A vaccine diluent for pigs, its preparation method and application
By using a swine vaccine diluent containing sodium thiosulfate and recombinant porcine interferon α, γ, and λ, the problems of poor blocking rate and immunization effect of existing diluents for classical swine fever virus were solved, resulting in higher vaccine efficacy and lower adverse reactions, thereby improving the immune level and production performance of the swine herd.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HARBIN PHARM GRP BIO-VACCINE CO LTD
- Filing Date
- 2024-12-30
- Publication Date
- 2026-06-30
AI Technical Summary
Existing swine vaccine diluents are not very effective in improving the vaccine's blocking rate against classical swine fever virus, and are unlikely to effectively improve the immune effect of pig herds and reduce adverse reactions.
A swine vaccine diluent composed of sodium thiosulfate and different proportions of recombinant porcine interferon α, γ, and λ was prepared by optimizing its coding gene and mixing it with water. This diluent can significantly improve the immune response of swine herds and reduce adverse reactions.
It significantly improved the blocking rate of swine vaccines against classical swine fever virus, enhanced the immunization effect, significantly reduced the adverse reactions of live classical swine fever vaccines, and improved the production performance of pig herds.
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Figure CN122297659A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to an animal vaccine diluent and its application, particularly to a swine vaccine diluent and its application, and belongs to the field of animal vaccine diluents. Background Technology
[0002] The pig farming industry is gradually moving towards a highly large-scale and intensive model. Under this model, various viral diseases require prevention. Simultaneously, the overuse of antibiotics during the farming process has led to the emergence of drug-resistant strains and pollution of the local ecosystem.
[0003] Currently, the main method for preventing swine fever is freeze-dried live vaccine preparation. The advantages of live vaccines include good immunization efficacy and a longer duration of action. Because live vaccines contain active viral antigens, they induce an autoimmune process in the body similar to viral infection, enhancing the body's immunity after injection. Furthermore, adverse absorption of live vaccines is generally not a problem after injection.
[0004] Existing swine vaccine diluents are not very effective in improving the vaccine's blocking rate against the virus. Therefore, there is an urgent need to develop swine vaccine diluents with higher blocking rates to dilute freeze-dried live vaccines and improve the blocking rate of swine vaccines against classical swine fever virus. Summary of the Invention
[0005] One of the objectives of this invention is to provide a diluent for a swine vaccine.
[0006] The second objective of this invention is to provide a method for preparing a diluent for swine vaccines.
[0007] The third objective of this invention is to apply the aforementioned swine vaccine diluent to improve the blocking rate of swine fever vaccine against swine fever virus or enhance the immunization effect of swine fever vaccine.
[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include:
[0009] One aspect of this invention is to provide a diluent for a swine vaccine.
[0010] In a preferred embodiment of the present invention, the swine vaccine diluent is composed of sodium thiosulfate, porcine interferon, and water; the amounts of each component by mass percentage are: sodium thiosulfate 0.5%-1.8%, porcine interferon 2.2%-4.2%, and the remainder is water.
[0011] In a preferred embodiment of the present invention, the amounts of each component in the swine vaccine diluent are: sodium thiosulfate 0.875% and swine interferon 4.125%.
[0012] In a preferred embodiment of the present invention, the porcine interferon is selected from any one or more of porcine interferon α, porcine interferon γ, or porcine interferon λ, forming a complex interferon.
[0013] In a preferred embodiment of the present invention, the porcine interferon is a composite interferon composed of porcine interferon α, porcine interferon γ and porcine interferon λ.
[0014] In a preferred embodiment of the present invention, the ratio of porcine interferon α: porcine interferon γ: porcine interferon λ in the composite interferon is 9:13:11 by mass.
[0015] In a preferred embodiment of the present invention, the nucleotide sequences of the encoding genes for porcine interferon α, porcine interferon γ, or porcine interferon λ are optimized based on Escherichia coli codon bias.
[0016] In a preferred embodiment of the present invention, the swine vaccine is a live swine fever vaccine.
[0017] Another aspect of the present invention provides a method for preparing a diluent for a swine vaccine, comprising: (1) adding sodium thiosulfate pentahydrate to water for injection, adjusting the pH value to 6.0, sterilizing, and obtaining an aqueous solution of sodium thiosulfate; (2) mixing the aqueous solution of sodium thiosulfate with swine interferon evenly, filtering, and obtaining the solution.
[0018] Another aspect of the present invention is to apply the aforementioned swine vaccine diluent to improve the blocking rate of swine vaccines against classical swine fever virus or enhance the immunizing effect of classical swine fever vaccines.
[0019] In a preferred embodiment of the present invention, the swine vaccine diluent is used to improve the blocking rate of swine vaccine against classical swine fever virus.
[0020] In a preferred embodiment of the present invention, the swine vaccine diluent is used to prepare a drug that enhances the immune efficacy of swine fever vaccine or increases the blocking rate of swine vaccine against swine fever virus.
[0021] This invention utilizes *Escherichia coli* as a genetically engineered host bacterium. Based on the codon bias of *E. coli*, the porcine interferon α, porcine interferon λ, and porcine interferon γ genes are optimized. The recombinant porcine interferon α, porcine interferon λ, and porcine interferon γ genes with different codon optimizations are added as immunostimulants in a 9:13:11 ratio to a sodium thiosulfate solution to obtain a porcine vaccine diluent. This porcine vaccine diluent is used to improve the blocking rate of porcine vaccines against classical swine fever virus or to enhance the immunization effect of classical swine fever vaccines. It not only improves the immune regulation of the pig herd but also significantly reduces adverse reactions after immunization with live classical swine fever vaccine and improves the production performance of pigs. The porcine vaccine diluent of this invention can significantly improve the cellular immune response in pigs and enhance the immunity of the pig herd. Attached Figure Description
[0022] Figure 1 This is a statistical chart showing the blocking rate of different dilution formulations against classical swine fever virus over 21 days. Detailed Implementation
[0023] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.
[0024] Aseptic room preparation
[0025] Turn on the Class 100 air conditioning 30 minutes before packaging; after packaging, irradiate the room with ultraviolet lamps for 2 hours (30 minutes during lunch break); after packaging, clean the machine and floor with disinfectant, and conduct a thorough cleaning once a week; fumigation is required every time the product is changed, and fumigation is also required if the product is not changed for more than a week.
[0026] Personnel preparation
[0027] Before starting work, employees must undergo pre-job training and pass the test; upon entering a Class 100,000 cleanroom, they must change into sterile clothing and wear sterile masks as required (sterile clothing must be changed daily after being cleaned and sterilized); sterile aloe vera gloves must be worn before operation and disinfected with 75% alcohol; personnel must disinfect their hands every 30 minutes.
[0028] Inactivation with diluent
[0029] During the heating phase, start stirring at 0-100℃, stop stirring above 100℃, sterilize at 116℃ for 40 minutes, and after sterilization, cool with drinking water below 100℃ and start stirring again, stop cooling at 50℃, maintain pressure at 0.1MPa for storage, and filter through a 0.1μm filter.
[0030] Diluent Dispensing
[0031] The vaccine is packaged according to the required dosage for factory delivery.
[0032] Diluent testing
[0033] (1) Sterility test: Refer to the sterility test or purity test method in Chinese Veterinary Pharmacopoeia 3306.
[0034] (2) pH value detection: The pH meter is calibrated before the test to ensure the accuracy of the test results. During the test, the pH meter is inserted into the diluent and the pH value is read after a period of stabilization.
[0035] Example 1: Preparation of diluent for swine vaccine
[0036] 1. Preparation of recombinant porcine interferon
[0037] Porcine interferon α (IFN-α), porcine interferon λ (IFN-λ), and porcine interferon γ (IFN-γ) were synthesized using gene synthesis methods (GenBank accession numbers: α: NM_214393.1; λ: NM_001142837.1; γ: NM_213948.1). The three fragments were optimized according to the codon bias of *E. coli* and ligated into pMal-c5X to construct recombinant plasmids. The recombinant plasmids were transformed into recipient cells *E. coli* BL21(D3) using a thermal transformation method. After extensive induction with 1.0 mmol / L IPTG, the recombinant proteins rIFN-α / λ / γ were obtained after purification using an MBP tag.
[0038] UV spectrophotometer A 280 The concentrations of recombinant protein rIFN-α ranged from 35.3 g / L to 50.6 g / L; recombinant protein rIFN-λ ranged from 47.3 g / L to 68.6 g / L; and recombinant protein rIFN-γ ranged from 55.3 g / L to 68.6 g / L.
[0039] 2. Vaccine diluent for experimental use
[0040] Diluent Formulation 1: Weigh 35g of sodium thiosulfate decahydrate and add water for injection to 1000mL, adjusting the pH to approximately 6.0. Autoclave, adding 10% of the evaporated amount. After sterilization, mix with rIFN-α (45g / L) at a 1:1 volume ratio and filter through a 0.1μm filter.
[0041] Diluent Formulation 2: Weigh 35g of sodium thiosulfate pentahydrate and add water for injection to 1000mL, adjusting the pH to approximately 6.0. Autoclave, adding 10% of the evaporated amount. After sterilization, mix with rIFN-λ (55g / L) at a 1:1 volume ratio and filter through a 0.1μm filter.
[0042] Diluent Formulation 3: Weigh 35g of sodium thiosulfate decahydrate and add water for injection to 1000mL, adjusting the pH to approximately 6.0. Autoclave, adding 10% of the evaporated volume as needed. After sterilization, mix with rIFN-γ (65g / L) at a 1:1 volume ratio and filter through a 0.1μm filter.
[0043] Diluent Formulation 4: Weigh 35g of sodium thiosulfate pentahydrate and add it to 1000mL of water for injection, adjusting the pH to approximately 6.0. Autoclave, adding 10% of the evaporated volume as needed. After sterilization, mix with rIFN-α (45g / L) and rIFN-λ (55g / L) at a 1:1:1 volume ratio, and filter through a 0.1μm filter.
[0044] Diluent Formulation 5: Weigh 35g of sodium thiosulfate pentahydrate and add it to 1000mL of water for injection, adjusting the pH to approximately 6.0. Autoclave, adding 10% of the evaporated amount. After sterilization, mix with rIFN-α (45g / L) and rIFN-γ (65g / L) at a 1:1:1 volume ratio, and filter through a 0.1μm filter.
[0045] Diluent Formulation 6: Weigh 35g of sodium thiosulfate decahydrate and add it to 1000mL of water for injection, adjusting the pH to approximately 6.0. Autoclave, adding 10% of the evaporated volume as needed. After sterilization, mix with rIFN-γ (65g / L) and rIFN-λ (55g / L) at a 1:1:1 volume ratio, and filter through a 0.1μm filter.
[0046] Diluent Formulation 7: Weigh 35g of sodium thiosulfate decahydrate and add it to 1000mL of water for injection, adjusting the pH to approximately 6.0. Autoclave, adding 10% of the evaporated volume as needed. After sterilization, mix with rIFN-α (45g / L), rIFN-γ (65g / L), and rIFN-λ (55g / L) in a 1:1:1:1 volume ratio, and filter through a 0.1μm filter.
[0047] Diluent formula 8: Weigh 35 g of sodium thiosulfate pentahydrate and add injection water to 1000 mL, adjust the pH value to about 6.0. Sterilize by high-pressure steam, and 10% of the evaporation needs to be supplemented. After sterilization, mix it with rIFN-α (45 g / L), rIFN-γ (65 g / L) and rIFN-λ (55 g / L) in a volume ratio of 2:1:1:1, and filter through a 0.1 μm filter element.
[0048] Test Example 1 Inspection Test of Porcine Vaccine Diluent
[0049] 1 Potency Test
[0050] According to the number of doses indicated on the vial label, dilute the classical swine fever vaccine with diluents of different new formulas to 1 / 3000 doses / mL, and intramuscularly inject 5 healthy and susceptible pigs with negative classical swine fever virus antibodies (experimental group), 1.0 mL per pig. 10 to 14 days after inoculation, together with 5 pigs in the control group, inject 1.0 mL of classical swine fever Shimen strain virus (not less than 10 5.0 MLD) to each pig, and observe for 16 days. The results of the potency test are shown in Table 1. All pigs in the control group died; all pigs in the experimental group survived healthily without clinical symptoms of classical swine fever.
[0051] Table 1 Comparison of Vaccine Potency Tests
[0052]
[0053] 2 Safety Test
[0054] Use healthy pigs with negative classical swine fever virus antibodies. Observe for 5 - 7 days before inoculation, and measure the body temperature once in the morning and afternoon every day. Select pigs with normal body temperature, spirit and appetite for use. Dilute each batch of classical swine fever vaccine according to the number of doses indicated on the vial label with diluents of different formulas to 6 doses per milliliter, and intramuscularly inject 5 pigs, 5.0 mL per pig (containing 30 doses). After inoculation, observe and measure the body temperature once in the morning and afternoon every day, and observe for 21 days. If there is no obvious change in body temperature, spirit and appetite compared with before inoculation; or the body temperature rises by more than 0.5 °C but does not exceed 1.0 °C, and the retention does not exceed 4 temperature readings; or the food intake decreases for no more than 1 day, the vaccine can be judged as qualified. If the body temperature of 1 pig rises by more than 1 °C but does not exceed 1.5 °C, and the retention does not exceed 2 temperature readings, the vaccine can also be judged as qualified. If the reaction of 1 pig exceeds the above standard; or there are other suspicious body temperature reactions and other abnormal phenomena, use 5 pigs for re-inspection once. If the same reaction still occurs in the pigs during re-inspection, the vaccine should be judged as unqualified. It is also possible to collect blood from pigs during the high-temperature period and inject 5.0 mL of the original blood of the suspicious pigs into 2 pigs by intramuscular injection, and measure the temperature and observe for 16 days. If there is no reaction in both pigs, the vaccine can be judged as qualified. If the first test result has confirmed that the vaccine is unsafe, re-inspection should not be carried out. The results of the safety test are shown in Table 2. The vaccine diluents of 8 formulas are all safe.
[0055] Table 2 Vaccine Safety Test
[0056]
[0057] Experiment Example 2: Experiment on improving the blocking rate of classical swine fever vaccine against classical swine fever virus using swine vaccine diluent.
[0058] While conducting vaccine safety testing experiments, the level of classical swine fever (CSF) serum antibodies was monitored. Specific procedures are detailed in the safety testing section of Experiment Example 1. The CSF virus blocking rate of the CSF vaccine was detected using the CSF virus ELISA antibody method. Specifically, the IDEXX CSFV Ab detection kit (batch number AE561) was used for testing. Sample diluent, positive and negative controls, washing buffer, and stop solution were all provided by the kit.
[0059] All reagents should be brought to 18-25°C before use. The reagents should be gently rotated or vortexed to mix thoroughly.
[0060] (1) Add 50 μL of sample dilution solution to the control well and the corresponding detection well, respectively.
[0061] (2) Add 50 μL of positive control and negative control to the corresponding control wells, two wells in each case.
[0062] (3) Add 50 μL of the test sample to the corresponding test well, and note that a different pipette tip should be used for each sample. Gently tap the microplate or shake it with a shaker to mix the solution in the reaction plate.
[0063] (4) Incubate at 18-25℃ for 2 hours (±5 minutes) or overnight (12-18 hours). The microplate should be sealed with a cover or incubated at room temperature in a humidifier to avoid liquid evaporation.
[0064] (5) Discard the liquid from each well into the waste container. Wash each well three times with 300 μL of washing solution. After each wash, aspirate the liquid from each well. After the last wash, pat the remaining washing solution in the well dry on absorbent material. Prevent the wells from drying out before adding the next reagent.
[0065] (6) Add 100 μL of enzyme-labeled antibody to each well, seal the reaction plate with a cover plate or incubate in a humidified chamber at 18-25°C for 30 minutes (±2 minutes). Repeat step 5.
[0066] (7) Add 100 μL of substrate solution to each reaction well and place in the dark at 18-25°C for 10 minutes (±1 minute).
[0067] (8) Add 100 μL of stop solution to each reaction well to terminate the reaction.
[0068] (9) Measure the absorbance of the sample and the control at 450 nm. Calculate the average absorbance of the sample and the control.
[0069] The results of the ELISA antibody test for swine fever virus are as follows: Figure 1 As shown in Table 3, the experimental results indicate that the swine fever vaccine diluted with vaccine diluent formulation 7 has a significantly higher blocking rate against the swine fever virus than the other 7 vaccine diluent formulations.
[0070] Table 3. Blocking rates of different dilution formulations against classical swine fever virus within 21 days.
[0071]
Claims
1. A vaccine diluent for swine, characterized in that, It is composed of sodium thiosulfate, porcine interferon and water; by mass percentage, the amounts of each component are: sodium thiosulfate 0.5%-1.8%, porcine interferon 2.2%-4.2%, and the balance is water.
2. The vaccine diluent for swine according to claim 1, characterized in that, The dosage of each component is as follows: sodium thiosulfate 0.875%, porcine interferon 4.125%.
3. The vaccine diluent for swine according to claim 1, characterized in that, The porcine interferon is selected from any one or more of porcine interferon α, porcine interferon γ, or porcine interferon λ, forming a complex interferon.
4. The vaccine diluent for swine according to claim 1, characterized in that, The porcine interferon is a complex interferon composed of porcine interferon α, porcine interferon γ and porcine interferon λ.
5. The vaccine diluent for swine according to claim 4, characterized in that, Based on mass ratio, the ratio of porcine interferon α: porcine interferon γ: porcine interferon λ in the composite interferon is 9:13:
11.
6. The vaccine diluent for swine according to claim 3, characterized in that, The nucleotide sequences of the encoding genes for porcine interferon α, porcine interferon γ, or porcine interferon λ are optimized based on E. coli codon bias.
7. The vaccine diluent for swine according to claim 1, characterized in that, The swine vaccine mentioned is a live swine fever vaccine.
8. The method of claim 1, wherein the vaccine diluent for swine is prepared by the steps of: include: (1) Add sodium thiosulfate pentahydrate to water for injection, adjust the pH to 6.0, sterilize, and obtain sodium thiosulfate aqueous solution; (2) Mix sodium thiosulfate aqueous solution with porcine interferon evenly, filter, and obtain the final product.
9. The use of the swine vaccine diluent according to any one of claims 1-7 in the dilution of classical swine fever vaccine.
10. The use of the swine vaccine diluent according to any one of claims 1-7 in the preparation of a medicament for improving the immunizing effect of swine fever vaccine or improving the blocking rate of swine fever vaccine against swine fever virus.