A preparation of PEDV yolk antibody and a preparation method thereof

Abstract

The application discloses a PEDV yolk antibody preparation, which is an antibody preparation for feed, is a high-temperature-resistant and stomach-passing antibody preparation, and comprises an antibody core, a stomach-passing layer and a high-temperature-resistant layer; the antibody core comprises PEDV antibody dry powder and starch; the stomach-passing layer comprises polyvinyl alcohol phthalate and calcium stearate; and the high-temperature-resistant layer comprises ethyl cellulose and calcium stearate. The PEDV yolk antibody preparation is more stable, the double-layer coating can better protect the activity of the antibody, the antibody preparation is not prone to deterioration and failure in a harsh environment such as high temperature and humidity, the antibody is released in the intestinal tract, and the PED prevention and treatment effect is directly, durably and effectively exerted, the antibody preparation is combined with the feed, the functional feed is prepared, and the occurrence of PED is prevented and treated. In clinical use, the application is more convenient and more suitable for actual production requirements.

Description

Technical Field

[0001] This invention relates to a high-temperature resistant, gastric-passing PEDV egg yolk antibody preparation for feed and its preparation process, belonging to the fields of biotechnology and feed. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is the pathogenic microorganism that causes porcine epidemic diarrhea (PED). It belongs to the family Coronaviridae and the genus Alviridae. It can affect pigs of all ages, characterized by lethargy, weight loss, vomiting, and watery diarrhea. In particular, it can cause mortality rates as high as 100% in suckling piglets under 7 days old.

[0003] PEDV is now widespread worldwide, and its prevalence in my country is increasing year by year. It has a long duration of illness and is prone to recurrent infections. During its global spread, PEDV has produced many variant strains. If a highly pathogenic strain becomes prevalent, it will have an immeasurable impact on my country's pig farming industry.

[0004] To date, there are no effective treatments for PEDV, and vaccination remains the most common preventative measure. However, traditional vaccine injections rarely induce mucosal immunity, resulting in a lack of IgA antibodies secreted by the intestinal mucosa and thus poor immunization efficacy. Oral vaccines have the advantage of generating an immune response compared to other routes of administration, but they also have disadvantages such as reduced effectiveness. Clinically, feed regurgitation is often used to control PEDV, which is effective in the short term but carries the risk of virus transmission and can easily lead to the spread of other diseases. Oral antibodies not only eliminate the risk of virus transmission, but the antibody manufacturing process also eliminates the presence of other pathogenic microorganisms, thus compensating for the shortcomings of feed regurgitation. Heat-resistant antibodies can be directly used in feed production while maintaining their activity. Antibodies treated through the stomach can be released slowly in the intestine, better inhibiting the proliferation of PEDV in the intestine and thus exerting a good preventative effect. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention aims to provide a heat-resistant, gastric-passing PEDV egg yolk antibody preparation for feed and its preparation process. This allows for convenient and rapid clinical application for the prevention and treatment of PED. The antibody preparation obtained by this invention effectively protects the antibody, and its special acid-resistant layer allows it to pass through the stomach, thus achieving intestinal release. The heat-resistant layer helps maintain the antibody's activity during feed pelleting, facilitating clinical use.

[0006] This invention provides a PEDV egg yolk antibody preparation, which is a feed-grade antibody preparation. The antibody preparation is a heat-resistant, gastric-passing antibody preparation, comprising an antibody core, a gastric-passing layer, and a heat-resistant layer. The antibody core comprises PEDV antibody powder and starch. The gastric-passing layer comprises polyvinyl phthalate and calcium stearate. The heat-resistant layer comprises ethyl cellulose and calcium stearate.

[0007] Preferably, the antibody core comprises PEDV antibody powder and starch in a mass ratio of 9-10:1-1.5. Further, the mass ratio of PEDV antibody powder to starch is preferably 6-10:1, more preferably 6:1, 7:1, 8:1, 9:1, or 10:1.

[0008] Preferably, in any of the above embodiments, the gastric layer comprises polyvinyl phthalate (PVAP) and calcium stearate in a mass ratio of 2.5–3:1–1.5. Further, the mass ratio of PVAP to calcium stearate is preferably 5–9:3, more preferably 5:3, 2:1, 7:3, 8:3, or 3:1.

[0009] In any of the above-mentioned preferred embodiments, the high-temperature resistant layer comprises ethyl cellulose and calcium stearate in a mass ratio of 1–2:1–1.5. Further, the mass ratio of ethyl cellulose to calcium stearate is preferably 2:1-3, more preferably 2:1, 1:1, or 2:3.

[0010] In any of the above-mentioned preferred embodiments, the PEDV antibody dry powder is obtained by immunizing laying hens with porcine epidemic diarrhea virus CV777 strain, collecting highly immunized eggs, extracting antibodies from the egg yolks, and then freeze-drying them.

[0011] Preferably, the neutralizing titer of the PEDV egg yolk antibody preparation is 1:7 TCID. 50 above.

[0012] In any of the above-mentioned preferred embodiments, the coating amount of the PEDV antibody dry powder in the PEDV egg yolk antibody preparation can reach 80% to 86% of the total mass.

[0013] The gastric-passing layer, polyvinyl phthalate, is an enteric-coated material. Calcium stearate possesses high-temperature resistance, acid resistance, and water resistance; their combined use ensures that the antibody passes through the stomach and is released into the intestines. Ethyl cellulose, with its good film-forming properties and insolubility in water, along with calcium stearate, serves as a high-temperature resistant layer, increasing the shelf life of the formulation and ensuring that the antibody can withstand high-temperature processes during preparation without losing its activity.

[0014] This invention also provides a method for preparing the above-mentioned PEDV egg yolk antibody preparation, comprising the following steps:

[0015] Preparation of antigen α:

[0016] The porcine epidemic diarrhea virus was revived and amplified, and the viral titer was determined to be 1x10⁻⁶. 7 If the TCID is above 50, add 0.1% formaldehyde and inactivate at 37°C for 2 hours to obtain the desired immunogenic antigen.

[0017] The specific steps for formaldehyde inactivation are conventional techniques in this field, with a preferred formaldehyde:virus liquid volume ratio of 1:1000 for inactivation.

[0018] b antibody preparation:

[0019] The antigen obtained in step a was emulsified to prepare a vaccine for immunizing laying hens. Each immunization was performed 14 days apart, with three immunizations. Seven days after the third immunization, eggs were collected, and after caprylic acid extraction, filtration for sterilization, and concentration, the desired antibody solution was obtained. The neutralizing titer of PEDV yolk antibodies in the concentrated antibody solution was all within 1:10 TCID. 50 above;

[0020] Preferably, the antigen obtained in step a is emulsified. The emulsion oil phase contains 94 parts white oil, 6 parts Span-80, and 2 parts aluminum stearate; the aqueous phase contains 96 parts virus solution and 4 parts Tween 80. 3 parts of the oil phase are placed in an emulsification tank and stirred at 7000 rpm. 2 parts of the aqueous phase are slowly added and stirred at 12000 rpm for 3-5 minutes to prepare the vaccine for immunizing laying hens. The first immunization is 1.5 mL / hen, followed by a second immunization 14 days later at 2 mL / hen. A third immunization 14 days later at 2 mL / hen is administered. On the 7th day after the third immunization, highly immunized eggs are collected. The solution is then extracted with 0.1% caprylic acid, concentrated 4-5 times using a hollow fiber ultrafiltration column with a molecular weight cutoff of 10 kD, and sterilized by filtration with a 0.22 μm cartridge filter to obtain the desired antibody solution. The neutralizing titer of the PEDV yolk antibody in the concentrated antibody solution is 1:10 TCID. 50 above;

[0021] c antibody lyophilization:

[0022] Add 3% trehalose, 1% mannitol and 1% astragalus polysaccharide to the antibody solution obtained in step b, stir well, aseptically dispense and quickly freeze-dry under vacuum to obtain PEDV egg yolk antibody lyophilized powder.

[0023] d-antibody core:

[0024] After mixing PEDV lyophilized powder with starch evenly, pelleting, drying, and sieving can yield particles with a diameter of 0.6-1 mm, which are the antibody cores.

[0025] e passes through the gastric layer:

[0026] Polyvinyl phthalate and calcium stearate are dissolved in an 80%–85% ethanol solution, with ethanol accounting for approximately 70%–82%, to obtain a gastric coating solution. The gastric coating solution is sprayed onto the antibody core, dried, and sieved through a 30-mesh sieve to obtain a granule formulation, in which the gastric coating layer accounts for 2.5%–3.5% of the total mass.

[0027] f. High-temperature resistant layer:

[0028] Ethyl cellulose and calcium stearate are dissolved in an 80%–85% ethanol solution, with ethanol accounting for approximately 70%–82%, to obtain a high-temperature resistant coating solution. The high-temperature resistant coating solution is sprayed onto the granules obtained in step e, dried, and sieved through a 30-mesh sieve. The resulting granules contain a high-temperature resistant layer that accounts for 2.5%–3.5% of the total mass.

[0029] In the product obtained after the antibody core, gastric layer and high-temperature resistant layer are dried during the preparation process, the ethanol is completely evaporated and the water content is about 6% of the total mass.

[0030] In a preferred embodiment of the present invention, the mass ratio of PEDV antibody dry powder to starch is 10:1, the mass ratio of polyvinyl phthalate (PVAP) to calcium stearate is 3:1 or 2:1, and the mass ratio of ethyl cellulose to calcium stearate is 2:1 or 1:1.

[0031] In a preferred embodiment of the present invention, the mass ratio of PEDV antibody dry powder to starch is 10:1, the mass ratio of polyvinyl phthalate (PVAP) to calcium stearate is 3:1, and the mass ratio of ethyl cellulose to calcium stearate is 2:1.

[0032] In a preferred embodiment of the present invention, the PEDV egg yolk antibody preparation is prepared according to the following method:

[0033] antigen preparation

[0034] Porcine epidemic diarrhea virus was revived and amplified using Vero cells, and the viral titer was determined to be 1x10⁻⁶. 7 If the TCID is above 50, add 0.1% formaldehyde and inactivate at 37°C for 2 hours to obtain the desired immunogenic antigen.

[0035] b antibody preparation

[0036] The antigen obtained in step a was emulsified. The oil phase of the emulsion contained 94 parts white oil, 6 parts Span-80, and 2 parts aluminum stearate; the aqueous phase contained 96 parts virus solution and 4 parts Tween 80. Three parts of the oil phase were placed in an emulsification tank and stirred at 7000 rpm. Two parts of the aqueous phase were slowly added and stirred at 12000 rpm for 3-5 minutes to prepare the vaccine for immunizing laying hens. The first immunization was 1.5 mL / hen, followed by a second immunization 14 days later at 2 mL / hen. A third immunization 14 days later at 2 mL / hen was administered. On the 7th day after the third immunization, highly immunized eggs were collected. The solution was extracted with 0.1% caprylic acid, concentrated 4-5 times using a hollow fiber ultrafiltration column with a molecular weight cutoff of 10 kD, and then sterilized by filtration with a 0.22 μm cartridge filter to obtain the desired antibody solution. The neutralizing titer of the PEDV egg yolk antibody in the concentrated antibody solution was above 1:10 TCID50.

[0037] c antibody lyophilized

[0038] Add 3% trehalose, 1% mannitol, and 1% astragalus polysaccharide to the antibody solution obtained in step b, stir well, aseptically dispense, and then rapidly freeze-dry under vacuum. This yields the PEDV egg yolk antibody lyophilized powder.

[0039] The trehalose used in this process can form a unique protective film at high temperatures, which adheres to the surface of the antibody to protect the antibody structure from damage. This further reduces the loss of antibody activity during the high-temperature preparation process.

[0040] d antibody core

[0041] After mixing PEDV lyophilized powder with starch evenly, pellets are made using a centrifugal pelletizing machine. After drying and sieving, particles with a diameter of 0.6-1 mm are obtained, which are the antibody cores.

[0042] e through the gastric layer

[0043] Polyvinyl phthalate (PVAP) and calcium stearate were dissolved in an 80%–85% ethanol solution, with ethanol accounting for approximately 70%–82%, to obtain a gastric coating solution.

[0044] Using a fluidized bed, the gastric coating solution is sprayed onto the antibody core, dried, and sieved through a 30-mesh sieve to obtain granule formulations, in which the gastric coating layer accounts for 2.5% to 3.5% of the total mass.

[0045] f High temperature resistant layer

[0046] Ethyl cellulose and calcium stearate are dissolved in an 80%–85% ethanol solution, with ethanol accounting for approximately 70%–82%, to obtain a high-temperature resistant coating solution.

[0047] Using a fluidized bed, the high-temperature resistant coating solution is sprayed onto the granules obtained in step e. After drying and sieving through a 30-mesh sieve, the high-temperature resistant layer accounts for 2.5% to 3.5% of the total mass of the granules.

[0048] Compared with existing technologies, the high-temperature resistant, gastric-passing PEDV egg yolk antibody preparation for feed and its preparation process described in this invention have the following advantages:

[0049] (1) The present invention provides a high-temperature resistant, gastric-passing PEDV egg yolk antibody preparation for feed and its preparation process. The obtained PEDV antibody preparation has more stable performance compared to traditional antibody preparations. The double-layer coating provides better protection for antibody activity than the single-layer coating. This allows the antibody preparation to withstand harsh environments such as high temperature and humidity without easily deteriorating or becoming ineffective.

[0050] (2) The feed-grade high-temperature resistant, gastric-passing PEDV egg yolk antibody preparation and its preparation process described in this invention have a gastric-passing layer, which enables the antibody preparation to pass through the stomach, avoiding the destruction of antibodies by gastric acid during oral administration of traditional antibodies, and allowing the antibodies to be released in the intestines, so as to play a more direct, long-lasting and effective role in preventing and treating PED.

[0051] (3) The high-temperature resistant, gastric-passing PEDV egg yolk antibody preparation for feed and its preparation process described in this invention have a high-temperature resistant coating layer, which can ensure that the antibody retains its activity during high-temperature feed preparation. This property enables the combination of antibody preparation with feed to produce functional feed and prevent the occurrence of PED. It is more convenient in clinical use and more in line with actual production needs. Attached Figure Description

[0052] Figure 1 The effect of high temperature on the neutralizing titer of PEDV egg yolk antibody preparations in preferred embodiments 1-5 of the present invention.

[0053] Figure 2 The effects of acidic environment on the neutralizing titer of PEDV egg yolk antibody preparations in preferred embodiments 1-5 of this invention.

[0054] Figure 3 The results of the challenge protection test using PEDV egg yolk antibody in preferred embodiment 2 of the present invention. Detailed Implementation

[0055] The present invention will be further described through the following specific embodiments to illustrate its advantages and features. However, this is not intended to limit the scope of the invention.

[0056] Example 1

[0057] PEDV egg yolk antibody preparation

[0058] A heat-resistant, gastric-passing PEDV egg yolk antibody formulation for feed, comprising an antibody core, a gastric-passing layer, and a heat-resistant layer. The antibody core comprises PEDV antibody powder and starch in a 10:1 mass ratio. The gastric-passing layer comprises polyvinyl phthalate (PVAP) and calcium stearate in a 3:1 mass ratio. The heat-resistant layer comprises ethyl cellulose and calcium stearate in a 2:1 mass ratio.

[0059] PEDV antibody powder is obtained by immunizing laying hens with the CV777 strain of porcine epidemic diarrhea virus, collecting highly immunized eggs, extracting antibodies from the egg yolks, and then freeze-drying them.

[0060] Furthermore, the neutralizing titer of the PEDV egg yolk antibody preparation is all within 1:7 TCID. 50 above.

[0061] The preparation method of the PEDV egg yolk antibody preparation in Example 1 is as follows:

[0062] antigen preparation

[0063] Porcine epidemic diarrhea virus was revived and amplified using Vero cells, and the viral titer was determined to be 1x10⁻⁶. 7 If the TCID is above 50, add 0.1% formaldehyde and inactivate at 37°C for 2 hours to obtain the desired immunogenic antigen.

[0064] b antibody preparation

[0065] The antigen obtained in step a was emulsified. The oil phase of the emulsion contained 94 parts white oil, 6 parts Span-80, and 2 parts aluminum stearate; the aqueous phase contained 96 parts virus solution and 4 parts Tween 80. Three parts of the oil phase were placed in an emulsification tank and stirred at 7000 rpm. Two parts of the aqueous phase were slowly added and stirred at 12000 rpm for 3-5 minutes to prepare the vaccine for immunizing laying hens. The first immunization was 1.5 mL / hen, followed by a second immunization 14 days later at 2 mL / hen. A third immunization 14 days later at 2 mL / hen was administered. On the 7th day after the third immunization, highly immunized eggs were collected. The solution was extracted with 0.1% caprylic acid, concentrated 4-5 times using a hollow fiber ultrafiltration column with a molecular weight cutoff of 10 kD, and then sterilized by filtration with a 0.22 μm cartridge filter to obtain the desired antibody solution. The neutralizing titer of the PEDV egg yolk antibody in the concentrated antibody solution was above 1:10 TCID50.

[0066] c antibody lyophilized

[0067] Add 3% trehalose, 1% mannitol, and 1% astragalus polysaccharide to the antibody solution obtained in step b, stir well, aseptically dispense, and then rapidly freeze-dry under vacuum. This yields the PEDV egg yolk antibody lyophilized powder.

[0068] d antibody core

[0069] PEDV lyophilized powder, starch, and ultrapure water are mixed evenly in a ratio of 10:1:1. The mixture is then pelletized using a centrifugal pelletizing machine. After drying and sieving, particles with a diameter of 0.6-1 mm are obtained, which are the antibody cores.

[0070] e through the gastric layer

[0071] Polyvinyl phthalate (PVAP) and calcium stearate were dissolved in an 80%–85% ethanol solution, with ethanol accounting for approximately 70%–82%, to obtain a gastric coating solution.

[0072] Using a fluidized bed, the gastric coating solution is sprayed onto the antibody core, dried, and sieved through a 30-mesh sieve to obtain granule formulations, in which the gastric coating layer accounts for 2.5% to 3.5% of the total mass.

[0073] f High temperature resistant layer

[0074] Ethyl cellulose and calcium stearate are dissolved in an 80%–85% ethanol solution, with ethanol accounting for approximately 70%–82%, to obtain a high-temperature resistant coating solution.

[0075] Using a fluidized bed, the high-temperature resistant coating solution is sprayed onto the granules obtained in step e. After drying and sieving through a 30-mesh sieve, the high-temperature resistant layer accounts for 2.5% to 3.5% of the total mass of the granules.

[0076] In the PEDV egg yolk antibody preparation obtained in Example 1, the final coating amount of the PEDV antibody dry powder can reach 80% to 86% of the total mass. The neutralizing titer of the PEDV egg yolk antibody preparation is all within 1:7 TCID. 50 above.

[0077] The specific steps for achieving antibody neutralization titer in the PEDV egg yolk antibody preparation are as follows:

[0078] I. 96-well cell plating

[0079] The Vero cell monolayer was digested with trypsin, centrifuged at 1500 rpm, and then cell culture medium was added for cell counting. The cell density was adjusted to 1 x 10⁻⁶ cells / mL. 5 Cells / mL. Then, 200 μL / well of the cell suspension was added to a 96-well plate and incubated overnight in a cell culture incubator. The cells can grow into a monolayer within 24 hours.

[0080] II. Virus TCID 50 Detection

[0081] The PEDV virus solution obtained in step a of Example 1 was diluted 10-fold. -1 ~10 -10For later use. Take a 96-well plate from section I, discard the cell culture medium, wash with PBS, add 50 μL of diluted virus solution to each well, perform 8 replicates for each dilution, and the rest are normal controls. Add 50 μL of cell maintenance medium to the normal controls, incubate in a cell culture incubator for 2 hours, discard all liquid, wash with PBS, add 150 μL of cell maintenance medium to each well, and culture and observe. Record the number of diseased wells each day, and continue to observe for 1 week. Calculate the TCID of the virus solution according to the Reed-Muench method. 50 .

[0082] III. Determination of antibody neutralization titer

[0083] 10g of the PEDV egg yolk antibody preparation was added to 100ml of sterile PBS, homogenized, and the supernatant was collected. The supernatant was centrifuged and filtered to obtain an antibody solution. The antibody solution was serially diluted, and an equal volume of the diluted antibody was mixed with the virus solution labeled in section II. The mixture was incubated in a cell culture incubator for 1 hour. The solution was then transferred to a 96-well plate prepared in section I, 50µL / well, with 8 replicates. After incubation for 2 hours, the plate was discarded, and the cells were washed with PBS. 150µL of cell maintenance medium was added to each well. Observations were recorded daily for one week, and the neutralizing titer of the antibody preparation was calculated.

[0084] Example 2

[0085] The PEDV egg yolk antibody preparations obtained in Example 1 were subjected to high-temperature resistance testing. Equal amounts of 20g of each of the PEDV egg yolk antibody preparations prepared in Example 1 were weighed and placed in an 85℃ oven for heat treatment for 10 minutes. After heat treatment, the samples were removed and allowed to return to room temperature. Three parallel tests were performed for each test.

[0086] The antibody neutralizing titer of the above-treated samples was determined according to the detection method in Example 1, Section III. The test results are as follows: Figure 1 As shown.

[0087] Example 3

[0088] The PEDV egg yolk antibody preparation obtained in Example 1 was tested for acid resistance. PBS (pH 7.4) was prepared by adjusting the pH to 2.5 with 2M HCl using 100 mL of PBS. 10 g of each of the PEDV egg yolk antibody preparations obtained in Example 1 were weighed and placed into separate 250 mL Erlenmeyer flasks. 100 mL of pH 2.5 PBS was added to each flask, and the flasks were incubated at 37°C for 30 min. After treatment, the flasks were filtered through gauze and washed 2-3 times with pH 7.4 PBS. Finally, the flasks were washed with 100 mL of pH 7.4 PBS into new Erlenmeyer flasks. Three parallel tests were performed for each assay.

[0089] The antibody neutralizing titer of the above-treated samples was determined according to the detection method in Example 1, Section III. The test results are as follows: Figure 2 As shown.

[0090] Example 4

[0091] The PEDV egg yolk antibody preparation obtained in Example 1 was subjected to a PEDV challenge protection test.

[0092] Sixty 30-day-old non-immunized weaned piglets were randomly divided into three groups of 20 piglets each. The specific steps are as follows:

[0093] Blank control group: fed with conventional pelleted feed.

[0094] Vaccine control group: fed with regular pelleted feed, each pig was injected with 2 mL of a live trivalent vaccine containing porcine transmissible gastroenteritis, porcine epidemic diarrhea (attenuated CV777), and porcine rotavirus (G5).

[0095] PEDV antibody group: fed pelleted feed supplemented with the A1 PEDV yolk antibody preparation in Example 1 (100g of PEDV yolk antibody preparation added to 100kg of feed).

[0096] Five days after the start of the experiment, a virulent strain of porcine epidemic diarrhea virus CV777 (containing 100 LD) was used. 50 The piglets were challenged with the virus, and each piglet was injected with 2 mL. The morbidity and mortality rates of each group of piglets were observed and recorded, and the protection rate was calculated. The experimental results are as follows: Figure 3 .

[0097] The experimental results showed that feeding pelleted feed containing PEDV egg yolk antibody preparation had a good protective effect, with a protection rate of up to 95%, while the mortality rate of the blank control was as high as 100%.

[0098] In summary, for PEDV egg yolk antibody preparations, the higher the proportion of lyophilized antibody powder in the core component, the higher the overall antibody titer. When the mass ratio of polyvinyl phthalate (PVAP) to calcium stearate in the gastric layer is 3:1, and the mass ratio of ethyl cellulose to calcium stearate in the heat-resistant layer is 2:1, the PEDV antibody preparation can maintain good antibody efficacy under high temperature and acidic environments. Furthermore, this protective effect is significantly better than that of monolayer-coated antibody preparations. This provides a basis for the clinical use of PEDV antibody preparations, specifically their formulation into functional feeds for the prevention and treatment of PED.

[0099] Example 5

[0100] The method for preparing the PEDV egg yolk antibody preparation in Example 5 was similar to that in Example 1, and the detection method for the obtained PEDV egg yolk antibody preparation was similar to that in Examples 1-4. The difference was that the components of the antibody core, gastric pass layer, and high-temperature resistant layer were adjusted in Example 5, and the composition ratios of the antibody core, gastric pass layer, and high-temperature resistant layer were studied and verified in groups A, B, C, and D, respectively.

[0101] Group A was used to produce 100g of PEDV egg yolk antibody preparation. The antibody core contained different mass ratios of antibody to starch (10:1, 7:1, and 4:1), the gastric pass layer contained a 3:1 mass ratio of polyvinyl phthalate (PVAP) to calcium stearate, and the heat-resistant layer contained a 2:1 mass ratio of ethyl cellulose to calcium stearate. PEDV antibody preparations A1, A2, and A3 were prepared separately. Table 1 shows the distribution ratios and dosages for each group. Groups B, C, and D were similar to Group A, except that the core was the same as A1. The mass ratios of polyvinyl phthalate (PVAP) to calcium stearate in the gastric pass layer of groups B, C, and D were 2:1, 3:1, and 4:1, respectively, and the mass ratios of ethyl cellulose to calcium stearate in the heat-resistant layer were 1:1, 2:1, and 3:1, respectively. Specific formulations are shown in Tables 2, 3, and 4 below. The comparative results of Example 5 are as follows: Figure 1 , Figure 2 As shown.

[0102] Table 1 Raw Material Formulation Group A

[0103]

[0104]

[0105] Note: The ratio of antibody lyophilized powder to starch is 10:1 and 9:1; the mass ratio of polyvinyl phthalate (PVAP) to calcium stearate in the gastric layer is 3:1; and the mass ratio of ethyl cellulose to calcium stearate in the high-temperature resistant layer is 2:1.

[0106] Table 2 Raw Material Formulation Group B

[0107]

[0108] Note: The ratio of starch to antibody lyophilized powder is 10:1; the mass ratio of polyvinyl phthalate (PVAP) to calcium stearate in the gastric layer is 2:1, 3:1, or 4:1; the mass ratio of ethyl cellulose to calcium stearate in the high-temperature resistant layer is 1:1.

[0109] Table 3 Raw material formulation group C

[0110]

[0111] Note: The ratio of starch to antibody lyophilized powder is 10:1; the mass ratio of polyvinyl phthalate (PVAP) to calcium stearate in the gastric layer is 2:1, 3:1, or 4:1; the mass ratio of ethyl cellulose to calcium stearate in the high-temperature resistant layer is 2:1.

[0112] Table 4 Raw Material Formulation Group D

[0113]

[0114]

[0115] Note: The ratio of starch to antibody lyophilized powder is 10:1; the mass ratio of polyvinyl phthalate (PVAP) to calcium stearate in the gastric layer is 2:1, 3:1, or 4:1; the mass ratio of ethyl cellulose to calcium stearate in the high-temperature resistant layer is 3:1.

[0116] Comparative Example

[0117] Group E was set up as a comparative example. The PEDV egg yolk antibody preparation and its preparation method in the comparative example differed from those in Example 1 in that Group E was a single-layer coating. The mass ratio of polyvinyl phthalate (PVAP), ethyl cellulose, and calcium stearate in the coating layer was 3:2:1, and the thickness of the coating layer was the same as the total thickness of the gastric-passing layer and the heat-resistant layer in Example 1. The specific ratios are shown in Table 4.

[0118] Table 4 Raw Material Formulation Group E

[0119]

[0120] Note: The ratio of antibody lyophilized powder to starch is 10:1, and the mass ratio of the coating layer polyvinyl phthalate (PVAP), ethyl cellulose, and calcium stearate is 3:2:1.

[0121] The effects of the comparative examples and the groups in Example 5 are compared, such as... Figure 1 , Figure 2 As shown, the neutralizing titer of the PEDV egg yolk antibody preparation obtained in the comparative example was significantly lower than that in Example 5.

[0122] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A PEDV yolk antibody preparation, the antibody preparation being an antibody preparation for feed, characterized by, The antibody preparation is a high-temperature resistant, gastro-resistant antibody preparation, which comprises an antibody core, a gastro-resistant layer and a high-temperature resistant layer; the antibody core comprises PEDV antibody dry powder and starch; the gastro-resistant layer comprises polyvinyl alcohol phthalate and calcium stearate with a mass ratio of 2.5-3:1-1.5; the high-temperature resistant layer comprises ethyl cellulose and calcium stearate with a mass ratio of 1-2:1-1.5; the gastro-resistant layer coating liquid is sprayed onto the antibody core, and the gastro-resistant layer accounts for 2.5%-3.5% of the total mass; the high-temperature resistant layer coating liquid is sprayed onto the antibody core and the gastro-resistant layer coated granular preparation obtained in the previous step, and the high-temperature resistant layer accounts for 2.5%-3.5% of the total mass, and the PEDV antibody dry powder is obtained by immunizing laying hens with porcine epidemic diarrhea virus CV777 strain, collecting high-immune hen eggs, extracting antibodies from the yolk, and freeze-drying the obtained antibodies.

2. The antibody formulation of claim 1, wherein The antibody core comprises PEDV antibody dry powder and starch with a mass ratio of 9-10:1-1.

5.

3. The antibody formulation of claim 1, wherein The neutralization titers of the PEDV yolk antibody preparations were all 1:7 TCID 50 The above.

4. A preparation method of the PEDV yolk antibody preparation according to any one of claims 1-3, comprising the following steps: a. Antigen preparation: The porcine epidemic diarrhea virus was recovered and amplified, and the titer reached 1x10 7 TCID 50 The above was added with 0.1% formaldehyde, and inactivated at 37°C for 2h to obtain the required immunizing antigen. b. Antibody preparation: The antigen obtained in step a is emulsified to prepare a vaccine, which is used to immunize laying hens. The immunization is performed three times with an interval of 14 days each time. On the 7th day after the third immunization, the eggs are collected, extracted with caprylic acid, filtered to remove bacteria, and concentrated to obtain the desired antibody solution. The PEDV yolk antibody neutralization titer of the antibody solution after concentration is all 1:10 TCID 50 The above; c. Antibody freeze-drying: In the antibody solution obtained in step b, 3% trehalose, 1% mannitol and 1% astragalus polysaccharide are added and stirred uniformly, and then sterilely packaged and rapidly freeze-dried under vacuum to obtain PEDV yolk antibody freeze-dried powder; d. Antibody core: After the PEDV freeze-dried powder and starch are uniformly mixed, they are prepared into pills, dried, and sieved to obtain granules with a diameter of 0.6-1 mm, which are the antibody core; e. Gastro-resistant layer: Polyvinyl alcohol phthalate and calcium stearate are dissolved in an 80%-85% ethanol solution, and the ethanol accounts for 70%-82% to obtain the gastro-resistant layer coating liquid; the gastro-resistant layer coating liquid is sprayed onto the antibody core, and the obtained granular preparation is dried and sieved, and the gastro-resistant layer accounts for 2.5%-3.5% of the total mass; f. High-temperature resistant layer: Ethyl cellulose and calcium stearate are dissolved in an 80%-85% ethanol solution, and the ethanol accounts for 70%-82% to obtain the high-temperature resistant layer coating liquid; the high-temperature resistant layer coating liquid is sprayed onto the granular preparation obtained in step e, and the obtained granular preparation is dried and sieved, and the high-temperature resistant layer accounts for 2.5%-3.5% of the total mass.

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