combination immunization composition of claim 7, wherein, The Edwardsiellaictaluri OmpC protein comprises the amino acid of SEQ ID NO.

4.

9. The combination immunization composition of claim 7, wherein, The mass ratio of the protein subunit vaccine adjuvant to the Edwardsiellaictaluri OmpC protein is 1:

1.

10. The protein subunit vaccine adjuvant of claim 1 or 2 or the combined immunization composition of any one of claims 7-9 is used in one of the following applications: application in preparing, screening or preventing a drug for fish bacterial pathogenic bacteria caused diseases; application in preparing, screening or preventing a vaccine for fish bacterial pathogenic bacteria caused diseases; application in preparing, screening or preventing a detection kit related to fish bacterial pathogenic bacteria.

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