Vibrio harveyi oral vaccine, its preparation method and application
By expressing the Vibrio harveyi OmpU protein in Lactococcus lactis NZ9000, a recombinant engineered bacterium NZ9000-pMG36e-OmpU vaccine was constructed, solving the problem of highly effective immune protection against Vibrio harveyi infection and achieving green and healthy aquaculture with highly effective immunization.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-02-06
- Publication Date
- 2026-06-23
AI Technical Summary
Vibrio harveyi infection currently leads to high mortality rates in marine aquaculture. Existing antibiotic control methods have resulted in the emergence of drug-resistant strains and drug residues, and there is a lack of efficient and environmentally friendly immune protection strategies.
Using Lactococcus lactis NZ9000 as a vector, the OmpU protein fragment of Vibrio harveyi was expressed, recombinant engineered bacteria were constructed, and an oral vaccine was developed. By improving the electroporation parameters to increase the plasmid transformation efficiency, the outer membrane protein OmpU of Vibrio harveyi was expressed, forming the Lactococcus lactis NZ9000-pMG36e-OmpU vaccine.
It effectively protects marine fish against Vibrio harveyi infection, reduces antibiotic dependence, promotes green and healthy aquaculture, overcomes the inconvenience of traditional vaccine administration, and improves the rate of immune protection.
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Figure CN122255232A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, specifically to an oral vaccine for Vibrio harveyi, its preparation method, and its application. Background Technology
[0002] Vibrio harveyi is a Gram-negative pathogen commonly found in marine aquaculture, causing high mortality rates and significant economic losses. Current control methods primarily rely on antibiotics during outbreaks, leading to the emergence of drug-resistant strains and a host of problems including drug residues and environmental pollution. Therefore, developing a control strategy that can replace antibiotics, provides highly effective immunoprotection, and is suitable for herd immunity has become an urgent need for the sustainable development of the industry.
[0003] Using intestinal probiotics as antigen delivery carriers is not only environmentally friendly but also enhances the disease resistance of farmed organisms. Lactococcus lactis NZ9000 is a safe probiotic for expression; its genetic manipulation system is mature, it grows rapidly, is non-pathogenic, and its expression process is simple and low-cost, making it suitable for constructing oral vaccine vectors to enhance disease resistance in aquatic animals.
[0004] The urgent task is to find ideal targets that have good immunogenicity, can effectively stimulate the host to produce specific antibodies, and may provide cross-immune protection against different strains by activating innate and cellular immune responses. Summary of the Invention
[0005] To address the aforementioned issues, this invention provides an oral vaccine for Vibrio harveyi, its preparation method, and its application, offering a new technical solution for the manufacture of genetically engineered drugs and vaccines, and promoting the development of the biopharmaceutical industry.
[0006] The first objective of this invention is to provide a Vibrio harveyi OmpU protein fragment, the amino acid sequence of which is shown in SEQ ID NO.2.
[0007] A second objective of this invention is to provide a gene encoding the aforementioned OmpU protein fragment.
[0008] Preferably, the nucleotide sequence is as shown in SEQ ID NO.1.
[0009] A third objective of this invention is to provide a recombinant expression vector containing the aforementioned genes.
[0010] Preferably, the recombinant expression vector comprises the nucleotide sequence of the Vibrio harveyi OmpU protein fragment, the hexahistine tag 6×His, and the vector pMG36e, the nucleotide sequence being shown in SEQ ID NO.1.
[0011] A fourth objective of this invention is to provide recombinant engineered bacteria containing the aforementioned genes or the aforementioned recombinant expression vectors.
[0012] Preferably, the recombinant engineered bacteria is a lactococcus lactis engineered bacteria containing the above-mentioned recombinant expression vector, and the lactococcus lactis is lactococcus lactis strain NZ9000.
[0013] A fifth objective of this invention is to provide a method for preparing an oral vaccine against Vibrio harveyi, comprising using the above-described OmpU protein fragment, the above-described gene, the above-described recombinant expression vector, or the above-described recombinant engineered bacteria.
[0014] A sixth object of the present invention is to provide an oral vaccine against Vibrio harveyi prepared by the above-described preparation method.
[0015] A seventh object of the present invention is to provide the application of the above-mentioned OmpU protein fragment, the above-mentioned gene, the above-mentioned recombinant expression vector, the above-mentioned recombinant engineered bacteria, or the above-mentioned oral vaccine in the prevention of Vibrio harveyi infection.
[0016] This invention provides a method for constructing an oral vaccine of *Lactococcus lactis* (NZ9000-pMG36e-OmpU) expressing the outer membrane protein OmpU of *Vibrio harveyi*, and for use in the prevention and control of *Vibrio harveyi* infection in marine economic animals. The pMG36e-OmpU recombinant vector plasmid consists of two parts: the *Vibrio harveyi* outer membrane protein gene OmpU and a hexahistine tag (6×His). First, these two sequences are ligated using PCR, then ligated to a vector using a seamless cloning method. The vaccine is constructed after transformation with *E. coli*. The plasmid is extracted from *E. coli*, and recombinant *Lactococcus lactis* is constructed by electroporation. By modifying the electroporation parameters to 2.5 kV and 5.0 ms, the transformation efficiency of the recombinant plasmid pMG36e-OmpU in the host bacterium NZ9000 is significantly improved, providing a stable and efficient experimental protocol for the construction of this recombinant bacterium. The recombinant strain NZ9000-pMG36e-OmpU is an engineered Lactococcus lactis (NZ9000) strain that expresses the outer membrane protein (OmpU) of Vibrio harveyi. It can effectively protect marine fish from Vibrio harveyi infection and provides a new solution for dealing with the harm of Vibrio harveyi infection and antibiotic resistance.
[0017] This invention utilizes the Lactococcus lactis NZ9000 expression system and improves transformation parameters to effectively express the Vibrio harveyi OmpU protein, developing an oral vaccine against Vibrio harveyi infection. The aim is to reduce Vibrio harveyi infection in marine aquaculture animals through oral administration, promote the reproduction of fish fry and fingerlings, and drive the development of bio-agriculture and related industries. This strategy not only has the potential to overcome the problems of inconvenient operation and high stress associated with traditional injection vaccines, but also provides a new technical solution for reducing antibiotic dependence in aquaculture and promoting the establishment of green and healthy aquaculture models. Attached Figure Description
[0018] Figure 1 The colony morphology of recombinant lactococcus NZ9000-pMG36e-OmpU on GM17 plates.
[0019] Figure 2 Electrophoresis images of recombinant plasmid PCR products. M is the DL2000 marker; 1-6 are PCR products of pMG36e-OmpU.
[0020] Figure 3 This is a Western blot diagram identifying the protein expression of the recombinant strain. M is the protein marker (10-180 kDa); 1 is Lactococcus lactis NZ9000; 2 is the recombinant expression strain.
[0021] Figure 4 The results show the immunoprotective efficacy of the recombinant strain. Detailed Implementation
[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.
[0023] Example 1:
[0024] I. Experimental Materials
[0025] 1. LB medium: Dissolve 10.0 g tryptone, 5.0 g yeast extract, and 10.0 g NaCl in water, bring the volume to 1 L, and autoclave at 121°C for 20 min. If preparing a solid medium, add 15 g agar powder before sterilization.
[0026] 2. M17 basal medium: Dissolve 5.0 g tryptone, 5.0 g soybean peptone, 5.0 g beef extract, 2.5 g yeast extract, 0.5 g ascorbic acid, 0.25 g magnesium sulfate, and 19.0 g sodium β-glycerophosphate in water, bring the volume to 1 L, adjust the pH to 6.9 ± 0.2, and autoclave at 121℃ for 15 min. If preparing a solid medium, add 15 g agar powder before sterilization.
[0027] 3. GM17 medium: Take 1 L of M17 basal medium, sterilize and cool it, then add 25 mL of 20% glucose stock solution under aseptic conditions, mix well, and make the final glucose concentration 0.5%. If preparing solid medium, add 15 g of agar powder before sterilization.
[0028] 4. GSGM17 medium: Take 1 L of M17 basal medium, add 171.15 g of sucrose and 25.0 g of glycine, bring the volume to 975 mL, adjust the pH to 6.9±0.2, and autoclave at 121℃ for 15 min. After the medium cools, add 25 mL of 0.5 M glucose stock solution under aseptic conditions and mix well.
[0029] 5. GM17-MC recovery medium: Take 1 L of M17 basal medium, add 171.15 g sucrose, 4.07 g MgCl2 and 0.22 g CaCl2, and bring the volume to 975 mL. Adjust the pH to 6.9±0.2, and autoclave at 121℃ for 15 min. After the medium cools, add 25 mL of 0.5 M glucose stock solution under aseptic conditions and mix well.
[0030] 6. 0.5 M EDTA solution (pH 8.0): Dissolve 55.83 g Na2EDTA·2H2O in 200 mL of water, stir thoroughly, bring the volume to 300 mL, adjust the pH to 8.0, autoclave at 121℃ for 20 min, and then dilute 10 times with sterile water for later use.
[0031] 7. Washing solution I: Dissolve 100 mL of glycerol and 171.15 g of sucrose in water, bring the volume to 1 L, and autoclave at 121℃ for 20 min before use.
[0032] 8. Washing solution II: Dissolve 100 mL of glycerol and 171.15 g of sucrose in water, add 50 mM EDTA to a final volume of 1 L, and autoclave at 121℃ for 20 min before use.
[0033] 9. Strains and plasmids: Lactococcus lactis NZ9000 was purchased from Guangzhou Newprobio Biotechnology Co., Ltd.; TOP10 competent cells were purchased from Guangzhou Xinkailai Biotechnology Co., Ltd.; Vibrio harveyi strain and expression plasmid pMG36e were both preserved in our laboratory.
[0034] 10. Primers: Primers were designed based on the Vibrio harveyi OmpU gene sequence (GenBank No. KP659344.1) and the characteristics of the pMG36e vector. They were synthesized by Guangzhou Branch of Beijing Qingke Biotechnology Co., Ltd. The sequences are shown in Table 1.
[0035] II. Experimental Methods
[0036] 1. DNA extraction from Vibrio harveyi
[0037] (1) Incubate Vibrio harveyi overnight in LB liquid medium.
[0038] (2) Collect 2 mL of bacterial solution, centrifuge at 4000 g for 10 min at room temperature to precipitate bacteria.
[0039] (3) Discard the culture medium, add 100 µL of TE Buffer to resuspend the bacteria, then add 10 µL of Lysozyme, and incubate in a water bath at 37°C for 10 min.
[0040] (4) Add 100 µL BTL Buffer and 20 µL Proteinase K Solution and vortex to mix. Place the sample in a 55°C water bath shaker to allow for complete lysis.
[0041] (5) Add 5 µL of RNase A and mix by inverting repeatedly. Incubate at room temperature for 5 min.
[0042] (6) Centrifuge at 10000 g for 2 min at room temperature. Precipitate the undissolved substance and carefully transfer the supernatant to a new centrifuge tube, avoiding aspirating any undissolved precipitate.
[0043] (7) Add 220 µL BDL Buffer, vortex to mix, and incubate at 65°C for 10 min.
[0044] (8) Add 220 µL of anhydrous ethanol and vortex at high speed for 20 seconds. If a precipitate appears at this time, blow it repeatedly with the tip of a pipette 10 times to break up the precipitate.
[0045] (9) Place the centrifuge column on a 2 mL collection tube, transfer all the obtained liquid into the column, centrifuge at 10000g for 1 min at room temperature, and discard the filtrate.
[0046] (10) Add 500 µL of HBC Buffer to the centrifuge column, centrifuge at 10000 g for 1 min at room temperature, and discard the filtrate.
[0047] (11) Add 700 µL DNA Wash Buffer, centrifuge at 10000 g for 1 min at room temperature, discard the filtrate, and repeat once.
[0048] (12) Put the centrifuge column back onto the collection tube and centrifuge at 10000 g for 2 min.
[0049] (13) Place the centrifuge column on a 1.5 mL centrifuge tube, add 50-100 µL of Elution Buffer preheated to 65 °C, and let it stand at room temperature for 3-5 min. Then centrifuge at 12000 g for 1 min, collect the liquid, and measure the concentration on a Nanodrop spectrophotometer.
[0050] 2. PCR amplification of homologous sequences
[0051] The purified Vibrio harveyi DNA extracted in step 1 was used as a template to amplify the fragment sequence with homologous arms using OmpU-F and OmpU-R primers (Table 1). The PCR reaction system and procedure are shown in Tables 2 and 3. The OmpU fragment is approximately 1008 bp in length and is expected to encode approximately 336 amino acid residues, as shown in SEQ ID NO.1 and SEQ ID NO.2.
[0052] Simultaneously, homologous sequences were amplified using primers pMG36e-F2 and pMG36e-R2 with plasmid pMG36e as a template. The PCR reaction system and procedure are shown in Tables 2 and 3, and the corresponding pMG36e fragments were obtained.
[0053] 3. Gel recovery and purification
[0054] (1) Perform DNA electrophoresis on the PCR products obtained in step 2. After the electrophoresis, cut out the corresponding fragments of OmpU and pMG36e under a UV lamp with a clean blade and then place them in a 1.5 mL centrifuge tube.
[0055] (2) Add 600 μL of Binding Buffer to the centrifuge tube and then place it in a 60°C water bath for 5-10 minutes until the gel is completely dissolved.
[0056] (3) Add the gel liquid into a centrifuge column with a sleeve, centrifuge at 10000 g for 1 min, and discard the waste liquid.
[0057] (4) Add 700 μL of DNA Wash Buffer to the centrifuge column, centrifuge at 12000 g for 1 min, and discard the waste liquid. Repeat once.
[0058] (5) Transfer the centrifuge column to a new sleeve, centrifuge at 12000 g for 2 min, discard the waste liquid, then transfer the centrifuge column to a new 1.5 mL centrifuge tube, open the centrifuge column cap and let it stand at room temperature for 2 min.
[0059] (6) Add 30 μL of preheated ddH2O without nucleic acid at 65℃ to the centrifuge column, let it stand at room temperature for 2 min, then centrifuge at 12000g for 1 min, collect the liquid and measure the nucleic acid concentration on a Nanodrop spectrophotometer.
[0060] 4. Recombination of fragments
[0061] The OmpU and pMG36e fragments obtained in step 3 were used to construct the recombinant plasmid pMG36e-OmpU using seamless cloning technology. The reaction system is shown in Table 4.
[0062] 5. Transformation and Identification
[0063] (1) Take 100 μL of TOP10 Escherichia coli competent cells and place them on ice to thaw for 5-10 min.
[0064] (2) Immediately after melting, add 10 μL of the ligation product obtained in step 4 to the competent cells, mix gently, and incubate on ice for 30 min.
[0065] (3) Place competent cells in a 42°C water bath for 90 seconds, and then immediately place them on ice for 2-3 minutes.
[0066] (4) Add 1000 μL of antibiotic-free LB liquid medium to competent cells and activate them in a shaker at 37°C for 1 h.
[0067] (5) Centrifuge the activated bacterial solution at 4000 rpm for 5 min, discard the supernatant until about 100 μL remains, resuspend the precipitate, mix well, and take an appropriate amount of the resuspended bacterial solution to spread evenly on an LB medium plate containing erythromycin (300 μg / mL). Place the plate in an incubator at 37℃ and incubate upside down overnight.
[0068] (6) Add 1 mL of LB liquid medium containing 300 μg / mL erythromycin to a 1.5 mL centrifuge tube. In a clean bench, use a pipette tip to pick up the single clones cultured on the plate and inoculate them into the medium in the centrifuge tube. Incubate at 37°C and 200 rpm for 5 h.
[0069] (7) The bacterial culture was identified by PCR. The positive clone was identified using universal primers pMG36e-F1 and pMG36e-R1 for the vector pMG36e. The PCR product was about 1500 bp. The positive clone bacterial culture was sequenced.
[0070] 6. Extraction of recombinant plasmids
[0071] (1) Take 2 mL of recombinant plasmid bacterial culture in the logarithmic growth phase (positive clone bacterial culture in step 5) and add it to 200 mL of liquid LB medium containing 300 μg / mL erythromycin. Incubate overnight at 37°C in a shaker.
[0072] (2) Distribute the bacterial culture evenly into 50 mL centrifuge tubes, centrifuge at 5000 g for 10 min, and discard the supernatant.
[0073] (3) Add 250 μL of Solution I / RNase A solution to the precipitate and repeatedly pipette to suspend the precipitate.
[0074] (4) Add 250 μL of Solution II to the centrifuge tube, mix well and let stand at room temperature for 2 min.
[0075] (5) Add 250 μL of Solution III to the centrifuge tube, mix immediately, and centrifuge at 12000 g for 10 min after observing the appearance of a white flocculent precipitate. After centrifugation, transfer the supernatant to the purification column and centrifuge at 10000 g for 1 min.
[0076] (6) Discard the liquid, add 500 μL of HB Buffer to the purification column, and centrifuge at 10000 g for 1 min.
[0077] (7) Discard the liquid and add 700 μL DNA Wash Buffer to the purification column. Centrifuge at 10000 g for 1 min and repeat twice.
[0078] (8) Continue with 10,000 g empty space for 2 min, then open the purification column and let it stand for 3-5 min.
[0079] (9) Transfer the purification column to a new tube, add 30 μL of 65℃ deionized water, let stand for 5 min, centrifuge at 10000g for 1 min to obtain the recombinant plasmid pMG36e-OmpU, and measure its concentration on a spectrophotometer.
[0080] 7. Preparation of Lactococcus lactis NZ9000 competent cells
[0081] (1) Streak the NZ9000 strain on GM17 solid medium, pick a single colony and culture it in 5 mL of GSGM17 liquid medium at 30℃ for about 10 h.
[0082] (2) Incubate 5 mL of the cultured bacterial solution in 50 mL of GSGM17 liquid medium at 30℃ for about 10 hours.
[0083] (3) Take 12.5 mL of the cultured bacterial solution and culture it in 100 mL of GSGM17 liquid medium at 30℃, and let it stand until OD. 600 It is around 0.4.
[0084] (4) Cool the cultured bacterial solution in an ice-water mixture. Shake the solution during cooling to ensure it is fully cooled. Dispense the bacterial solution into pre-cooled 50 mL centrifuge tubes, centrifuge at 4000 rpm and 4°C for 15 min, discard the supernatant, add 25 mL of pre-cooled washing buffer I, resuspend the bacterial pellet, and let it stand on an ice-water mixture for 15 min.
[0085] (5) Centrifuge at 4℃ for 15 min at 4000 rpm, discard the supernatant, add 25 mL of pre-cooled washing solution II, resuspend the bacterial precipitate, and place it on an ice-water mixture for 15 min.
[0086] (6) Centrifuge at 4℃ for 15 min at 4000 rpm, discard the supernatant, add 25 mL of pre-cooled washing solution I, resuspend the bacterial precipitate, and place it on an ice-water mixture for 15 min.
[0087] (7) Centrifuge at 4℃ for 15 min at 4000 rpm, discard the supernatant, resuspend the bacterial precipitate in 200 µL of washing solution I in each tube, dispense into pre-cooled sterile centrifuge tubes, and store in an ultra-low temperature freezer at -80℃.
[0088] 8. Transformation of recombinant plasmid pMG36e-OmpU in Lactococcus lactis NZ9000
[0089] (1) Take Lactococcus lactis NZ9000 competent cells out of the -80℃ freezer and place them on ice to thaw.
[0090] (2) Prepare the electric shock cup and pre-cool it on ice for more than 30 minutes. Using voltage as the variable, select three levels: 1.8 kV, 2.2 kV, and 2.5 kV. Using time as the variable, select three levels: 3.0 ms, 5.0 ms, and 6.0 ms. Set up 9 test groups.
[0091] (3) Add 100 ng of pMG36e-OmpU recombinant plasmid to 80 μL of competent cells and mix carefully. Aspirate the mixture into a pre-cooled electroconversion cup, avoiding air bubbles as much as possible, and gently shake to bring the solution to the bottom of the electroconversion cup. Quickly place the electroconversion cup into the electroconverter and set the electroconversion program for electro-excitation.
[0092] (4) Quickly remove the electroconversion cup, immediately add GM17-MC recovery medium, gently mix the bacterial solution with the medium, transfer to a 1.5 mL centrifuge tube, and incubate at 30°C for 1 h.
[0093] (5) Centrifuge at 4000 rpm for 5 min, discard half of the culture medium supernatant, and thoroughly mix the remaining bacterial solution by pipetting. Take 100 μL and spread it onto GM17 plates containing erythromycin (4 μg / mL) resistance. Incubate overnight at 30°C. The colony morphology of the recombinant bacteria on the plate is shown in the figure. Figure 1 .
[0094] (6) In a clean bench, pick up a single colony cultured on a plate using a pipette tip and place it in a centrifuge tube containing GM17 liquid medium with 4 μg / mL erythromycin. Incubate at 30°C and 200 rpm for 5-6 h. Perform PCR identification on the cultured bacterial solution. Sequencing is performed on positive clones; correct sequencing indicates the recombinant strain NZ9000-pMG36e-OmpU. The universal primers pMG36e-F1 and pMG36e-R1 for the vector pMG36e were used for positive clone identification in this experiment. The PCR identification results of the positive clones are shown below. Figure 2 .
[0095] (7) Among the above-tested electro-conversion parameters, 2.5 kV and 5.0 ms have the highest conversion efficiency and are the most stable.
[0096] 9. Western blot detection of protein expression in recombinant strain NZ9000-pMG36e-OmpU
[0097] (1) The logarithmic growth phase of the recombinant lactococcus strain containing plasmid pMG36e-OmpU (NZ9000-pMG36e-OmpU) and the control strain (Lactococcus lactis NZ9000) were inoculated at a volume ratio of 1% into 200 mL of GM17 medium containing 4 μg / mL erythromycin and blank GM17 medium, respectively, and incubated at 30℃ overnight.
[0098] (2) After collecting the bacterial cells by centrifugation (10000 rpm, 10 min, 4℃), wash twice with sterile PBS solution, resuspend in a small amount of PBS, and place the recombinant expression bacteria NZ9000-pMG36e-OmpU and lactococcus lactis NZ9000 suspensions on ice and sonicate for 10 min at 250 W. Repeat the operation for 2 s and stop for 3 s until the suspension is clear.
[0099] (3) After centrifuging the lysed samples (10,000 rpm, 10 min, 4℃), 20 μL of lysed recombinant Lactococcus lactis containing plasmid pMG36e-OmpU and 20 μL of lysed Lactococcus lactis NZ9000 were mixed with 5 μL of 5×SDS gel loading buffer and boiled for 5 min. Electrophoresis was performed at 60V for 30 min using a 10% separating gel and a 5% stacking gel. After all the samples had entered the separating gel, the voltage was increased to 110V. Electrophoresis was stopped when the bromophenol blue migrated to the bottom of the glass plate. The separated proteins were then transferred from the gel to a polyvinylidene fluoride (PVDF) membrane using a wet transfer method at a constant current of 100 mA for 55 min. After transfer, the PVDF membrane was washed three times with PBST, 5 min each time. The PVDF membrane was then transferred to an antibody incubation cassette, blocked overnight at 4°C with 5% skim milk powder prepared with PBST, and washed three times with PBST, 5 min each time. Anti-His antibody (1:5000) prepared with antibody dilution buffer was added, and the membrane was incubated on a low-speed shaker at room temperature for 2 h. After incubation, the membrane was washed three times with PBST. Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (proteintech, Cat No. SA00001-1) diluted with 5% skim milk was added, and the membrane was incubated at room temperature for 1 h. After three washes with PBST, the PVDF membrane was placed in the chromogenic solution for color development and photographed for analysis. The detection results are shown below. Figure 3 .
[0100] (4) The target protein was expressed in the recombinant strain protein, and the protein electrophoresis yielded a band of about 40 kDa, which was consistent with the size of the target protein.
[0101] 10. Immunization and challenge tests
[0102] Pearl grouper with a body weight of 6.83±0.36 g after temporary acclimatization were selected and treated with PBS (PBS group), NZ9000 Lactococcus lactis (NZ9000 group), Lactococcus lactis containing plasmid pMG36e (pMG36e group), and Lactococcus lactis containing recombinant plasmid pMG36e-OmpU (pMG36e-OmpU group) (all bacterial concentrations were 1.0×10⁻⁶). 7Oral immunization with CFU / mL, administered for the first 4 days of each week by mixing recombinant lactococci with feed (ensuring a bacterial content of 10 CFU / mL per gram of feed). 7 CFU was administered to the grouper, and for the next three days, they were fed regular feed at 1% of their body weight twice daily (8:00 AM and 5:00 PM). After four weeks of rearing, they were challenged with Vibrio harveyi H-5 strain. The intraperitoneal injection concentration for each grouper was 10... 7 CFU / mL Vibrio harveyi H-5 was administered in 100 μL volumes. The patient was observed for 14 days, and the relative percent survival (RPS) was calculated as follows: RPS = [1 - (mortality rate in the immunized group / mortality rate in the control group)] × 100%. The results showed that the relative immunoprotection rate of the recombinant strain was approximately 45.5%. (See attached table for details.) Figure 4 .
[0103] SEQ ID NO.1 (OmpU nucleotide sequence) ATGAAGAAAACTCTGATTGCTCTTTCTGTATCTGCAGCAGCTATGGCAACTGGCGTTAACGCAGCTGAACTTTACAACCAAGACGGCACTTCTCTAGAAATGGGCGGTCGTGCTGAAGCACGTCTATCTATGAAAGATGGCGACGTAGCGGACAACTCTCGTATCCGTCTAAACTTCCTTGGCACACAAGCAATCAACGACAATCTATACGGTGTTGGTTTCTGGGAAGGTGAATTTACTACTGCTGAAGAAGGCGGCGTAGACGGCAACAGCAACCTTGACACTCGTTACGCATACGCTGGTCTAGGCGGTGCATTCGGTGAAGTTACTTACGGTAAAAACGACGGTGCGCTAGGCGTTATCACTGACTTCACAGATATCATGGCGTACGCTGGTAACTCTGCTGCTGACAAACTAGCTGCAGCTGACCGTTCAGACAACATGCTGTCTTACAAAGGTCAATTCGAAAACCTAGCAGTTAAAGCTAGCTACCGTTTCGCTGACCGCGTAGAAAACGCAGCTGGTACTGAGTACACTGATAACGGCGAAGATGGCTACTCTCTATCTGCTATCTACACTCTAGGCGACACTGGTCTTGACCTAGGTGCTGGTTACGCAGACCAATCTGACGCTAACGAATACATGCTTGCTGCTTCTTACACAATGAACGACCTATACTTTGCAGGTCTATTCACTGACGGTGAAAAAGAAGCAACATTCAAGAAAACTGTAGATTACACTGGTTACGAACTAGCAGGTGCTTACACTCTAGGTCAAACAGTGTTCACAACGACGTACAACAACGCAGAAACTAACAACGAAACTTCTGCAAACAACTTCGCAGTTGACGCGTCTTACTACTTCAAGCCTAACTTCCGTGGTTACGTATCGTACAACTTCAACCTAATTGATGCTGGCGATGCTATGGGCTCAACGACTTCTGCTAACTACAAAGCAACGAAGATCGATTCTGAAGACGAGCTAGC TCTCGGTCTACGTTACGACTTC 。
[0104] SEQ ID NO.2 MKKTLIALSVSAAAMATGVNAAELYNQDGTSLEMGGRAEARLSMKDGDVADNSRIRLNFLGTQAINDNLYGVGFWEGEFTTAEEGGVDGNSNLDTRYAYAGLGGAFGEVTYGKNDGALGVITDFTDIMAYAGNSAADKLAAADRSDNMLSYKGQFENLAVKASYRFAD RVENAAGTEYTDNGEDGYSLSAIYTLGDTGLDLGAGYADQSDANEYMLAASYTMNDLYFAGLFTDGEKEATFKKTVDYTGYELAGAYTLGQTVFTTTYNNAETNNETSANNFAVDASYYFKPNFRGYVSYNFNLIDAGDAMGSTTSANYKATKIDSEDELALGLRYDF.
[0105] Table 1
[0106] Table 2
[0107] Table 3
[0108] Table 4
[0109] Nanjing Jiancheng Guide Beijing Jiancheng Bioengineering Research Institute Co., Ltd.
Claims
1. A Vibrio harveyi OmpU protein fragment, characterized in that, The amino acid sequence is shown in SEQ ID NO.
2.
2. A gene encoding the OmpU protein fragment of claim 1.
3. The gene according to claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID NO.
1.
4. A recombinant expression vector containing the gene as described in claim 2 or 3.
5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector contains the nucleotide sequence of the Vibrio harveyi OmpU protein fragment, the hexahydristidine tag 6×His, and the vector pMG36e, the nucleotide sequence of which is shown in SEQ ID NO.
1.
6. Recombinant engineered bacteria containing the gene of claim 2 or 3 or the recombinant expression vector of claim 4 or 5.
7. The recombinant engineered bacteria according to claim 6, characterized in that, The recombinant engineered bacteria is a lactococcus engineered bacteria containing the recombinant expression vector of claim 5, wherein the lactococcus is lactococcus strain NZ9000.
8. A method for preparing an oral vaccine against Vibrio harveyi, characterized in that, This includes using the OmpU protein fragment of claim 1, the gene of claim 2 or 3, the recombinant expression vector of claim 4 or 5, or the recombinant engineered bacteria of claim 6 or 7.
9. An oral vaccine against Vibrio harveyi prepared by the preparation method of claim 8.
10. The use of the OmpU protein fragment of claim 1, the gene of claim 2 or 3, the recombinant expression vector of claim 4 or 5, the recombinant engineered bacteria of claim 6 or 7, or the oral vaccine of claim 9 in the prevention of Vibrio harveyi infection.