A food-grade lactic acid bacteria drinking water vaccine for porcine epidemic diarrhea and a preparation method and application thereof

Abstract

This invention belongs to the field of veterinary biological products technology, and discloses a food-grade lactic acid bacteria drinking water vaccine for porcine epidemic diarrhea (PED), its preparation method, and its application. The invention uses recombinant lactococcus carrying the PEDV C-COE gene as the core, combined with skim milk powder-glucose adjuvant to prepare the drinking water vaccine. The vaccine has no antibiotic resistance markers and high safety. A three-stage drinking water immunization program can induce high levels of PEDV-specific antibodies and secretory IgA in pigs, while simultaneously activating cellular immunity, effectively blocking PEDV infection. Furthermore, the immunization procedure is simple and stress-free, suitable for large-scale pig farms, providing a new and efficient solution for PED control.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, and relates to a food-grade lactic acid bacteria drinking water vaccine for porcine epidemic diarrhea, its preparation method and application, specifically relating to a genetically engineered vaccine that expresses protective antigens of porcine epidemic diarrhea virus (PEDV) using food-grade lactic acid bacteria as a carrier, its preparation method, and its application in the prevention and control of porcine epidemic diarrhea. Background Technology

[0002] Porcine epidemic diarrhea (PED) can spread among pigs at all stages and is most likely to cause piglet mortality. The virus is characterized by rapid spread, high morbidity and mortality, bringing huge disasters to farmers. PED has gradually developed into one of the most serious diseases in the pig industry.

[0003] Early-developed inactivated and attenuated vaccines are collectively known as traditional vaccines, which carry the risk of secondary infection. Therefore, creating new, healthy, reliable, and highly effective vaccines is imperative. New vaccines primarily include those based on lactic acid bacteria and nucleic acids. Lactic acid bacteria, being beneficial gut bacteria, have promising applications. Lactic acid bacteria (LAB), as an edible fungus, are widely used in food production, and many lactococci and lactobacilli are generally considered safe. International research teams have pioneered the use of lactic acid bacteria as engineered bacteria to construct expression systems. Through this vector, the target gene can rapidly penetrate the mucous membrane and enter the bloodstream. Because this vector is food-grade, it does not contain any antibiotic genes or other harmful components. Zhao De et al.'s lactic acid bacteria vaccine, administered to mice via various methods, showed the best efficacy with a 10-fold increase in oral administration. This vaccine can stimulate both humoral and cellular immunity.

[0004] Nisin is a small antimicrobial peptide composed of 34 amino acids, produced by LAB. It has virtually no toxic side effects on humans or animals and is currently used as a natural preservative in the food industry in over 50 countries worldwide. NICE is a Nisin-controlled gene expression system, one of the most commonly used expression systems for food induction. Numerous reports have demonstrated the versatility and effectiveness of using the NICE system to express heterologous proteins in lactic acid bacteria.

[0005] PEDV is an RNA virus with a single, upright strand approximately 28 kb in length. It has a cap-like structure at its 5' end and a tail-like structure called Poly(A) at its 3' end. The gene sequence from 5' to 3' is 5'UTR-Replace(ORF1a and ORF1b)-S-ORF3-E-MN-3'UTR. The PEDV genes encode the S, M, E, and N proteins, with the S protein, located on the outermost layer of the viral particle, being most beneficial for early diagnosis. However, current technology lacks a stable, effective, and commercially viable PEDV lactic acid bacteria drinking water vaccine, necessitating the development of related technologies and products. Summary of the Invention

[0006] To address the shortcomings of existing PEDV vaccines, this invention aims to provide a food-grade lactic acid bacteria drinking water vaccine for PEDV that is highly safe, requires no injection, and can induce good mucosal and humoral immunity. It also provides a standardized preparation method and application scheme to solve the problems of poor vaccine safety, cumbersome immunization procedures, and insufficient immunization effects in PEDV prevention and control.

[0007] The technical solution adopted by this invention to solve its technical problem is as follows: 1. Construction and identification of recombinant engineered strains The starting strain used in this invention is a food-grade Lactococcus lactis carrying the PEDV protective antigen C-COE gene preserved in the laboratory. Its recombinant plasmid pNZ8149 / C-COE was correctly identified by double enzyme digestion. The inserted target gene fragment is 530 bp in size, the full length of the plasmid is 2591 bp, and there is no antibiotic marker, which meets the food-grade safety requirements.

[0008] Stability verification method of strain: The strain was inoculated into M17 medium containing 0.5% lactose and cultured overnight at 30°C. The plasmid was extracted by alkaline lysis method, and after double digestion with Sac I and Nco I, it was detected by 1% agarose gel electrophoresis. The corresponding size of the insertion fragment was visible, which proved that the target gene was stably present during the passage.

[0009] Target protein expression validation method: Take the logarithmic growth phase bacterial culture (OD) 600 (≈0.4), add 1 ng / mL Nisin to induce for 3~6 h, after sonication, take the supernatant for 15% SDS-PAGE electrophoresis, a specific band of about 20 kDa can be seen, which is consistent with the theoretical size of C-COE protein, proving that the recombinant strain can achieve the correct expression of foreign protein.

[0010] 2. Preparation process of drinking water vaccine (1) Preparation of vaccine adjuvant: Take 200 mL of skim milk powder and 10 g of glucose according to the mass-volume ratio, mix and dissolve them, sterilize at 121℃ for 20 min, and then cool to obtain lactic acid bacteria protectant (adjuvant).

[0011] (2) Optimization of bacterial culture and adjuvant ratio: The recombinant lactic acid bacteria were fermented and cultured until the viable count was ≥10. 9 The CFU / mL solution is mixed with adjuvant at a volume ratio of 4:1 to obtain the drinking water formulation vaccine. This ratio results in the highest survival rate and optimal activity of lactic acid bacteria.

[0012] 3. Immunization Application Methods (1) Grouping of experimental animals: The pigs to be immunized were randomly divided into experimental group and control group, and the feeding and management conditions of the two groups were completely the same.

[0013] (2) Immunization program: Before immunization, the water supply to the pigs was withheld for 2 hours. Each pig in the experimental group was given 50 mL of drinking water containing the above-mentioned vaccine and was required to drink it within 5 minutes. Then, normal water supply was restored. The control group was given only the same amount of clean water. After the first immunization, a booster immunization was given every 7 days for a total of 3 immunizations.

[0014] (3) Monitoring of immunization efficacy: The weight changes of the pig herd were monitored weekly during the immunization period; serum was collected at 1, 3 and 5 weeks after the first immunization, and the levels of PEDV-specific antibodies and secretory IgA antibodies were detected by ELISA; anticoagulated blood was collected at 5 weeks after immunization, and peripheral blood CD8 was detected by flow cytometry. + The percentage of T cells is used to assess the level of cellular immunity.

[0015] Compared with the prior art, the beneficial effects of the present invention include: (1) Excellent safety: The carrier lactic acid bacteria are food-grade microorganisms, the recombinant plasmid has no antibiotic resistance marker, there is no injection stress when immunized orally, there is no risk of virulence reversion and virus shedding, and it has no adverse effects on the growth of pigs. The weight gain of the experimental group pigs is better than that of the control group.

[0016] (2) Comprehensive immune effect: Humoral immunity: The level of PEDV-specific antibodies in the experimental group was higher than that in the control group from the 3rd week of immunization, and the difference was extremely significant in the 5th week (P<0.01). Mucosal immunity: The level of secretory IgA in the experimental group was higher than that in the control group from the first week of immunization, and the difference was extremely significant from the third week (P<0.01), which can effectively block PEDV invasion through the intestine; Cellular immunity: Experimental group CD8 + The proportion of T cells reached 15.3%, which was 1.11 percentage points higher than that of the control group (14.19%), and could help clear virus-infected cells.

[0017] (3) Convenient application: Water immunization does not require injection, is simple to operate, and is suitable for large-scale pig farms to immunize the entire herd, greatly reducing labor costs. Attached Figure Description

[0018] Figure 1 This is a flowchart of the ELISA method for detecting PEDV-specific antibodies; Figure 2 This is the detection of the PEDV lactic acid bacteria recombinant expression plasmid C-COE gene. M represents DNA Marker DL 2 000 (2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp); 1 represents the pNZ8149 / C-COE plasmid, with an insert size of 530 bp and a vector size of 2591 bp, and the plasmid was digested with SacⅠ and NcoⅠ enzymes; 2 represents the recombinant plasmid that has not been digested. Figure 3 This is an SDS-PAGE assay used to detect the expression of the C-COE gene in recombinant PEDV bacteria. M represents a low molecular weight protein marker (97.2 kDa, 66.4 kDa, 43.0 kDa, 31.0, 20.1 kDa); 1-2 represent the pNZ8149 / C-COE expression band, with the target protein size approximately 20 kDa; 3 represents the uninduced control strain. Figure 4 The weight gain values ​​for each group are the average weight gain of all pigs in the group during the trial period, and the PEDV food-grade lactic acid bacteria vaccine is the effect of increased pig weight. Figure 5 The test was performed using ELISA to detect PEDV antibodies produced in pigs stimulated by lactic acid bacteria vaccine. ** represents the difference between the experimental group and the control group, which was very significant (P<0.01). Figure 6 This is a vaccine-stimulated mucosal immune assay (IgA). **This represents a significant difference between the experimental group and the control group (P<0.01). Figure 7 This is a flow cytometry test for CD8. A represents the CD8 detection in the control group; B represents the CD8 detection in the experimental group; C represents the ratio of the control group to the experimental group. The CD8 values ​​in each group are the mean values ​​within the group. Detailed Implementation

[0019] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0021] Operating procedures summarized as follows Figure 1 As shown.

[0022] Example 1: Detection of the stability of PEDV food-grade lactic acid bacteria 1. Revival of food-grade lactic acid bacteria from porcine PEDV (1) After sterilizing 30 mL of PNZ8149 screening medium, add lactose to a final concentration of 0.5% and BP to a final concentration of 0.004%, and pour into plates; (2) Take the inoculum out of the ultra-low temperature freezer, streak it, and let it stand overnight at 30°C; (3) Observe and find mature single colonies, and inject them together with the pipette tip into 5 mL of liquid culture medium and grow overnight at 30°C; (4) Take 100 μL of bacterial culture into an EP tube and store the culture in a -80°C refrigerator.

[0023] 2. Stability testing of PEDV food-grade lactic acid bacteria (1) Inoculate the lactic acid bacteria onto M17 medium containing lactose and grow overnight at 30 degrees Celsius; (2) Collect 3-5 μL of bacterial precipitate at 12000 rpm for 3 min 30 s; (3) Add 125 μL of THMS-buffer to the precipitate. Before use, add lysozyme at a concentration of 4 mg / mL and then suspend the precipitate. (4) Water bath (1 hour, 37°C); (5) Add freshly prepared 0.2N NaOH and 1% SDS, and mix thoroughly. (6) Ice bath for 5 minutes; (7) Add 189 μL of ice-cold 3 mol / L potassium acetate at pH 5.5 and shake gently; (8) Ice bath for 5 min, centrifuge (12000 rpm) for 5 min; (9) Take the supernatant and inject it into another sterile EP tube, add an equal volume of phenol: chloroform: isoamyl alcohol, shake, and centrifuge (12000 rpm for 5 minutes). (10) Use a pipette to take the supernatant and inject it into another sterile EP tube. Add 600 μL of isoamyl alcohol using a pipette, shake, and centrifuge. (11) Discard the supernatant, add 1 mL of medical alcohol, and centrifuge (12000 rpm) for 5 min; (12) Use a pipette to take the supernatant and inject it into another sterile EP tube, add isoamyl alcohol, shake, and centrifuge; (13) Air dry completely; (14) Add 20 μL H2O, of which 0.5 μL of RNase at a concentration of 20 μg / mL has already been added; (15) The enzyme-lysed sample was detected by agarose gel electrophoresis at 110V, and the gel after electrophoresis was detected by a gel imaging instrument to verify the presence of the porcine epidemic diarrhea virus C-COE gene.

[0024] The bacteria were placed in a lactic acid bacteria medium containing lactose and incubated statically at 30 degrees Celsius overnight. The PEDV vector recombinant plasmid was then isolated using an alkaline lysis method. Sac I. Nco I. Double enzyme digestion was used to verify whether the PEDV C-COE gene still existed. The results showed that the inserted PEDV protective antigen fragment C-COE was stably present. (See...) Figure 2 .

[0025] 3. Detection of protective antigen gene expression in PEDV food-grade lactic acid bacteria 3.1 Processing of proteins loaded onto SDS-PAGE; (1) Take the liquid culture of lactic acid bacteria and inoculate it into the lactic acid bacteria culture medium at a ratio of 1:10000, and incubate at 30 degrees Celsius overnight. (2) Take the lactic acid bacteria culture that has been cultured overnight and inoculate it into the culture medium at a ratio of 1:25. Culture for about 4 hours until the absorbance is about 0.4 at 600. (3) The culture was terminated after inducing with Nisin for 3-6 hours; (4) Take the sample and centrifuge at 12000 rpm for 3 min 30 s, then discard the supernatant (take twice). (5) Suspend with sterile water, centrifuge at 12,000 rpm for 3 min 30 s, and discard the supernatant; (6) Add 100 mg / mL of lysozyme and mix by pipetting. Place at 37°C for half an hour. (7) Take a 5K protein concentration tube, centrifuge the bacterial solution at 6000 rpm and 4 degrees Celsius for half an hour, discard the supernatant, and prepare for ultrasonic disruption. (8) Collect the bacterial cells at low temperature, suspend them in 1×PBS (DTT, 1%), and sonicate them (sonicate for 6 seconds, intermittently for 12 seconds, 99 times each, power 250W). Observe whether there is precipitation; if so, stir and continue sonication. (9) Centrifuge (15,000 rpm, 15 minutes, 4 degrees Celsius) to remove lactic acid bacteria fragments, and wash with washing buffer (100 mL + 100 μL DTT, 1‰) three times. (10) Each ultrasound session: 4 seconds, 10 seconds, 25 times, 250W. The ultrasound conditions should be adjusted appropriately according to the instrument. (11) After ultrasonication, centrifuge (15,000 rpm for 20 minutes) and wash thoroughly with washing buffer to remove residual fragments of lactic acid bacteria as much as possible; (12) Centrifuge (15,000 rpm, 20 minutes) and resuspend in resuspension buffer (60 ml + 60 μL DTT, 1‰); (13) Centrifuge (15,000 rpm for 20 minutes), weigh the bacterial cells, and dissolve them in dissolution buffer (1% DTT) at 30 mg / mL. Stir and mix at 4°C (overnight). (14) Centrifuge (15,000 rpm for 15 minutes), take the upper liquid and dispense it into 1 mL / tube, and store the sample in a -20℃ or -80℃ refrigerator.

[0026] 3.2 Protein decolorization (1) Rinse the glass plate and comb with tap water and detergent, clean with hydrogen peroxide several times, then wipe with alcohol cotton balls and let dry at room temperature; (2) Leak test: Install a clean glass plate, then use ultrapure water to check if the airtightness is good. If it is good, pour out the water and then use absorbent paper to dry it completely. (3) Prepare approximately 10 mL of a separating gel with a concentration of 15%, and mix well:

[0027] (4) Inject the separating adhesive into the clean plate space, cover with isoamyl alcohol liquid seal, and let stand for about 20 minutes to coagulate; (5) Prepare approximately 3 mL of a 5% concentrated gel and mix well:

[0028] (6) Use a syringe to draw up the upper layer of isoamyl alcohol, rinse with distilled water, use absorbent paper to absorb the water to ensure cleanliness, then quickly inject the concentrated gel with a pipette, insert the comb into the concentrated gel between the two plates, let it stand for about half an hour to allow the concentrated gel to solidify, remove the comb, and use a needle to straighten the gel hole. (7) Add 1 mL of the analyte to the EP tube, add 40 μL of water and 10 μL of SDS gel loading buffer (containing 2-mercaptoethanol), and mix well; (8) Take a large beaker, fill it with water, bring it to a boil in the oven, and place the EP tube in the boiling water bath for about ten minutes; (9) Centrifuge at 12000 rpm for 3 min, and collect the supernatant for sample loading; (10) Install the electrophoresis apparatus, add electrophoresis buffer to the electrophoresis apparatus, load the samples, 10 μL each of the sample and control, and 5 μL of protein marker; (11) When bromophenol blue is in the stacking gel, adjust the voltage to 80V. After passing through the stacking gel, immediately adjust it to 120V. After reaching the bottom of the gel, turn off the electrophoresis apparatus. It takes about 1-3 hours. (12) Remove the plate and use a blade to remove the separating gel, taking care not to damage it. Place the separating gel into a petri dish and pour the staining solution into the petri dish to cover the separating gel. Start the shaker (80 rpm) and stain for 1-2 hours. After that, pour out the staining solution and pour the decolorizing solution into the petri dish. Place the petri dish on the shaker (80 rpm) for decolorization. Change the decolorizing solution every half hour (the decolorizing solution formula is 45 ml of ultrapure water; 10 ml of anhydrous acetic acid; 45 ml of ethanol) until the color is completely removed.

[0029] PEDV food-grade lactic acid bacteria, induced by Nisin, were analyzed by SDS-PAGE electrophoresis, which showed correct C-COE expression consistent with the target size of 20 kDa. Figure 3 This indicates that it can be used for the next step of lactic acid bacteria vaccine preparation and immunization.

[0030] Example 2: Development of adjuvants and dosage forms for PEDV lactic acid bacteria vaccine 1. Preparation of lactic acid bacteria adjuvant A protective agent, also known as an adjuvant, for lactic acid bacteria vaccines was prepared using skim milk powder, glucose, and other ingredients in a specific ratio. Experiments involving heating and high pressure confirmed the stability and effectiveness of this protective agent, particularly its ability to prolong the survival time of lactic acid bacteria. In this example, the adjuvant consisted of 200 mL of milk and 10 g of glucose.

[0031] 2. Ratio of lactic acid bacteria to adjuvants After culturing PEDV lactic acid bacteria, the bacterial count was determined using a calculator. Different bacterial counts per milliliter were mixed with adjuvants in varying proportions, and the bacterial activity was then measured. The results showed that when the bacterial count reached 10 per milliliter... 9 When the ratio of the two is 4:1, the activity of lactic acid bacteria is the strongest, thus forming the drinking water formulation of PEDV lactic acid bacteria vaccine.

[0032] Example 3: Detection of the Immunization Efficacy of PEDV Lactic Acid Bacteria Vaccine 1. Setting up the experimental and control groups The purchased piglets were placed in pens No. 1 and No. 2 for rearing. Pen No. 1 was set up as the control group, and pen No. 2 was the experimental group. According to the principle of the only variable, the piglets were immunized with bacteria No. 2. The piglets in pen No. 1 did not need to be immunized. All other rearing conditions were exactly the same.

[0033] 2. Processing of immune and blood samples (1) Before immunization, feed normally while controlling water intake; (2) Add water to pen 1 2 hours after feeding, and add the prepared drinking water vaccine to pen 2, about 50 mL per piglet; (3) Ensure that the piglets completely ingest the aqueous vaccine within 5 minutes, and then provide water normally; (4) Boost the immunization every seven days, and a total of three immunizations are required; (5) Blood should be drawn from the jugular vein one week after immunization and before the second immunization; (6) Blood samples were collected in parallel from the control group; (7) After blood collection, take out 2 mL of blood sample and let it stand for 30 minutes. Then centrifuge at 4 degrees and 4000 rpm for 10 minutes. After the sample is separated into layers, use a pipette to take about 1 mL of serum into an EP tube and store it at -20℃. Try not to freeze and thaw it repeatedly. Anticoagulants are required when collecting blood samples. In this experiment, heparin is used as the anticoagulant, with the following formula: 0.1 g heparin + 5 mL normal saline. Then, use a pipette to add 200 μL to a 10 mL sterile test tube to allow the anticoagulant to adhere to the tube wall. This generally anticoagulates 3-5 mL of human blood.

[0034] 3. Weight monitoring One week apart, the weight of the experimental group and the control group was measured using an electronic scale to monitor the growth of the pigs in real time through changes in weight, for a total of 5 weeks.

[0035] After five weeks of measurement and calculation, it was found that the mass changes of rings 1 and 2 were not significantly different, but ring 2 gained slightly more weight than ring 1. Figure 3 This indicates that the PEDV lactic acid bacteria food-grade vaccine not only does not affect the weight gain of pigs, but may also be beneficial to the weight gain of pigs by stimulating appetite and regulating intestinal balance.

[0036] 4. ELISA detection of PEDV-specific antibodies Pigs in the experimental group were immunized with PEDV lactic acid bacteria vaccine via drinking water, and booster immunizations were administered three times at seven-day intervals. Blood samples were then collected at weeks 1, 3, and 5 post-immunization. The blood was centrifuged, and the serum was collected. PEDV antibody ELISA kits were used to test the control and experimental groups of pigs.

[0037] The steps are as follows: (1) Reduce the concentration of the original standard according to the instructions provided; (2) Sample addition: Set up two control wells (without analyte or enzyme-labeled reagent), one standard well, and one sample well. Add 50 μL of standard to each well, add 40 μL of diluent to the sample well, and then add 10 μL of serum. Care should be taken to avoid touching the well walls when adding the sample, and the well should be shaken to ensure even mixing after addition. (3) Incubation: Cover the board with the sealing film and keep it at 37°C for half an hour; (4) Solution preparation: Reduce the concentration of the concentrated washing solution by 30 times with double-distilled water; (5) Washing: Tear off the film, pour out the liquid, add the diluted washing solution, let it stand for 30 seconds and then pour it out. It needs to be washed 5 times. Use gauze to pat the board clean. (6) Add enzyme: Add 50 μL of enzyme-labeled reagent using a pipette; (7) Incubation: The procedure is the same as (3); (8) Washing: Tear off the film, pour out the liquid, add the diluted washing solution, let it stand for 30 seconds and then pour it out. It needs to be washed 5 times. Use gauze to pat the plate clean. (9) Color development: The color development agent is divided into A and B. Add A and B to the well plate in sequence, with each amount being 50 μL. Shake gently to mix evenly and develop color in the dark (room temperature, 10 minutes). (10) Termination: Add 50 μL of stop solution to the well to stop the reaction; (11) Measurement: Use a spectrometer (λ=450) to measure the OD value of all wells. The measurement should be completed within 15 minutes after the previous step.

[0038] Pigs in the experimental group immunized with PEDV lactic acid bacteria vaccine administered via drinking water were given booster immunizations three times, seven days later. Blood samples were then collected at weeks 1, 3, and 5 post-immunization. Serum was collected by centrifugation, and ELISA kits were used to detect porcine epidemic diarrhea virus antibodies in samples from both the control and experimental groups. Statistical analysis revealed that the PEDV antibody levels in the experimental group were higher than those in the control group at all monitoring points, but the antibody levels in the experimental group were significantly higher than those in the control group at week 5. Figure 4 The results indicate that food-grade lactic acid bacteria in drinking water can stimulate the production of certain antibodies in pigs, and antibody levels began to show significant changes after week 5.

[0039] 5. ELISA detection of vaccine-stimulated IgA levels The IgA levels produced by immunized pig herds were detected using an IgA ELISA kit, with a control group included. The procedure is similar to that for detecting PEDV antibodies.

[0040] Similar to the ELISA detection of PEDV-specific antibodies, an IgA ELISA kit was used to detect IgA levels in immunized pig herds, with a control group included. Statistical analysis revealed that the overall IgA level in the experimental group was significantly higher than that in the control group starting from week 3. IgA represents mucosal immunity, indicating that the vaccine effectively stimulated mucosal immunity. (See [link to relevant documentation]). Figure 6 .

[0041] 6. Assay for cellular immunity in porcine herds Five weeks after immunization, anticoagulated blood was collected, and the CD8 content in the control and experimental groups was detected using CD8 monoclonal antibodies.

[0042] The sample processing steps are as follows (all operations are performed under low temperature conditions): (1) Take whole blood and add diluent at a ratio of 1:1, mix by pipetting, and add 10 ml to each test tube; (2) First add the separation solution, 1:1 (5mL), take the blood containing the diluent and add it to the centrifuge tube containing the separation solution, 5mL in each tube (add slowly). (3) Centrifuge (20 min, 2200 rpm, 4℃); (4) After centrifugation, the blood sample will be separated into 4 layers. Take the lymphatic fluid and transfer it to an EP tube. (5) Perform centrifugation (5-10 min, 1800 rpm, 4℃); (6) Aspirate the supernatant, add 2-3 mL of lysis buffer to the precipitate using a pipette, and centrifuge; (7) Discard the supernatant, add washing solution to the precipitate, mix well, and centrifuge (1600 rpm, 15 min, 4℃). (8) Cell counting, adjust the nucleated cell count to 110. 6 / 110μL; (9) Add monoclonal antibody (per 110 cells) 6 Add 1 microgram of monoclonal antibody to 110 μL of water, mix well, and incubate in the dark for 1 hour. (10) Wash cells: Centrifuge (1500 rpm, 10 min) and discard the supernatant; (11) Add 500 μL of PBS to each tube, mix well, and filter through a 300-mesh copper screen; (12) Upstream flow cytometer: Measure the CD8+ T cells in each sample, with 10,000 counts per specimen.

[0043] Five weeks after immunization, anticoagulated blood was collected, and the CD8 levels in the control and experimental groups were measured using CD8 monoclonal antibodies. The results showed that the average CD8+ T cell count in the experimental group reached 15.3%, while that in the control group reached 14.19%. Figure 6 In comparison, the experimental group had slightly higher CD8+ T levels than the control group, but the difference was not significant. This indicates that the vaccine can stimulate some cellular immunity in pigs, but the level of stimulation is not significant.

[0044] Currently, porcine epidemic diarrhea (PEDV) continues to spread widely in countries around the world. This virus causes severe diarrhea and dehydration in piglets by attacking the intestinal villi, leading to mass mortality and significant economic losses to the pig farming industry. At present, research on PEDV prevention and control is not particularly extensive worldwide; therefore, developing effective, rapid, and efficient methods has become a crucial issue. Immunization experiments using lactic acid bacteria vaccines conducted by Zhao De et al. have demonstrated that lactic acid bacteria vaccines can stimulate the animal's intestines, thereby inducing humoral and cellular immunity. Lactic acid bacteria, as edible fungi and beneficial bacteria naturally present in the gut, do not produce toxins, their expressed products are directly related to human health, and they can be administered orally, making them a suitable direct-consumption vaccine with excellent research prospects.

[0045] As Gram-positive bacteria, lactic acid bacteria have thick cell walls. In this invention, lysozyme was initially used, but it was not possible to successfully separate the protein for a long time. Later, lysozyme and ultrasonic disruption were used in combination, and the protein was successfully separated, proving the existence of the protective immune response antigen gene of the experimental bacteria.

[0046] Lactic acid bacteria are edible fungi that can be taken orally without intravenous injection, avoiding the harm caused by syringes. In addition, the adjuvant for lactic acid bacteria has been adjusted. Skim milk powder and glucose are selected and formulated into a protective agent for lactic acid bacteria vaccines in a certain ratio. Water is controlled before immunization so that piglets can ingest all the lactic acid bacteria in the shortest time and complete the immunization process, eliminating the cumbersome injection process.

[0047] Lactic acid bacteria can directly enter the bloodstream via the small intestine, which is beneficial for generating mucosal immunity. This aligns perfectly with the characteristic of PEDV infecting through the intestines and stimulating mucosal immunity. ELISA is used to detect IgA antibodies to assess mucosal immunity. Additionally, the vaccine can induce the production of PEDV antibodies, which can be directly measured using ELISA. Flow cytometry is used to compare the CD8+ T cell counts in the two groups of pigs to determine the level of cellular immunity.

[0048] Based on the above data, the following conclusions can be drawn: This food-grade lactic acid bacteria vaccine in drinking water can induce significant mucosal immunity in pigs and stimulate the production of specific PEDV antibodies after week 5, thereby protecting pigs from PEDV infection and preventing disease. In addition, this vaccine can also stimulate cellular immunity.

[0049] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A food-grade lactic acid bacteria drinking water vaccine for porcine epidemic diarrhea, characterized in that, The recombinant food-grade Lactococcus lactis liquid mixed with vaccine adjuvant in volume ratio (3-5):1, the recombinant Lactococcus lactis carries the recombinant plasmid pNZ8149 / C-COE without antibiotic resistance marker, the recombinant plasmid contains PEDV protective antigen C-COE gene fragment, the size of the inserted fragment is 530bp; the vaccine adjuvant is prepared by skimmed milk powder and glucose, and the viable bacteria in the vaccine are not less than 10 9 CFU / mL.

2. The food-grade lactic acid bacteria drinking water vaccine for porcine epidemic diarrhea according to claim 1, characterized in that, The vaccine adjuvant was prepared by mixing 200 mL of skim milk powder and 10 g of glucose in ultrapure water, sterilizing at 121 °C for 20 min, and then cooling; the volume ratio of the recombinant lactococcus bacterial solution to the adjuvant was 4:

1.

3. The food-grade lactic acid bacteria drinking water vaccine for porcine epidemic diarrhea according to claim 1, characterized in that, The recombinant food-grade Lactococcus lactis was induced to express PEDV C-COE protein with a final concentration of 1 ng / mL Nisin. The target protein has a molecular weight of approximately 20 kDa.

4. A method for preparing a food-grade lactic acid bacteria drinking water vaccine for porcine epidemic diarrhea according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) The frozen recombinant food-grade lactococcus lactis was streaked onto a PNZ8149 screening plate containing 0.5% lactose and 0.004% BP, and incubated at 30°C overnight. Single colonies were picked and inoculated onto M17 liquid medium containing lactose, and incubated at 30°C with shaking overnight. (2) Inoculate 1% into fresh lactose-containing M17 liquid medium, and cultivate at 30°C with shaking until the OD 600 0.3-0.5, and then add Nisin to a final concentration of 1 ng / mL to induce expression for 3-6 h, and collect the bacterial cells by centrifugation. (3) Resuspend the bacterial cells in sterile PBS and adjust the viable count to ≥10⁻⁶. 9 CFU / mL, to obtain lactic acid bacteria culture; (4) Mix the lactic acid bacteria liquid with the vaccine adjuvant described in claim 2 at a volume ratio of 4:1 to obtain a food-grade lactic acid bacteria drinking water vaccine for swine epidemic diarrhea.

5. The preparation method according to claim 4, characterized in that, It also includes a step to verify the stability of the recombinant plasmid: take the bacterial culture obtained in step (1), extract the recombinant plasmid by alkaline lysis, perform double digestion with restriction endonucleases SacI and NcoI, detect by 1% agarose gel electrophoresis, and if bands corresponding to the 530bp target fragment and the 2591bp vector fragment appear, the recombinant plasmid is determined to be stable.

6. The preparation method according to claim 4, characterized in that, It also includes a step to detect the expression of the PEDV C-COE protective antigen gene: take the bacterial culture induced by Nisin, centrifuge to collect the bacterial cells, add THMS buffer containing 4 mg / mL lysozyme and incubate at 37°C for 1 h, sonicate to break up the bacteria, take the supernatant and perform 15% SDS-PAGE electrophoresis, and if a specific protein band appears at about 20 kDa, it is determined that the target protein is correctly expressed.

7. The preparation method according to claim 5, characterized in that, The specific steps for extracting plasmids using the alkaline lysis method include: suspending the bacterial cells in a THMS-buffer containing lysozyme, incubating in a 37°C water bath for 1 hour, adding 0.2N NaOH and 1% SDS for lysis, incubating on ice, adding 3mol / L, pH 5.5 potassium acetate to precipitate the protein, extracting with phenol-chloroform-isoamyl alcohol, precipitating with isopropanol, washing with 70% ethanol, and dissolving in ultrapure water containing RNase A.

8. A method for immunizing the porcine epidemic diarrhea food-grade lactic acid bacteria drinking water vaccine according to any one of claims 1 to 3, characterized in that, For the prevention and control of porcine epidemic diarrhea, the method of immunization via drinking water is adopted, specifically including: (1) Before immunization, control the water intake of the pigs to be immunized for 1.5 to 2.5 hours; (2) Add the vaccine to the drinking water at a dose of 50 mL per pig or administer it orally in a measured amount. The pigs should finish ingesting the vaccine within 5 minutes, and then resume normal water supply. (3) Administer booster immunizations on the 7th and 14th days after the first immunization, for a total of 3 immunizations.

9. The method for applying immunity according to claim 8, characterized in that, Pig herd weight changes were monitored weekly during immunization. Serum samples were collected at weeks 1, 3, and 5 after the initial immunization. PEDV-specific antibody and IgA antibody levels were detected using ELISA. Heparin-anticoagulated blood was collected at week 5 after immunization, and peripheral blood CD8 counts were detected using flow cytometry. + The percentage of T cells is used to assess the level of cellular immunity.

10. The use of the immunization application method according to claim 8 in the preparation of mucosal and humoral immunization agents for porcine epidemic diarrhea.

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