Mycoplasma gallisepticum inactivated vaccine and preparation method thereof

Abstract

The invention relates to the technical field of veterinary drug vaccines, in particular to a mycoplasma gallisepticum inactivated vaccine and a preparation method thereof.The preparation method comprises the following steps that mycoplasma gallisepticum is cultured and harvested in a culture medium, and a bacterial solution is obtained; centrifuging and resuspending the bacterial liquid, and inactivating to obtain an inactivated bacterial liquid; adding muramyl dipeptide, trehalose, thiomersalate and tween-80 into the inactivated vaccine solution to obtain a water phase; taking 94 parts of white oil for injection and 1 part of aluminum stearate, uniformly mixing under a water bath heating condition of 80 DEG C, then slowly adding 5 parts of span-80, carrying out high-pressure steam sterilization at 115 DEG C, maintaining for 40 minutes, and cooling for later use to obtain an oil phase; taking the oil phase and the water phase according to a mass ratio of 3: 2, and emulsifying to obtain an inactivated vaccine; preparing lipidosome by using a film dispersion method or a reverse evaporation method, wherein the average particle size is controlled to be 100-200nm; a part of 10%-20% of inactivated vaccine bacterial liquid is wrapped in a liposome to form an inactivated vaccine with a targeted delivery system.

Description

[0001] The present invention relates to the technical field of veterinary vaccines, in particular to an inactivated Mycoplasma gallisepticum vaccine and a preparation method thereof. Background Art

[0002] Mycoplasma gallisepticum (MG) is the primary pathogen causing chronic respiratory disease (CRD) in chickens and turkeys. Clinical manifestations include coughing, runny nose, rales during breathing, and, in severe cases, open mouth breathing. The incidence of CRD has increased annually with the expansion of poultry farming, changes in feeding practices, and increased stocking density. According to statistics, infection with MG in chickens increases the rate of weak chicks by approximately 10%, decreases the egg production rate of laying hens by 10-20%, and reduces the weight of broilers by 38%. The market life is prolonged, feed conversion efficiency is reduced, immune suppression occurs, and other diseases can occur, resulting in significant economic losses for the poultry industry.

[0003] Currently, the prevention and treatment of mycoplasma infections primarily rely on drug therapy, vaccination, and the establishment of healthy livestock herds. While drug treatment is effective for CRD, it cannot eliminate the carrier state and can also cause drug residues. Therefore, effective vaccination has become a crucial means of disease prevention and control, and vaccine research has become a significant area of research.

[0004] In traditional Mycoplasma gallisepticum vaccine production, culture medium components and culture processes limit the proliferation of Mycoplasma gallisepticum, resulting in low bacterial concentrations for vaccine production (typically only up to 1.0×10^9 CCU / ml). This necessitates the concentration of semi-finished products during inactivated vaccine production, increasing production costs and operational complexity while also compromising the protective efficacy of the finished vaccine.

[0005] To overcome these problems, it is urgent to develop a new vaccine that can efficiently reproduce Mycoplasma gallisepticum, simplify the production process, reduce costs, and provide high protection. In addition, with the continuous development of the poultry farming industry, higher requirements are placed on the safety and effectiveness of vaccines. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention provides an inactivated Mycoplasma gallisepticum vaccine and a preparation method thereof, which solve the problems of the above-mentioned background technology.

[0007] According to a first aspect of the present invention, there is provided a method for preparing an inactivated Mycoplasma gallisepticum vaccine, comprising the following steps:

[0008] Cultivating and harvesting Mycoplasma gallisepticum in a culture medium to obtain a bacterial liquid;

[0009] The bacterial solution is centrifuged and resuspended, and then inactivated to obtain an inactivated bacterial solution;

[0010] Add muramyl dipeptide, trehalose, thimerosal, and Tween-80 to the inactivated vaccine solution to obtain an aqueous phase; take 94 parts of white oil for injection and 1 part of aluminum stearate, mix them evenly under heating conditions at 80°C in a water bath, then slowly add 5 parts of Siben-80, sterilize with high-pressure steam at 115°C for 40 minutes, cool and set aside to obtain an oil phase; emulsify the oil phase and aqueous phase in a mass ratio of 3:2 to obtain an inactivated vaccine;

[0011] Liposomes were prepared using thin film dispersion or reverse evaporation methods, with an average particle size of 100-200 nm.

[0012] Encapsulate 10%-20% of the inactivated vaccine solution in liposomes to form an inactivated vaccine with a targeted delivery system;

[0013] A cold-adapted protein is added to the inactivated vaccine with a targeted delivery system at a concentration of 0.5 mg / mL to obtain the Mycoplasma gallisepticum inactivated vaccine.

[0014] According to an embodiment of the present invention, the Mycoplasma gallisepticum strain is a CR strain or an R strain.

[0015] The vaccine also contains pharmaceutically acceptable excipients or adjuvants, such as Tween-80 and Sperm-80. A special emulsification technique is employed during its preparation to form a stable oil-in-water emulsion. The oil phase consists of veterinary white oil, aluminum stearate, and Sperm-80, while the aqueous phase is a mixture of inactivated vaccine solution and Tween-80. This inactivated vaccine offers minimal stress, high safety, good absorption, and excellent preventive efficacy, making it particularly suitable for immunization of poultry.

[0016] Muramyl dipeptide (MDP) is added to the inactivated vaccine solution as an immune enhancer with a final concentration of 10 μg / mL to activate macrophages, enhance the body's immune response to vaccine antigens, and improve protective efficacy.

[0017] Trehalose is added to the aqueous phase as a stabilizer with a final concentration of 5% (w / v) to prevent the vaccine from inactivating during storage; at the same time, thimerosal is added as a preservative with a final concentration of 0.01% (w / v) to ensure the long-term stability and safety of the vaccine.

[0018] Combined with nanoliposome technology, part of the inactivated vaccine is encapsulated in liposomes to form a targeted delivery system, which enables the vaccine to be more effectively delivered to target immune cells, thereby enhancing the immune effect and reducing the required dose. Liposomes can be prepared by thin film dispersion or reverse evaporation, with the average particle size controlled at 100-200nm. Cold-adaptive proteins (ColdShock Proteins, CSPs) are added to a final concentration of 0.5mg / mL. These proteins can maintain the integrity of the protein structure at low temperatures, helping to maintain the activity of inactivated vaccines during cold chain transportation.

[0019] According to an embodiment of the present invention, the culture medium comprises:

[0020] PPLO broth 23.0-26.0 g;

[0021] Glucose 8.0-10.0g;

[0022] 10 mL of 1% thallium acetate solution;

[0023] 25% yeast extract 100 mL;

[0024] 1% phenol red solution 1.0-2.0 mL;

[0025] Add deionized water to a volume of 790-840 mL, sterilize at 115°C for 20 minutes, and after cooling, aseptically add:

[0026] 10% arginine solution (filter sterilized) 10 mL;

[0027] 150-200 mL of porcine serum;

[0028] Penicillin 800,000 to 1.2 million units;

[0029] Adjust the pH to 7.6-7.8 with sodium hydroxide solution.

[0030] According to an embodiment of the present invention, centrifuging and resuspending the bacterial solution includes:

[0031] The seeds for Mycoplasma gallisepticum production were inoculated into the culture medium at 10% of the volume, cultured at 36-37°C for 16-18 hours, and harvested;

[0032] Select 10% of the harvested bacterial liquid volume and subculture it once more using the same volume of culture medium and the same method;

[0033] The culture medium was inoculated with 10% of the subcultured bacterial solution, cultured at 36-37° C. for 24-48 hours, and harvested when the pH of the culture medium dropped to about 7.0.

[0034] According to an embodiment of the present invention, centrifuging and resuspending the bacterial solution includes:

[0035] The cultured bacterial suspension was centrifuged at 8000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS.

[0036] According to an embodiment of the present invention, the inactivation comprises:

[0037] Add formaldehyde solution to the resuspended bacterial solution to make the final concentration reach 0.02% to 0.04%, and let it stand at 37°C for 24 to 36 hours to completely inactivate the bacteria;

[0038] After the inactivation is completed, the pH value is adjusted to 7.4-7.6 with sodium hydroxide solution to remove the residual inactivator to obtain an inactivated bacterial solution.

[0039] According to an embodiment of the present invention, the concentration of the muramyl dipeptide in the aqueous phase is 10 μg / mL.

[0040] According to an embodiment of the present invention, the concentration of trehalose in the aqueous phase is 5% (w / v);

[0041] The concentration of the thimerosal in the aqueous phase was 0.01% (w / v).

[0042] According to an embodiment of the present invention, the concentration of Tween-80 in the aqueous phase is 5% (w / v).

[0043] According to an embodiment of the present invention, the step of emulsifying the oil phase and the water phase in a mass ratio of 3:2 to obtain an inactivated vaccine comprises:

[0044] Add 3 parts of the oil phase into a sterile container, start the homogenizer at 13000 r / min and stir for 90 seconds, repeat 3-5 times, then slowly add 2 parts of the water phase, and then stir at 15000 r / min for 90 seconds, repeat 3-5 times, until a milky white uniform phase is obtained, and the inactivated vaccine is obtained.

[0045] According to a second aspect of the present invention, an inactivated Mycoplasma gallisepticum vaccine prepared by the above-mentioned preparation method is provided.

[0046] The preparation method of the present invention not only improves the safety and effectiveness of the vaccine, but also gives it higher innovative value, enabling it to demonstrate better performance in practical applications. By integrating existing high-efficiency inactivation and emulsification technologies, it ensures a significant improvement in the safety and effectiveness of the vaccine.

[0047] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. DETAILED DESCRIPTION

[0048] The present invention provides an inactivated Mycoplasma gallisepticum vaccine and a preparation method thereof.

[0049] Example 1: CR strain inactivated vaccine prepared by optimized scheme

[0050] method:

[0051] The CR strain (Mycoplasma gallisepticum CR strain) is propagated using a modified culture medium and inactivated according to the steps of claim 1.

[0052] Muramyl dipeptide (MDP) was added to the inactivated vaccine solution as an immunopotentiator with a final concentration of 10 μg / mL.

[0053] Trehalose was added as a stabilizer (5% w / v) and thimerosal as a preservative (0.01% w / v).

[0054] The water-in-oil emulsion is prepared by a multi-stage emulsification process, and the average particle size of the liposome is controlled at 100-200nm.

[0055] Cold-adaptive proteins (CSPs) were added to the culture medium at a final concentration of 0.5 mg / mL.

[0056] Example 2: R strain inactivated vaccine prepared by optimized scheme

[0057] method:

[0058] The R strain (Mycoplasma gallisepticum R strain) is propagated using a modified culture medium and inactivated according to the steps of claim 1.

[0059] Other conditions are the same as in Example 1.

[0060] Example 3: CR strain inactivated vaccine introduced into targeted delivery system

[0061] method:

[0062] Same as Example 1, but additionally using nanoliposome technology to encapsulate part of the inactivated vaccine in liposomes to form a targeted delivery system.

[0063] Example 4: Inactivated CR strain vaccine with introduction of cold-adaptive protein

[0064] method:

[0065] Same as Example 1, except that cold-adaptive proteins (CSPs) were additionally added to the culture medium at a final concentration of 0.5 mg / mL.

[0066] Comparative Example

[0067] Comparative Example 1: CR strain inactivated vaccine prepared by traditional method

[0068] method:

[0069] The CR strain was propagated using traditional culture medium and inactivated according to traditional methods without adding any improved ingredients or processes.

[0070] Comparative Example 2: CR strain inactivated vaccine without added immunopotentiator

[0071] method:

[0072] Same as Example 1, but without adding muramyl dipeptide (MDP) as an immunopotentiator.

[0073] Comparative Example 3: CR strain inactivated vaccine without multi-stage emulsification process

[0074] method:

[0075] The same as Example 1, but instead of adopting the multi-stage emulsification process, only single-stage emulsification is used.

[0076] Comparative Example 4: CR strain inactivated vaccine without adding cold-adaptive protein

[0077] method:

[0078] Same as Example 1, but without adding cold-adaptive proteins (CSPs) to the culture medium.

[0079] Experimental example

[0080] The vaccines of Examples 1-4 and Comparative Examples 1-4 were tested. In order to more comprehensively evaluate the effect of the veterinary drug composition for preventing and treating coccidiosis of the present invention, we added more experimental data and conducted the following tests:

[0081] (1) Detection of peak antibody levels:

[0082] On the 7th, 14th, 21st, 28th, 35th, 42nd, 49th and 56th days after immunization, 20 geese were randomly selected for blood sampling and serum separation.

[0083] Antibody levels were measured using plate agglutination and hemagglutination inhibition tests. 25 μL of antigen and 25 μL of serum were added to a white porcelain reaction plate, shaken for two minutes, and observed for agglutination. Antibody titers were determined using the hemagglutination inhibition test according to standard operating procedures.

[0084] The antibody levels after each blood draw were recorded, and the peak antibody levels and their occurrence times were determined. The results are shown in Table 1.

[0085] Table 1. Vaccines tested in Examples 1-4 and Comparative Examples 1-4

[0086]

[0087] (2) Virus protection rate test:

[0088] Healthy, 7-day-old goslings of similar weight and negative for MG antibodies were selected as experimental animals. They were artificially infected via tracheal challenge with a logarithmically growing strain of MG containing at least 5 × 10^8 CFU / mL, 0.1 mL per dose, twice daily for 5 consecutive days.

[0089] The clinical symptoms of the geese after infection were recorded, such as open mouth breathing, respiratory rales, sneezing, mucous nasal discharge in the nasal cavity, conjunctival flushing, increased tearing, and swollen eyelids.

[0090] Fourteen days after infection, all geese were dissected to check for the presence of excessive viscous fluid or catarrhal secretions in the nostrils, air sacs, trachea and bronchi, for edema of the tracheal wall, for thickening of the air sac membrane and its grayish-white opaque appearance, for the presence of cheesy exudates on the air sac wall, and for congestive pulmonary lesions.

[0091] Take a throat swab from the experimental animal and put it in 0.5 mL of normal saline. After shaking thoroughly, take out the throat swab and extract DNA, which is used as a template for PCR identification.

[0092] To assess the serum plate agglutination test, 25 μL of antigen and 25 μL of serum are added to a white porcelain plate, shaken for two minutes, and observed for the appearance of granular or clumping agglutination. Agglutination usually begins to appear and concentrates at the periphery of the mixture. A negative reaction is determined by the absence of agglutination.

[0093] The geese were evaluated comprehensively based on clinical symptoms, autopsy, PCR testing, and antibody levels (plate agglutination). The incidence rate and protection rate were calculated. Protection rate = (incidence rate of the control group - incidence rate of the experimental group) / incidence rate of the control group × 100%. The results are shown in Table 2.

[0094] Table 2. Challenge test results of vaccines of Examples 1-4 and Comparative Examples 1-4

[0095] Experimental groups Protection rate against virus (%) Peak antibody levels Example 1 85 27 Example 2 83 26 Example 3 90 30 Example 4 87 28 Comparative Example 1 30 18 Comparative Example 2 70 22 Comparative Example 3 75 24 Comparative Example 4 82 26

[0096] (3) Weight gain detection

[0097] Before vaccination, the initial body weight of each goose was weighed and recorded.

[0098] The geese were weighed every 7 days until the end of the experiment.

[0099] The average weight gain of geese in each group during the entire experimental period was calculated, and the t-test was used to compare the significance of differences (P values) between different groups.

[0100] (4) Absorption test at the injection site

[0101] After vaccination, observe the injection site daily for adverse reactions such as redness, swelling, tissue liquefaction, and granulation tissue hyperplasia.

[0102] After the experiment, the injection site was dissected to check for abnormal pathological changes, such as inflammation, abscesses, etc.

[0103] Vaccine absorption was assessed based on the appearance and anatomy of the injection site.

[0104] If there are no adverse reactions such as redness, swelling, tissue liquefaction, and granulation tissue hyperplasia at the injection site, and the injection site is well absorbed, the vaccine is considered to have good absorption properties. The results are shown in Table 3.

[0105] Table 3. Weight gain and injection site absorption of vaccines in Examples 1-4 and Comparative Examples 1-4

[0106] Experimental groups Weight gain (P value) Injection site absorption Example 1 P>0.05 Good, no abnormal reaction Example 2 P>0.05 Good, no abnormal reaction Example 3 P>0.05 Good, no abnormal reaction Example 4 P>0.05 Good, no abnormal reaction Comparative Example 1 P>0.05 Good, no abnormal reaction Comparative Example 2 P>0.05 Good, no abnormal reaction Comparative Example 3 P>0.05 Good, no abnormal reaction Comparative Example 4 P>0.05 Good, no abnormal reaction

[0107] The challenge protection rates of Examples 1 to 4 were significantly higher than those of the vaccine prepared by the conventional method in Comparative Example 1, reaching 85%, 83%, 90%, and 87%, respectively, while Comparative Example 1 was only 30%. This indicates that the improvements of the present invention significantly enhance the protective effect of the vaccine.

[0108] Peak antibody levels:

[0109] The peak antibody levels of Examples 1 to 4 were 27, 26, 30, and 28, respectively, which were significantly higher than those of Comparative Example 2 (22), Comparative Example 3 (24), and Comparative Example 4 (26). In particular, Example 3, which introduced the targeted delivery system, had the highest peak antibody level, reaching 30.

[0110] Weight gain:

[0111] There was no significant difference in weight gain between the geese in all examples and comparative examples and the control group (P>0.05), indicating that the vaccines had no negative impact on the growth and development of geese.

[0112] Absorption at the injection site:

[0113] The injection site absorption of all examples and comparative examples was good without abnormal reactions, indicating that the vaccine was safe and had excellent absorption performance.

[0114] In summary, the inactivated Mycoplasma gallisepticum vaccine and its preparation method provided by the present invention not only improve the safety and effectiveness of the vaccine, but also greatly enhance the protective efficacy of the vaccine, and have important application value and broad market prospects.

[0115] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0116] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing an inactivated Mycoplasma gallisepticum vaccine, characterized in that: The following steps are involved: Cultivating and harvesting Mycoplasma gallisepticum in a culture medium to obtain a bacterial liquid; The bacterial solution is centrifuged and resuspended, and then inactivated to obtain an inactivated bacterial solution; adding muramyl dipeptide, trehalose, thimerosal and Tween-80 to the inactivated seedling solution to obtain an aqueous phase; Take 94 parts of white oil for injection and 1 part of aluminum stearate, mix them evenly under heating conditions at 80°C in a water bath, then slowly add 5 parts of Siben-80, sterilize with high-pressure steam at 115°C for 40 minutes, cool and set aside to obtain an oil phase; emulsify the oil phase and the aqueous phase in a mass ratio of 3:2 to obtain an inactivated vaccine; Liposomes were prepared using thin film dispersion or reverse evaporation methods, with an average particle size of 100-200 nm. Encapsulate 10%-20% of the inactivated vaccine solution in liposomes to form an inactivated vaccine with a targeted delivery system; A cold-adapted protein is added to the inactivated vaccine with a targeted delivery system at a concentration of 0.5 mg / mL to obtain the Mycoplasma gallisepticum inactivated vaccine.

2. The preparation method according to claim 1, wherein: The culture medium comprises: PPLO broth 23.0-26.0 g; Glucose 8.0-10.0g; 10 mL of 1% thallium acetate solution; 25% yeast extract 100 mL; 1% phenol red solution 1.0-2.0 mL; Use deionized water to make up to 790-840 mL, sterilize at 115°C for 20 minutes, and after cooling, add aseptically: 10% arginine solution 10 mL; 150-200 mL of porcine serum; Penicillin 800,000 to 1.2 million units; Adjust the pH to 7.6-7.8 with sodium hydroxide solution.

3. The preparation method according to claim 1, wherein: The centrifuging and resuspending the bacterial solution comprises: The seeds for Mycoplasma gallisepticum production were inoculated into the culture medium at 10% of the volume, cultured at 36-37°C for 16-18 hours, and harvested; 10% of the harvested bacterial suspension was selected and subcultured once more using the same volume of culture medium and the same method. 10% of the subcultured bacterial suspension was inoculated into the culture medium and cultured at 36-37°C for 24-48 hours. The cells were harvested when the pH of the culture medium dropped to approximately 7.

0.

4. The preparation method according to claim 1, wherein: The centrifuging and resuspending the bacterial solution comprises: The cultured bacterial suspension was centrifuged at 8000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS.

5. The preparation method according to claim 1, wherein: The inactivation comprises: Add formaldehyde solution to the resuspended bacterial solution to make the final concentration reach 0.02% to 0.04%, and let it stand at 37°C for 24 to 36 hours to completely inactivate the bacteria; After the inactivation is completed, the pH value is adjusted to 7.4-7.6 with sodium hydroxide solution to remove the residual inactivator to obtain an inactivated bacterial solution.

6. The preparation method according to claim 1, wherein: The concentration of the muramyl dipeptide in the aqueous phase was 10 μg / mL.

7. The preparation method according to claim 1, wherein: The concentration of trehalose in the aqueous phase is 5% w / v; The concentration of the thimerosal in the aqueous phase was 0.01% w / v.

8. The preparation method according to claim 1, wherein: The concentration of Tween-80 in the aqueous phase was 5% w / v.

9. The preparation method according to claim 1, wherein: The method of emulsifying the oil phase and the water phase in a mass ratio of 3:2 to obtain an inactivated vaccine comprises: Add 3 parts of the oil phase into a sterile container, start the homogenizer at 13000 r / min and stir for 90 seconds, repeat 3-5 times, then slowly add 2 parts of the water phase, and then stir at 15000 r / min for 90 seconds, repeat 3-5 times, until a milky white uniform phase is obtained, and the inactivated vaccine is obtained.

10. An inactivated Mycoplasma gallisepticum vaccine prepared by the preparation method according to any one of claims 1 to 9.

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