Preparation method of chicken mycoplasmosis inactivated vaccine

Abstract

The invention relates to the technical field of animal vaccine preparation, and discloses a chicken mycoplasma disease inactivated vaccine preparation method, which comprises: culturing a mycoplasma strain: inoculating a chicken mycoplasma strain to an SP4 culture medium, culturing at a culture temperature of 35-39 DEG C for 48-72 h, and collecting mycoplasma thalli; inactivation and antigen protection: adding an inactivator and an antigen protective agent into the mycoplasma suspension to carry out inactivation and antigen protection reaction at the reaction temperature of 25-37 DEG C for 3-6 hours; and preparation of a nano-delivery system: preparing the nano-particle delivery system through a composite structure of chitosan and sodium alginate. According to the technical scheme of cooperation of dynamic inactivation and antigen protection, an antigen protection system of polylysine, glutamine and arginine is synchronously introduced in the inactivation process, so that damage of an inactivating agent to mycoplasma antigen epitopes is remarkably avoided.

Description

Technical Field

[0001] The invention relates to the technical field of animal vaccine preparation, in particular to a method for preparing an inactivated vaccine for chicken mycoplasma disease. Background Art

[0002] Mycoplasmosis is an important respiratory disease of poultry caused by mycoplasma infection, mainly Mycoplasma gallisepticum and Mycoplasma synoviae. The disease is highly contagious and often causes problems such as slow growth, decreased egg production, and reduced feed conversion rate in poultry, which seriously affects the economic benefits of the poultry industry. In order to prevent and control the disease, inactivated vaccines are widely used. The core of the vaccine is to retain immunogenicity by inactivating pathogens so that it can effectively induce specific immune responses in the body after vaccination. At present, most of the inactivated Mycoplasma vaccines on the market are treated with chemical inactivators (such as formaldehyde or β-propiolactone) and combined with traditional mineral oil adjuvants. However, these vaccines have shown problems such as insufficient immune efficacy, poor persistence, and low antigen delivery efficiency in actual applications.

[0003] In the prior art, traditional chicken mycoplasma disease inactivated vaccines often rely on high concentrations of chemical inactivators, such as formaldehyde or β-propiolactone, during the inactivation process. These inactivators achieve inactivation effects by cross-linking pathogen proteins or destroying nucleic acid structures. However, while high concentrations of inactivators completely inactivate pathogens, they often inevitably destroy the three-dimensional structure of mycoplasma surface proteins, resulting in damage to antigenic epitopes, and ultimately leading to a significant reduction in the immunogenicity of the vaccine. On the other hand, if the inactivator concentration is reduced, it is difficult to completely inactivate the pathogen, and there may be a risk of residual live pathogens, which poses a hidden danger to the safety of the vaccine. Therefore, it is difficult to achieve an effective balance between the inactivator concentration and the integrity of the antigen in the prior art.

[0004] Traditional inactivated vaccines lack efficient antigen delivery technology and mostly use mineral oil adjuvants or aluminum hydroxide gel as adjuvants. These adjuvants can only prolong the immune effect by slowly releasing antigens, but cannot accurately deliver antigens to key cells of the immune system, such as dendritic cells or macrophages, resulting in low antigen utilization. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for preparing an inactivated vaccine for chicken mycoplasma disease, which solves the problems of incomplete inactivation or impaired antigen immunogenicity and unsustainable immune effect in the existing vaccines.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for preparing an inactivated vaccine for chicken mycoplasma disease, comprising the following steps: Cultivation of mycoplasma strains: inoculate the Mycoplasma galli strains into SP4 medium at a temperature of 35 to 39°C for 48 to 72 hours, and collect the mycoplasma cells; Inactivation and antigen protection: Add inactivator and antigen protection agent to the mycoplasma suspension for inactivation and antigen protection reaction, the reaction temperature is 25-37°C, and the reaction time is 3-6 hours; Preparation of nano-delivery system: Preparation of nanoparticle delivery system through the composite structure of chitosan and sodium alginate; Antigen encapsulation and adjuvant loading: encapsulating inactivated mycoplasma antigens into nano-delivery particles and adding immune-enhancing adjuvants; Homogenous mixing of vaccine preparation: The antigen-adjuvant nanoparticles are mixed with a stabilizer and a diluent and the vaccine is prepared after homogenous mixing.

[0007] Furthermore, Mycoplasma galli is a microorganism that lacks a cell wall. The culture conditions must provide a nutritional environment rich in amino acids and serum to maintain the proliferation of the mycoplasma. SP4 medium is a commonly used selective culture medium for mycoplasma. The culture temperature and time ensure that the bacteria reach the maximum growth state and avoid the decrease in metabolic antigenicity caused by over-culture.

[0008] β-propiolactone is a commonly used inactivator that ensures that microorganisms lose their infectivity by alkylating mycoplasma nucleic acids, but its inactivation effect may destroy the epitope structure of mycoplasma surface protein antigens. Adding polylysine, glutamine and arginine as protective agents, based on the chemical cross-linking of amino acids and the combination with antigens, can stabilize the three-dimensional structure of mycoplasma surface antigens, prevent the destruction of inactivators, and maintain the immunogenicity of antigens.

[0009] Antigens encapsulated in nanoparticles can avoid rapid degradation in the body and improve the delivery efficiency of antigens in the immune system. Immunoenhancing adjuvants CpG oligonucleotides and Poly (I:C) act as TLR9 and TLR3 agonists, respectively, which can effectively activate dendritic cells and macrophages, promote antigen presentation, and further enhance the immunogenicity of the vaccine.

[0010] Preferably, the inactivator is β-propiolactone, and its mass fraction accounts for 0.02 to 0.1; the antigen protective agent includes the following components, calculated by mass fraction: Polylysine: 0.1-0.3; Glutamine: 0.1-0.5; Arginine: 0.05~0.2.

[0011] Furthermore, β-propiolactone inactivates mycoplasma through epoxidation reaction, but its high efficiency may lead to antigen degradation. Polylysine, as a cross-linking agent, can form a protein protection network to reduce the risk of degradation during the inactivation process. Glutamine and arginine further stabilize the tertiary structure of the antigen through hydrogen bonds or electrostatic effects.

[0012] Preferably, the nano delivery system comprises the following components, in parts by mass: Chitosan: 0.1-0.3; Sodium alginate: 0.05~0.2; Calcium phosphate: 0.2~1.0.

[0013] Furthermore, ionic crosslinking between chitosan and sodium alginate forms nanoparticles, and chitosan provides cationic protection while improving mucosal adhesion; sodium alginate provides biocompatibility as an outer layer structure, and calcium phosphate can stabilize the nanoparticle structure while also assisting in antigen delivery.

[0014] Preferably, the culture temperature of the mycoplasma strain is 37° C. and the culture time is 48 hours.

[0015] Furthermore, 37°C is the optimal growth temperature for mycoplasma, and 48 hours can ensure that the maximum bacterial concentration is reached without producing excessive metabolic waste, thus ensuring the antigenic integrity of the bacteria.

[0016] Preferably, in the inactivation and antigen protection steps, the order of adding the inactivator and the antigen protection agent is to first add β-propiolactone and then add polylysine, glutamine and arginine, and gradually adjust the pH to 7.0-7.5.

[0017] Furthermore, the inactivator first acts on the mycoplasma to destroy the nucleic acid, and then the protective agent is added to form a protective shell, which can reduce the damage of the inactivator to the protein antigen; adjusting the pH to 7.0-7.5 can further stabilize the protein structure.

[0018] Preferably, during the preparation of the nano-delivery particles, chitosan is dispersed by ultrasound with an ultrasound power of 50 to 100 W for 20 to 60 seconds.

[0019] Furthermore, ultrasonic treatment disperses chitosan evenly through sonication to avoid the formation of large particle agglomerates, while improving the binding efficiency of chitosan and antigens.

[0020] Preferably, in the antigen encapsulation and adjuvant loading steps, the immune enhancing adjuvant comprises the following components, in parts by mass: CpG oligonucleotide: 0.0001-0.001; Poly:0.0002~0.003.

[0021] Furthermore, CpG oligonucleotides, as TLR9 agonists, can activate B cells and dendritic cells; Poly (I:C), as TLR3 agonists, can enhance antiviral cellular immune responses. The combination of the two can significantly increase the intensity of the immune response.

[0022] Preferably, in the homogenous mixing step of the vaccine preparation, the mixing time is 10 to 20 minutes and the mixing temperature is 4 to 10°C.

[0023] Furthermore, controlling the homogenization time and temperature can avoid antigen and adjuvant denaturation while ensuring the uniformity of the preparation.

[0024] Preferably, the stabilizer comprises the following components, in parts by mass: Glycerol: 0.05~0.2; Gelatin: 0.3~0.7.

[0025] Furthermore, glycerol, as a low molecular weight stabilizer, can prevent the inactivation of antigens during the vaccine freezing process; gelatin can further enhance the stability of the vaccine by combining with antigens to form a protective barrier.

[0026] Preferably, the pH range of the PBS buffer is 7.2 to 7.4, and is used for dilution of vaccine preparations.

[0027] Furthermore, glycerol, as a low molecular weight stabilizer, can prevent the inactivation of antigens during the vaccine freezing process; gelatin can further enhance the stability of the vaccine by combining with antigens to form a protective barrier.

[0028] The present invention provides a method for preparing an inactivated vaccine for chicken mycoplasma disease, which has the following beneficial effects: 1. The present invention adopts a dynamic inactivation and antigen protection synergistic technical solution, and introduces an antigen protection system of polylysine, glutamine and arginine simultaneously during the inactivation process, which significantly avoids the destruction of mycoplasma antigen epitopes by the inactivator. Compared with the existing practice of simply relying on high-concentration inactivators, it solves the problem of reduced vaccine immunogenicity due to damaged antigen structure, and at the same time improves the immune effect of the vaccine.

[0029] 2. The present invention achieves precise encapsulation and stable release of inactivated mycoplasma antigens by designing a chitosan-sodium alginate nanodelivery system. The nanodelivery system has double-layer protection and pH responsiveness, which can significantly improve the delivery efficiency of antigens in the body. Compared with traditional emulsified adjuvants, it solves the problems of low antigen delivery efficiency and insufficient persistence of immune response, and at the same time improves the immune persistence and specificity of the vaccine.

[0030] 3. The present invention introduces CpG oligonucleotides and Poly (I:C) as TLR agonists in immune adjuvants to construct an efficient immune enhancement system. This design can significantly activate the body's cellular and humoral immune responses. Compared with the prior art using a single adjuvant, the present invention effectively solves the problem of insufficient vaccine immune induction, while ensuring the diversity of adjuvant effects and the comprehensiveness of immune responses.

[0031] 4. The present invention introduces CpG oligonucleotides and Poly (I:C) as TLR agonists in the immune adjuvant to construct a highly efficient immune enhancement system. This design can significantly activate the body's cellular immunity and humoral immune response. Compared with the prior art scheme of using a single adjuvant, the present invention effectively solves the problem of insufficient vaccine immune induction, while ensuring the diversity of adjuvant effects and the comprehensiveness of immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The figure is a flow chart of the method of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Please see attached Figure 1 The present invention provides a method for preparing an inactivated vaccine for mycoplasma disease in chickens, comprising the following steps: Cultivation of mycoplasma strains: inoculate the Mycoplasma galli strains into SP4 medium at a temperature of 35 to 39°C for 48 to 72 hours, and collect the mycoplasma cells; Inactivation and antigen protection: Add inactivator and antigen protection agent to the mycoplasma suspension for inactivation and antigen protection reaction, the reaction temperature is 25-37°C, and the reaction time is 3-6 hours; Preparation of nano-delivery system: Preparation of nanoparticle delivery system through the composite structure of chitosan and sodium alginate; Antigen encapsulation and adjuvant loading: encapsulating inactivated mycoplasma antigens into nano-delivery particles and adding immune-enhancing adjuvants; Homogenous mixing of vaccine preparation: The antigen-adjuvant nanoparticles are mixed with a stabilizer and a diluent and the vaccine is prepared after homogenous mixing.

[0035] Example 1: Basic preparation method of mycoplasma inactivated vaccine Strain culture The Mycoplasma gallisepticum strain was inoculated into SP4 medium, the culture temperature was controlled at 37°C, the culture time was set to 48 hours, and the cells proliferated to an OD600 value of 1.0, the culture was terminated, the cells were collected by centrifugation (5000×g, 15 minutes), washed 3 times with PBS, and the mycoplasma suspension was prepared at a concentration of 10 9 CFU / mL.

[0036] Inactivation and Antigen Protection β-Propiolactone was added to the mycoplasma suspension at a final concentration of 0.05% (v / v). The reaction temperature was set to 25°C and inactivated for 3 hours under stirring conditions (400 rpm). Subsequently, antigen protectants were added: polylysine 0.2%, glutamine 0.3% and arginine 0.1%. The pH of the system was adjusted to 7.3 and the reaction was continued for 30 minutes to complete antigen protection. No mycoplasma growth was detected by post-inactivation culture, confirming that the inactivation was complete.

[0037] Preparation of nanodelivery particles Inactivated mycoplasma antigen was added to the chitosan solution (0.2% w / v, dissolved in 1% acetic acid solution) at a concentration of 2 mg / mL. Ultrasonic dispersion (power 70 W, time 30 seconds) was used to evenly distribute the antigen. 1% (w / v) CaCl2 solution was added to form primary chitosan particles. Subsequently, sodium alginate solution (0.1% w / v) was added dropwise and slowly stirred (200 rpm) for 20 minutes to complete the preparation of double-layer nanoparticles.

[0038] Vaccine preparation mixing Mix the nanoparticle solution with the stabilizer: 0.1% (w / v) glycerol, 0.5% (w / v) gelatin, and PBS to a volume of 100 mL. Homogenize for 15 minutes (10°C), then aseptically dispense into glass bottles and store at 2-8°C for later use.

[0039] Example 2: Preparation of optimized long-acting immune vaccine Strain culture and inactivation The steps of culturing the Mycoplasma galli strain were the same as those in Example 1, and the final bacterial concentration was 10 9 CFU / mL. Add 0.08% (v / v) β-propiolactone to the mycoplasma suspension, inactivate at 30°C for 4 hours. In order to avoid high temperature damage to the antigen, magnetic stirring was used throughout the process, and the speed was controlled at 300rpm. After inactivation, 0.3% polylysine, 0.5% glutamine, and 0.2% arginine were added in sequence. Adjust the pH to 7.2 and protect for 45 minutes.

[0040] Nanoparticle preparation The inactivated antigen was added to the chitosan solution (0.25% w / v) and dispersed by ultrasound (power 100 W, time 40 seconds) to form a uniform antigen distribution solution. Then, sodium alginate solution (0.2% w / v) was added dropwise and cross-linked with CaCl2 (1% w / v) to generate chitosan-sodium alginate composite nanoparticles. The reaction time was 30 minutes and the particle size was controlled at 200 nm (detected by dynamic light scattering).

[0041] Adjuvant loading and mixing with vaccine formulation Add 0.001% (w / v) CpG oligonucleotide and 0.003% (w / v) Poly (I:C) to the nanoparticles and mix well by stirring (300 rpm). Then add 0.1% glycerol and 0.7% gelatin and adjust to 100 mL with PBS. The homogenization time is 15 minutes (8°C). Then divide and store in a refrigerator.

[0042] Example 3: Adjuvant combination vaccine for improving immune induction efficacy Strain culture and inactivation The Mycoplasma gallinarum strain was cultured at 37°C for 48 hours until the bacterial concentration reached 10 9 CFU / mL. β-propiolactone was used in the inactivation step at a concentration of 0.03% (v / v), the reaction temperature was controlled at 37°C, the reaction time was 6 hours, and after the inactivation, 0.15% polylysine, 0.3% glutamine and 0.1% arginine were added in sequence, the pH of the system was adjusted to 7.0, and the protection time was 30 minutes.

[0043] Double-layer optimization of nanodelivery particles Mycoplasma antigen was added to 0.2% (w / v) chitosan solution and ultrasonic dispersion (power 80 W, time 30 seconds) was used to form chitosan particles. Subsequently, 0.1% (w / v) sodium alginate solution was added to cross-link to form double-layer particles. Calcium phosphate (0.5% w / v) was introduced to enhance stability. The static reaction time was set to 25 minutes.

[0044] Immunoadjuvant loading CpG oligonucleotide 0.0008% (w / v) and Poly (I:C) 0.0015% (w / v) were added to the surface of the nanoparticles, both of which served as TLR9 and TLR3 agonists, respectively, to ensure the dual activation capabilities of cellular immunity and humoral immunity.

[0045] Vaccine preparation mixing and packaging The nanoparticles were mixed evenly with glycerol (0.05%), gelatin (0.6%), and PBS at 4°C for 20 min, and finally packaged and refrigerated.

[0046] Example 4: Preparation of antigen-protective vaccine for stable storage Strain inactivation and antigen protection The culture and inactivation steps were the same as those in Example 1. After inactivation, antigen protective agents were added at concentrations of: 0.2% polylysine, 0.4% glutamine, and 0.15% arginine. The reaction temperature was 30° C. and the reaction time was 30 minutes.

[0047] Nanoparticles enhance stability The preparation of chitosan-alginate nanoparticles was consistent with that in Example 2, but 0.5% (w / v) calcium phosphate was additionally introduced into the outer layer of the particles to enhance storage stability. The nanoparticles were formed by standing for 35 minutes.

[0048] Stabilizer Optimization and Mixing In the vaccine mixing step, glycerol was added in an amount of 0.2%, gelatin was added in an amount of 0.7%, the mixing time was controlled to 10 minutes, and the mixing temperature was maintained at 5°C. The preparation was refrigerated and stored after packaging, and the test showed that the antigen stability was maintained after 3 months of storage.

[0049] Example 5: Preparation of vaccines for long-term immune efficacy Strain culture and inactivation The mycoplasma strain culture step was the same as that in Example 1. In the inactivation step, the concentration of β-propiolactone was 0.04% (v / v), the temperature was set to 28° C., and the time was 5 hours. After inactivation, antigen protective agents were added: polylysine 0.25%, glutamine 0.35%, and arginine 0.1%, the pH was adjusted to 7.4, and the protection reaction was carried out for 40 minutes.

[0050] Enhanced Nanoparticles The preparation of double-layer nanoparticles of chitosan (0.25% w / v) and sodium alginate (0.15% w / v) was the same as in Example 3, and 0.001% (w / v) of CpG oligonucleotide and 0.002% (w / v) of Poly (I:C) were added to the nanoparticles to ensure a long-lasting immune enhancement effect.

[0051] Formulation Optimization The prepared nanoparticles were mixed with 0.1% glycerol and 0.6% gelatin, and the volume was adjusted to 100 mL with PBS. The mixing and homogenization time was 18 min (4°C), and the mixture was stored in aliquots.

[0052] Comparative Example 1: Unoptimized inactivation scheme based on Example 1 Strain culture The Mycoplasma gallisepticum strain was inoculated into SP4 medium at 37°C for 48 hours. 9 The operation was the same as in Example 1.

[0053] Inactivation and Antigen Protection β-Propiolactone was directly added to the mycoplasma suspension with a final concentration of 0.1% (v / v). The reaction temperature was 37°C and the reaction time was 6 hours. No antigen protection agents such as polylysine, glutamine and arginine were added during the inactivation process. The inactivation effect was directly detected after the inactivation was completed, and there was no antigen protection link.

[0054] Nanoparticle preparation and mixing with vaccine formulation Nanoparticles and vaccine preparations were prepared in the same manner as in Example 1, with all steps and parameters being the same. The preparations were finally packaged and stored.

[0055] Comparative Example 2: Based on Example 2 without the introduction of nano delivery system Strain culture and inactivation The steps and parameters for culturing and inactivating the strain were consistent with those in Example 2, including the addition of 0.08% β-propiolactone and antigen protective agents (polylysine, glutamine and arginine).

[0056] Direct mixing of vaccine formulations The inactivated antigen is not encapsulated in nanoparticles, but is directly mixed with immune adjuvants (CpG oligonucleotide 0.001% and Poly (I:C0) 003%) and stabilizers (glycerol 0.1%, gelatin 0.7%). The homogenization time of the mixing process is 15 minutes, the temperature is 10°C, and the antigen is directly packaged and refrigerated for storage. There is no nano delivery system design.

[0057] Comparative Example 3: Low-efficiency adjuvant design based on Example 3 Strain culture and inactivation The process of culturing strains and inactivation is consistent with that in Example 3, and the inactivation conditions and the parameters for using the antigen protective agent are the same.

[0058] Nanoparticle preparation The double-layer nanoparticles were prepared by composite design of chitosan (0.2%) and sodium alginate (0.1%), and the steps were completely consistent with those in Example 3.

[0059] Adjuvant use No CpG oligonucleotide and Poly (I:C) were used. Only aluminum hydroxide gel was added as an immune adjuvant with a concentration of 0.1% (w / v). The homogenization time after mixing was 15 minutes, the mixing temperature was 8°C, and the preparation was packaged and stored in a refrigerator.

[0060] Comparative Example 4: Unoptimized stability design based on Example 4 Strain culture and inactivation The steps of culturing the strain and inactivating the strain were consistent with those in Example 4, and the inactivation conditions and the amount of antigen protective agent added were the same.

[0061] Nanoparticle preparation The chitosan-sodium alginate-calcium phosphate composite nanoparticle design in Example 4 was adopted, and the steps were exactly the same, and the particle size of the prepared particles was controlled at 200 nm.

[0062] Stabilizer design No glycerol was added to the vaccine preparation, only gelatin was added at a concentration of 0.5%. The protection system was not optimized, and the vaccine was packaged and stored after mixing.

[0063] Comparative Example 5: Unoptimized inactivator concentration based on Example 5 Strain culture Mycoplasma strains were inoculated into SP4 medium at 37°C for 48 hours until the bacterial concentration reached 10 9 CFU / mL were collected.

[0064] Inactivation and Antigen Protection A lower concentration of β-propiolactone was used during the inactivation process, with a final concentration of 0.02% (v / v). The reaction temperature was maintained at 28°C and the time was set to 5 hours. Antigen protectants were added: polylysine 0.2%, glutamine 0.3%, and arginine 0.1%. The pH was adjusted to 7.4 and the protection reaction time was 40 minutes.

[0065] Nanodelivery system preparation and adjuvant loading The nano-delivery system of Example 5 was used, with chitosan concentration of 0.25%, sodium alginate concentration of 0.15%, and the steps of particle preparation and addition of immune adjuvants (CpG oligonucleotide, Poly (I:C)) were the same.

[0066] Vaccine preparations The final mixing step was made up to 100 mL with 0.1% glycerol, 0.6% gelatin, and PBS. The homogenization time was 18 minutes and the mixing temperature was 4°C.

[0067] Experiment 1: Comparison of antigen protection effects during inactivation Purpose The effect of introducing antigen protective agents (polylysine, glutamine, arginine) during the inactivation process in Example 1 on antigen epitope protection was verified.

[0068] Experimental methods Sample preparation The inactivation and antigen protection scheme of Example 1 was used as the test group.

[0069] Comparative Example 1 was used as a control group (no antigen protective agent was added, and only β-propiolactone was used for inactivation).

[0070] Three parallel samples were prepared for each group.

[0071] Experimental procedures The inactivated antigen proteins were extracted from the samples of the experimental group and the control group respectively.

[0072] The integrity of the antigen protein was checked by SDS-PAGE.

[0073] Enzyme-linked immunosorbent assay (ELISA) was used to determine the binding ability of antigen and monoclonal antibody, reflecting the integrity of antigen epitope.

[0074] Experimental setup Sample number: 3 parallel samples per group.

[0075] Detection indicators: antigen protein integrity (SDS-PAGE) and antigen binding ability (ELISAOD value).

[0076] Data analysis: Independent sample t-test was used to analyze the differences between the two groups.

[0077] Experiment 2: Comparison of antigen delivery effects of nano-delivery systems Purpose Evaluate the effect of nanodelivery system on improving antigen delivery efficiency.

[0078] Experimental methods Sample preparation The nanoparticle-encapsulated antigen in Example 2 was used as the test group.

[0079] Comparative Example 2 (no introduction of nanoparticle delivery, only direct mixing of antigen and adjuvant) was used as a control group.

[0080] Experimental procedures The vaccines of the experimental group and the control group were injected into SPF chickens (7 days old, 5 in each group).

[0081] Blood samples were collected on days 7, 14, and 21 after vaccination.

[0082] ELISA was used to detect the antibody levels (IgG concentration) in the sera of the two groups and compare the antigen delivery effects.

[0083] Experimental setup Animal model: 7-day-old SPF chickens, 5 in each group.

[0084] Vaccination dose: 0.5 mL of vaccine was injected subcutaneously into each chicken.

[0085] Detection indicators: changes in antibody concentration (IgG concentration) over time.

[0086] Data analysis: Draw the antibody titer change curves of the two groups and compare the immune effects of the two groups at each time point.

[0087] Experiment 3: Comparison of the effects of adjuvant design on immune activation Purpose The optimized adjuvant design in Example 3 (CpG oligonucleotide + Poly (I:C)) was verified to enhance the immune activation effect.

[0088] Experimental methods Sample preparation The experimental group adopted the scheme of nanoparticles combined with CpG oligonucleotide and Poly (I:C) in Example 3.

[0089] The control group adopted the scheme of Comparative Example 3 (using only aluminum hydroxide gel as an adjuvant).

[0090] Experimental procedures Each group of chickens (5 chickens in each group) were injected with the vaccine at 7 days old, with a vaccination dose of 0.5 mL / chicken.

[0091] Blood samples were collected on days 7, 14, and 21 after vaccination.

[0092] Detect the levels of IFN-γ (reflecting cellular immune response) and IgG (reflecting humoral immune response) in the blood.

[0093] Experimental setup Animal model: 7-day-old SPF chickens, 5 in each group.

[0094] Detection indicators: IFN-γ level and IgG concentration.

[0095] Data analysis: The immune response intensity of the two groups was compared at different time points.

[0096] Experiment 4: Vaccine stability test Purpose Test the effect of optimized stabilizers (glycerol + gelatin) on vaccine stability.

[0097] Experimental methods Sample preparation The vaccine to which glycerol and gelatin were added in Example 4 was used as the test group.

[0098] Comparative Example 4 (only gelatin was added without glycerol) was used as a control group.

[0099] The two groups of samples were packaged separately and stored at 4°C.

[0100] Experimental procedures The antigen stability was tested at 0, 1, and 3 months of storage.

[0101] SDS-PAGE was used to detect the extent of antigen protein degradation.

[0102] ELISA was used to detect the binding ability of antigen and monoclonal antibody.

[0103] Experimental setup Sample number: 3 parallel samples per group.

[0104] Storage condition: 4℃.

[0105] Detection indicators: antigen protein degradation degree (SDS-PAGE) and binding ability (ELISAOD value).

[0106] Data analysis: Compare the changes in antigen stability between the two groups over time.

[0107] Experiment 5: Effect of inactivator concentration on immune effect Purpose Verify the effect of inactivator concentration on antigen immunogenicity and inactivation effect.

[0108] Experimental methods Sample preparation The vaccine with the optimized inactivator concentration (0.04% β-propiolactone) in Example 5 was used as the test group.

[0109] The vaccine of Comparative Example 5 (low concentration inactivator, 0.02% β-propiolactone) was used as the control group.

[0110] Experimental procedures Each group of vaccines was inoculated into 7-day-old SPF chickens (5 chickens in each group) with a vaccination dose of 0.5 mL / chicken.

[0111] Blood samples were collected on the 7th and 21st days after vaccination to test IgG concentration and evaluate the intensity of the immune response.

[0112] The inactivation effect was verified by culturing and testing the number of viable bacteria.

[0113] Experimental setup Animal model: 7-day-old SPF chickens, 5 in each group.

[0114] Detection indicators: antibody titer (IgG concentration) and inactivation thoroughness.

[0115] Data analysis: Compare the immune response intensity and inactivation effect of the two groups of vaccines.

[0116] Experiment 1: Comparison of antigen protection effects during inactivation Experimental Description Purpose The effect of introducing antigen protective agents (polylysine, glutamine, arginine) during the inactivation process in Example 1 on antigen epitope protection was verified.

[0117] Experimental Materials Mycoplasma gallinarum (culture concentration is 10 9 CFU / mL) β-Propiolactone (inactivator) Antigen protectants: polylysine, glutamine, arginine SDS-PAGE electrophoresis reagents ELISA kit (monoclonal antibodies against mycoplasma antigens) PBS buffer Experimental equipment: constant temperature stirrer, gel electrophoresis instrument, microplate reader Experimental procedures Sample preparation Experimental group: The inactivation and antigen protection scheme in Example 1 was adopted, 0.05% β-propiolactone was added to 10 mL of mycoplasma suspension, the inactivation temperature was 25° C., the time was 3 hours, after inactivation, 0.2% polylysine, 0.3% glutamine and 0.1% arginine were added in sequence, the pH was adjusted to 7.3, and the protection reaction was carried out for 30 minutes.

[0118] Control group: the inactivation scheme in comparative example 1 was adopted, 0.1% β-propiolactone was added to 10 mL of mycoplasma suspension, the inactivation temperature was 37° C., the time was 6 hours, and no antigen protective agent was added.

[0119] SDS-PAGE detection Take 20 μL of the inactivated antigen samples from the test group and the control group, and mix them with the protein denaturation buffer.

[0120] Electrophoresis was performed on 10% SDS-PAGE gel to observe the integrity of the antigen protein band.

[0121] ELISA test The binding ability of the two groups of antigens and antibodies was detected using an ELISA kit with monoclonal antibodies as capture antibodies.

[0122] Add 100 μL of diluted inactivated antigen (concentration 10 μg / mL), incubate for 1 hour, and measure the 450 nm OD value with an enzyme reader.

[0123] Experimental data Table name: Antigen integrity and binding ability test results of the experimental group and the control group Summary The addition of antigen protectants during the inactivation process significantly improved the integrity of the antigen protein. These protectants stabilized the tertiary structure of the mycoplasma surface protein by forming hydrogen bonds and electrostatic interactions with the antigen molecules. In the SDS-PAGE test, the bands of the test group samples were clearer and more complete, indicating that the antigen was not excessively damaged during the inactivation process. In contrast, the bands of the control group were blurred, and the antigen was significantly degraded.

[0124] The role of antigen protectants is not limited to physical protection, but also has a chemical stabilization effect. Polylysine can bind to the protein surface when inactivated, preventing β-propiolactone from over-modifying the antigen epitope, while glutamine and arginine provide additional molecular stability. This stability is directly reflected in the ELISA test results. The OD value of the experimental group was significantly higher than that of the control group. The enhanced binding ability indicates that the antigen epitope is better preserved and can induce an immune response more effectively.

[0125] Antigen degradation is a major bottleneck in traditional inactivation technology. This experiment verified the theoretical basis of multi-component synergistic protection. By adjusting the concentration of the inactivator and introducing the protective agent, the problem of antigen epitope damage was solved while maintaining immunogenicity. Combined with experimental data, this innovative design not only reflects the scientific nature of the method, but also provides significant practical application value.

[0126] Experiment 2: Comparison of antigen delivery effects of nano-delivery systems Experimental Description Purpose The enhancement effect of the nanoparticle delivery system in Example 2 on antigen delivery efficiency and immune response was verified by comparing it with the traditional scheme of Comparative Example 2 (no nanoparticles, only direct mixing of antigen and adjuvant).

[0127] Experimental Materials SPF chickens (7 days old, 5 per group) Nanoparticle vaccine (prepared in Example 2) Non-nanoparticle vaccine (prepared in Comparative Example 2) ELISA kit (for detecting antibody titer) PBS solution Constant temperature box, centrifuge, microplate reader Experimental procedures Vaccination Experimental group: Each SPF chicken was subcutaneously injected with 0.5 mL of the nanoparticle vaccine prepared in Example 2.

[0128] Control group: each SPF chicken was subcutaneously injected with 0.5 mL of the non-nanoparticle vaccine prepared in Comparative Example 2.

[0129] Each group had 5 chickens, and the breeding environment was consistent. The condition of the chickens was observed daily after vaccination.

[0130] Blood sample collection Blood samples were collected from the chicken wing vein on the 7th, 14th and 21st days after inoculation, with each blood sample volume of approximately 0.5 mL.

[0131] The blood samples were centrifuged after standing, and the serum was separated and stored in a -20°C refrigerator for later use.

[0132] Antibody titer testing The diluted serum samples were added to the ELISA plate to detect the antigen-specific IgG levels.

[0133] Enzyme-labeled secondary antibody and colorimetric substrate were added, and after incubation, the OD value was read at a wavelength of 450 nm using a microplate reader.

[0134] The blood sample of each chicken was tested three times, and the mean value was taken as the antibody titer of the individual.

[0135] Experimental data Table name: Antibody titer test results of nano-delivered vaccines and non-nano-delivered vaccines Summary The effect of the nanoparticle delivery system was intuitively verified in the experiment. The antibody titer in the test group was significantly higher than that in the control group at each time point. From a mechanistic point of view, this is related to the design of the double-layer structure of chitosan and sodium alginate. The chitosan on the surface of the nanoparticles has cationic properties and can form a stable bond with the antigen to delay the degradation of the antigen. Sodium alginate, as the outer layer material, further provides protection to prevent the rapid clearance of the antigen when it is delivered in the body. This design enables the antigen to effectively reach the immune active site.

[0136] In the control group, the non-nanoparticle vaccine showed lower antibody levels. This is because when the nanodelivery system is not used, the antigen is directly exposed to the body fluids and is easily degraded or eliminated. The lack of protection from particle encapsulation causes the antigen to be unable to continuously stimulate the immune system. In addition, the combination of adjuvant and antigen is not as efficient as in the experimental group. The delivery efficiency of the traditional direct mixing method is insufficient, especially in maintaining long-term immunity.

[0137] The pH responsiveness of the nanodelivery system is a highlight of the present invention. In the acidic microenvironment of the target site, the particles can gradually release antigens, so that the immune cells are continuously stimulated. This mechanism is in sharp contrast to the instantaneous release of non-nanoparticles, and also explains why the antibody titer of the test group increased significantly on the 14th and 21st days. This experiment combines dynamic data and delivery mechanism to prove the advantages of the present invention in antigen delivery efficiency, especially its superiority in sustained immune stimulation.

[0138] Experiment 3: Comparison of the effects of adjuvant design on immune activation Experimental Description Purpose The effect of the optimized adjuvant combination (CpG oligonucleotide + Poly (I:C) in Example 3 on the immune activation effect was verified and compared with the solution of using only the traditional aluminum hydroxide gel adjuvant in Comparative Example 3.

[0139] Experimental Materials SPF chickens (7 days old, 5 per group) Nanoparticle vaccine (prepared in Example 3) Non-nanoparticle vaccine (prepared in Comparative Example 3, containing only aluminum hydroxide adjuvant) ELISA kit (for IgG antibody detection) IFN-γ detection kit (ELISA method) Constant temperature incubator, microplate reader, centrifuge Experimental procedures Vaccination Experimental group: Each SPF chicken was subcutaneously injected with 0.5 mL of the vaccine prepared in Example 3 (containing CpG oligonucleotide + Poly (I:C) adjuvant).

[0140] Control group: each SPF chicken was subcutaneously injected with 0.5 mL of the vaccine prepared in Comparative Example 3 (containing only aluminum hydroxide gel adjuvant).

[0141] Each group consisted of 5 chickens, which were raised under the same conditions and their health conditions were observed.

[0142] Collecting blood samples On the 7th, 14th and 21st days after inoculation, blood samples were collected from the chicken wing vein, with a collection volume of 0.5 mL per chicken.

[0143] The blood samples were centrifuged after standing, and the serum was separated and stored in a -20°C refrigerator.

[0144] Antibody and cytokine detection IgG antibody detection: ELISA method was used. Diluted serum samples were added and monoclonal antibodies against mycoplasma antigens were used to detect IgG levels. OD values ​​were read at a wavelength of 450 nm.

[0145] IFN-γ detection: ELISA kit was used to detect the IFN-γ level in serum, which reflects the cellular immune activity.

[0146] Experimental data Table name: Comparison of immune stimulation effects between optimized adjuvant combination and traditional adjuvant Summary The introduction of optimized adjuvants significantly stimulated higher levels of immune response. The IgG and IFN-γ concentrations in the experimental group were higher than those in the control group at each time point. This effect came from the synergistic effect of CpG oligonucleotides and Poly (I:C). CpG oligonucleotides strongly stimulated B cell proliferation and promoted antibody secretion by activating Toll-like receptor 9 (TLR9), while Poly (I:C) simulated viral RNA, activated the TLR3 pathway, induced dendritic cells and macrophages to release IFN-γ, and further enhanced the cellular immune response. In contrast, the aluminum hydroxide used in the control group as a single adjuvant only provided antigen sustained release function and could not effectively activate cellular immunity.

[0147] From the 7th day to the 21st day, the IFN-γ level in the experimental group continued to increase, showing a significant time dependence. This shows that the optimized adjuvant can not only quickly initiate the immune response, but also prolong the time window of the immune response through continuous stimulation of the delivery system. This long-term immune induction ability gives the vaccine a greater advantage in practical applications. In the control group, the IFN-γ concentration stabilized after 14 days, indicating that traditional adjuvants cannot provide continuous immune stimulation. This difference further reflects the scientificity and innovation of the adjuvant combination design in the present invention.

[0148] In addition, the IgG titer of the test group was close to saturation on the 21st day, while the titer of the control group was still far behind. This indicates that the optimized adjuvant can enhance antigen presentation and immune cell activity, enabling the body to produce higher levels of humoral immunity more quickly. This advantage can significantly improve the protection efficiency, especially in a highly pathogenic virus environment. Combining the results and mechanisms, the adjuvant design of the present invention clearly solves the problem of insufficient immune induction of traditional vaccines.

[0149] Experiment 4: Stability test of vaccine formulation Experimental Description Purpose The effect of the optimized stabilizer (glycerol + gelatin) on improving the stability of vaccine antigens at different storage times was tested to verify the superiority of the optimized stabilizer formula in Example 4 and to compare it with Comparative Example 4 (using only gelatin as a stabilizer).

[0150] Experimental Materials Example 4 Vaccine preparation (containing glycerol + gelatin stabilizer) Comparative Example 4 Vaccine Preparation (Containing Gelatin Stabilizer Only) SDS-PAGE Kit ELISA kit (detection of antigen binding ability) 4℃ storage environment, ELISA reader, gel electrophoresis instrument Experimental procedures Sample preparation Take 20 mL of each vaccine preparation of Example 4 and Comparative Example 4, and dispense them into sterile glass bottles, and mark them as the test group and the control group, respectively.

[0151] The samples were stored at 4°C and were taken after 0, 1 and 3 months of storage.

[0152] Antigen degradation test (SDS-PAGE) At each time point, 1 mL of sample was taken, protein denaturation buffer was added, boiled for 5 minutes, and the supernatant was collected after centrifugation.

[0153] Run the samples on a 12% SDS-PAGE gel to observe the integrity of the antigen protein bands and record the band intensity and cleavage conditions.

[0154] Antigen binding ability test (ELISA) Samples were taken at each storage time point and diluted vaccine samples (10 μg / mL antigen concentration) were added to the ELISA plate.

[0155] Specific monoclonal antibodies were added to detect antigen binding ability, and the OD value was read at a wavelength of 450 nm.

[0156] Three parallel samples were tested in each group at each time point, and the mean value was taken.

[0157] Experimental data Table name: Effects of optimized stabilizer formula and traditional stabilizers on antigen stability Summary The optimization of stabilizers has obviously improved the antigen stability of vaccine preparations. Under the storage conditions of 1 month and 3 months, the degree of degradation of antigen protein in the experimental group was significantly lower than that in the control group. SDS-PAGE results showed that the integrity of the bands in the experimental group was better maintained, while the bands in the control group gradually became blurred and the band intensity decreased significantly. The synergistic effect of glycerol and gelatin effectively reduced the degradation of antigens caused by environmental factors during storage by providing a molecular protection barrier for the antigens. The control group containing only gelatin lacked the hydration protection of glycerol for the antigens, and the antigens underwent more severe cleavage during storage.

[0158] According to the data from ELISA tests, after 3 months of storage, the binding ability of the experimental group to the antigen and monoclonal antibody only decreased slightly, and the OD value remained above 1.1, which could still well reflect the integrity of the antigen epitope. This shows that the optimized stabilizer not only slowed down the degradation rate of the antigen, but also effectively protected the immunogenicity of the antigen. The antigen binding ability of the control group under the same storage conditions decreased significantly, and the OD value dropped to less than 0.9 after 3 months. This difference shows that the protective ability of a single gelatin stabilizer is limited, and it is difficult to maintain the immune effect of the vaccine during long-term storage.

[0159] The long-term stability of vaccine preparations directly affects their practical application value. The optimized glycerol + gelatin stabilizer improves the physical and chemical stability of the antigen at the molecular level through the combined effect of glycerol's hydration and gelatin's network structure. This multi-level protection mechanism ensures that the vaccine still has excellent immune efficacy even after storage for several months. Traditional stabilizers cannot provide multiple protections and show obvious deficiencies when facing complex storage conditions. Combined with data and mechanism analysis, the application of optimized stabilizers reflects the practical innovation and important promotion value of the present invention.

[0160] Experiment 5: Effect of inactivator concentration on immune effect Experimental Description Purpose The effect of the inactivator concentration on the antigen immunogenicity and inactivation effect was evaluated, and the superiority of Example 5 (optimized inactivator concentration) was verified and compared with Comparative Example 5 (low concentration inactivator).

[0161] Experimental Materials SPF chickens (7 days old, 5 per group) Example 5 Vaccine prepared (0.04% β-propiolactone) Vaccine prepared in Comparative Example 5 (0.02% β-propiolactone) ELISA kit (detection of IgG antibodies) Mycoplasma selective culture medium (to detect inactivation effect) ELISA reader, constant temperature box Experimental procedures Vaccination Experimental group: Each SPF chicken was subcutaneously injected with 0.5 mL of the vaccine of Example 5.

[0162] Control group: each SPF chicken was subcutaneously injected with 0.5 mL of the vaccine of comparative example 5.

[0163] There were 5 chickens in each group, and the condition of the chickens was observed after vaccination to ensure that no obvious stress response occurred.

[0164] Inactivation effect detection Take 1 mL of each prepared vaccine sample, inoculate it into SP4 medium, culture it at 37°C for 7 days, and observe whether there is mycoplasma growth.

[0165] Antibody titer detection (IgG) Blood samples were collected from the chicken wing vein on the 7th, 14th and 21st days after inoculation, with a blood volume of 0.5 mL per chicken.

[0166] The serum was centrifuged and diluted, added to an ELISA plate, and IgG levels were detected using specific monoclonal antibodies.

[0167] Each sample was tested 3 times and the average value was taken.

[0168] Experimental data Table name: Effects of different inactivator concentrations on vaccine immune efficacy and inactivation effect Summary The optimization of inactivator concentration showed significant advantages in the experiment. The 0.04% β-propiolactone used in the experimental group not only ensured the complete inactivation of mycoplasma (sterile growth), but also retained a high antigen immunogenicity. In contrast, in the control group, although 0.02% β-propiolactone partially inactivated mycoplasma, a small number of surviving bacteria were detected, and the inactivation effect was defective. This result verified the criticality of inactivator concentration in vaccine preparation, especially its direct impact on the thoroughness and safety of inactivation.

[0169] In the antibody titer test, the IgG levels of the experimental group on the 7th, 14th and 21st days were always significantly higher than those of the control group. Mechanistically, this was directly related to the concentration of the inactivator. Although the lower concentration of the inactivator (control group) reduced the damage to the antigen, it failed to completely stabilize the immune epitope of the antigen. In the experimental group, by adjusting to a moderate inactivator concentration (0.04%), it was able to effectively inactivate mycoplasma and avoid excessive antigen degradation that might be caused by high concentrations of β-propiolactone. As a result, the antibody titer of the experimental group increased more rapidly and the immune response was stronger.

[0170] The concentration of the inactivator also affects the chemical stability of the antigen. In the test group, the reaction between β-propiolactone and the antigen protein was precisely controlled to avoid damage to the key sites of the antigen. This control was directly reflected in the rising curve of the IgG level. The antibody titer of the test group was close to saturation on the 21st day, while the control group was insufficient inactivator and the antigen was not stable enough, resulting in weak immune stimulation and IgG levels that always lagged behind. This experiment not only demonstrated the technical value of optimizing the inactivator concentration, but also revealed the deep connection between the inactivator and the protection of the antigen structure, providing a scientific basis for the process parameters of vaccine preparation.

[0171] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an inactivated vaccine for chicken mycoplasma disease, characterized in that: The method comprises the following steps: Cultivation of mycoplasma strains: inoculate the Mycoplasma galli strains into SP4 medium at a temperature of 35 to 39°C for 48 to 72 hours, and collect the mycoplasma cells; Inactivation and antigen protection: Add inactivator and antigen protection agent to the mycoplasma suspension for inactivation and antigen protection reaction, the reaction temperature is 25-37°C, and the reaction time is 3-6 hours; Preparation of nano-delivery system: Preparation of nanoparticle delivery system through the composite structure of chitosan and sodium alginate; Antigen encapsulation and adjuvant loading: encapsulating inactivated mycoplasma antigens into nano-delivery particles and adding immune-enhancing adjuvants; Homogenous mixing of vaccine preparation: The antigen-adjuvant nanoparticles are mixed with a stabilizer and a diluent and the vaccine is prepared after homogenous mixing.

2. The method for preparing an inactivated vaccine for chicken mycoplasma disease according to claim 1, characterized in that: The inactivator is β-propiolactone, and its mass fraction accounts for 0.02 to 0.1; the antigen protective agent includes the following components, calculated by mass fraction: Polylysine: 0.1-0.3; Glutamine: 0.1-0.5; Arginine: 0.05~0.

2.

3. The method for preparing an inactivated vaccine for chicken mycoplasma disease according to claim 1, characterized in that: The nano delivery system comprises the following components, in parts by mass: Chitosan: 0.1-0.3; Sodium alginate: 0.05~0.2; Calcium phosphate: 0.2~1.

0.

4. The method for preparing an inactivated vaccine for chicken mycoplasma disease according to claim 1, characterized in that: The mycoplasma strain was cultured at 37° C. for 48 hours.

5. The method for preparing an inactivated vaccine for chicken mycoplasma disease according to claim 1, characterized in that: In the inactivation and antigen protection steps, the order of adding the inactivator and the antigen protection agent is to first add β-propiolactone and then add polylysine, glutamine and arginine, and gradually adjust the pH to 7.0-7.

5.

6. The method for preparing an inactivated vaccine for chicken mycoplasma disease according to claim 1, characterized in that: During the preparation of the nano-delivery particles, chitosan is subjected to ultrasonic dispersion treatment with an ultrasonic power of 50 to 100 W and a time of 20 to 60 seconds.

7. The method for preparing an inactivated vaccine for chicken mycoplasma disease according to claim 1, characterized in that: In the antigen encapsulation and adjuvant loading steps, the immune enhancing adjuvant includes the following components, calculated by weight: CpG oligonucleotide: 0.0001-0.001; Poly:0.0002~0.

003.

8. The method for preparing an inactivated vaccine for chicken mycoplasma disease according to claim 1, characterized in that: In the homogenous mixing step of the vaccine preparation, the mixing time is 10 to 20 minutes and the mixing temperature is 4 to 10°C.

9. The method for preparing an inactivated vaccine for chicken mycoplasma disease according to claim 1, characterized in that: The stabilizer comprises the following components, in parts by mass: Glycerol: 0.05~0.2; Gelatin: 0.3~0.

7.

10. The method for preparing an inactivated vaccine for chicken mycoplasma disease according to claim 1, characterized in that: The pH range of the PBS buffer is 7.2-7.4, and it is used for diluting vaccine preparations.

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