Preparation method of porcine acute diarrhea syndrome coronavirus yolk antibody and application thereof

Abstract

This invention utilizes an insect-baculovirus expression system to prepare the recombinant protein SADS-CoV-S of porcine acute diarrhea syndrome coronavirus, which is then used to prepare egg yolk antibodies. By optimizing the composition of the subunit vaccine composition for immunizing laying hens, IL-2 and ZnCl2 solution are added as immunostimulants to the conventional adjuvant, increasing the antibody concentration in the egg yolk antibody extract and improving the neutralizing antibody titer and protective efficacy. When IL-2 and zinc chloride aqueous solution are added to the immunization composition simultaneously, the resulting egg yolk antibody aqueous solution exhibits the highest protein concentration, and at the same protein concentration, the egg yolk antibody has the highest neutralizing titer. Compared to the immunization composition using Freund's complete adjuvant alone, the neutralizing titer is increased by more than 60 times.

Description

Technical Field

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

[0002] Currently, there is a lack of effective means to control SADS-CoV. No commercially available vaccines have been approved for clinical use, and there are no specific treatments available. Farmers can only rely on traditional methods such as biosecurity, enhanced feeding management, and symptomatic supportive treatment. Biosecurity measures can only block the virus transmission route from the outside, and are difficult to effectively control the shedding of the virus in asymptomatic infected pigs or the spread of residual virus in the environment. Symptomatic treatment mainly focuses on fluid replacement and correction of electrolyte imbalances, which can only alleviate dehydration symptoms in sick pigs but cannot inhibit viral replication and proliferation in the body. This approach is extremely ineffective for piglets with rapidly progressing disease and severe symptoms, and is unlikely to reduce mortality. Regarding vaccine development, although inactivated vaccines, live attenuated vaccines, and genetically engineered vaccines are relatively mature technologies in the field of coronaviruses, there is a lack of specific prevention and control measures for SADS-CoV.

[0004] Antibiotics have been widely used in the prevention and control of animal diseases. However, as SADS-CoV is a viral infectious disease, antibiotics are ineffective. Furthermore, the overuse of antibiotics not only leads to drug residues in animal products, endangering human health, but also increases bacterial resistance to antibiotics, disrupts the microecological balance of the breeding environment, and contradicts the trend of green farming. Therefore, finding safe and efficient alternatives in livestock and poultry farming is urgently needed. Against this backdrop, finding efficient, environmentally friendly, and residue-free alternative prevention and control methods has become a research hotspot in the field of animal husbandry and veterinary medicine. Egg yolk antibodies (Immunoglobulin Y, IgY) have gradually attracted attention due to their unique advantages. Egg yolk antibodies are specific immunoglobulins enriched in egg yolks after immunization with specific antigens in hens. They have the characteristics of simple preparation process, low production cost, high safety, non-activation of the complement system, and low likelihood of cross-reaction. Compared with mammalian-derived antibodies, they are more suitable as biological agents for the prevention and control of animal diseases and have shown good application prospects in the prevention and control of various animal diseases in aquaculture, livestock, and poultry. Currently, researchers have conducted some research on the application of egg yolk antibodies against livestock and poultry viruses, such as classical swine fever virus, porcine epidemic diarrhea virus, and Newcastle disease virus in chickens. However, the types of viruses studied are still relatively limited. In particular, there are currently no research reports on SADS-CoV-specific egg yolk antibodies at home and abroad. There is a lack of egg yolk antibody prevention and control technologies and products for SADS-CoV, which makes it impossible to provide effective biological control measures for SADS-CoV infection.

[0005] Egg yolk antibodies, as green and efficient biological agents, have broad application prospects in the prevention and control of SADS-CoV infection. Therefore, researching and developing methods for preparing specific egg yolk antibodies against SADS-CoV and their application technologies, filling the technological gap in this field, and providing a safe and effective SADS-CoV prevention and control solution for the pig industry are of great significance for promoting the development of green pig farming and reducing breeding losses. Summary of the Invention

[0006] The purpose of this invention is to provide an egg yolk antibody against porcine acute diarrhea syndrome coronavirus, its preparation method, and its application, aiming to solve the problem of the lack of egg yolk antibody prevention and treatment methods against porcine acute diarrhea syndrome coronavirus (SADS-CoV) in the prior art.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: A yolk antibody against porcine acute diarrhea syndrome coronavirus S protein is disclosed. The yolk antibody is prepared by constructing a recombinant porcine acute diarrhea syndrome coronavirus protein SADS-CoV-S in an insect-baculovirus expression system, purifying it, adding Freund's adjuvant and other components 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 method for preparing the egg yolk antibody includes the following steps: (1) Preparation of antigen: The recombinant protein SADS-CoV-S of porcine acute diarrhea syndrome coronavirus was constructed in the insect-baculovirus expression system, the protein was expressed and purified, and the purified recombinant protein SADS-CoV-S was harvested after dialysis and concentration; (2) Preparation of immune composition: The purified SADS-CoV-S protein solution, IL-2 aqueous solution and zinc chloride aqueous solution were mixed and stirred evenly to obtain a mixed solution. Then, the mixed solution was mixed with an equal volume of Freund's complete adjuvant and emulsified at 6000 rpm for 5 min to obtain the corresponding immune composition. (3) Immunizing laying hens: Immunize laying hens with an immunization composition. First, immunize laying hens, and then perform a second, third and fourth booster immunization. Collect eggs. (4) Separate and purify the egg yolk to obtain egg yolk antibody.

[0009] In step (1), the preparation of the antigen is as follows: using the S protein of SADS-CoV as the target antigen protein, the open reading frame of the protein S gene (sequence shown as SEQ ID No: 3) is inserted into the vector pYBDM-IM, transformed into Ac MultiBac / rSW106 / asd- / inv+ competent cells, and an engineered bacterium expressing the recombinant protein SADS-CoV-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 SADS-CoV-S is harvested, which is the target antigen protein.

[0010] In step (2), the concentration of the SADS-CoV-S protein solution is 1.1 mg / mL, the IL-2 aqueous solution contains 10,000 units / mL, and the concentration of the zinc chloride aqueous solution is 3.2 mg / mL.

[0011] In step (2), the ratio of SADS-CoV-S protein solution: IL-2 aqueous solution: zinc chloride aqueous solution is 1:0.04-0.06:0.06-0.04; preferably, the volume ratio of IL-2 aqueous solution: zinc chloride aqueous solution is 6:4-4:6; more preferably, the volume ratio of IL-2 aqueous solution: zinc chloride aqueous solution is 4:6.

[0012] In step (3), the immunization of laying hens specifically involves administering the immunization composition to the laying hens four times, injecting 1 mL into the subpectoral muscle of each hen each time, with a one-week interval between each immunization, and collecting eggs starting one week after the third immunization.

[0013] In step (4), 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 egg 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 to prepare crude extract of egg yolk antibodies.

[0014] In step (4), 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 fully dialyzed with a dialysis bag to obtain anti-SADS-CoV-S protein egg yolk antibody.

[0015] The present invention also claims protection for the use of the above-mentioned anti-swine acute diarrhea syndrome coronavirus S protein egg yolk antibody in the preparation of medicaments or feed additives for the prevention and / or treatment of diseases caused by swine acute diarrhea syndrome coronavirus.

[0016] On the other hand, the present invention also provides a method for detecting the titer of egg yolk antibodies against porcine acute diarrhea syndrome coronavirus (SADS-CoV). The method uses an indirect enzyme-linked immunosorbent assay (ELISA) to detect the titer of egg yolk antibodies against SADS-CoV S protein. The specific steps are as follows: 1) Coat an ELISA plate with 1 μg / mL SADS-CoV S protein (100 μL per well), incubate overnight at 4°C, wash three times with PBST, add blocking buffer containing 5% BSA, and block at 37°C for 2 hours; 2) After washing three times, add serially diluted egg yolk antibodies against SADS-CoV-S protein, and simultaneously set up a blank control and a negative control group, incubate at 37°C for 2 hours; 3) After washing three times, add horseradish peroxidase-labeled goat anti-chicken secondary antibody, and incubate at 37°C for 1 hour; 4) After washing four times, add TMB chromogenic solution, and develop color at 37°C in the dark for 15-30 minutes; 5) Add 2M... 6) Use 100 μL of H2SO4 stop solution to terminate the reaction; 7) Use an enzyme-linked immunosorbent assay (ELISA) reader to read OD450nm. If the ratio of the absorbance of the test sample to the negative control is greater than 2.1, it is considered positive. The maximum positive dilution is the antibody titer.

[0017] The present invention has the following beneficial effects: First, the egg yolk antibody against porcine acute diarrhea syndrome coronavirus (SADS-CoV) S protein of this invention can be obtained from eggs, eliminating the need for animal slaughter and complying with animal rights protection. It also exhibits high specificity, is non-toxic, has no side effects, and is environmentally friendly, making it suitable for the prevention and treatment of diseases caused by SADS-CoV. Furthermore, the egg yolk antibody against SADS-CoV S protein of this invention has higher safety because there is no cross-serological reaction between the immunoglobulins of poultry and mammals due to the phylogenetic distance between them. Moreover, the egg yolk antibody can serve as a novel biocontrol agent to replace antibiotics, is suitable for piglet farming scenarios, and provides a green solution for SADS-CoV infection control.

[0018] Secondly, this invention optimizes the composition of a subunit vaccine composition for immunizing laying hens by adding IL-2 and ZnCl2 solutions as immunomodulators to the conventional adjuvant. This increases the antibody concentration in the egg yolk antibody extract, thereby improving the neutralizing antibody titer and protective efficacy. When IL-2 and zinc chloride aqueous solution are added simultaneously to the immunization composition, the resulting egg yolk antibody aqueous solution exhibits the highest protein concentration, and at the same protein concentration, the egg yolk antibody exhibits the highest neutralizing titer, representing a more than 60-fold increase in neutralizing titer compared to immunization compositions using Freund's complete adjuvant alone. The highest titer of the extracted egg yolk antibody, reaching 213.6, is achieved when the volume ratio of IL-2 aqueous solution to zinc chloride aqueous solution is 4:6. Attached Figure Description

[0019] Figure 1 : PCR diagram of SADS-CoV-S gene cloning and vector construction, lane M: DNA molecular weight standard marker, lane 1: SADS-CoV-S gene.

[0020] Figure 2 Coomassie Brilliant Blue Spectrum of induced purified SADS-CoV-S protein. Lane M: Protein molecular weight standard marker. 1-2: 50 mM imidazole elution. Lane 3: 250 mM imidazole elution.

[0021] Figure 3 SDS-PAGE image of SADS-CoV-S egg yolk antibodies.

[0022] Figure 4 Serological titer of egg yolk antibody against porcine acute diarrhea syndrome coronavirus S protein.

[0023] Figure 5 : Neutralizing titer of egg yolk antibody against porcine acute diarrhea syndrome coronavirus S protein. Detailed Implementation

[0024] 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.

[0025] The egg yolk antibody against porcine acute diarrhea syndrome coronavirus (SADS-CoV-S) protein of this invention was prepared by constructing a recombinant SADS-CoV-S protein in an insect-baculovirus expression system, purifying it, adding Freund's adjuvant, and injecting it into laying hens. Eggs from these immunized hens were collected, and the yolk was isolated and purified from the yolk. Specificity was detected by Western blotting, antibody fluctuations were detected by indirect ELISA, and antibody neutralization experiments were used to determine the effectiveness, ultimately yielding a highly efficient and pure egg yolk antibody.

[0026] Example 1: Preparation of S protein antigen of porcine acute diarrhea syndrome coronavirus 1.1 Cloning of the SADS-CoV-S target gene Using cDNA from cell infection fluid containing the complete SADS-CoV genome preserved in the laboratory as a template, specific primers were designed using snapgene software for PCR amplification of the target gene. Homologous arms were designed based on the multiple cloning site region of the pYBDM-IM vector. The target gene (SEQ ID NO. 3) was amplified by PCR using the designed specific primers. The PCR reaction system was prepared according to the 2×pfu Mix instructions (Table 1). The primer sequences are as follows: Upstream primer: 5' gcattctgcctttgcgATGAAACTTTTTACAGTTTTC 3' (SEQ ID NO. 1); Downstream primer: 5' agctcgtcgacgtaggcctttTTGGACGTGGACCTTTTCAAT 3' (SEQ ID NO.2); PCR reaction program: 94℃, 30s; 98℃, 10s; 65℃, 5s; 72℃, 15s; ×34 cycles; 72℃, 5min; store at 4℃. PCR products were then subjected to gel electrophoresis. The electrophoresis program was: constant voltage 160V, 15min. If the product size was correct and the concentration was high, the gel was excised and recovered.

[0027] Table 1 PCR reaction system

[0028] The results are as follows Figure 1 As shown, the amplified gene size is approximately 3400 bp, which is in line with expectations and can be used for the next step of the experiment.

[0029] 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) Separate the target gene fragment using agarose gel electrophoresis and cut out the target gene band; 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. 3) Add 300 μL XP2 Binding Buffer to the adsorption column, centrifuge at 13000g for 1 min, and discard the waste liquid; 4) Add 700 μL of SPW Buffer to the adsorption column, centrifuge at 10000g for 1 min, and discard the waste liquid; 5) Repeat step 4). 6) Centrifuge at 13000g for 2 minutes to remove any remaining liquid from the adsorption column; 7) Transfer the adsorption column to a clean 1.5 mL centrifuge tube, add 30 μL of Elution Buffer (or TE buffer). Buffer), incubate at room temperature for 1 min, then centrifuge at 13000g for 1 min to elute DNA; 8) Measure the concentration of the recovered DNA for the next step of the experiment.

[0030] 1.3 Double digestion of the vector The following systems were prepared according to the TaKaRa endonuclease instructions (Table 2), and the gels were incubated at 37°C for 15 min, and then the gels were cut and recovered.

[0031] Table 2 Double enzyme digestion reaction system

[0032] 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; 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; 3) Randomly pick single colonies into centrifuge tubes containing 1 mL of LB liquid medium containing Amp, and 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. 4) Store the bacterial culture with correct test results at 80℃. After successful sequencing, extract the recombinant plasmid for later use.

[0033] 1.5 Obtaining Recombinant Baculovirus Two μL of recombinant plasmid pYBDM-IM-GP67-SADS-CoV-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 red fluorescence appeared, it indicated that the recombinant baculovirus was successfully constructed.

[0034] 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 a 1:5000 dilution of HRP-6×His-tagged antibody, and the cells were developed using ECL chemiluminescence buffer.

[0035] 1.6 Expression and purification of recombinant SADS-CoV-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 SADS-CoV-S protein was determined using a BCA protein quantification kit.

[0036] The results are as follows Figure 2 As shown, the target band size of the induced purified SADS-CoV-S protein is 130 kDa, which can be used for the next step of the experiment.

[0037] Example 2. Preparation of egg yolk antibody against porcine acute diarrhea syndrome coronavirus S protein 2.1 Preparation of Freund's adjuvanted vaccine Freund's complete adjuvant was mixed with an equal volume of SADS-CoV-S protein solution (1 mg / mL) and emulsified at 6000 rpm for 5 min. The emulsified vaccine was milky white and did not separate into layers after centrifugation at 3000 rpm for 15 min, indicating that the Freund's adjuvant was a water-in-oil emulsion. A drop of vaccine placed on the surface of water spread rapidly, but a second drop did not spread, indicating that the water-in-oil effect was initially achieved; the final concentration of the antigen protein was 0.5 mg / mL.

[0038] 2.2 Immunize laying hens and collect eggs Sixty healthy laying hens were randomly divided into two groups of 30 each: a control group and an experimental group. They were provided with ample feed and water for one week and observed. Hens exhibiting good growth and appetite, laying 5-7 eggs per week per hen, were then immunized. Booster immunizations were administered one week apart (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.

[0039] Table 3. Immune Process in Hens

[0040] 2.3 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.

[0041] The isolated egg yolk antibodies were purified using ammonium sulfate precipitation: based on the measured volume of crude egg yolk antibody extract, saturated ammonium sulfate solution was slowly added until the final saturation was 55%, 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.

[0042] Example 3. Identification of egg yolk antibodies against porcine acute diarrhea syndrome coronavirus 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 12.6 mg / mL. 20 μL of the diluted protein solution was mixed with 5 μL of 5× loading buffer, boiled in a water bath for 10 min, and then subjected to 12% reducing SDS-PAGE electrophoresis.

[0043] The results are as follows Figure 3 As shown, the heavy chain and light chain are located at the 70kDa and 25kDa bands, respectively, with a total molecular weight of approximately 180kDa and a purity of about 90%.

[0044] 3.2 The fluctuations in detection patterns using indirect enzyme-linked immunosorbent assay (ELISA) Porcine acute diarrhea syndrome coronavirus (PAC) S protein 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 hours. 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 hours, followed by three washes. 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 hour, followed by four washes. 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.

[0045] like Figure 4 ELISA results showed that the titer of egg yolk antibodies gradually increased after the second immunization, reaching its maximum between the 8th and 10th week after the initial immunization, and then tending to decline after the 14th week. The titer in the control group showed no significant change.

[0046] 3.3 Antibody Neutralization Assay 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 vertically in the 96-well plate for a total of 12 gradients. Then add 50 μL of pre-diluted 200 TCID50 SADS-CoV / 2023 / HNNY virus solution (isolated and preserved by our laboratory) to each well, and set up positive and negative controls. In the positive control column, add 50 μL of serum-free DMEM medium and 50 μL of diluted virus solution to each well. In 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 Vero cells, which have reached 90% confluence, from the incubator, add sterile PBS, wash three times along the well wall, and then discard the PBS.

[0047] After incubation for 1 h, the mixture was added to a pre-washed Vero cell culture plate and incubated at 37°C for 2 h. The mixture was then discarded, and the plate was washed three times with PBS. DMEM containing 8 μg / mL trypsin was added to each well. Microscopic observation was used to systematically observe the cytopathic effects of the cells in this experiment.

[0048] During the experiment, morphological changes in cells were observed daily under a microscope, with a focus on typical pathological features such as cell shrinkage, fusion, and detachment. To ensure the reliability of the experimental results, positive and negative controls were set up: when significant cytopathic effects were observed in the positive control column and normal cell morphology was maintained in the negative control column, the lesion wells appearing in the test sample 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.

[0049] like Figure 5 Antibody neutralization experiments showed that egg yolk antibodies against porcine acute diarrhea syndrome coronavirus (SADS-CoV) S protein could significantly inhibit the growth of SADS-CoV. The neutralizing titer of egg yolk antibodies gradually increased after the second immunization, reaching its highest level at week 8 after the first immunization, and then showed a downward trend after week 14. The control group showed no neutralizing effect.

[0050] Example 4: Optimization of Egg Yolk Antibody Preparation Process 4.1 Preparation of Immunoassay Preliminary experiments conducted by the inventors show that simply injecting subunit vaccines produces egg yolk antibodies with low titers, with the highest neutralizing antibody titer reaching only 26. This is insufficient for the large-scale production of SADS-CoV-S protein egg yolk antibody drugs. Therefore, improving the titer of egg yolk antibodies is a key technical challenge in the development and production of related drugs.

[0051] To improve the preventive and therapeutic efficacy against SADS-CoV infection, this invention prepares different immune compositions to obtain high-titer SADS-CoV-S protein yolk antibodies. Through screening a large number of molecular adjuvants, the inventors discovered that adding IL-2 aqueous solution, ZnCl2 aqueous solution, and Astragalus polysaccharide aqueous solution to the vaccine can enhance the immunogenicity of the composition. Based on this, this invention uses Freund's complete adjuvant as the base adjuvant and adds multiple molecular adjuvants to enhance the immunogenicity of the immune composition. Further optimization of the components has resulted in the design of various immune compositions, as detailed in Table 4 below.

[0052] To facilitate vaccine preparation, this invention prepares IL-2, zinc chloride, and astragalus polysaccharide into aqueous solutions using sterile PBS, wherein the IL-2 aqueous solution contains 10,000 units / mL; the zinc chloride aqueous solution has a concentration of 3.2 mg / mL; and the astragalus polypeptide aqueous solution has a concentration of 3.2 mg / mL.

[0053] Table 4 Composition (volume ratio) of the immunomodulatory composition

[0054] Preparation of the immune composition: According to the composition in Table 4, the SADS-CoV-S protein solution (1.1 mg / mL) was mixed with IL-2, zinc chloride and / or astragalus polysaccharide aqueous solution and stirred evenly to obtain a mixed solution. Then, Freund's complete adjuvant was mixed with an equal volume of the mixed solution and emulsified at 6000 rpm for 5 min to obtain the corresponding immune composition, wherein the final concentration of antigen protein was 0.5 mg / mL.

[0055] 4.2 Immunize laying hens and collect eggs Immunize laying hens and collect eggs according to the method in Example 2, Section 2.2.

[0056] 4.3 Isolation and purification of egg yolk antibodies Egg yolk antibodies from eggs produced in weeks 8-10 were isolated and purified according to the method described in section 2.3 of Example 2.

[0057] 4.4 Concentration Determination The protein concentration in the egg yolk antibody solution of each group during weeks 8-10 was determined according to the method in 3.1 of Example 3, as shown in Table 5.

[0058] 4.5 Determination of antibody neutralizing titer The protein concentration of each group of egg yolk antibody solutions was adjusted to 10 mg / mL; the average neutralizing antibody titer of each group of egg yolk antibody solutions was determined according to the method in 3.3 of Example 3, as shown in Table 5.

[0059] Table 5. Effects of the composition of the immunoassay on antibody yield and neutralizing titer.

[0060] Based on the above experimental results, it can be seen that when IL-2 and zinc chloride aqueous solution are added to the immune composition at the same time, the protein concentration in the prepared egg yolk antibody aqueous solution is the highest, and the neutralizing titer of the egg yolk antibody is the highest at the same protein concentration. Compared with the immune composition using Freund's complete adjuvant alone, the neutralizing titer is increased by more than 60 times.

[0061] Furthermore, the inventors conducted optimization experiments on the dosage of IL-2 and zinc chloride, and determined the milk concentration and neutralizing antibody titer according to the above method, as shown in Table 6 below.

[0062] Table 6 Composition optimization of immune compositions (volume ratio)

[0063] Based on the above experiments, it can be seen that when the volume ratio of IL-2 aqueous solution to ZnCl2 aqueous solution is 6:4-4:6, the antibody titer is the highest, which is higher than 211.

[0064] 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 egg yolk antibody against the S protein of porcine acute diarrhea syndrome coronavirus, characterized in that: The egg yolk antibody was prepared by using the recombinant protein SADS-CoV-S of porcine acute diarrhea syndrome coronavirus, constructed in an insect-baculovirus expression system. Its amino acid sequence is shown in SEQ ID No:

4. After purification, adjuvants and other components were added to prepare a subunit vaccine, which was injected into laying hens for immunization. Eggs from the immunized laying hens were collected, and the yolks were isolated and purified from the immunized eggs.

2. The method for preparing the egg yolk antibody against porcine acute diarrhea syndrome coronavirus S protein according to claim 1, characterized in that, Includes the following steps: (1) Preparation of antigen: The recombinant protein SADS-CoV-S of porcine acute diarrhea syndrome coronavirus was constructed in the insect-baculovirus expression system, the protein was expressed and purified, and the purified recombinant protein SADS-CoV-S was harvested after dialysis and concentration; (2) Preparation of immune composition: The purified SADS-CoV-S protein solution, IL-2 aqueous solution and zinc chloride aqueous solution were mixed and stirred evenly to obtain a mixed solution. Then, the mixed solution was mixed with an equal volume of Freund's complete adjuvant and emulsified at 6000 rpm for 5 min to obtain the corresponding immune composition. (3) Immunizing laying hens: Immunize laying hens with an immunization composition. First, immunize laying hens, and then perform a second, third and fourth booster immunization. Collect eggs. (4) Separate and purify the egg yolk to obtain egg yolk antibody.

3. The preparation method according to claim 2, characterized in that, In step (1), the preparation of the antigen is as follows: using the S protein of SADS-CoV as the target antigen protein, the open reading frame of the protein S gene, the sequence of which is shown in SEQ ID No: 3, is inserted into the vector pYBDM-IM and transformed into Ac MultiBac / rSW106 / asd- / inv+ competent cells to construct an engineered bacterium expressing the recombinant protein SADS-CoV-S; 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 SADS-CoV-S is harvested, which is the target antigen protein.

4. The preparation method according to claim 2, characterized in that, In step (2), the concentration of the SADS-CoV-S protein solution is 1.1 mg / mL, the IL-2 aqueous solution contains 10,000 units / mL, and the concentration of the zinc chloride aqueous solution is 3.2 mg / mL.

5. The preparation method according to claim 2, characterized in that, In step (2), the ratio of SADS-CoV-S protein solution: IL-2 aqueous solution: zinc chloride aqueous solution is 1:0.04-0.06:0.06-0.04; preferably, the volume ratio of IL-2 aqueous solution: zinc chloride aqueous solution is 6:4-4:6; more preferably, the volume ratio of IL-2 aqueous solution: zinc chloride aqueous solution is 4:

6.

6. The preparation method according to claim 2, characterized in that, In step (3), the immunization of laying hens specifically involves administering the immunization composition to the laying hens four times, injecting 1 mL into the subpectoral muscle of each hen each time, with a one-week interval between each immunization, and collecting eggs starting one week after the third immunization.

7. The preparation method according to claim 2, characterized in that, In step (4), 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 pH 5.3 acidified water, vortex to mix, and let stand overnight at 4℃ to prepare crude extract of egg yolk antibodies.

8. The preparation method according to claim 2, characterized in that, In step (4), 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 fully dialyzed with a dialysis bag to obtain anti-SADS-CoV-S protein egg yolk antibody.

9. The use of the anti-swine acute diarrhea syndrome coronavirus S protein yolk antibody prepared by any one of claims 2-8 in the preparation of drugs or feed additives for the prevention and / or treatment of diseases caused by swine acute diarrhea syndrome coronavirus.

10. A method for detecting the titer of egg yolk antibodies against porcine acute diarrhea syndrome coronavirus, characterized in that: The titer of egg yolk antibodies against porcine acute diarrhea syndrome coronavirus S protein was detected using an indirect enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows: 1) Coat an ELISA plate with 1 μg / mL SADS-CoV S protein, 100 μL per well, at 4°C overnight. After washing three times with PBST, add blocking buffer containing 5% BSA and block at 37°C for 2 h. The amino acid sequence of SADS-CoV S protein is shown in SEQ ID No:

4. 2) After washing three times, add serially diluted egg yolk antibody against SADS-CoV-S protein, and set up blank control and negative control groups. Incubate at 37℃ for 2 hours. 3) After washing 3 times, add horseradish peroxidase-labeled goat anti-chicken secondary antibody and incubate at 37°C for 1 hour; 4) After washing 4 times, add TMB colorimetric solution and develop the color at 37°C in the dark for 15-30 minutes; 5) Add 100 μL of 2M H2SO4 to terminate the reaction; 6) Read OD using an ELISA reader 450nm A positive result is defined as a ratio of the absorbance of the test sample to the negative control greater than 2.1, with the maximum positive dilution being the antibody titer.

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