Pasteurella egg yolk antibody for sheep and preparation method thereof
By preparing high-titer, high-purity ovine Pasteurella egg yolk antibodies, the problem of ovine Pasteurella infection control in existing technologies has been solved, enabling rapid prevention and efficient treatment, reducing side effects and production costs, and making it suitable for large-scale application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XINJIANG ACAD OF ANIMAL SCI
- Filing Date
- 2025-12-19
- Publication Date
- 2026-04-28
AI Technical Summary
Among the existing technologies for the prevention and control of ovine pasteurellosis, vaccines have long immunization cycles and poor immunization effects in young animals. Antibiotic treatment is prone to drug resistance and drug residues. Existing egg yolk antibodies cover only one serotype, have low purity and unstable titers, resulting in high rates of clinical side effects and insufficient protective efficacy. Moreover, the preparation process is complex and difficult to scale up.
Inactivated vaccines were prepared by reviving and culturing frozen Pasteurella bacteria of various pathogenic serotypes (A, D, and F). Hens were immunized and eggs were collected. Egg yolk antibodies were purified by centrifugation, ammonium sulfate precipitation, and dialysis. Quality testing was performed to prepare high-titer, high-purity egg yolk antibodies.
It achieves rapid prevention and efficient treatment of ovine pasteurellosis. The antibody covers a wide range of serotypes, has high purity, low side effect rate, low cost, is suitable for large-scale production, has stable potency, and avoids drug resistance and drug residues.
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Figure CN121930337A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of veterinary biological products technology, specifically to a Pasteurella multocida egg yolk antibody for sheep and its preparation method. Background Technology
[0002] Pasteurella multocida infection in sheep is an acute, febrile infectious disease caused by pathogenic Pasteurella multocida (mainly types A and D in China). Clinically, it is characterized by high fever, pneumonia, swollen throat, and septicemia. It can lead to acute death in lambs and a significant decline in the productive performance of adult sheep (such as reduced milk production and slower weight gain). This disease is widespread in livestock-producing areas globally, especially in large-scale sheep farms, where outbreaks can cause morbidity rates of 20%-50% and mortality rates of 10%-30%.
[0003] Currently, the industry's prevention and control methods for ovine pasteurellosis are mainly divided into two categories:
[0004] Vaccine prevention: Most commercially available vaccines are inactivated or attenuated Pasteurella vaccines, requiring immunization 2-4 weeks in advance. This results in a long immunization cycle and poor efficacy in young animals (lambs) under one month old (due to interference from maternal antibodies). Furthermore, their therapeutic effect is minimal. They are inadequate for responding to sudden outbreaks of disease.
[0005] Antibiotic treatment: Florfenicol, enrofloxacin and other antibiotics are commonly used in clinical practice, but long-term use has led to a year-on-year increase in the proportion of drug-resistant Pasteurella bacteria, and there is also the risk of drug residues, which affects the food safety of mutton and dairy products.
[0006] Egg yolk antibodies (IgY) are specific antibodies produced in the yolk of hens after immunization with specific antigens. They have advantages such as wide availability, low cost, no drug resistance, and high safety, and have been used in the prevention and control of diseases in pigs and poultry. However, there are still key technical deficiencies in the preparation of egg yolk antibodies against Pasteurella multocida.
[0007] Existing products mostly use a single serotype of Pasteurella for immunization, which cannot cover pathogenic A, D, and F strains in sheep, resulting in insufficient cross-protection. Moreover, the preparation process lacks standardized extraction and purification procedures, and the antibody purity is only 60%-70%. Impurities (such as vitellin) can easily cause adverse reactions such as local injection swelling in sheep, leading to stress and affecting the immunization effect or even having the opposite effect. In addition, the antibody titer is unstable (fluctuating by ±40% between batches), which cannot guarantee the clinical prevention and control effect.
[0008] Therefore, developing a sheep Pasteurella egg yolk antibody that covers the main pathogenic serotypes, has high purity, stable titer, and a simple preparation process is of great significance for solving the current problem of sheep Pasteurella infection prevention and control. Summary of the Invention
[0009] The purpose of this invention is to provide a Pasteurella egg yolk antibody for sheep and its preparation method, in order to solve the problems of existing Pasteurella egg yolk antibodies having single serotype coverage, low purity, unstable titer, high clinical side effect rate, insufficient protective efficacy, and poor efficacy of egg yolk antibodies prepared from strains isolated from non-native animals; the existing Pasteurella egg yolk antibody preparation process is complex, requires high equipment investment, and is difficult to scale up.
[0010] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of the present invention provides a method for preparing Pasteurella multocida egg yolk antibodies for sheep, comprising the following steps:
[0011] S1, Immunogen preparation:
[0012] Take frozen bacterial suspensions of Pasteurella multocida types A, D, and F, revive and culture the strains using modified Martin broth medium, then inactivate the bacterial suspensions, and finally add adjuvants to prepare vaccines.
[0013] S2, Hen Immunization:
[0014] Select healthy 20-week-old Leghorn chickens, inject them intramuscularly with inactivated bacterial vaccine, test the yolk antibody titer in the eggs, and start collecting eggs after they pass the test. Eggs are then pre-treated.
[0015] S3, crude extraction of egg yolk antibodies:
[0016] Separate the egg yolk, collect the yolk fluid, add PBS buffer in a certain proportion to fully dissolve the egg yolk, let stand and centrifuge to obtain crude extract;
[0017] S4, Egg yolk antibody purification:
[0018] The crude body fluid was precipitated with a 38% saturated ammonium sulfate solution, the antibody precipitate was collected, dissolved in PBS buffer, dialyzed once, then a 28% saturated ammonium sulfate solution was added for a second precipitation, and finally dialyzed twice to obtain purified egg yolk antibody solution.
[0019] S5, Antibody Quality Testing and Packaging:
[0020] The antibodies undergo quality testing, and once they pass the test, they are repackaged.
[0021] Preferably, the specific steps for preparing the S1 immunogen are as follows:
[0022] S11, strain recovery and culture:
[0023] Take frozen bacterial cultures of Pasteurella multocida types A, D, and F, revive them with modified Martin broth medium, and incubate at 37°C and 160 rpm / min for 18 h;
[0024] The revived bacterial culture was inoculated into fresh modified Martin broth medium at a volume ratio of 1:100 and cultured at 37°C with shaking at 160 rpm / min for 18-24 h.
[0025] The bacterial concentration was adjusted to 1×10⁻⁶ using McFarland turbidimetric tubes and plate counts. ^9 CFU / mL:
[0026] S12, bacterial inactivation:
[0027] Slowly add 37% formaldehyde solution to the bacterial culture until the final concentration is 0.3% (v / v), and inactivate by shaking at 37°C for 24 hours;
[0028] Spread 0.1 mL of the inactivated bacterial solution onto a modified Martin agar plate and incubate at 37°C for 48 h to confirm sterile growth.
[0029] S13, Adding adjuvants to prepare the vaccine:
[0030] An inactivated bacterial vaccine and aluminum hydroxide adjuvant were mixed at a volume ratio of 5:1 to prepare an immunization vaccine.
[0031] Preferably, the specific steps for immunizing the S2 hen are as follows:
[0032] Chicken selection:
[0033] Select healthy 20-week-old Leghorn chickens with an egg production rate of ≥90%, with 50 chickens per group. Test the serum of Pasteurella antibodies in advance, with a titer of ≤1:8 to exclude interference from maternal antibodies.
[0034] Immunization regimen:
[0035] Day 0, initial immunization: intramuscular injection of 0.5 mL of inactivated vaccine per animal;
[0036] Day 14, first booster immunization: 0.5 mL of inactivated vaccine per animal via intramuscular injection;
[0037] On day 28, the second booster immunization was administered: 1.0 mL of inactivated vaccine was injected intramuscularly per animal.
[0038] Egg collection:
[0039] Seven days after the second booster immunization, the titer of egg yolk antibodies in the eggs was tested to see if it was ≥1:128. If it was qualified, eggs were collected and collected continuously for 4 weeks.
[0040] Egg pretreatment:
[0041] After rinsing the collected eggs with clean water, immerse them in a 0.1% benzalkonium chloride solution for 10 minutes for disinfection, then rinse them three times with sterile distilled water and air dry them for later use.
[0042] Preferably, the specific steps for crude extraction of S3 egg yolk antibodies are as follows:
[0043] S31, yolk separation:
[0044] On a sterile operating table, crack open the sterilized egg, separate the yolk with sterile tweezers, remove the egg white and yolk membrane, and collect the yolk fluid;
[0045] S32, Dilution and Stirring:
[0046] Mix egg yolk solution and PBS buffer at a ratio of 1:3 (v / v), pour into a sterile mixing vessel, and stir at 25°C and 60 rpm for 30 minutes to fully dissolve the egg yolk.
[0047] S33, Settling and Centrifugation:
[0048] The mixture was placed in a 4°C refrigerator and allowed to stand for 12 hours to allow the impurities to precipitate fully. Then it was transferred to a centrifuge tube and centrifuged at 4°C and 4000 rpm for 20 minutes. The supernatant, which is the crude extract of egg yolk antibodies, was collected, and the bottom precipitate was discarded.
[0049] Preferably, the specific steps for purifying the S4 egg yolk antibody are as follows:
[0050] S41, initial sedimentation:
[0051] The crude extract was placed in a 4°C water bath, and a 38% saturated ammonium sulfate solution was slowly added while stirring at a dropping rate of 1 mL / min to avoid excessively high local concentrations.
[0052] After adding the antibody, continue stirring at 4°C for 4 hours to allow the antibody to precipitate fully. Centrifuge at 4°C and 4000 rpm for 20 minutes, discard the supernatant, and collect the antibody precipitate at the bottom.
[0053] S42, one-step dissolution and dialysis:
[0054] Dissolve the antibody precipitate in an equal volume of PBS buffer and stir until completely clear;
[0055] The solution was placed into a dialysis bag and then into a container containing PBS dialysis solution. Dialysis was performed at 4°C for 24 hours, with the dialysis solution being changed every 6 hours to remove any residual ammonium sulfate.
[0056] S43, secondary precipitation and dialysis:
[0057] After dialysis, slowly add 28% saturated ammonium sulfate solution to the antibody solution, stir at 4°C for 4 hours, centrifuge, and collect the precipitate;
[0058] After dissolving the precipitate in PBS buffer, dialyze again for 24 hours to obtain purified egg yolk antibody solution.
[0059] Preferably, the antibody quality detection in S5 includes, but is not limited to, the following items:
[0060] ①Valence testing:
[0061] The purified antibody was diluted using the tube agglutination method at ratios of 1:16, 1:32, 1:64, 1:128, and 1:256, with 100 μL of diluent added to each well, followed by 100 μL of 1×10⁻⁶ precipitate. ^8 The highest dilution at which obvious agglutination occurs after incubating Pasteurella multocida culture at 37°C for 24 hours is the titer, which should be ≥1:128.
[0062] ②Purity testing:
[0063] Perform SDS-PAGE electrophoresis, stain with Coomassie Brilliant Blue after electrophoresis, and calculate antibody purity using grayscale analysis software; the purity must be ≥90%.
[0064] ③ Sterility test:
[0065] Spread 0.1 mL of antibody solution onto a modified Martin agar plate and incubate at 37°C for 48 h. No bacterial growth indicates the plate is ready.
[0066] ④ Heat source detection:
[0067] According to the "Regulations for Veterinary Biological Products", antibody solution was injected into the marginal ear vein of rabbits and observed for 3 days. The rabbits were considered qualified if their body temperature increased by ≤0.6℃.
[0068] Preferably, the specific steps of the dispensing in S5 are as follows:
[0069] The qualified antibody solution is filtered through a 0.22μm sterile filter membrane, dispensed into sterile vials, sealed, and stored at 2-8℃.
[0070] Preferably, the Martin broth culture medium has the following composition:
[0071] 10g peptone, 5g yeast extract, 2g glucose, 5g sodium chloride, 2g dipotassium hydrogen phosphate, 1000mL distilled water;
[0072] The pH value of the Martin broth medium is 7.2-7.4.
[0073] Preferably, the PBS buffer solution has the following composition:
[0074] Potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 2.9g, sodium chloride 8.0g, distilled water 1000mL;
[0075] The pH value of the PBS buffer is 7.4.
[0076] A second aspect of the present invention provides a Pasteurella multocida egg yolk antibody for sheep, wherein the Pasteurella multocida egg yolk antibody is prepared by the method described in the first aspect of the present invention.
[0077] This invention has at least the following beneficial effects:
[0078] (1) The technical solution provided by the present invention has the advantages of comprehensive serotype coverage, strong protective efficacy, use of animal-isolated strains with strong specificity, high antibody purity, low side reaction rate, fast onset of action, no drug resistance and residue, etc., and can be used for prevention and treatment.
[0079] (2) The preparation process of this invention is simple and low-cost. It does not require complex column chromatography equipment. Purification can be achieved simply by centrifugation, precipitation and dialysis. The equipment investment cost is reduced by 60%. The raw material is eggs, which are readily available and inexpensive. When produced on a large scale, the cost per milliliter of antibody is only 50% of that of existing products, resulting in significant economic benefits.
[0080] (3) The ovine Pasteurella egg yolk antibody prepared by the present invention has stable titer and long shelf life. Through standardized immunization procedures and purification processes, the antibody titer fluctuation between batches is ≤15%. After being stored at 2-8℃ for 12 months, the titer decrease rate is ≤10%, which is convenient for long-term storage in farms. Attached Figure Description
[0081] Figure 1 PCR specificity identification of Pasteurella A, D, and F serotypes in Example 1;
[0082] Figure 2 For the SDS-PAGE electrophoresis antibody purity detection in Example 1;
[0083] Figure 3 This is for monitoring antibody levels in vivo after injection of Pasteurella A, D, and F egg yolk antibodies in Example 1. Detailed Implementation
[0084] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0085] Existing vaccines have long immunization cycles and poor immunization effects in young animals, making them unable to cope with sudden outbreaks of disease; antibiotic treatment is prone to drug resistance and drug residues, endangering food safety.
[0086] Existing Pasteurella egg yolk antibodies cover only a single serotype (currently only rabbit-derived Pasteurella D egg yolk antibodies), have low purity (≤70%), unstable titers (large batch-to-batch variations), high clinical adverse reaction rates (≥15%), and insufficient protective efficacy (≤70%). Egg yolk antibodies prepared from strains isolated from non-native animals are less effective. Existing preparation processes are complex (requiring multiple column chromatography steps), require high equipment investment, and are difficult to scale up for production.
[0087] In summary, the present invention aims to provide a sheep Pasteurella egg yolk antibody that covers all epidemic serotypes (A, D, and F serotypes), has a purity ≥90%, a titer ≥1:128, a simple preparation process, and no side effects, to achieve rapid prevention (effective within 24 hours) and highly effective treatment (cure rate ≥80%) of sheep Pasteurella infection, while avoiding drug resistance and drug residue problems.
[0088] The method for preparing Pasteurella multocida egg yolk antibodies of the present invention includes five core steps: "immunogen preparation → chicken immunization → egg yolk antibody extraction → purification → quality detection".
[0089] The Pasteurella multocida egg yolk antibody of the present invention consists of two parts: an "immunogen formula" and an "antibody extraction and purification reagent formula," as detailed below:
[0090] 1. Immunogen formulation (used to immunize hens to produce specific antibodies)
[0091] (1) Pasteurella strain combination
[0092] Select one strain each of Pasteurella multocida (serotypes A, D, and F) isolated from sheep. Mix them in a volume ratio of 1:1:1.
[0093] (2) Culture medium formula for the strain (Martin broth medium, 1L)
[0094] Table 1
[0095]
[0096] In addition, the final pH of the culture medium is 7.2-7.4, which is suitable for the growth of Pasteurella multocida.
[0097] (3) Immune adjuvant formulation
[0098] Immunoadjuvant: Inactivated bacterial vaccine and aluminum hydroxide adjuvant are mixed at a volume ratio of 5:1.
[0099] (4) Inactivated vaccine concentration
[0100] After incubation at 37℃ and 160 rpm / min for 18 h on a shaker, the concentrations of the three bacterial strains were adjusted to 1×10⁻⁶. ^9 CFU / mL, mixed at a ratio of 1:1:1, then formaldehyde solution was added to a final concentration of 0.3% (v / v) for inactivation, thus preparing an inactivated vaccine for immunization.
[0101] 2. Antibody extraction and purification reagent formulation
[0102] (1) Egg yolk dilution buffer (PBS buffer, 0.01 mol / L, pH 7.4)
[0103] Table 2
[0104]
[0105] (2) Ammonium sulfate precipitate (used for antibody purification)
[0106] Primary precipitation: Ammonium sulfate saturation 38% (calculated based on a saturated ammonium sulfate solution at 20℃ (541 g / L), add 69.2 mL of 38% saturated ammonium sulfate solution per 100 mL of crude extract);
[0107] Secondary precipitation: Ammonium sulfate saturation 28% (45.2 mL of 38% saturated ammonium sulfate solution is added to every 100 mL of the first purification solution).
[0108] (3) Dialysate
[0109] Similar to "egg yolk dilution buffer" (0.01 mol / L PBS, pH 7.4), it is used to remove ammonium sulfate residue.
[0110] 3. Final yolk antibody product specifications
[0111] Potency: ≥1:128 (test tube agglutination method, for Pasteurella multocida types A / C);
[0112] Purity: ≥90% (SDS-PAGE electrophoresis grayscale analysis);
[0113] Sterility: Meets the sterility testing requirements of the "Regulations for Veterinary Biological Products of the People's Republic of China" (2020 Edition);
[0114] Pyrogenicity: Detected using the rabbit method, no pyrogenic reaction was observed;
[0115] Stability: After 12 months of storage at 2-8℃, the potency decrease rate is ≤10%.
[0116] Based on the above technical solutions, the present invention provides the following embodiments:
[0117] Example 1
[0118] This embodiment provides a method for preparing Pasteurella multocida egg yolk antibodies for sheep, including the following steps:
[0119] Step 1: Immunogen Preparation (Inactivated Pasteurella M. A, D, and F Types Vaccine)
[0120] Strains resuscitation and culture:
[0121] Take frozen bacterial cultures of Pasteurella multocida types A, D, and F and revive them with modified Martin broth medium (incubate at 37°C for 160 rpm / min for 18 h).
[0122] The revived bacterial culture was inoculated into fresh modified Martin broth medium at a volume ratio of 1:100 and cultured at 37°C with shaking at 160 rpm / min for 18 h.
[0123] The bacterial concentration was adjusted to 1×10⁻⁶ using McFarland turbidimetric tubes and plate counts. ^9 CFU / mL.
[0124] Bacterial inactivation:
[0125] Slowly add 37% formaldehyde solution to the bacterial culture until the final concentration is 0.3% (v / v), and inactivate by shaking at 37°C for 24 hours;
[0126] Spread 0.1 mL of the inactivated bacterial solution onto a modified Martin agar plate and incubate at 37°C for 48 h to confirm sterile growth (inactivation qualified).
[0127] Adding adjuvants to prepare vaccines:
[0128] Immunization vaccine: Inactivated bacterial vaccine and aluminum hydroxide adjuvant are mixed at a volume ratio of 5:1 to prepare immunization vaccine.
[0129] Step Two: Hen Immunization Program (To Obtain High-Immune Eggs)
[0130] Chicken selection: Select healthy Leghorn chickens at 20 weeks of age (egg production rate ≥90%), with 50 chickens per group. Test serum Pasteurella antibodies in advance (titer ≤1:8, to exclude maternal antibody interference).
[0131] Immunization regimen:
[0132] Day 0 (initial immunization): Intramuscular injection of 0.5 mL of inactivated vaccine per animal (injection site: leg muscle);
[0133] Day 14 (first booster immunization): Intramuscular injection of 0.5 mL of inactivated vaccine per animal;
[0134] Day 28 (Second booster immunization): Intramuscular injection of 1.0 mL of inactivated vaccine per animal;
[0135] Egg collection: Seven days after the second booster immunization, the antibody titer of egg yolks is tested (≥1:128). Once qualified, eggs are collected and the collection continues for 4 weeks (during which the antibody titer remains stable).
[0136] Egg pretreatment: After rinsing the collected eggs with clean water, immerse them in 0.1% benzalkonium chloride solution for 10 minutes for disinfection, then rinse them 3 times with sterile distilled water and air dry for later use.
[0137] Step 3: Crude extraction of egg yolk antibodies
[0138] Egg yolk separation:
[0139] On a sterile operating table, crack open the sterilized egg, separate the yolk with sterile tweezers (remove the egg white and yolk membrane), and collect the yolk fluid;
[0140] Dilution and stirring:
[0141] Mix egg yolk solution and PBS buffer at a ratio of 1:3 (v / v), pour into a sterile mixing vessel, and stir at 25°C and 60 rpm for 30 minutes to fully dissolve the egg yolk.
[0142] Settling and centrifugation:
[0143] The mixture was placed in a 4°C refrigerator and allowed to stand for 12 hours to allow the impurities to precipitate fully. Then it was transferred to a centrifuge tube and centrifuged at 4°C and 4000 rpm for 20 minutes. The supernatant (i.e., crude extract of egg yolk antibodies) was collected, and the bottom precipitate (mainly egg yolk lipoprotein) was discarded.
[0144] Step 4: Purification of egg yolk antibodies (double ammonium sulfate precipitation + dialysis)
[0145] One settling:
[0146] The crude extract was placed in a 4°C water bath, and a 38% saturated ammonium sulfate solution was slowly added while stirring (dropping rate 1 mL / min to avoid excessively high local concentrations).
[0147] After adding the ingredients, continue stirring at 4°C for 4 hours to allow the antibody to precipitate fully.
[0148] Centrifuge at 4℃ and 4000rpm for 20min, discard the supernatant (containing contaminating proteins), and collect the antibody precipitate at the bottom.
[0149] One-time dissolution and dialysis:
[0150] The antibody precipitate was dissolved in an equal volume of PBS buffer (the same volume as the crude extract) and stirred until completely clear;
[0151] The solution was placed into a dialysis bag (molecular weight cutoff 8000-14000 Da), and then placed in a container containing PBS dialysis solution. Dialysis was performed at 4°C for 24 hours, and the dialysis solution was changed every 6 hours (4 times in total) to remove ammonium sulfate residue.
[0152] Secondary precipitation and dialysis:
[0153] After dialysis, slowly add 28% saturated ammonium sulfate solution to the antibody solution, stir at 4°C for 4 hours, centrifuge (in the same dissolution and dialysis cycle), and collect the precipitate;
[0154] After dissolving the precipitate in PBS buffer, dialyze again for 24 hours (changing the buffer 4 times) to obtain purified egg yolk antibody solution.
[0155] Step 5: Antibody quality testing and packaging
[0156] Potency assay: The purified antibody was serially diluted (1:16, 1:32, 1:64, 1:128, 1:256) using the tube agglutination method. 100 μL of diluent was added to each well, followed by 100 μL of 1×10⁻⁶ HCl. ^8 The highest dilution that produces obvious agglutination (a decrease in liquid turbidity ≥50%) in a CFU / mL Pasteurella bacterial suspension incubated at 37°C for 24 hours is the potency, which should be ≥1:128.
[0157] Purity testing: SDS-PAGE electrophoresis was performed (separating gel concentration 12%, stacking gel concentration 5%). After electrophoresis, Coomassie brilliant blue staining was used, and antibody purity was calculated using grayscale analysis software. It must be ≥90%.
[0158] Aseptic and pyrogen detection:
[0159] Sterility test: Spread 0.1 mL of antibody solution onto a modified Martin agar plate and incubate at 37°C for 48 h. No bacterial growth indicates that the test is successful.
[0160] Pyrogen detection: In accordance with the "Regulations for Veterinary Biological Products", antibody solution (10mL / kg body weight) was injected into the marginal ear vein of rabbits and observed for 3 days. The rabbits were considered qualified if their body temperature increased by ≤0.6℃.
[0161] Dispensing: After filtering the qualified antibody solution through a sterile filter membrane (0.22μm), dispense it into sterile vials (10mL / vial), seal and store at 2℃.
[0162] Example 2
[0163] This embodiment provides a method for preparing Pasteurella multocida egg yolk antibodies for sheep, which is the same as in Embodiment 2, except that:
[0164] The revived bacterial culture was inoculated into fresh modified Martin broth medium at a volume ratio of 1:100 and cultured at 37°C with shaking at 160 rpm / min for 20 h.
[0165] The qualified antibody solution is filtered through a sterile filter membrane (0.22μm), dispensed into sterile vials (20mL / vial), sealed, and stored at 5℃.
[0166] Example 3
[0167] This embodiment provides a method for preparing Pasteurella multocida egg yolk antibodies for sheep, which is the same as in Embodiment 2, except that:
[0168] The revived bacterial culture was inoculated into fresh modified Martin broth medium at a volume ratio of 1:100 and cultured at 37°C with shaking at 160 rpm / min for 24 h.
[0169] After the qualified antibody solution is filtered through a sterile filter membrane (0.22μm), it is dispensed into sterile vials (10mL / vial), sealed, and stored at 8℃.
[0170] A series of characterizations were performed on Example 1 above, and the results are as follows:
[0171] like Figure 1 As shown, Pasteurella multocida serotypes A, D, and F were identified by specific PCR. Type A: 1045 bp, Type D: 648 bp, Type F: 851 bp.
[0172] Figure 2 For SDS-PAGE electrophoresis antibody purity detection.
[0173] Figure 3 This study monitors antibody levels in the body after injection of Pasteurella A, D, and F yolk antibodies. The graphs from left to right correspond to A, D, and F, respectively.
[0174] In addition, bar charts were used to compare the titers of egg yolk antibodies in multiple batches, and the results are shown in Table 3 below:
[0175] Table 3. Bar chart comparing antibody titers in multiple batches of egg yolk.
[0176]
[0177] Compared with existing technologies for the prevention and control of ovine pasteurellosis and for the preparation of egg yolk antibodies, this invention has the following significant advantages:
[0178] ①Comprehensive serotype coverage, strong protective efficacy:
[0179] For the first time, immunization with three strains of type A, D, and F can simultaneously address the main pathogenic serotypes of ovine pasteurellosis, achieving a clinical protection rate of 90% (far higher than the 70% of existing single serotype antibodies), and the protective effect on lambs is equally stable (avoiding interference from maternal antibodies).
[0180] ②Using native animals to isolate strains, resulting in high specificity:
[0181] These strains retain the natural pathogenicity characteristics of sheep and are highly homologous in genotype and antigenicity to the prevalent strains that cause pasteurellosis outbreaks in large-scale farms. This avoids the problem of large antigenic differences between non-native animal strains (such as rabbit-derived and avian-derived Pasteurella) and sheep pathogenic strains, laying the foundation for the subsequent production of specific antibodies.
[0182] ③ High antibody purity and low side reaction rate:
[0183] Through the "double ammonium sulfate precipitation + dialysis" process, the antibody purity is increased to over 90%, and more than 90% of impurities such as yolk lipoprotein are removed. Clinical trials show that after injection into 1-month-old lambs, the local swelling rate is ≤2%, which is far lower than the 15% of existing products, and the safety is significantly improved.
[0184] ④ It takes effect quickly and has no drug resistance or residue.
[0185] The antibodies prepared by this invention can be applied to, but are limited to, the following fields:
[0186] Preventive application: Specific protection is produced within 24 hours after injection, which can quickly respond to sudden outbreaks (current vaccines take 2-4 weeks to take effect).
[0187] Therapeutic applications: The cure rate for sick sheep reaches 80%, and the antibody mechanism of action is "specific binding to bacterial antigens", with no risk of antibiotic resistance and no residues in meat or milk, ensuring food safety.
[0188] In summary, the preparation process of this invention is simple and low-cost: it eliminates the need for complex column chromatography equipment, achieving purification solely through centrifugation, precipitation, and dialysis, reducing equipment investment costs by 60%; the raw material is eggs (easily available and inexpensive), and the cost per milliliter of antibody in large-scale production is only 50% of existing products, resulting in significant economic benefits. Furthermore, the antibodies obtained by this invention exhibit stable titers and a long shelf life: through standardized immunization procedures and purification processes, batch-to-batch antibody titer fluctuations are ≤15% (compared to ≥40% for existing products); after storage at 2-8℃ for 12 months, the titer decline rate is ≤10%, facilitating long-term storage by farms.
[0189] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.
[0190] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for preparing Pasteurella multocida egg yolk antibodies for sheep, characterized in that, Includes the following steps: S1, Immunogen preparation: Take frozen bacterial suspensions of Pasteurella multocida types A, D, and F, revive and culture the strains using modified Martin broth medium, then inactivate the bacterial suspensions, and finally add adjuvants to prepare vaccines. S2, Hen Immunization: Select healthy 20-week-old Leghorn chickens, inject them intramuscularly with inactivated bacterial vaccine, test the yolk antibody titer in the eggs, and start collecting eggs after they pass the test. Eggs are then pre-treated. S3, crude extraction of egg yolk antibodies: Separate the egg yolk, collect the yolk fluid, add PBS buffer in a certain proportion to fully dissolve the egg yolk, let stand and centrifuge to obtain crude extract; S4, Egg yolk antibody purification: The crude body fluid was precipitated with a 38% saturated ammonium sulfate solution, the antibody precipitate was collected, dissolved in PBS buffer, dialyzed once, then a 28% saturated ammonium sulfate solution was added for a second precipitation, and finally dialyzed twice to obtain purified egg yolk antibody solution. S5, Antibody Quality Testing and Packaging: The antibodies undergo quality testing, and once they pass the test, they are repackaged.
2. The method for preparing Pasteurella multocida egg yolk antibody according to claim 1, characterized in that, The specific steps for preparing the S1 immunogen are as follows: S11, strain recovery and culture: Take frozen bacterial cultures of Pasteurella multocida types A, D, and F, revive them with modified Martin broth medium, and incubate at 37°C and 160 rpm / min for 18 h; The revived bacterial culture was inoculated into fresh modified Martin broth medium at a volume ratio of 1:100 and cultured at 37°C with shaking at 160 rpm / min for 18-24 h. The bacterial concentration was adjusted to 1×10⁻⁶ using McFarland turbidimetric tubes and plate counts. ^9 CFU / mL: S12, bacterial inactivation: Slowly add 37% formaldehyde solution to the bacterial culture until the final concentration is 0.3% (v / v), and inactivate by shaking at 37°C for 24 hours; Spread 0.1 mL of the inactivated bacterial solution onto a modified Martin agar plate and incubate at 37°C for 48 h to confirm sterile growth. S13, Adding adjuvants to prepare the vaccine: An inactivated bacterial vaccine and aluminum hydroxide adjuvant were mixed at a volume ratio of 5:1 to prepare an immunization vaccine.
3. The method for preparing Pasteurella multocida egg yolk antibody according to claim 2, characterized in that, The specific steps for immunizing S2 hens are as follows: Chicken selection: Select healthy 20-week-old Leghorn chickens with an egg production rate of ≥90%, with 50 chickens per group. Test the serum of Pasteurella antibodies in advance, with a titer of ≤1:8 to exclude interference from maternal antibodies. Immunization regimen: Day 0, initial immunization: intramuscular injection of 0.5 mL of inactivated vaccine per animal; Day 14, first booster immunization: 0.5 mL of inactivated vaccine per animal via intramuscular injection; On day 28, the second booster immunization was administered: 1.0 mL of inactivated vaccine was injected intramuscularly per animal. Egg collection: Seven days after the second booster immunization, the titer of egg yolk antibodies in the eggs was tested to see if it was ≥1:
128. If it was qualified, eggs were collected and collected continuously for 4 weeks. Egg pretreatment: After rinsing the collected eggs with clean water, immerse them in a 0.1% benzalkonium chloride solution for 10 minutes for disinfection, then rinse them three times with sterile distilled water and air dry them for later use.
4. The method for preparing Pasteurella multocida egg yolk antibody according to claim 3, characterized in that, The specific steps for crude extraction of S3 egg yolk antibodies are as follows: S31, yolk separation: On a sterile operating table, crack open the sterilized egg, separate the yolk with sterile tweezers, remove the egg white and yolk membrane, and collect the yolk fluid; S32, Dilution and Stirring: Mix egg yolk solution and PBS buffer at a ratio of 1:3 (v / v), pour into a sterile mixing vessel, and stir at 25°C and 60 rpm for 30 minutes to fully dissolve the egg yolk. S33, Settling and Centrifugation: The mixture was placed in a 4°C refrigerator and allowed to stand for 12 hours to allow the impurities to precipitate fully. Then it was transferred to a centrifuge tube and centrifuged at 4°C and 4000 rpm for 20 minutes. The supernatant, which is the crude extract of egg yolk antibodies, was collected, and the bottom precipitate was discarded.
5. The method for preparing Pasteurella multocida egg yolk antibody according to claim 4, characterized in that, The specific steps for purifying the S4 egg yolk antibody are as follows: S41, initial sedimentation: The crude extract was placed in a 4°C water bath, and a 38% saturated ammonium sulfate solution was slowly added while stirring at a dropping rate of 1 mL / min to avoid excessively high local concentrations. After adding the antibody, continue stirring at 4°C for 4 hours to allow the antibody to precipitate fully. Centrifuge at 4°C and 4000 rpm for 20 minutes, discard the supernatant, and collect the antibody precipitate at the bottom. S42, one-step dissolution and dialysis: Dissolve the antibody precipitate in an equal volume of PBS buffer and stir until completely clear; The solution was placed into a dialysis bag and then into a container containing PBS dialysis solution. Dialysis was performed at 4°C for 24 hours, with the dialysis solution being changed every 6 hours to remove any residual ammonium sulfate. S43, secondary precipitation and dialysis: After dialysis, slowly add 28% saturated ammonium sulfate solution to the antibody solution, stir at 4°C for 4 hours, centrifuge, and collect the precipitate; After dissolving the precipitate in PBS buffer, dialyze again for 24 hours to obtain purified egg yolk antibody solution.
6. The method for preparing Pasteurella multocida egg yolk antibody according to claim 5, characterized in that, The antibody quality detection in S5 includes, but is not limited to, the following items: ①Valence testing: The purified antibody was diluted using the tube agglutination method at ratios of 1:16, 1:32, 1:64, 1:128, and 1:256, with 100 μL of diluent added to each well, followed by 100 μL of 1×10⁻⁶ precipitate. ^8 The highest dilution at which obvious agglutination occurs after incubating Pasteurella multocida culture at 37°C for 24 hours is the titer, which should be ≥1:
128. ②Purity testing: Perform SDS-PAGE electrophoresis, stain with Coomassie Brilliant Blue after electrophoresis, and calculate antibody purity using grayscale analysis software; the purity must be ≥90%. ③ Sterility test: Spread 0.1 mL of antibody solution onto a modified Martin agar plate and incubate at 37°C for 48 h. No bacterial growth indicates the plate is ready. ④ Heat source detection: According to the "Regulations for Veterinary Biological Products", antibody solution was injected into the marginal ear vein of rabbits and observed for 3 days. The rabbits were considered qualified if their body temperature increased by ≤0.6℃.
7. The method for preparing Pasteurella multocida egg yolk antibody according to claim 1, characterized in that, The specific steps for dispensing in S5 are as follows: The qualified antibody solution is filtered through a 0.22μm sterile filter membrane, dispensed into sterile vials, sealed, and stored at 2-8℃.
8. The method for preparing Pasteurella multocida egg yolk antibody according to claim 1, characterized in that, The composition of the Martin broth culture medium is as follows: 10g peptone, 5g yeast extract, 2g glucose, 5g sodium chloride, 2g dipotassium hydrogen phosphate, 1000mL distilled water; The pH value of the Martin broth medium is 7.2-7.
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9. The method for preparing Pasteurella multocida egg yolk antibody according to claim 1, characterized in that, The PBS buffer solution has the following composition: Potassium dihydrogen phosphate 0.27g, disodium hydrogen phosphate 2.9g, sodium chloride 8.0g, distilled water 1000mL; The pH value of the PBS buffer is 7.
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10. A Pasteurella multocida egg yolk antibody for sheep, characterized in that, The Pasteurella multocida egg yolk antibody for sheep is prepared by the method described in any one of claims 1 to 9.