A protein subunit vaccine adjuvant for pathogenic bacteria of yellow catfish, preparation method and application

Abstract

The application discloses a protein subunit vaccine adjuvant for pathogenic bacteria of Pelteobagrus fulvidraco, a preparation method and application, wherein the protein subunit vaccine adjuvant is encoded by a nucleic acid sequence, the nucleic acid sequence comprises SEQ ID NO. 1, the encoded protein of the nucleic acid sequence is the protein subunit vaccine adjuvant, and the protein subunit vaccine adjuvant comprises amino acids shown as SEQ ID NO. 2. The protein subunit vaccine adjuvant is an IL6 protein of the Pelteobagrus fulvidraco, can be used as a vaccine adjuvant, significantly improves the immune effect of a vaccine for the Pelteobagrus fulvidraco, and specifically shows that serum lysozyme activity and complement C3 content are improved, immune-related genes (such as IL-1β and TNF-α) are enhanced in expression, and the protection rate of the vaccine after an attack of Edwardsiellaictaluri is significantly improved. The application further discloses a combined immunization composition composed of the IL6 protein and the OmpC protein of the Edwardsiellaictaluri and application of the combined immunization composition in preparation of an Edwardsiellaictaluri subunit vaccine.

Description

Technical Field

[0001] This invention belongs to the field of vaccine prevention and control technology, specifically relating to a protein subunit vaccine adjuvant, preparation method and application for pathogenic bacteria against yellow catfish. Background Technology

[0002] Yellow catfish ( Tachysurus fulvidraco Yellow catfish (Pelteobagrus fulvidraco), also known as yellow croaker or catfish, is an important freshwater aquaculture fish. It is prized for its delicious flesh and lack of intramuscular bones, making it popular with fish farmers and consumers. However, with the development of intensive aquaculture, diseases frequently occur in yellow catfish fry and during long-term cultivation. Edwardsiella tarda and Aeromonas vera are two major bacterial pathogens, causing significant economic losses to the yellow catfish farming industry.

[0003] To effectively improve the immunity of seedlings against pathogenic bacteria, the industry uses antibiotics extensively, raising public concerns about aquatic food safety. The development and application of aquatic vaccines urgently need to be addressed. Several challenges exist in the application of aquatic vaccines. For example, applying vaccines during the seedling stage can achieve resistance to specific pathogens at minimal cost; however, how to further improve the efficacy and duration of immunity is an important area requiring further research and development in the field of aquatic vaccines. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the present invention aims to provide a protein subunit vaccine adjuvant, its preparation method, and its application against pathogens causing yellow catfish. The IL6 protein of this invention can serve as an adjuvant for vaccines against other specific pathogens, enhancing the vaccine's immunogenicity and protective efficacy.

[0005] To achieve the above objectives, the present invention provides the following solution: In a first aspect, the present invention provides a protein subunit vaccine adjuvant against pathogenic bacteria of yellow catfish, wherein the protein subunit vaccine adjuvant is encoded by a nucleic acid sequence, the nucleic acid sequence including as shown in SEQ ID NO.1, and the protein encoded therein is the protein subunit vaccine adjuvant, wherein the protein subunit vaccine adjuvant includes amino acids as shown in SEQ ID NO.2.

[0006] Preferably, the pathogenic bacterium is Edwardsiella tarda.

[0007] It should be noted that the aforementioned protein subunit vaccine adjuvant is actually interleukin-6 protein (IL6 protein for short).

[0008] Secondly, the present invention provides a method for preparing the above-mentioned protein subunit vaccine adjuvant, the method comprising the following steps: Construction of expression plasmid: Using a cDNA library from yellow catfish liver tissue, the homologous gene fragment of yellow catfish and whiteleg 6 was obtained by primer amplification and ligated into an expression vector to obtain the required expression plasmid; Protein expression and purification to obtain the desired protein subunit vaccine adjuvant: The obtained expression plasmid is transformed into the expression strain to obtain positive recombinant bacteria, the positive recombinant bacteria are induced to express, extracted and purified to obtain the desired protein.

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

[0010] Preferably, the expressed bacterial strain is *Escherichia coli* Rosetta.

[0011] Preferably, the protein expression and purification step specifically includes the following operations: Positive clones containing plasmid pET-21d-IL6 were cultured overnight at 37°C in LB medium containing Amp, and then inoculated into LB medium containing Amp and cultured at 37°C until OD. 600 The concentration was 0.4~0.8. IPTG was added to a final concentration of 1mM and cultured for a while. The bacterial culture was then centrifuged, and the bacterial cells were collected and resuspended in buffer. Lysis buffer was added, and the cells were sonicated and centrifuged to collect the precipitate. The urea process causes the precipitate to denature and overlap, which is then refolded by dialysis using a dry dialysis bag, purified by Ni-NTA affinity chromatography, and then concentrated to obtain the desired protein, which is the protein subunit vaccine adjuvant.

[0012] Thirdly, the present invention provides a combined immunization composition comprising the above-mentioned protein subunit vaccine adjuvant and Edwardsiella tarda OmpC protein.

[0013] Preferably, the Edwardsiella tarda OmpC protein comprises the amino acids shown in SEQ ID NO. 4, and the coding sequence of the Edwardsiella tarda OmpC protein gene is shown in SEQ ID No. 3.

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

[0015] Fourthly, the present invention proposes the application of the above-described protein subunit vaccine adjuvant or the combined immunization composition as described above in one of the following applications: Application in the preparation, screening, or prevention of diseases caused by pathogenic bacteria in fish; Applications in the preparation, screening, or prevention of vaccines for diseases caused by bacterial pathogens in fish; Applications of diagnostic kits for the preparation, screening, or prevention of bacterial pathogens in fish.

[0016] In the technical solution described in this invention, the method for preparing yellow catfish IL6 protein using a prokaryotic expression system includes constructing a prokaryotic expression plasmid vector, in vitro induction of expression, and purification and isolation of IL6 protein.

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

[0018] During prokaryotic expression, the bacterial culture OD 600 The concentration was 0.4-0.8. IPTG was added to a final concentration of 1 mM, and the mixture was induced overnight at 37 °C and 180 rpm. The recombinant protein was obtained by inclusion body refolding.

[0019] Compared with the prior art, the present invention discloses the following technical effects: (1) This invention provides a yellow catfish IL6 protein, which can be used as a vaccine adjuvant in yellow catfish vaccines. By increasing the activity of lysozyme and complement C3 in seedlings, the expression of IL-1β and TNFα is enhanced, thereby improving the vaccine's immune effect and protective efficiency.

[0020] (2) This invention also provides an OmpC protein derived from Edwardsiella tarda, which can be used as a subunit vaccine against Edwardsiella tarda. The expression system was further optimized, and the recombinant protein was purified to a concentration of 2.1 mg / L in bacterial culture. Attached Figure Description

[0021] 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.

[0022] Figure 2 The results of SDS-PAGE validation of purified IL6 protein from yellow catfish are shown, where M represents Marker and IL6 represents the SDS-PAGE electrophoresis results after IL6 purification.

[0023] Figure 3 The results of Western blot validation of purified IL6 protein from yellow catfish are shown, where M represents Marker and IL6 represents the WB result after IL6 purification.

[0024] Figure 4 The results of the 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.

[0025] Figure 5SDS-PAGE validation results for the purification of OmpC protein from Edwardsiella tarda.

[0026] Figure 6 Western blot validation results for the purification of OmpC protein from Edwardsiella tarda.

[0027] Figure 7 Figure 1 shows the serum lysozyme activity and serum complement C3 content of yellow catfish after immunization.

[0028] Figure 8 This diagram shows the expression levels of immune-related genes in the liver, head kidney, and spleen of yellow catfish after immunization.

[0029] Figure 9 The graph shows the survival rate of yellow catfish after immunization against Edwardsiella tarda. Detailed Implementation

[0030] The following examples are merely illustrative of the invention and should not be construed as limiting it. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. Experimental methods not specifically described in the examples, and reagents and materials not specified in the examples, are performed according to conventional conditions in the art, and all reagents used are commercially available.

[0031] The LB medium formula is as follows: 5 g yeast extract, 10 g sodium chloride, and 10 g tryptone, dissolved in 1 L deionized water and autoclaved at 121°C for 15 min.

[0032] Example 1: Construction of recombinant expression plasmid (1) Construction of pET-21d-IL6 expression plasmid Extraction of yellow catfish using the TriZol method ( Pelteobagrus fulvidraco Total RNA was collected from liver tissue, and a cDNA library of yellow catfish liver tissue was synthesized using M-MuLV Reverse Transcriptase. 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, the yellow catfish IL6 homologous gene was cloned. Pf- IL6 (as shown in SEQ ID NO. 1); its encoded sequence is 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 the 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 Greens 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 combined immunization composition, characterized in that, The combined immunization composition comprises a protein subunit vaccine adjuvant and Edwardsiella tarda OmpC protein; the amino acids of the protein subunit vaccine adjuvant are shown in SEQ ID NO.

2.

2. The combined immunization composition according to claim 1, characterized in that, The amino acid sequence of the Edwardsiella tarda OmpC protein is shown in SEQ ID NO.

4.

3. The combined immunization composition according to claim 1, characterized in that, The protein subunit vaccine adjuvant has a mass ratio of 1:1 to Edwardsiella tarda OmpC protein.

4. The use of the combined immunizing composition according to any one of claims 1-3 in the preparation of a medicament for preventing diseases caused by pathogenic bacteria of yellow catfish; wherein the pathogenic bacteria of yellow catfish is Edwardsiella tarda.

5. The use of the combined immunization composition according to any one of claims 1-3 in the preparation of a vaccine for the prevention of diseases caused by pathogenic bacteria of yellow catfish; wherein the pathogenic bacteria of yellow catfish is Edwardsiella tarda.

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