Preparation of nano MOF-SIP-coated HP55 oral vaccine applied to streptococcus agalactiae infection of nile tilapia
Through the preparation method of nano-MOF-SIP@HP55 oral vaccine, combining nanomaterials and antigens, and using acid-base sensitive coating materials, the shortcomings of traditional vaccines in preventing and treating streptococci infection with Nile tilapia were solved, and the results were achieved. Efficient and safe immune protection effects.
Patent Information
- Application Number
- CN202411526738.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2025-02-18
AI Technical Summary
The prior art is difficult to effectively prevent and treat Nile tilapia Streptococcus infection, especially due to the emergence of highly resistant bacterial strains, traditional vaccines are low in safety, high in cost and difficult to achieve herd immunity.
The preparation method of nano-MOF-SIP@HP55 oral vaccine is adopted to achieve protective delivery and sustained release of antigen by combining MOF nanomaterials with antigen SIP, and using acid-base-sensitive enteric coating material HP55.
This method provides a simplified preparation process that can effectively protect antigen from digestion by gastric juice, gradually release antigen in the intestine, enhance immune response, reduce mortality, and have high commercial value.
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Figure CN119454923A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of vaccines, and in particular relates to the preparation of a nano MOF-SIP@HP55 oral vaccine for Nile tilapia Streptococcus agalactiae infection. Background Art
[0002] As an omnivorous fish, Nile tilapia has significant advantages in temperature adaptability, stress resistance, reproductive capacity and development speed compared with other freshwater fish. With the further improvement of productivity in modern society, the freshwater fish consumption market is becoming increasingly broad, which has led to unprecedented development of tilapia farming. However, in order to meet the rapid expansion of market demand, the aquaculture industry cultivates Nile tilapia through high-density farming technology, which has caused the aquaculture water environment to gradually deteriorate and highly lethal bacteria and viruses to run rampant. Among them, Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common and most harmful bacterial pathogen in tilapia farming, causing extremely serious economic losses to the aquaculture industry. Practitioners urgently need strategies that can effectively control its infection and spread. In the embryonic stage of GBS research, aquaculture practitioners fed antibiotics or supplemented vitamins to fish to prevent and treat diseases, but the use of large amounts of antibiotics led to the abuse of antibiotics, increased aquaculture costs and a cliff-like deterioration of water bodies. However, the more serious situation is that the emergence of highly resistant GBS strains has significantly reduced the therapeutic effect of antibiotics. Therefore, strategies that can effectively prevent and control GBS infection and spread have attracted much attention from scientific researchers and aquaculture practitioners.
[0003] Tilapia is a teleost fish with a relatively complete immune system, which provides a theoretical basis for the development and market promotion of GBS vaccines. From traditional inactivated vaccines to new subunit vaccines that have emerged with the development of genetic engineering technology, researchers have designed and developed a variety of vaccines. However, traditional vaccines have low safety, high cost, single vaccination methods, and difficulty in achieving herd immunity. Some teams have turned their attention to subunit vaccines with high safety, low cost, and excellent immunogenicity. At the same time, nanomaterials have also developed rapidly and cross-integrated with vaccine immunology. Nanomaterials are widely used in protein delivery, drug transportation, and molecular diagnosis. These include inorganic mesoporous silica nanomaterials and organic polymer metal organic framework nanomaterials. Therefore, the use of nanoparticles combined with antigens to prepare diversified vaccination dosage vaccines, deliver and target the release of antigens while achieving herd immunity, has become one of the breakthroughs in the prevention and treatment of GBS in tilapia. Summary of the invention
[0004] The purpose of the present invention is to provide a method for preparing a nano MOF-SIP@HP55 oral vaccine for Nile tilapia Streptococcus agalactiae infection.
[0005] The object of the present invention is achieved by a method for preparing a nano MOF-SIP@HP55 oral vaccine for Nile tilapia infection with Streptococcus agalactiae, comprising the following steps:
[0006] 1) Preparation of MOF: 7.5 mL of 1.843 M 2-methylimidazole and 5 mL of 1.925 mM hexadecyltrimethylammonium p-toluenesulfonate were stirred at room temperature and a speed of 500 r for 5 min. During the stirring process, 2.5 mL of 97.5 mM cobalt nitrate hexahydrate was added, and the mixture was allowed to stand in a refrigerator at 4°C for 1 h to obtain the core ZIF-67 of MOF; then 10 mL of 0.9625 M hexadecyltrimethylammonium p-toluenesulfonate and 5 mL of 97.5 mM zinc nitrate hexahydrate were added to the reaction system, and the mixture was allowed to stand in a refrigerator at 4°C for 1 h. Then, 40 mL of H2O was added and allowed to stand for 12 h to obtain a hollow MOF. The mixture was centrifuged at room temperature and a speed of 8000 r for 15 min, and the precipitate was collected and vacuum dried at room temperature to obtain the finished MOF;
[0007] 2) Preparation of MOF-SI: Add 1 mg of the MOF material prepared in step 1) to 1 ml of the SIP protein solution, and incubate at a constant temperature of 4°C on a rocking table for 12 h to avoid denaturation and degradation of the protein components; perform solid-liquid separation in a refrigerated centrifuge to obtain a freeze-dried sample of MOF-SIP after loading and binding;
[0008] 3) Preparation of MOF-SIP@HP55 nanovaccine: Take 2.5 mL of MOF-SIP prepared in step 2), add 5 μL PEG400 and 50 μL Span 80, mix well, add 25 mL of ethyl acetate: dichloromethane with a volume ratio of 1:1, ultrasonically emulsify at a power of 160 W for 3 to 5 minutes, transfer the emulsion to 250 mL of 0.1 wt% PVA, and ultrasonically react at a power of 160 W for 3 to 5 minutes. After the ultrasonic reaction, quickly transfer the emulsion to 250 mL of 0.1 wt% PVA aqueous phase; slowly add 0.5 wt% hydroxypropyl methylcellulose phthalate HP55 solution to the system in a fume hood, wait for the organic solvent to evaporate until there is no obvious odor, and then centrifuge to collect the precipitate to obtain the encapsulated MOF-SIP@HP55 nanovaccine.
[0009] The nano MOF-SIP@HP55 oral vaccine prepared by the method of the present invention is used as a drug for preparing a drug for protecting Nile tilapia from infection by Streptococcus agalactiae.
[0010] Beneficial effects of the present invention:
[0011] 1) Provide a Nile tilapia nano oral vaccine with a simplified preparation process and the ability to resist digestion of protective antigens by gastric juice.
[0012] 2) Provide a method for protecting Nile tilapia from infection with Streptococcus agalactiae, reducing the mortality of tilapia after infection with Streptococcus agalactiae, and having a good immune protection effect.
[0013] 3) Provided are an immune tilapia feed prepared by a tilapia Streptococcus agalactiae nano oral vaccine and a method for preparing the immune tilapia feed.
[0014] 4) The MOF-SIP nanomaterials described in the present invention have an extremely high loading rate, which can basically achieve a loading rate of 20.41%. The higher loading rate can reduce the amount of MOF nanomaterials used while maintaining a high concentration of antigens, thereby reducing the cytotoxicity and stimulation of the finished nanovaccine to fish.
[0015] 5) The use of acid-base sensitive enteric coating material HP55 protects the MOF-SIP system from digestion by gastric juice. At the same time, it can gradually release the protective antigen SIP under intestinal conditions, providing an effective and sustained antigen concentration for gastric dendritic cells to take up antigens, which can achieve the effect of continuously stimulating the immune system, enhancing the immune response, and improving the immune protection rate.
[0016] 6) The preparation method of MOF-SIP is simpler and the cost is extremely low. The protective antigen protein SIP has excellent immune prototype and conservation. The nanovaccine prepared using SIP has high commercial value.
[0017] 7) MOF-SIP@HP55 can achieve herd immunity through oral administration, reducing the difficulty of vaccination and stress death caused by vaccination. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is the MOF loading rate curve of the gradient concentration SIP of the present invention.
[0019] Figure 2 Thermogravimetric test diagram of MOF and MOF-SIP.
[0020] Figure 3 This is the SIP release diagram of MOF-SIP@HP55 under artificial gastrointestinal fluid.
[0021] Figure 4 The figure is a comparison of cell survival rates under treatment with samples of different concentrations. DETAILED DESCRIPTION
[0022] The present invention is described in detail below with reference to the accompanying drawings and embodiments.
[0023] Example
[0024] Test process:
[0025] 1) Preparation of MOF: 7.5 mL of 1.843 M 2-methylimidazole and 5 mL of 1.925 mM hexadecyltrimethylammonium toluenesulfonate (CTATos) were stirred at room temperature and speed of 500 r for 5 min. During the stirring process, 2.5 mL of 97.5 mM cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was added, and the reaction system was placed in a refrigerator at 4°C for 1 h to obtain the core ZIF-67 of MOF. Subsequently, 10 mL of 0.9625 M hexadecyltrimethylammonium toluenesulfonate (CTATos) and 5 mL of 97.5 mM zinc nitrate hexahydrate (Zn(NO3)2·6H2O) were added to the reaction system, and the reaction system was placed in a refrigerator at 4°C for 1 h. 40 mL of dd H2O was added and the reaction system was placed in a refrigerator for 12 h to obtain a hollow MOF. The reaction system was centrifuged at room temperature and speed of 8000 r for 15 min, and the precipitate was collected and vacuum dried at room temperature to obtain the finished MOF.
[0026] 2) Protein expression and purification:
[0027] Expression of recombinant protein: thaw the frozen pET32a-SIP genetic engineering strain in a clean bench, take 100μL and inoculate it into 20mL liquid LB containing a final concentration of 200μg / mL Amp antibiotic, activate and culture for 3h at 37℃240r, transfer the bacterial solution to 1L liquid LB medium containing the same concentration of Amp, and proliferate and culture under the same activation conditions for 4h. After the end, let it stand on a shaker for 30min and slowly cool to 16℃, add 300μL 1M IPTG, culture at 16℃180r for 14h, and collect the bacterial precipitate by centrifugation. The collected bacteria per liter of bacterial solution are resuspended with 25mL Protein Buffer with pH 8.0, add 11.4μL Tween:β-ME volume ratio of 50:7 solution, mix well, and then add 50μL PMSF protease inhibitor. PMSF not only inhibits the degradation of SIP by protease, but also eliminates the foam of the resuspension and reduces the generation of foam, thereby improving the lysis effect. The bacterial suspension was lysed by using a pressure homogenizer at a pressure of 750 mPa for 5 minutes to break the bacterial cells, and the supernatant containing the target recombinant protein SIP was collected after centrifugation at 15000r for 30 minutes.
[0028] Purification of recombinant protein SIP (surface immune protein)
[0029] The SIP protein was purified according to the scheme screened and optimized by our research group. 2+ Column purification of protein, Ni 2+ Before adding the protein solution, the column was first rinsed once with ddH2O and ProteinBuffer at full speed, and then the supernatant of the lysate centrifuged was added to control the Ni 2+The column flow rate was 7s / drop, so that the 6-His tag of SIP was tightly bound to NiSO4. After all the supernatant passed through the column, Protein Buffer containing 20mM imidazole was added for washing, and the flow rate was controlled at 4-5s / drop. When there was no visible color change after mixing 20μL of flow-through with 100μL G-250 Coumarin, stop collecting and drain the liquid in the column. Finally, Protein Buffer containing 300mM imidazole was added to elute SIP, and the flow rate was controlled at 6-7s / drop. The flow-through of 300mM imidazole was collected in a 50mL clean plastic centrifuge tube.
[0030] The flow-through was placed in a dialysis bag with a minimum permeation size of 14 kDa after being bathed in boiling water for 30 minutes. Based on a volume ratio of 50:1 between the flow-through and TEA enzyme, a 1 mg / mL TEA protein solution was added to the dialysis bag. 透析液 :V SIP蛋白溶液 Take 30:1, prepare dialysis solution, and dialyze at 4℃ for at least 8h. After dialysis, add SIP protein solution to the regenerated Ni 2+ The flow rate was controlled at 4-5 s / drop m, the flow-through was collected and the Ni was washed with Protein Buffer containing 20 mM imidazole. 2+ Stop when there is no obvious blue color in the test column.
[0031] 3) Protein loading: 1 mg of the corresponding MOF nanomaterial was added to 1 ml of SIP protein solution with gradient concentrations, and incubated at a low speed at 4 °C on a rocking table for 12 h to avoid denaturation and degradation of protein components. After loading, solid-liquid separation was performed in a refrigerated centrifuge, and the supernatant protein concentration was determined according to the prepared standard curve to obtain the loading result (see Figure 1 ). In addition, MOF nanomaterials were used to combine with SIP concentrations with high loading rates in the loading curve. The freeze-dried sample MOF-SIP after loading and combination was taken, and a quantitative sample was taken for thermogravimetric testing. The protein loading rate was further determined by passing air and slowly heating from room temperature to 900°C at a constant rate. The loading rate calculation formula is: Loading rate (%) = (initial total protein - total supernatant protein) / [(initial total protein - total supernatant protein) + nanomaterial mass] × 100%.
[0032] 4) Vaccine encapsulation: In order to achieve sustained release of vaccine targeting the intestinal tract of Nile tilapia, a layer of HP55 (hydroxypropyl methylcellulose phthalate) acid-base sensitive material was encapsulated on the surface of MOF-SIP prepared in step 3) by double emulsion method. Take 2.5mL MOF-loaded SIP material (i.e. MOF-SIP prepared in step 3) and add 5μL PEG400 and 50μL Span 80, mix well, add 25mL ethyl acetate: dichloromethane = 1:1 (v:v) system, and ultrasonic emulsify at a power of 160W for 3-5min. Transfer the emulsion to 250mL 0.1wt% PVA, and perform ultrasonic reaction at a power of 160W for 3-5min. After the ultrasonic reaction, quickly transfer the double emulsion to 250mL 0.1wt% PVA aqueous phase. Slowly add 0.5 wt% HP55 (hydroxypropyl methylcellulose phthalate) solution to the system in a fume hood, wait for the organic solvent to evaporate until there is no obvious odor, and then collect the precipitate by centrifugation to obtain the encapsulated MOF-SIP@HP55 nanovaccine.
[0033] 5) Acid-base release test of sustained-release effect of nanovaccine: Artificial tilapia gastric juice (pH 1.5) and artificial tilapia intestinal juice (pH 7.4) were prepared according to the specifications of the "Chinese Pharmacopoeia", filtered with a 0.22μm filter head and refrigerated for later use. The MOF-SIP@HP55 nanovaccine prepared in step 4) was treated with artificial intestinal juice, gastric juice and pure water with pH 7.0 for 6 hours, and samples were taken every 30 minutes to measure the protein concentration. At the same time, the artificial intestinal-gastric juice group was treated with MOF-SIP@HP55 nanovaccine in gastric juice for 3 hours, and samples were taken every 30 minutes to measure the protein concentration. After that, the artificial gastric juice was replaced with artificial intestinal juice by centrifugation, and the vaccine particles in the EP tube were gently blown and dispersed using a pipette. The protein concentration was tested every 30 minutes within 3 hours after the replacement of the artificial intestinal juice (see Figure 3 ).
[0034] 6) Combining the nano vaccine with the feed: Stir the ordinary feed with the adhesive according to the instructions of the adhesive and mix it with the MOF-SIP@HP55 nano vaccine. Wrap the feed with gauze and quickly put it into a solution containing 2.5wt% triethyl citrate, 5wt% acetic acid and 0.5wt% chitosan. After it is completely soaked, transfer it to pure water and soak it quickly. Apply pressure to remove excess water and place it in an ultra-low temperature refrigerator for at least more than 24 hours. Use a freeze dryer set to 0.01mbar, preheat the vacuum pump, and quickly transfer the prepared oral vaccine feed from the ultra-low temperature refrigerator to the freeze dryer for rapid freeze drying until it is completely dry, and store it in a frozen state for use.
[0035] 7) Biological safety test: After 293T cells were revived, they were cultured in a 25T cell culture flask with DMEM high-glucose medium containing 10wt% fetal bovine serum for 48h. When the cell density reached 90% under an inverted microscope, the cells were subcultured for 48h. After trypsin digestion, DMEM complete medium was added to terminate the digestion. After centrifugation to collect the cells, 1ml of complete medium was used to gently blow and mix the cells. Then 5μl of the culture solution was mixed with 0.4wt% trypan blue. After counting with an automatic cell counter, the cells were diluted with complete medium to a cell concentration of 5×10 4 / ml, take 100μl of diluted cells and add them to the edge wells of a 96-well plate with 0.1wt% PBS and culture for 48h. After setting the drug gradient, add 5μl of the corresponding drug to each well and culture for 24h. Set three replicates for each gradient concentration. Add 10μl CCK-8 ( Cell counting The reagent was incubated for 2 hours, and the absorbance at a wavelength of 450 nm was measured by an ELISA instrument to obtain the test results. The cell survival rate was calculated using the formula: Cell survival rate (%) = [(As-Ab) / (Ac-Ab)] × 100%, where As is the absorbance of the drug group, Ab is the absorbance of the blank group without cells, and Ac is the absorbance of the control group without drugs (see Figure 4 ).
[0036] 8) Pre-challenge test: After streaking and culturing on BHI plates for 18 hours, pick a single colony for enrichment culture at 37°C and mix the bacterial solution with 500 μL of 30 wt% sterilized glycerol and store in a -80°C refrigerator. Take 2 μL of the strain and add it to 1 ml of BHI medium (brain heart infusion medium) for activation for 12 hours, then take 40 μL of the activated bacterial solution and add it to 20 ml of BHI medium and culture for 8 hours until the OD reaches 0. 600 = 1. Dilution was performed in a 10-fold gradient, from the original bacterial solution to 10 -5 Take 4 μL of each diluted bacterial solution and spot it on a solid BHI plate and set up 6 sets of parallel. Set 1×10 6 The challenge test was carried out at a dose of 1×10 cfu / ml, and the original bacterial solution was diluted 2000 times with sterile PBS to obtain 1×10 6 CFU / ml of the challenge bacterial solution. It was observed that tilapia began to die from the third day, and the mortality rate reached the highest point on the sixth day, reaching 80%. By obtaining liver tissue with lesions from dead tilapia for colony PCR verification, it was proved that Nile tilapia died of visceral hemorrhage caused by infection with Streptococcus agalactiae, which was consistent with the typical symptoms of visceral hemorrhage and exophthalmos obtained by autopsy.
[0037] 9) Challenge and protection test: 20±10g Nile tilapia were randomly divided into five groups, namely blank control group, MOF-SIP@HP55 nanovaccine group, SIP group, and challenge and protection group, with 20 test individuals in each group. Except for the blank control group, the rest groups were intraperitoneally injected with 0.2ml of 1×10 6 The relative protection rate of MOF nanovaccine was (Table 1).
[0038] Table 1 shows the relative protection rate of nano vaccines
[0039]
[0040] in conclusion
[0041] 1. Loading rate determination: The SIP loading rate of MOF nanomaterials gradually increased with the initial SIP concentration gradient, and the loading rate of the material entered a plateau at the measured SIP concentration. MOF reached a maximum saturated loading rate of 21.41% at 2 mg / mL. In addition, the loading capacity of MOF for SIP was further verified by thermogravimetric tests. The temperature was raised at a constant rate from room temperature to 900°C. As the temperature gradually increased, the SIP protein gradually turned into ash at high temperature. Compared with MOF, the thermogravimetric curve of MOF-SIP decreased linearly, while MOF decreased significantly at 600°C, with a decrease of about 30%, indicating that the loading rate of MOF is about 30% (see Figure 2 ).
[0042] 2. Acid-base sustained release results: The protective antigen release of MOF-SIP@HP55 under artificial gastrointestinal fluid conditions is as follows Figure 3 As shown in Figure 2, under the conditions of gastric fluid simulation and pure water at pH 1.5 and 7.0, MOF-SIP@HP55 only weakly released SIP protein. In the intestinal fluid simulation environment, HP55 rapidly expanded and dissolved to release SIP, among which the release rate was the highest within the first hour of intestinal fluid treatment, and there was no significant increase in the release of SIP over time (see Figure 3 ).
[0043] 3. Biological safety test: The cell survival rate of MOF-SIP at 100 μg / mL was 81.68%, but it dropped to 65.47% at 120 μg / mL, which indicates that 120 μg / mL of MOF-SIP is the critical value of the cytotoxicity test. The biosafety of MOF-SIP is the highest when the concentration is below 100 μg / mL, and the toxicity to cells is low (see Figure 4 ).
Claims
1. A method for preparing a nano-MOF-SIP@HP55 oral vaccine for Nile tilapia infection with Streptococcus agalactiae, comprising the following steps: 1) Preparation of MOF: 7.5 mL of 1.843 M 2-methylimidazole and 5 mL of 1.925 mM hexadecyltrimethylammonium p-toluenesulfonate were stirred at room temperature and 500 r for 5 min. During the stirring process, 2.5 mL of 97.5 mM cobalt nitrate hexahydrate was added, and the mixture was placed in a refrigerator at 4 °C for 1 h to obtain the core ZIF-67 of MOF. Subsequently, 10 mL of 0.9625 M hexadecyltrimethylammonium p-toluenesulfonate and 5 mL of 97.5 mM zinc nitrate hexahydrate were added to the reaction system. After the mixture was placed in a refrigerator at 4 °C for 1 h, 40 mL of H2O was added and the mixture was placed in a refrigerator for 12 h to obtain a hollow MOF. The mixture was centrifuged at 8000 r for 15 min at room temperature, and the precipitate was collected and vacuum dried at room temperature to obtain the finished MOF. 2) Preparation of MOF-SI: Add 1 mg of the MOF material prepared in step 1) to 1 ml of SIP protein solution, and incubate at a constant temperature and low speed of 4 °C on a rocking table for 12 h to avoid denaturation and degradation of protein components; perform solid-liquid separation in a refrigerated centrifuge to obtain a freeze-dried sample of MOF-SIP after loading and binding; 3) Preparation of MOF-SIP@HP55 nanovaccine: Take 2.5 mL of MOF-SIP prepared in step 2), add 5 μL PEG400 and 50 μL Span 80, mix well, add 25 mL of ethyl acetate: dichloromethane with a volume ratio of 1: 1, ultrasonically emulsify at a power of 160 W for 3 to 5 min, transfer the emulsion to 250 mL 0.1wt% PVA, and ultrasonically react again at a power of 160 W for 3 to 5 min. After the ultrasonic reaction, quickly transfer the emulsion to 250 mL 0.1wt% PVA aqueous phase; slowly add 0.5wt% hydroxypropyl methylcellulose phthalate HP55 solution to the system in a fume hood, wait for the organic solvent to evaporate until there is no obvious odor, and then centrifuge to collect the precipitate to obtain the encapsulated MOF-SIP@HP55 nanovaccine.
2. Use of the nano MOF-SIP@HP55 oral vaccine prepared by the method of claim 1 as a drug for preparing a drug for protecting Nile tilapia from infection with Streptococcus agalactiae.