An immunological preparation for stimulating hens to produce low allergenic eggs and a method of use thereof

Abstract

The application relates to the technical field of pet allergy intervention, in particular to an immunological preparation for stimulating laying hens to produce low-allergen eggs and an application method thereof. The immunological preparation is an oil-in-water emulsion, contains recombinant Fel d1 protein or specific peptide segments with immunogenicity as immunogens, an adjuvant system is composed of 30-50% of squalene or white oil oil phase, Tween-80 or Span-80 emulsifier and 50-200 micrograms of Quil A immunostimulant, and a buffer system is phosphate buffer with pH 7.2-7.4. When prepared, the recombinant Fel d1 protein is dissolved in the phosphate buffer and mixed with the oil phase, high-speed shearing emulsification is carried out to form the oil-in-water emulsion, the oil-in-water emulsion is sterilized and packaged after filtration, and is stored at 2-8 DEG C. The mild and safe adjuvant combination is adopted, the oil-in-water emulsion has good physical stability, the content of Fel d1 in the eggs can be reduced by more than 60%, and the oil-in-water emulsion can provide core raw materials for low-allergen pet food.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products, specifically an immunomodulator for stimulating laying hens to produce low-allergen eggs and its application method. Background Technology

[0002] With the continued growth of pet ownership worldwide, cat allergy has become one of the major health problems troubling many pet-owning families. Fel d1 protein is a major allergen secreted by felines, widely present in cat saliva, dander, and secretions. It can be transmitted through the air and trigger allergic reactions in humans, including symptoms such as sneezing, nasal congestion, tearing, itchy skin, and even asthma, severely impacting the quality of life for people with allergies.

[0003] Traditional solutions for cat allergies mainly include environmental cleaning, human antihistamines, and interventions for the pet itself (such as regular bathing and the use of Fel d1 inhibitors). However, these methods have significant limitations: environmental cleaning only temporarily reduces the concentration of allergens in the air and cannot fundamentally solve the problem; human antihistamines require long-term use and may cause side effects such as drowsiness and drug dependence; and frequent bathing or the use of chemical inhibitors on pets is not only inconvenient but may also cause stress reactions in the pets. The effectiveness of these traditional solutions is generally limited, and their inconvenience makes them difficult to meet the long-term needs of pet owners with allergies.

[0004] In recent years, research on reducing the allergenicity of pets through dietary interventions has gained increasing attention, becoming a new approach to solving feline allergies. One strategy involves adding egg yolk antibodies (IgY) capable of neutralizing Fel d1 to pet food, attempting to reduce the allergens ingested by pets through the action of exogenous antibodies. However, this approach has significant drawbacks: exogenously added antibodies may be inactivated in the pet's digestive tract by gastric acid and digestive enzymes, making it difficult to guarantee effective neutralization; furthermore, the production cost of egg yolk antibodies is high, making large-scale application economically impractical.

[0005] Another more promising strategy is "source control," which involves using immunization to enable hens to produce eggs containing high titers of anti-Fel d1 antibodies (i.e., "hypoallergenic eggs"), using these as a core ingredient in pet food production. The theoretical basis for this approach is that after being stimulated by specific antigens, hens accumulate large amounts of specific IgY antibodies in the yolk through a humoral immune response. When pets ingest eggs containing these antibodies, the anti-Fel d1 antibodies can bind to the allergen in the pet's body, thereby neutralizing its sensitizing activity. Compared to exogenously adding antibodies, the "source control" strategy has potentially better efficacy and lower long-term costs, and is therefore considered a more promising technological direction.

[0006] However, existing immunization programs face the core problem of unstable immunization efficacy in practical applications. The antibody titers and inhibitory effects induced by different immunogens (including whole proteins and specific peptides) combined with different adjuvants vary significantly, making it difficult to establish stable and reproducible production processes. This results in inconsistent levels of anti-Fel d1 antibodies in the produced eggs, failing to guarantee the consistency and reliability of different batches. This fundamental deficiency severely restricts the large-scale production and commercial promotion of hypoallergenic eggs.

[0007] In view of the current state of the technology, there is an urgent need to develop a highly efficient, safe, stable, and suitable immunomodulator for large-scale immunization of laying hens, in order to produce low-allergen eggs with a stable reduction in Fel d1 content, thereby providing high-quality core raw materials for the low-allergenic pet food industry. This invention addresses the technical problem of unstable immunization effects in existing technologies by providing a compound immunomodulator specifically designed to stimulate laying hens to produce anti-Fel d1 antibodies, along with its application method. Summary of the Invention

[0008] This invention addresses the technical problems of existing technologies that produce hypoallergenic eggs by immunizing laying hens, such as unstable immunization effects, unclear impact on the health and egg production performance of laying hens, and difficulty in balancing cost and safety. It provides an immunizing agent specifically designed to stimulate laying hens to produce hypoallergenic eggs and its application method.

[0009] This invention provides an immunomodulator for stimulating laying hens to produce low-allergen eggs. The immunomodulator is an oil-in-water emulsion, characterized by comprising an immunogen, an adjuvant system, and a buffer system; the immunogen is recombinant Fel d1 protein or a specific peptide with immunogenicity thereof; the adjuvant system comprises an oil phase, an emulsifier, and an immunostimulant; and the buffer system is a phosphate buffer.

[0010] Furthermore, the immunogen, which is the effective component of the immunomodulator of the present invention, is recombinant Fel d1 protein or its immunogenic specific peptide. The recombinant Fel d1 protein is preferably produced using an E. coli expression system or a yeast expression system, and its purity is greater than 90%.

[0011] Fel d1 is a major allergenic protein in felines, secreted by the sebaceous and salivary glands of cats and found in their dander, saliva, and tears. This invention uses recombinantly expressed Fel d1 protein as an immunogen, which can effectively stimulate laying hens to produce specific antibodies against Fel d1. These antibodies accumulate in egg yolks, thereby neutralizing and reducing the residual Fel d1 allergen content in eggs.

[0012] Furthermore, the adjuvant system of the immunomodulator of the present invention is used to enhance the immunogenicity of the immunogen and improve the antibody response level, and comprises three components: an oil phase, an emulsifier, and an immunostimulant. The oil phase is a metabolizable natural oil, preferably squalene and / or white oil, accounting for 30% to 50% of the total volume of the immunomodulator.

[0013] Squalene is a naturally occurring organic compound with high safety, and it can be normally metabolized in humans and animals. White oil, as a commonly used pharmaceutical excipient, has good chemical stability and safety. Using metabolizable natural oils as the oil phase ensures the stability of the emulsion while reducing the risk of residues in edible animals.

[0014] Furthermore, the emulsifier in the adjuvant system of the present invention is Tween-80 and / or Span-80, used to stabilize the structure of the oil-in-water emulsion. Both Tween-80 and Span-80 are commonly used nonionic surfactants with good emulsifying properties, capable of forming stable oil-in-water emulsions. In a preferred embodiment of the present invention, a concentration of 5% Tween-80 is used as the emulsifier.

[0015] Furthermore, the immunostimulant in the adjuvant system of the present invention is Quil A or a similar safe adjuvant ingredient suitable for food animals, with a dosage of 50-200ug per dose.

[0016] Quil A is a mixture of natural saponins extracted from the bark of the soap tree. It has been widely used as an adjuvant in veterinary vaccines, exhibiting good immune-enhancing effects while maintaining high safety for food-consuming animals. Quil A can stimulate the body to produce stronger humoral and cellular immune responses, with low residual risk.

[0017] Furthermore, the buffer system of the immunomodulator of the present invention is a phosphate buffer solution, which is used to dissolve the immunogen and adjust the pH of the preparation to 7.2-7.4, while adjusting the osmotic pressure to a suitable range.

[0018] Phosphate buffer is one of the most commonly used buffer systems in biological products. It has a moderate pH buffering capacity, adjustable osmotic pressure, and no significant effect on bioactive components.

[0019] Furthermore, for multi-dose packaging, the immunomodulator of the present invention may contain a preservative, such as thimerosal or a safer alternative, at a concentration of 0.0001. For single-dose packaging, no preservative may be added.

[0020] According to a specific embodiment of the present invention, the contents of each component of the immunizing agent are as follows: the concentration of recombinant Fel d1 protein is 50-100 ug / mL for the first immunization and 25-50 g / mL for the booster immunization; squalene or white oil accounts for 30% to 50% of the total volume; Tween-80 accounts for 1% to 10%; Quil A is 50-200 ug / mL; phosphate buffer is made up to 100%; and thimerosal is 0.01% to 0.002%.

[0021] The present invention also provides a method for preparing the above-mentioned immunomodulator, characterized by comprising the following steps: dissolving recombinant Feld1 protein in phosphate buffer, adjusting the pH value to 7.2-7.4 to obtain an aqueous antigen solution; mixing the oil phase components, emulsifier and immunostimulant evenly to form an oil phase mixture; slowly adding the oil phase mixture to the aqueous antigen solution under high-speed shearing or ultrasonic emulsification conditions to form a stable oil-in-water emulsion; performing aseptic filtration and dispensing, and storing at 2.8°C.

[0022] Further, the aqueous antigen preparation step includes: dissolving recombinant Fel d1 protein in phosphate buffer, stirring and dissolving at 25°C to form a homogeneous aqueous antigen solution; adjusting the pH of the aqueous antigen solution to 7.2-7.4; and performing preliminary filtration of the aqueous antigen solution using a 0.22µm filter to remove any possible impurities and particles. The purity of the recombinant Fel d1 protein is greater than 90%, preferably produced using an E. coli expression system or a yeast expression system. Stirring and dissolving at 25°C avoids the influence of high temperature on protein activity, and a pH of 7.2-7.4 is a range with good protein stability.

[0023] Furthermore, the adjuvant oil phase preparation step includes: mixing the oil phase components, emulsifier, and immunostimulant in a specific ratio to form an oil phase mixture; homogenizing the oil phase mixture to ensure thorough integration of all components; and aseptically filtering the oil phase mixture to ensure sterility in subsequent preparation processes. The amount of Quil A added needs to be precisely controlled; too little may not achieve the expected immune-enhancing effect, while too much may cause local side effects. Homogenization can be performed using a magnetic stirrer or a high-speed homogenizer.

[0024] Further, the emulsification and post-processing steps include: slowly adding the oil phase mixture dropwise to the aqueous phase antigen solution under high-speed shear or ultrasonic emulsification conditions while maintaining stirring to form a stable oil-in-water emulsion; aseptically filtering using a 0.22µm filter to ensure the sterility of the final product; and aseptically dispensing the emulsion into suitable containers. High-speed shear emulsification is a key process step in the preparation of oil-in-water emulsions, and the shear rate and shear time directly affect the average particle size and stability of the emulsion. A high-speed shear emulsifier is preferably used, with a shear rate of 10,000-20,000 r / min and a shear time of 5-15 min.

[0025] Furthermore, the finished product storage steps include: sealing the dispensed formulation under nitrogen protection to prevent oxidation and deterioration; storing the finished product at 2-8°C in the dark; and determining the specific shelf life based on stability study results. Nitrogen protection eliminates oxygen from the container, reducing the risk of oxidation of the formulation; refrigerated storage at 2-8°C is the recommended storage condition for most protein-based biological products, ensuring the stability of the formulation within its shelf life.

[0026] The present invention also provides a method for using the above-mentioned immune agents in the production of hypoallergenic eggs, characterized by including: administering initial and booster immunizations to laying hens, and collecting the produced eggs starting 10-14 days after the booster immunization.

[0027] Furthermore, the initial immunization is administered via subcutaneous injection in the neck or intramuscular injection in the breast, with each chicken receiving 0.5-1.0 mL of an immunizing agent containing 50-100 μm Fel d1 protein. For subcutaneous injection in the neck, the needle is inserted into the subcutaneous tissue below the hen's neck to avoid damage to the carotid artery and trachea; for intramuscular injection in the breast, the needle is inserted into the breast muscle layer to a depth of approximately 1-2 cm. Both injection methods effectively deliver the immunizing agent.

[0028] Furthermore, the booster immunization is performed three to four weeks after the initial immunization, using the same injection method as the initial immunization, with each chicken injected with 0.5-1.0 mL of an immunizing agent containing 25-50 μM Fel d1 protein. The antigen dose for the booster immunization is lower than that for the initial immunization. This is based on the principle of memory response in immunology, that is, after the initial immunization, the body has already produced memory B cells, and the lower dose during the booster immunization can stimulate a high level of antibody response.

[0029] Furthermore, based on antibody monitoring results, maintenance immunization is administered every 8-12 weeks during the laying period, with the same injection dosage as the booster immunization. Maintenance immunization is used to continuously stimulate the body to produce antibodies, maintain a high titer of anti-Fel d1 IgY antibodies in the egg yolk, and ensure a continuous decrease in the Fel d1 content in the egg.

[0030] Furthermore, eggs from the immunized flock were collected 10-14 days after the booster immunization was completed. At this time, the titer of anti-Feld1 IgY antibody in the egg yolk had reached a high level, which could effectively neutralize the Feld1 allergen in the egg yolk.

[0031] Furthermore, the titer of anti-Fel d1 IgY antibody in egg yolk was detected using enzyme-linked immunosorbent assay (ELISA) to assess the immunization effect. For ELISA testing, egg yolk was diluted and coated into wells of a microplate, horseradish peroxidase-labeled anti-chicken IgY secondary antibody was added, and after substrate development, the OD450 value was measured to calculate the antibody titer.

[0032] Furthermore, the ELISA method was used to detect the residual Fel d1 allergen content in eggs or egg yolk powder to verify the effectiveness of the immunomodulatory agents. The goal was to reduce the Fel d1 content in the produced eggs by more than 60% compared to ordinary eggs.

[0033] Furthermore, based on the experimental data of the present invention, t-tests or ANOVA were used for inter-group comparisons. The results showed that the p-value for the control group compared with the first week after the first immunization was 0.0004, the p-value for the control group compared with the second week after the first immunization was 0.014, and the p-value for the control group compared with the third week after the first immunization was 0.3127. These results indicate that immunization with the immunizing agent of the present invention significantly reduces the Fel d1 content in eggs, and the immunization effect is stable and sustained.

[0034] Furthermore, the time progression of antibody response after immunization in laying hens according to the present invention is as follows: 0-3 weeks after the first immunization, the body produces an initial immune response, and the IgY antibody titer gradually increases but remains relatively low; 1-2 weeks after the booster immunization, memory B cells are activated, and the IgY antibody titer rapidly increases to its peak; 3-8 weeks after the booster immunization, the antibody titer remains at a high level, and this stage is the optimal window for egg production; 8-12 weeks after the booster immunization, the antibody titer gradually decreases, and maintenance immunization is required to maintain the effect.

[0035] The beneficial effects of this invention are as follows: Using recombinant Fel d1 protein as an immunogen, it can stimulate laying hens to produce specific high-titer antibodies, thereby reducing the allergen content in eggs from the source, with a target reduction of Fel d1 content of more than 60%. The adjuvant system uses squalene, Quil A, and other ingredients that have been proven to be relatively safe for poultry and have low residue risks, avoiding the use of strong adjuvants such as Freund's complete adjuvant, which may cause severe local reactions, thus ensuring the health of laying hens and the safety of egg products. The oil-in-water emulsion formulation has good physical stability; the immunogen is effectively encapsulated in the oil phase to prevent protein degradation. The mature preparation process facilitates large-scale production and quality control, ensuring consistent potency across different batches. This invention clearly defines the optimal ratio range of each component, optimizing production costs while ensuring immune efficacy. This invention directly produces hypoallergenic eggs as the core biological raw material, laying the foundation for the development of a series of end-product pet foods targeting human and pet allergies, creating a completely new product category and technological barrier.

[0036] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0037] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 This is a flowchart of the preparation process of the immunomodulator of the present invention, which shows the complete preparation process of the water-in-oil emulsion immunomodulator, including four main steps: antigen aqueous phase preparation, adjuvant oil phase preparation, emulsification and post-processing, and finished product storage.

[0038] Figure 2 This is a timeline diagram of the immunization program for laying hens according to the present invention. It shows the complete immunization program timeline from the first immunization, booster immunization to maintenance immunization. The horizontal axis represents the number of days after immunization, the vertical axis represents the antibody titer, and the time windows of the peak antibody titer and the optimal egg production period are marked.

[0039] Figure 3 This is a schematic diagram of the injection sites for immunization in laying hens, illustrating the location and anatomical structure of the injection sites for two injection methods: subcutaneous injection in the neck and intramuscular injection in the chest, including key anatomical landmarks such as the carotid artery, trachea, muscle layer, and subcutaneous tissue.

[0040] Figure 4 This is a comparison chart of the immune effect detection of the present invention. It uses a combination of box plot and scatter plot to show the changes in Feld1 content in the control group and the immunized group at different time points, and marks the p-value for statistical analysis, intuitively presenting the effect of significantly reducing Feld1 content after immunization. Detailed Implementation

[0041] The embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings.

[0042] The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the invention, and should not be construed as limiting the invention.

[0043] like Figure 1As shown in the figure, an immune agent for stimulating laying hens to produce low-allergen eggs according to an embodiment of the present invention is an oil-in-water emulsion. Its complete preparation process includes four main steps: antigen aqueous phase preparation, adjuvant oil phase preparation, emulsification and post-treatment, and finished product storage.

[0044] The immunomodulator of this invention is an oil-in-water emulsion, characterized by comprising three core components: an immunogen, an adjuvant system, and a buffer system. The immunogen is recombinant Fel d1 protein or its immunogenic specific peptide, preferably recombinant Fel d1 protein produced using an E. coli or yeast expression system with a purity greater than 90%. Fel d1 is a major allergenic protein in felines, secreted by the sebaceous and salivary glands of cats and present in cat dander, saliva, and tears. This invention uses recombinantly expressed Fel d1 protein as an immunogen, which can effectively stimulate laying hens to produce specific antibodies against Fel d1. These antibodies can accumulate in egg yolks, thereby neutralizing and reducing the residual Fel d1 allergen content in eggs.

[0045] The adjuvant system of the immunomodulator of this invention comprises three components: an oil phase, an emulsifier, and an immunostimulant, used to enhance the immunogenicity of the immunogen and improve the antibody response level. The oil phase is a metabolizable natural oil, preferably squalene and / or white oil, accounting for 30-50% of the total volume of the immunomodulator. Squalene is a naturally occurring organic compound with high safety and can be normally metabolized in humans and animals; white oil, as a commonly used pharmaceutical excipient, has good chemical stability and safety. Using a metabolizable natural oil as the oil phase ensures the stability of the emulsion and reduces the risk of residues in edible animals. The emulsifier is Tween-80 and / or Span-80, used to stabilize the structure of the oil-in-water emulsion. Tween-80 and Span-80 are both commonly used nonionic surfactants with good emulsifying properties, capable of forming stable oil-in-water emulsions. In a preferred embodiment of this invention, a concentration of 5% Tween-80 is used as the emulsifier. The immunostimulant is Quil A or a similar safe adjuvant suitable for food animals, with a dosage of 50-200 μg per dose. Quil A is a mixture of natural saponins extracted from the bark of the soapberry tree and has been widely used as an adjuvant in veterinary vaccines, exhibiting good immune-enhancing effects while maintaining high safety for food animals. Quil A can stimulate the body to produce stronger humoral and cellular immune responses with low residual risk.

[0046] The buffer system of the immunomodulator of this invention is a phosphate buffer solution, used to dissolve the immunogen and adjust the pH of the preparation to 7.2-7.4, while simultaneously adjusting the osmotic pressure to a suitable range. Phosphate buffer solution is one of the most commonly used buffer systems in biological products, with moderate pH buffering capacity, adjustable osmotic pressure, and no significant effect on bioactive components. For multi-dose packaging, a preservative can be added, using thimerosal or a safer alternative, at a concentration of 0.0001. For single-dose packaging products, no preservative is required.

[0047] The method for preparing the immunomodulator according to embodiments of the present invention includes the following specific steps.

[0048] The first step is the preparation of the aqueous antigen phase. Recombinant Fel d1 protein is dissolved in phosphate buffer and stirred continuously at 25°C for 30 minutes until completely dissolved, forming a homogeneous aqueous antigen solution. Stirring at 25°C avoids the influence of high temperature on protein activity. The pH of the aqueous antigen solution is adjusted to 7.2-7.4, which is the range with good protein stability. The aqueous antigen solution is preliminarily filtered using a 22µm filter to remove any possible impurities and particles. The purity of the recombinant Fel d1 protein is greater than 90%, preferably produced using an E. coli expression system or a yeast expression system. The filtrate is transferred to the next step for later use.

[0049] The second step is the preparation of the adjuvant oil phase. The oil phase components, namely squalene and / or white oil, emulsifier Tween-80, and immunostimulant Quil A, are mixed evenly in a specific ratio to form the oil phase mixture. The amount of Quil A added needs to be precisely controlled; too little may not achieve the expected immune-enhancing effect, while too much may cause local side effects. The oil phase mixture is then homogenized to ensure thorough integration of all components. Homogenization can be performed using a magnetic stirrer or a high-speed homogenizer, with a mixing time of no less than 10 minutes to ensure uniform distribution of all components. The oil phase mixture is then aseptically filtered using a 0.22µm filter to ensure sterility in subsequent preparation processes.

[0050] The third step is emulsification and post-processing. Under high-speed shearing or ultrasonic emulsification conditions, the oil phase mixture is slowly added dropwise to the aqueous phase antigen solution while stirring, forming a stable oil-in-water emulsion. High-speed shear emulsification is a key process step in the preparation of oil-in-water emulsions; the shear rate and shear time directly affect the average particle size and stability of the emulsion. A high-speed shear emulsifier is preferred, with a shear rate of 10000-2000 r / min and a shear time of 5-15 min. The dropwise addition process should be completed within 5-10 min, followed by continued shear emulsification for another 5-15 min. The average particle size of the resulting oil-in-water emulsion should be controlled within the range of 1-10 μm to ensure physical stability and sustained-release effect of the immunogen. Aseptic filtration is performed using a 0.22 μm filter to ensure the sterility of the final product. Under aseptic conditions, the emulsion is dispensed into suitable containers, with 1 mL, 5 mL, or 10 mL packaging options available.

[0051] The fourth step is finished product storage. The dispensed formulation is sealed using nitrogen protection to remove oxygen from the container and reduce the risk of oxidation. The finished product is stored at 2-8°C in the dark, which is the recommended storage condition for most protein-based biological products, ensuring the formulation remains stable within its shelf life. The specific shelf life is determined based on stability study results; using nitrogen protection can effectively extend the shelf life of the formulation.

[0052] The application method of the immunomodulator in the production of hypoallergenic eggs according to embodiments of the present invention includes the following specific steps.

[0053] The first step is the initial immunization. For example... Figure 3 As shown, the initial immunization of laying hens is administered via subcutaneous injection in the neck or intramuscular injection in the pectoral region. For the subcutaneous injection in the neck, the hen should be firmly restrained. Gently pinch the skin to create a fold in the subcutaneous tissue below the neck, insert the needle at a 30-45° angle, and after confirming no blood flow, slowly inject the solution, taking care to avoid damaging the carotid artery and trachea. For the intramuscular injection in the pectoral region, the needle is inserted vertically into the pectoral muscle layer to a depth of approximately 1-2 cm, penetrating into the muscle fibers. Each hen receives 0.5-1.0 ml of an immunizing agent containing 50-100 μg of Fel d1 protein. After the initial immunization, the laying hens develop an initial immune response, with IgY antibody titers gradually increasing but remaining relatively low. This phase lasts 0-3 weeks.

[0054] The second step is booster immunization. A booster immunization is administered three to four weeks after the initial immunization, using the same injection method as the initial immunization. Each chicken is injected with 0.5-1.0 mL of an immunomodulator containing 25-50 μg of Fel d1 protein. The antigen dose for the booster immunization is lower than that for the initial immunization. This is based on the principle of memory response in immunology; that is, after the initial immunization, the body has already produced memory B cells, and a lower dose during the booster immunization is sufficient to stimulate a high level of antibody response. One to two weeks after the booster immunization, memory B cells are activated, and IgY antibody titers rapidly rise to their peak. Three to eight weeks after the booster immunization, antibody titers remain at a high level; this stage is the optimal window for egg production.

[0055] The third step is maintenance immunization. Based on antibody monitoring results, maintenance immunization is administered every 8-12 weeks during the egg-laying period, with the same injection dosage as the booster immunization. Maintenance immunization is used to continuously stimulate the body to produce antibodies, maintain a high titer of anti-Fel d1 IgY antibodies in the egg yolk, and ensure a continuous decrease in the Fel d1 content in the egg.

[0056] The fourth step is egg collection. Eggs from the immunized flock are collected 10-14 days after booster immunization. At this time, the anti-Fel d1 IgY antibody titer in the yolks has reached a high level, effectively neutralizing the Fel d1 allergen in the yolks. Collection continues for 8-12 weeks. During this period, the anti-Fel d1 IgY antibody titer in the yolks remains at a high level, ensuring the continued production of low-allergen eggs.

[0057] This invention employs an enzyme-linked immunosorbent assay (ELISA) to detect the titer of anti-Fel d1 IgY antibody in egg yolks to assess the immunization effect. The specific procedure includes the following steps: Egg yolks are diluted 1:1000 with phosphate buffer. 100 μL of the diluted solution is coated onto the wells of an ELISA plate and incubated overnight at 4°C. The next day, the coating solution is removed, and the plate is washed three times with phosphate buffer containing 0.5% Tween-20 for 3 minutes each time. Horseradish peroxidase-labeled anti-chicken IgY secondary antibody is added to each well (100 mL), and the plate is incubated at 37°C for 1 hour. After incubation, the plate is washed three times again with washing buffer. Tetramethylbenzidine substrate solution is added to each well (100 μL), and the reaction is carried out at room temperature in the dark for 15 minutes. Finally, 50 μL of 2 mol / L sulfuric acid stop solution is added to each well, and the optical density of each well is measured at a wavelength of 450 nm to calculate the antibody titer.

[0058] Simultaneously, the ELISA method was used to detect the residual Fel d1 allergen content in eggs or egg yolk powder to verify the effectiveness of the immunomodulatory agents. The specific procedures included: taking 100 mg of the egg yolk sample to be tested, adding 1 mg of phosphate buffer, mixing thoroughly, centrifuging, and collecting the supernatant. The supernatant was diluted appropriately and coated onto the wells of an ELISA plate, incubated overnight at 4°C. After washing, mouse anti-Feld1 monoclonal antibody was added as the primary antibody, and incubated at 37°C for 1 h. After washing, horseradish peroxidase-labeled goat anti-mouse IgG was added as the secondary antibody, and incubated at 37°C for 1 h. After washing, tetramethylbenzidine substrate was added for color development, and the optical density value at a wavelength of 450 μm was measured. The Fel d1 content was calculated according to the standard curve.

[0059] like Figure 4 As shown in the figure, the comparison chart of the immunization effect detection in this embodiment of the invention uses a combination of box plots and scatter plots to display the changes in Fel d1 content in the control group and the immunized group at different time points, and the p-values ​​for statistical analysis are marked, intuitively presenting the effect of significantly reducing Fel d1 content after immunization. Based on the experimental data of this invention, t-tests or ANOVA were used for inter-group comparisons. The results showed that the p-value for the control group compared to the first week after the first immunization was 0.0004, the p-value for the control group compared to the second week after the first immunization was 0.0104, and the p-value for the control group compared to the third week after the first immunization was 0.3127. These results indicate that after immunization with the immunizing agent of this invention, the Fel d1 content in eggs is significantly reduced, and the immunization effect is stable and sustained.

[0060] The time progression of antibody response after immunization in laying hens according to this invention is as follows: 0-3 weeks after the first immunization, the body produces an initial immune response, and the IgY antibody titer gradually increases but remains relatively low; 1-2 weeks after the booster immunization, memory B cells are activated, and the IgY antibody titer rapidly increases to its peak; 3-8 weeks after the booster immunization, the antibody titer remains at a high level, and this stage is the optimal window for egg production; 8-12 weeks after the booster immunization, the antibody titer gradually decreases, and maintenance immunization is required to maintain its effectiveness.

[0061] Specific application examples One hundred healthy 18-week-old laying hens were randomly divided into an experimental group and a control group, with 50 hens in each group. The experimental group received their first immunization via subcutaneous injection in the neck, with each hen receiving 0.8 mL of an immunomodulator containing 80 μg of recombinant Fel d1 protein. The control group received an equal volume of phosphate-buffered saline. A booster immunization was administered three weeks after the first immunization, with the dose halved to 40 μg per hen. Eggs were collected starting two weeks after the booster immunization, and samples were collected every two weeks for ELISA testing.

[0062] The test results showed that the Fel d1 content in the control group eggs remained stable in the range of 3-4 ug / g, while the Fel d1 content in the experimental group eggs decreased to 1-1.5 ug / g in the second week after booster immunization, with a reduction rate of 60-70%; it decreased to 0.8-1.2 ug / g in the fourth week, with a reduction rate of 70-80%; and it decreased to 0.9-1.3 ug / g in the sixth week, with a reduction rate of 65-75%. The p-values ​​(p<0.25) at each time point compared with the control group were all less than 0.05, indicating statistically significant differences.

[0063] The results of anti-Fel d1 IgY antibody titer detection in egg yolks showed that no specific antibodies were detected in the control group, while the experimental group reached high titer levels of 1:12800, 1:25600, and 1:12800 in weeks 2, 4, and 6-5, respectively. Throughout the experimental period, the egg production rate of the hens remained at 80-92%, with no significant difference compared to before immunization and the control group, indicating that this immunization regimen had no adverse effects on the health and egg production performance of the hens.

[0064] Based on the data from the above embodiments, the content ranges of each component of the immunizing agent in this embodiment are as follows: the concentration of recombinant Feld1 protein is 50-100 ug / mL for the first immunization and 25-50 ug / mL for the booster immunization; squalene or white oil accounts for 30-50% of the total volume; Tween-80 accounts for 1-10%; Quil A is 50-200 ug / mL; phosphate buffer is replenished to 100%; and thimerosal is 0.01-0.02%.

[0065] This formulation range has been experimentally verified to ensure that it stimulates laying hens to produce high-titer anti-Fel d1 IgY antibodies, achieving the goal of reducing the Fel d1 allergen content in eggs by more than 60% from the source. Meanwhile, the selected adjuvant components, such as squalene and Quil A, are widely used in veterinary vaccines, and their safety has been fully verified.

[0066] In the preparation process, the parameters for high-speed shear emulsification are set to a shear rate of 10,000-20,000 r / min and a shear time of 5-15 min. Under these conditions, the average particle size of the oil-in-water emulsion prepared can be controlled within the range of 1-10 μm, effectively encapsulating immunogenic proteins and preventing their degradation. Aseptic filtration is performed using a 0.22 μm filter, and the final product fully meets aseptic requirements. The finished product is sealed under nitrogen protection and stored at 2-8℃ away from light, with a shelf life of over 12 months.

[0067] The immunization program for laying hens adopts an initial immunization dose of 20-100ug / bird, a booster immunization dose of 25-50ug / bird, a booster immunization 3-4 weeks after the initial immunization, and maintenance immunization every 8-12 weeks thereafter. This can maintain the anti-Fel d1 IgY antibody titer in egg yolks at a level of not less than 1:12800, effectively ensuring that the Fel d1 content in eggs is reduced by more than 60%.

[0068] The oil-in-water emulsion immunomodulator of this invention can be administered to laying hens via intramuscular or subcutaneous injection, making it simple to administer and suitable for large-scale immunization applications. The resulting hypoallergenic eggs can be further processed into egg yolk powder for use as a functional ingredient in pet food. For example, adding 20 to 30 kilograms of this hypoallergenic egg yolk powder to each ton of cat food can reduce the Fel d1 allergen content in the cat food by 70% to 85%, thereby effectively reducing allergic reactions to Fel d1 in cats.

[0069] The hypoallergenic eggs used in this invention serve as the core biological raw material, laying the foundation for the development of a series of end-product pet foods targeting human and pet allergies. They can be used to prepare pet staple food, snacks, nutritional supplements, and other products, creating entirely new product categories and technological barriers.

[0070] The specific embodiments of the present invention have been described in detail above, but these embodiments are only used to explain the present invention and not to limit its scope. Those skilled in the art can make various changes, modifications or combinations to these embodiments without departing from the principles of the present invention.

Claims

1. An immunomodulator for stimulating laying hens to produce hypoallergenic eggs, comprising an oil-in-water emulsion, characterized in that, It includes immunogens, adjuvant systems, and buffer systems; The immunogen is recombinant Fel d1 protein or a specific peptide with immunogenicity thereof; The adjuvant system comprises an oil phase, an emulsifier, and an immunostimulant; The buffer system is a phosphate buffer solution.

2. The immunomodulatory agent according to claim 1, characterized in that, The purity of the recombinant Fel d1 protein is greater than 90%; and / or, the concentration of the immunogen is 50-100 μg / mL in the initial immunization formulation and 25-50 μg / mL in the booster immunization formulation.

3. The immunomodulatory agent according to claim 1, characterized in that, The adjuvant system: The oil phase is squalene and / or white oil, accounting for 30% to 50% of the total volume of the immunomodulator; The emulsifier is Tween-80 and / or Span-80; The immunostimulant is Quil A, and the dosage is 50-200 μg / mL.

4. The immunomodulatory agent according to claim 3, characterized in that, The emulsifier is 5% Tween-80.

5. The immunomodulatory agent according to claim 1, characterized in that, The buffer system is a phosphate buffer solution with a pH of 7.2-7.

4.

6. The immunomodulatory agent according to claim 1, characterized in that, The immunomodulator also contains a preservative at a concentration of 0.01%-0.02%, wherein the preservative is thimerosal or a safe alternative thereof.

7. The immunomodulatory agent according to claim 1, characterized in that, The average particle size of the oil-in-water emulsion is 1-10 μm.

8. A method for using the immunomodulatory agent as described in any one of claims 1-7 in the production of hypoallergenic eggs, characterized in that, include: The laying hens were given their first and second immunizations, and the eggs were collected 10-14 days after the second immunization.

9. The application method according to claim 8, characterized in that, The initial immunization dose is 0.5-1.0 mL per laying hen, containing 50-100 μg of immunogen; the booster immunization is performed 3 to 4 weeks after the initial immunization, with an injection dose of 0.5-1.0 mL per laying hen, containing 25-50 μg of immunogen; the injection method is subcutaneous injection in the neck or intramuscular injection in the chest.

10. The application method according to claim 8, characterized in that, This also includes maintaining immunization of laying hens every 8-12 weeks during the egg-laying period, with the same injection dosage as booster immunization; and using the collected eggs to process and prepare hypoallergenic egg yolk powder, which is then used as a raw material in the production of pet food.

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