Preparation method of porcine delta coronavirus yolk antibody and application thereof

Abstract

This invention utilizes the recombinant porcine deltacoronavirus protein PDCoV-S constructed in an insect-baculovirus expression system, and uses it as an immunogen to prepare egg yolk antibodies. The egg yolk antibodies are further processed into oral formulations. Through optimization of emulsifiers and synergists, microcapsule formulations with high encapsulation efficiency, high gastric acid throughput, and good intestinal release performance are obtained. Furthermore, the addition of synergists enhances the therapeutic efficacy of the formulation. When the mass ratio of baicalin, Polygonatum odoratum polysaccharide, and nano-selenium is 2:1:2, the encapsulation efficiency of the microcapsules can be effectively improved, thus enhancing the drug's effect. This microcapsule formulation can be used as an active ingredient in the production of drugs or feed additives for the treatment or prevention of PDCoV infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-porcine deltacoronavirus egg yolk antibody, its preparation method, and its application. Background Technology

[0002] Porcine Delta coronavirus (PDCoV) belongs to the genus Coronavirus in the family Coronaviridae. It is an enveloped, single-stranded, positive-sense RNA virus closely related to coronaviruses of mammalian and avian origin. It is a newly emerging, highly pathogenic porcine enteric coronavirus. The virus was first discovered in pig herds in Hong Kong, my country in 2012. The first PDCoV epidemic was reported in Ohio, USA in 2014, where the virus was successfully isolated from diarrheal piglets. Subsequently, the virus rapidly spread to many major pig-producing countries worldwide, including Canada, South Korea, and Vietnam. In 2015, the presence of PDCoV was also confirmed in pig herds in East my country. Currently, PDCoV is widespread globally, becoming one of the major diseases threatening the development of the pig industry. Newborn suckling piglets are the main susceptible group, exhibiting particularly high pathogenicity and mortality. PDCoV primarily targets and infects the gastrointestinal tract of pigs, causing severe damage to the small intestine (especially the jejunum and ileum), inducing acute secretory enteritis. Affected piglets clinically exhibit watery diarrhea, frequent vomiting, and rapid dehydration. The mortality rate in newborn piglets is typically above 40%, and in some large-scale pig farms, the mortality rate can soar to 90%, causing significant economic losses to the pig farming industry and severely hindering its large-scale and healthy development. Furthermore, related studies have confirmed that PDCoV exhibits extremely broad cell tropism, replicating efficiently not only in porcine cell lines but also in various animal cell lines, including human, bovine, and chicken lines. In 2021, PDCoV was successfully isolated from plasma samples of three febrile children in Haiti. This discovery indicates the potential for cross-species transmission of the virus, posing a potential threat to public health. Developing efficient and safe PDCoV control technologies has become a crucial and urgent problem to be solved.

[0003] Currently, there is no mature and effective prevention and control system for PDCoV. There are no approved commercial vaccines for clinical control, and no specific drugs for treatment. In the livestock sector, the only options are traditional methods such as biosecurity, enhanced feeding management, and symptomatic supportive treatment to combat the virus. Specifically, biosecurity can only block the external transmission routes of the virus. It is difficult to effectively control issues such as the shedding of the virus from asymptomatic infected pigs and the spread of residual virus in the environment. Furthermore, the potential for viral mutation further reduces the effectiveness of biosecurity. Symptomatic supportive treatment, based on fluid replacement and correction of electrolyte imbalances, can only temporarily alleviate dehydration symptoms in sick piglets and cannot fundamentally inhibit the replication and proliferation of PDCoV in the pig's body. For newborn piglets with rapidly progressing disease and severe symptoms, the treatment effect is very limited, making it difficult to effectively reduce mortality after an outbreak. Regarding vaccine development, although inactivated vaccines, live attenuated vaccines, and genetically engineered vaccines have accumulated mature experience in the field of coronavirus prevention and control, and some research teams have also started the development of PDCoV mRNA vaccines and achieved phased breakthroughs, proving that such vaccines have good immune protection potential and commercial feasibility, as of now, all PDCoV-related vaccines are in the laboratory development or clinical trial stage and have not yet been industrialized and implemented, which cannot meet the actual prevention and control needs of pig farms. PDCoV prevention and control still faces the dilemma of lacking effective preparations.

[0004] In the field of animal disease prevention and control, antibiotics have been widely used. However, PDCoV infection is a viral disease, and antibiotics have no inhibitory or killing effect on this virus. The overuse of antibiotics not only fails to achieve the desired control effect but also leads to a series of problems such as the proliferation of drug-resistant strains and drug residues in animal products. This contradicts the current development concepts of green and healthy farming and is inconsistent with the trend of large-scale and standardized livestock and poultry farming. Under these circumstances, the development of efficient, environmentally friendly, and drug-residue-free new prevention and control methods has become a key research focus and hot topic in the field of animal husbandry and veterinary medicine. Egg yolk antibodies (Immunoglobulin Y, IgY), as a novel biological agent, are gradually attracting the attention of researchers due to their unique advantages. This antibody is a specific immunoglobulin that accumulates in large quantities in egg yolks after hens are immunized with a specific antigen. It has significant advantages such as simple preparation process, low production cost, high biosafety, no activation of the body's complement system, and low likelihood of inducing cross-immune reactions. Compared with antibodies derived from mammals, it is more suitable as a biological agent for the prevention and control of animal diseases. It has been applied in the prevention and control of various animal diseases in aquatic animals, livestock and poultry, and has shown excellent control effects, providing a new technical approach for the prevention and control of novel viral diseases such as PDCoV.

[0005] Egg yolk antibodies, as a green, efficient, and safe biological agent, possess irreplaceable advantages and broad application prospects in the prevention and control of PDCoV infection. Based on this, research on the preparation methods and application technologies of specific egg yolk antibodies against PDCoV will fill the technological gap in this field, providing a safe and effective PDCoV prevention and control solution for the pig industry. This will effectively address the current predicament of lacking effective agents for PDCoV prevention and control, and has significant theoretical and practical value for promoting the development of green pig farming, reducing economic losses in pig farming, ensuring the healthy and stable development of the pig industry, preventing the cross-species transmission of PDCoV, and safeguarding public health security. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-porcine deltacoronavirus egg yolk antibody, its preparation method, and its application, aiming to solve the problem of the lack of egg yolk antibody prevention and control methods against porcine deltacoronavirus (PDCoV) in the prior art.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: This invention discloses an anti-porcine deltacoronavirus S protein yolk antibody, which is prepared by constructing a recombinant porcine deltacoronavirus protein PDCoV-S in an insect-baculovirus expression system, purifying it, adding Freund's adjuvant to prepare a subunit vaccine, injecting it into laying hens, collecting the eggs from the immunized laying hens, and separating and purifying the yolk from the immunized eggs.

[0008] Specifically, the preparation steps of the egg yolk antibody are as follows: (1) Preparation of antigen: The recombinant protein PDCoV-S of porcine deltacoronavirus was constructed in the insect-baculovirus expression system, the protein was expressed and purified, and the purified recombinant protein was harvested after dialysis and concentration. (2) Immunizing laying hens: The purified recombinant protein PDCoV-S was mixed with an equal amount of Freund's adjuvant and emulsified into a water-in-oil emulsion. The laying hens were immunized for the first time, and then given a second, third and fourth booster immunization. Eggs were collected. (3) Separate and purify egg yolk to obtain egg yolk antibody.

[0009] In step (1), the preparation of the antigen is as follows: using the S protein of PDCoV as the target antigen protein, the open reading frame of the protein S gene is inserted into the vector pYBDM-IG, transformed into Ac MultiBac / rSW106 / asd- / inv+ competent cells, and an engineered bacterium expressing the recombinant protein PDCoV-S is constructed; the recombinant bacterium is infected with Sf9 to obtain recombinant baculovirus, and after further infection to the P3 generation, the supernatant is collected, centrifuged, and subjected to nickel ion affinity chromatography, washing, dialysis, and concentration. After identification, the purity reaches more than 90%, and the purified recombinant protein PDCoV-S is harvested, which is the target antigen protein.

[0010] In step (2), the immunization of laying hens specifically involves administering recombinant PDCoV-S protein to the hens four times, with 1 mL injected subpectorally into the pectoral muscle for each immunization, and an interval of one week between each immunization. Eggs are collected starting one week after the third immunization.

[0011] In step (3), the separation of egg yolk antibodies is as follows: wash the collected eggs with water, wipe them with 75% alcohol for disinfection, and let them air dry naturally; separate the egg yolk and egg white, remove the yolk membrane from the egg yolk with sterile filter paper, and then puncture the yolk with a sterile needle to collect the yolk liquid; pour the yolk liquid into a sterilized 50 ml centrifuge tube, 5 ml / tube, and add 45 ml of acidified water (pH 5.3), vortex to mix, and let stand overnight at 4°C.

[0012] In step (3), the purification of egg yolk antibodies is as follows: centrifuge the crude extract of egg yolk antibodies, collect the supernatant and dilute it with an equal volume of PBS, then add an equal volume of saturated ammonium sulfate to make the final concentration of saturated sulfuric acid 50%, vortex to mix, and let stand at 4°C; after the protein precipitates, centrifuge and discard the supernatant, dissolve the precipitate with PBS, add saturated ammonium sulfate again to the final concentration of 33%, stand at 4°C for 2 h, centrifuge and discard the supernatant, add a small amount of PBS solution to resuspend, and obtain egg yolk antibody extract. The obtained egg yolk antibody extract is dialyzed thoroughly with a dialysis bag to obtain anti-PDCoV-S protein egg yolk antibody.

[0013] On the other hand, the present invention also provides the use of the above-mentioned anti-porcine deltacoronavirus S protein yolk antibody in the preparation of medicaments or feed additives for the prevention and / or treatment of diseases caused by porcine deltacoronavirus.

[0014] This invention also claims protection for an oral formulation of an anti-porcine deltacoronavirus S protein yolk antibody, characterized in that the oral formulation is prepared by the following method: 1) Preparation of egg yolk antibody solution: The purified egg yolk antibody was diluted to 10 mg / mL with physiological saline, and a synergist was added at 2.5% (wt / v) and stirred evenly to prepare the egg yolk antibody solution. The synergist was prepared by mixing baicalin, Polygonatum polysaccharide and nano selenium in a mass ratio of 1:1:1 or 2:1:1 or 2:1:2. 2) Preparation of microcapsule emulsion: A 3% (w / w) gelatin solution and a 0.5% (w / w) sodium alginate solution were mixed evenly at a volume ratio of 1:1. The egg yolk antibody solution prepared in step 1) was then added, followed by an emulsifier with a final concentration of 0.5%. The mixture was stirred at 1500 rpm for 25 minutes at 55°C to emulsify and thus prepare the microcapsule emulsion. The volume ratio of egg yolk antibody solution: gelatin solution: sodium alginate was 1:50:50. The emulsifier consisted of Tween-80 and Span-20 in a mass ratio of 4:1. 3) Spray drying: The microcapsule emulsion prepared in step 2) is spray dried. The spray drying conditions are: inlet air temperature of 165℃, feed rate of 10mL / min, drying time of 10s, and outlet temperature of 55℃. Egg yolk antibody microcapsules are prepared by spray drying and sealed and packaged to obtain the anti-porcine deltacoronavirus S protein egg yolk antibody preparation.

[0015] The present invention has the following beneficial effects: The anti-porcine deltacoronavirus S protein egg yolk antibody of the present invention was prepared by constructing a recombinant porcine deltacoronavirus protein PDCoV-S in an insect-baculovirus expression system, purifying it, adding Freund's adjuvant to prepare a subunit vaccine, injecting it into laying hens, collecting the eggs from the immunized hens, and separating and purifying the yolk from the immunized eggs. Specificity was detected by Western blotting, antibody fluctuations were detected by indirect ELISA, and antibody neutralization experiments were used to determine the effectiveness, ultimately obtaining a highly efficient and high-purity egg yolk antibody.

[0016] The anti-porcine deltacoronavirus S protein egg yolk antibody of this invention can be obtained from eggs, eliminating the need for animal slaughter, thus complying with animal rights protection. It is highly specific, non-toxic, has no side effects, and is environmentally friendly, making it suitable for the prevention and treatment of diseases caused by porcine deltacoronavirus. Furthermore, due to the phylogenetic distance between poultry and mammals, there is no cross-serological reaction between their immunoglobulins, resulting in higher safety. In livestock and poultry farming, the overuse of antibiotics not only leads to drug residues in animal products, harming human health, but also increases bacterial resistance to antibiotics and disrupts the microecological balance of the farming environment. Finding safe and efficient alternatives is urgently needed. Egg yolk antibodies can serve as a novel biocontrol agent to replace antibiotics, suitable for piglet farming scenarios, providing a green solution for PDCoV infection control.

[0017] This invention further processes egg yolk antibodies into oral formulations. Through optimization of emulsifiers and synergists, a microcapsule formulation with high encapsulation efficiency, high gastric acid passage rate, and good intestinal release performance was obtained. Furthermore, the addition of synergists enhances the therapeutic efficacy of the formulation. When the mass ratio of baicalin, Polygonatum polysaccharide, and nano-selenium is 2:1:2, the encapsulation efficiency of the microcapsules can be effectively improved, thereby enhancing the drug's effect. This microcapsule formulation can be used as an active ingredient in the production of drugs or feed additives for the treatment or prevention of PDCoV infection. Attached Figure Description

[0018] Figure 1 PCR diagram for PDCoV-S gene cloning and vector construction: Lane M: DNA molecular weight standard marker, Lane 1: PDCoV-S original plasmid, Lanes 2-4: PDCoV-S double digestion.

[0019] Figure 2 Coomassie Brilliant Blue for induced purification of PDCoV-S protein. Lane M: protein molecular weight standard marker, 1: cell supernatant infected with sf9, 3: purified PDCoV-S.

[0020] Figure 3 This is an SDS-PAGE image of PDCoV-S egg yolk antibodies.

[0021] Figure 4 Serological titer of egg yolk antibody against porcine deltacoronavirus S protein.

[0022] Figure 5 The neutralizing titer of egg yolk antibodies against porcine deltacoronavirus S protein is shown in the figure. Detailed Implementation

[0023] To make the objectives and technical solutions of this invention clearer, the technical solutions of this invention will be described in detail below. However, the embodiments of this invention are not limited to the following examples, and therefore the scope of protection claimed by this invention is not limited to what is described.

[0024] Example 1: Preparation of porcine deltacoronavirus S protein antigen 1.1 Cloning of the PDCoV-S target gene Using cDNA from cell infection fluid containing the complete PDCoV genome preserved in the laboratory as a template, specific primers for PCR amplification of the target gene were designed using snapgene software. Homologous arms were designed based on the multiple cloning site region of the pYBDM-IG vector. The target gene (nucleotide sequence as shown in SEQ ID No: 3) was amplified by PCR using the designed specific primers, and the PCR reaction system was prepared according to the 2×pfu Mix instructions. The primer sequences are as follows: Upstream primer: 5' gcattctgcctttgcgATGCAGAGAGCTCTATTG 3' (SEQ ID NO. 1); Downstream primer: 5' agctcgtcgacgtaggcctttCCATTCCTTAAACTTAAA 3' (SEQ ID NO. 2); The PCR reaction system was as follows: template < 1 μg, forward and reverse primers 0.5 μL each, 2×pfu Mix 2 μL, ddH2O to a final volume of 20 μL; PCR reaction program: 94℃, 30 s; (98℃, 10 s; 65℃, 5 s; 72℃, 15 s) × 34 cycles; 72℃, 5 min; store at 4℃.

[0025] The PCR products were subjected to gel electrophoresis. The electrophoresis program was: constant voltage 160V, 15 min. If the product size was correct and the concentration was high, the gel was cut and recovered.

[0026] 1.2 Gel extraction and recovery of PCR products Following the instructions of the OMEGA Gel Extraction Kit, the PCR products were extracted and recovered using a gel. The specific steps are as follows: 1) Use agarose gel electrophoresis to separate the target gene fragment and cut out the target gene band.

[0027] 2) Add an equal volume of XP2 Binding Buffer, and after the gel is completely dissolved, transfer it to a DNA Mini adsorption column, centrifuge at 10000g for 1 min, and discard the waste liquid.

[0028] 3) Add 300 μL XP2 Binding Buffer to the adsorption column, centrifuge at 13000g for 1 min, and discard the waste liquid.

[0029] 4) Add 700 μL of SPW Buffer to the adsorption column, centrifuge at 10000g for 1 min, and discard the waste liquid.

[0030] 5) Repeat step 4).

[0031] 6) Centrifuge at 13000g for 2 minutes to remove any remaining liquid from the adsorption column.

[0032] 7) Transfer the adsorption column to a clean 1.5 mL centrifuge tube, add 30 μL of Elution Buffer (or TE Buffer), incubate at room temperature for 1 min, and centrifuge at 13000 g for 1 min to elute the DNA.

[0033] 8) Measure the concentration of the recovered DNA for the next step of the experiment.

[0034] 1.3 Double Enzyme Digestion of the Vector The double digestion reaction system was prepared according to the TaKaRa restriction enzyme instructions, with pYBDM-IG plasmid <1μg, BamHI and EcoRI restriction enzymes 1μL each, 10× Green Buffer 2μL, and ddH2O added to a final volume of 20μL. The mixture was incubated at 37℃ for 15min, and then the gel was cut and recovered separately.

[0035] 1.4 Construction of recombinant plasmids: The target DNA fragment is homologously recombined with the vector recovered by enzyme digestion and then transformed. The specific steps are as follows: 1) After preparing the recombination system, recombine it at 50℃ for 20 minutes.

[0036] 2) The recombinant product was transformed into 50 μL of E. coli DH5α competent cells, spread onto LB solid plates containing Amp, and incubated upside down in a 37°C incubator overnight.

[0037] 3) Randomly pick single colonies and place them in a centrifuge tube containing 1 mL of LB liquid medium containing Amp. Incubate at 37°C with shaking at 180 rpm for 4 h. Take 1 μL of the bacterial solution for PCR identification and then send it for testing.

[0038] 4) Store the bacterial culture with correct test results at 80℃. After successful sequencing, extract the recombinant plasmid for later use.

[0039] The recombinant plasmid was identified by double enzyme digestion, and the results are as follows: Figure 1 As shown, the amplified gene size is approximately 3500 bp, which is in line with expectations and can be used for the next step of the experiment.

[0040] 1.5 Obtaining Recombinant Baculovirus Two μL of recombinant plasmid pYBDM-IG-GP67-PDCoV-S was transformed into Ac MultiBac / rSW106 / asd- / inv+ competent cells, and screened using blue-white screening. White spots were picked and identified by bacterial culture PCR. Recombinant bacterial cultures that were successfully verified by PCR were transfected into sf9 insect cells. After 72 h, the cells were observed under an inverted fluorescence microscope. If specific green fluorescence appeared, it indicated that the recombinant baculovirus was successfully constructed.

[0041] After successfully constructing the recombinant baculovirus and passaged it to the third generation, the cell pellet and supernatant were collected by centrifugation. The cell pellet and supernatant were transferred to 1.5 mL EP tubes, 5× protein loading buffer was added, and the tubes were boiled in a water bath for 10 min. Western blot was used to detect protein expression. The primary antibody used was HRP-6×His-tagged antibody diluted 1:5,000, and the cells were developed using ECL chemiluminescence buffer.

[0042] 1.6 Expression and purification of recombinant PDCoV-S protein Recombinant baculovirus expressed by Western blot was inoculated at a ratio of 1.5% into culture flasks containing 300 mL of sf9 cells at an appropriate cell density. After 3 days, fluorescence was observed and cell death was recorded. When the cell death rate was between 30% and 40%, the cells were centrifuged at 8,000 g for 10 min, and the supernatant was collected. The collected supernatant was used for protein purification using Ni-NTA, eluting the protein with an imidazole gradient. The eluted protein was dialyzed using dialysis buffer (pH 7.4) containing 50 mmol / L Tris and 150 mmol / L NaCl at 4°C, 300 r / min for 12 h. The dialyzed protein was filtered through a 0.22 μm filter and identified by SDS-PAGE. The concentration of PDCoV-S protein was determined using a BCA protein quantification kit.

[0043] The results are as follows Figure 2 As shown, the target band size of the induced and purified PDCoV-S protein is 130 kDa, which can be used for the next step of the experiment.

[0044] Example 2: Preparation of porcine deltacoronavirus S protein yolk antibody 2.1 Immunize laying hens and collect eggs Sixty healthy laying hens were randomly divided into two groups: a control group and an experimental group, with 30 hens in each group. Both groups were provided with ample feed and water for one week and observed. Hens exhibited good growth and appetite, laying 5-7 eggs per week. They were then immunized, with the immunization site being the pectoral muscle. The immunization dose was 1 mL per hen per dose. Booster immunizations were administered every week (the control group received PBS), for a total of four immunizations. Eggs were collected daily from the fourth week after the first immunization, numbered, labeled, and stored at 4℃ for later use.

[0045] 2.2 Isolation and purification of egg yolk antibodies Wash the eggs with clean water to remove any dirt. After shelling, separate the yolks and whites using an egg separator. Pierce the yolk membrane with a syringe, extract the yolk, and record its volume. Transfer the yolk to an Erlenmeyer flask and dilute it 10 times with deionized water. Adjust the pH to 5.0 with 0.1 mol / L hydrochloric acid solution and freeze overnight at -20°C to remove fat. Thaw the yolk the next day at 4°C, transfer the diluted yolk to a centrifuge tube, and centrifuge at 10,000 rpm for 10 minutes at 4°C. Collect the supernatant as the crude yolk antibody extract.

[0046] The isolated egg yolk antibodies were purified using ammonium sulfate precipitation. Based on the measured volume of the crude egg yolk antibody extract, saturated ammonium sulfate solution was slowly added until a final saturation of 55% was achieved, and the mixture was thoroughly mixed. The mixture was centrifuged at 10000 rpm for 10 min at 4°C, the supernatant was discarded, and the solution was resuspended in an appropriate amount of PBS. Then, 33% ammonium sulfate solution was slowly added again, and the mixture was stirred and incubated overnight at 4°C. The mixture was then centrifuged at 10000 rpm for 15 min at 4°C, and the supernatant was discarded. The solution was resuspended in a small amount of PBS and transferred to a dialysis bag. The dialysis bag containing the protein solution was placed in the dialysis buffer and dialyzed at 4°C, with the dialysis buffer changed every 12 h. After dialysis, the solution was filtered through a 0.22 μm filter for sterilization. The final purified IgY solution was aliquoted and stored at -20°C for later use.

[0047] Example 3: Identification of egg yolk antibodies against porcine deltacoronavirus S protein 3.1 Concentration and Purity Determination The purified egg yolk antibody concentration was determined using a BCA protein concentration assay kit, and the protein concentration was found to be 11.8 mg / mL. 20 μL of the diluted protein solution was mixed with 5 μL of 5× loading buffer, incubated in a boiling water bath for 10 min, and then subjected to 12% reducing SDS-PAGE electrophoresis. The results are as follows: Figure 3 As shown, the heavy chain and light chain are located at the 60kDa and 35kDa bands, respectively, with a total molecular weight of approximately 180kDa and a purity of about 90%.

[0048] 3.2 The fluctuations in detection patterns using indirect enzyme-linked immunosorbent assay (ELISA) The porcine deltacoronavirus S protein prepared in Example 1 was diluted to 10 μg / mL with coating buffer, and 100 μL was added to each well. The plate was incubated overnight at 4°C. The next day, the coating buffer was aspirated, and the plate was washed three times with PBST. Then, 200 μL of blocking buffer containing 5% BSA was added to each well, and the plate was blocked at 37°C for 2 h. The blocking buffer was discarded, and the plate was washed three times. Egg yolk antibodies for different time periods were diluted 1:1000, and blank and negative control groups were also included. Each dilution was used in triplicate, with 100 μL added to each well. The plate was incubated at 37°C for 2 h, and washed three times after incubation. HRP-rabbit anti-chicken IgY was diluted 1:5000 with antibody dilution buffer, and 100 μL was added to each well. The plate was incubated at 37°C for 1 h, and washed four times after incubation. Add 100 μL of TMB chromogenic reagent to each well in the dark, gently tap to mix, and incubate at 37°C in the dark for 15-30 minutes. Stop the reaction by adding 100 μL of 2M H₂SO₄ to each well, and measure the absorbance at OD450 nm using a microplate reader.

[0049] ELISA results as follows Figure 3The results show that the titer of egg yolk antibodies gradually increased after the second immunization, while the titer of the control group did not change significantly.

[0050] 3.3 Determination of neutralizing titer of egg yolk antibodies Add 50 μL of serum-free DMEM to each well of a 96-well plate beforehand. After centrifuging the egg yolk antibody, take 50 μL and serially dilute it longitudinally in the 96-well plate to create 8 gradients. Then, add 50 μL of pre-diluted 200 TCID50 PDCoV / HNNY virus solution (isolated and prepared in our laboratory) to each well, and set up positive and negative controls. For the positive control column, add 50 μL of serum-free DMEM medium and 50 μL of diluted virus solution to each well. For the negative control column, add only serum-free DMEM. Gently mix the 96-well plate containing the test sample and virus dilution, and then incubate at 37°C for 1 h. Remove the pre-coated ST cells (reaching 90% confluence) from the incubator, add sterile PBS, wash three times along the well wall, and then discard the PBS. After incubation for 1 hour, the mixture was added to pre-washed ST cell culture plates and incubated at 37°C for 2 hours. The mixture was then discarded, and the cells were washed three times with PBS. DMEM containing 8 μg / mL trypsin was added to each well. Microscopic observation was used to systematically observe cytopathic effects. During the experiment, morphological changes in cells were observed daily under a microscope, focusing on typical pathological features such as cell rounding, fusion, and detachment. To ensure the reliability of the results, positive and negative controls were included: when significant cytopathic effects were observed in the positive control group and normal cell morphology was maintained in the negative control group, the wells showing pathological changes in the test samples were recorded. To further improve the accuracy and reproducibility of the data, the neutralizing titer of each test sample was determined three times independently, and the average of the three determinations was used as the final result to minimize experimental error and ensure the scientific validity and reliability of the data.

[0051] The results of the antibody neutralization experiment are as follows Figure 4 This indicates that egg yolk antibodies against porcine deltacoronavirus S protein can significantly inhibit the growth of PDCoV. The neutralizing titer of the egg yolk antibodies gradually increases after the second immunization, demonstrating high neutralizing activity, while the control group showed no neutralizing effect.

[0052] Example 4: Preparation of an anti-porcine deltacoronavirus S protein egg yolk antibody preparation For ease of use, the present invention further prepares the anti-porcine deltacoronavirus S protein egg yolk antibody obtained by spray drying to obtain an orally administered powder. The preparation method of the powder includes the following steps: 1) Preparation of egg yolk antibody solution: The egg yolk antibody purified in Example 2 was diluted with physiological saline to a 10 mg / mL egg yolk antibody solution; 2) Preparation of microcapsule emulsion: Mix 3% gelatin solution and 0.5% sodium alginate solution at a volume ratio of 1:1 until homogeneous. Then add the egg yolk antibody solution prepared in step 1), and add emulsifier with a final concentration of 0.5% (V / V). Emulsify at 55℃ and 1500 rpm for 25 min to obtain microcapsule emulsion. The volume ratio of egg yolk antibody solution: gelatin solution: sodium alginate is 1:50:50. 3) Spray drying: The microcapsule emulsion prepared in step 2) is spray dried. The spray drying conditions are: inlet air temperature of 165℃, feed rate of 10mL / min, drying time of 10s, and outlet temperature of 55℃. Egg yolk antibody microcapsules are prepared by spray drying and sealed and packaged to obtain the anti-porcine deltacoronavirus S protein egg yolk antibody preparation.

[0053] 4.1 Determination of encapsulation efficiency of microcapsule formulations: Take 2g of the microcapsules of this invention into a 10ml centrifuge tube, add 5ml of 0.05mol / L sodium citrate lysis solution, place in a constant temperature shaker at 37℃ and shake at 200rpm for 12h, centrifuge at 5000rpm for 10min, collect the supernatant, and determine the concentration of egg yolk antibody in the solution using the Folin phenol method. Calculate the encapsulation efficiency of the microcapsules using the following formula.

[0054] Encapsulation efficiency (%) = Actual mass of egg yolk antibody in microcapsules / Theoretical mass of egg yolk antibody in microcapsules × 100%.

[0055] 4.2 Release rate of microcapsule formulation in simulated gastric juice: Take 2g of the microcapsules of this invention into a 10ml centrifuge tube, add 5ml of artificial gastric fluid, place it in a constant temperature shaker at 37℃ and shake at 200rpm for 2h, centrifuge at 1000rpm for 5min, take the supernatant and stop the reaction in an ice bath, determine the concentration of egg yolk antibody in the supernatant using the Folin-phenol method, and calculate the release rate of the microcapsules according to the following formula.

[0056] Release rate (%) = Amount of egg yolk antibody released / Actual mass of egg yolk antibody in microcapsules × 100% 4.3 Release rate of microcapsule formulation in simulated intestinal fluid: Take 2g of the microcapsules of the present invention into a 10ml centrifuge tube, add 5ml of artificial intestinal fluid, and incubate, centrifuge and detect under the same conditions as in section 3.2, and calculate the release rate according to the following formula.

[0057] Release rate (%) = Amount of egg yolk antibody released / Actual mass of egg yolk antibody in microcapsules × 100%.

[0058] 4.4 Effect of emulsifiers on the performance of egg yolk antibody microcapsule formulations In this process, the inventors explored the effects of different emulsifiers on the stability and release properties of microcapsule formulations. The specific emulsifier composition used in the experiments and the related results are as follows: Table 1. Latex composition (final concentration) and its impact on the quality of microcapsule formulations.

[0059] Based on the above experiments, it can be seen that when the ratio of Tween-80 to Span-20 in the emulsifier composition is 4:1, the encapsulation rate of the egg yolk antibody microcapsule preparation is the highest. Moreover, the microcapsule has a low gastric juice release rate, which reduces the dissolution and inactivation of the microcapsule in gastric juice, allowing the egg yolk antibody to enter the intestine through the stomach and exert its effect.

[0060] 4.5 Optimization of Egg Yolk Antibody Microcapsule Formulation To enhance the efficacy and stability of the formulation, the inventors further added a synergist component to the egg yolk antibody to prepare an anti-porcine deltacoronavirus S protein egg yolk antibody formulation. The specific technical solution is as follows: 1) Preparation of egg yolk antibody solution: The egg yolk antibody purified in Example 2 was diluted to 10 mg / mL with physiological saline, and a synergist was added at 2.5% (wt / v) and stirred until homogeneous to prepare the egg yolk antibody solution. The synergist was prepared by mixing baicalein, Polygonatum odoratum polysaccharide and nano selenium in a certain proportion; baicalein and Polygonatum odoratum polysaccharide were purchased from Fufeng Sinote Biotechnology Co., Ltd.; nano selenium was purchased from Hebei Qiansheng Biotechnology Co., Ltd. 2) Preparation of microcapsule emulsion: Mix 3% gelatin solution and 0.5% sodium alginate solution at a volume ratio of 1:1 until homogeneous. Then add the egg yolk antibody solution prepared in step 1), and add emulsifier (Tween-80: Span-20 mass ratio of 4:1) to a final concentration of 0.5%. Emulsify by stirring at 1500 rpm for 25 min at 55℃ to prepare the microcapsule emulsion. The volume ratio of egg yolk antibody solution: gelatin solution: sodium alginate is 1:50:50. 3) Spray drying: The microcapsule emulsion prepared in step 2) is spray dried. The spray drying conditions are: inlet air temperature of 165℃, feed rate of 10mL / min, drying time of 10s, and outlet temperature of 55℃. Egg yolk antibody microcapsules are prepared by spray drying and sealed and packaged to obtain the anti-porcine deltacoronavirus S protein egg yolk antibody preparation.

[0061] Determination of the protective efficacy of anti-porcine deltacoronavirus S protein egg yolk antibody preparation: Thirty-five healthy, susceptible 3-day-old piglets (PDCoV neutralizing antibody titer ≤1:4) were randomly divided into 7 groups of 5 piglets each. Piglets in groups 1-6 were orally administered 1.0 mL / piglet of porcine deltacoronavirus PDCoV / HNNY virus solution (isolated and prepared in our laboratory, 108.0 TCID50 / 0.1 mL). After the piglets developed diarrhea symptoms, groups 1-5 were orally fed the anti-porcine deltacoronavirus S protein egg yolk antibody preparation listed in Table 2 below, 1.0 g / piglet, once daily. Group 6 received no treatment, and group 7 received neither challenge nor treatment. Piglets in each group were kept in isolation and observed for 14 consecutive days. Clinical symptoms were recorded, and the number of days until diarrhea symptoms disappeared was recorded as the number of days of recovery. The average number of days of recovery and the final survival rate were calculated. Details are shown in Table 2 below. Table 2 Optimization of Egg Yolk Antibody Microcapsule Formulation

[0062] Based on the above experimental results, it can be seen that when a synergist composed of baicalin, Polygonatum odoratum polysaccharide, and nano-selenium is added to the antibody solution in a certain proportion, it can effectively improve the therapeutic effect of the egg yolk antibody microcapsule preparation. Compared with the average cure time of 3.2 days without the addition, the addition of the above three components can shorten the cure time to 1.2-2.2 days, with an average reduction of more than 1 day. Furthermore, it has been determined that when the mass ratio of baicalin, Polygonatum odoratum polysaccharide, and nano-selenium is 2:1:2, it can also effectively improve the encapsulation efficiency of the microcapsules and enhance the drug efficacy. This microcapsule preparation can be used as an active ingredient in the production of drugs or feed additives for the treatment or prevention of PDCoV infection.

[0063] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An anti-porcine deltacoronavirus S protein yolk antibody, comprising: constructing a recombinant porcine deltacoronavirus protein PDCoV-S in an insect-baculovirus expression system; purifying the protein and adding Freund's adjuvant to prepare a subunit vaccine; injecting the vaccine into laying hens; collecting the eggs from the immunized hens; and separating and purifying the yolk from the immunized eggs. The antibody is characterized by: The preparation steps of the egg yolk antibody are as follows: (1) Preparation of antigen: The recombinant protein PDCoV-S of porcine deltacoronavirus was constructed in the insect-baculovirus expression system, the protein was expressed and purified, and the purified recombinant protein was harvested after dialysis and concentration. (2) Immunizing laying hens: The purified recombinant protein PDCoV-S was mixed with an equal amount of Freund's adjuvant and emulsified into a water-in-oil emulsion. The laying hens were immunized for the first time, and then given a second, third and fourth booster immunization. Eggs were collected. (3) Separate and purify egg yolk to obtain egg yolk antibody.

2. The anti-porcine deltacoronavirus S protein egg yolk antibody according to claim 1, characterized in that, In step (1), the preparation of the antigen is as follows: using the S protein of PDCoV as the target antigen protein, the open reading frame of the protein S gene with the sequence shown in SEQ ID No: 3 is inserted into the vector pYBDM-IG, transformed into Ac MultiBac / rSW106 / asd- / inv+ competent cells, and engineered bacteria expressing recombinant protein PDCoV-S are constructed; the recombinant bacteria are infected with Sf9 to obtain recombinant baculovirus, and after further infection to the P3 generation, the supernatant is collected, centrifuged, and subjected to nickel ion affinity chromatography, washing, dialysis, and concentration. After identification, the purity reaches more than 90%, and the purified recombinant protein PDCoV-S is harvested, which is the target antigen protein.

3. The anti-porcine deltacoronavirus S protein egg yolk antibody according to claim 1, characterized in that, In step (2), the immunization of laying hens is specifically as follows: The laying hens are immunized four times with recombinant protein PDCoV-S, with 1 mL injected subpectorally into the pectoral muscle each time, and the interval between each immunization is one week. Eggs are collected one week after the third immunization.

4. The anti-porcine deltacoronavirus S protein egg yolk antibody according to claim 1, characterized in that, In step (3), the separation of egg yolk antibodies is as follows: wash the collected eggs with water, wipe them with 75% alcohol for disinfection, and let them air dry naturally; separate the egg yolk and egg white, remove the yolk membrane from the egg yolk with sterile filter paper, and then puncture the yolk with a sterile needle to collect the yolk liquid; pour the yolk liquid into a sterilized 50 ml centrifuge tube, 5 ml / tube, add 45 ml of pH 5.3 acidified water, vortex to mix, and let stand overnight at 4°C.

5. The anti-porcine deltacoronavirus S protein egg yolk antibody according to claim 1, characterized in that, In step (3), the purification of egg yolk antibodies is as follows: centrifuge the crude extract of egg yolk antibodies, collect the supernatant and dilute it with an equal volume of PBS, then add an equal volume of saturated ammonium sulfate to make the final concentration of saturated sulfuric acid 50%, vortex to mix, and let stand at 4°C; after the protein precipitates, centrifuge and discard the supernatant, dissolve the precipitate with PBS, add saturated ammonium sulfate again to the final concentration of 33%, stand at 4°C for 2 h, centrifuge and discard the supernatant, add a small amount of PBS solution to resuspend, and obtain egg yolk antibody extract. The obtained egg yolk antibody extract is dialyzed thoroughly with a dialysis bag to obtain anti-PDCoV-S protein egg yolk antibody.

6. The use of the anti-porcine deltacoronavirus S protein yolk antibody according to claim 1 in the preparation of a medicament or feed additive for the prevention and / or treatment of diseases caused by porcine deltacoronavirus.

7. An oral formulation of an anti-porcine deltacoronavirus S protein egg yolk antibody, characterized in that, The oral formulation is prepared using the following method: 1) Preparation of egg yolk antibody solution: The purified egg yolk antibody of claim 1 was diluted to 10 mg / mL with physiological saline, and a synergist was added at 2.5% wt / v and stirred evenly to prepare the egg yolk antibody solution. The synergist was prepared by mixing baicalein, Polygonatum polysaccharide and nano selenium in a mass ratio of 1:1:1 or 2:1:1 or 2:1:

2. 2) Preparation of microcapsule emulsion: A 3% (w / w) gelatin solution and a 0.5% (w / w) sodium alginate solution were mixed evenly at a volume ratio of 1:

1. The egg yolk antibody solution prepared in step 1) was then added, followed by an emulsifier with a final concentration of 0.5%. The mixture was stirred at 1500 rpm for 25 minutes at 55°C to emulsify and thus prepare the microcapsule emulsion. The volume ratio of egg yolk antibody solution: gelatin solution: sodium alginate was 1:50:

50. The emulsifier consisted of Tween-80 and Span-20 in a mass ratio of 4:

1. 3) Spray drying: The microcapsule emulsion prepared in step 2) is spray dried. The spray drying conditions are: inlet air temperature of 165℃, feed rate of 10mL / min, drying time of 10s, and outlet temperature of 55℃. Egg yolk antibody microcapsules are prepared by spray drying and sealed and packaged to obtain the anti-porcine deltacoronavirus S protein egg yolk antibody preparation.

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