Preparation of TW-type IBV egg yolk antibodies, indirect ELISA detection method and antiviral application

Abstract

This invention discloses a method for preparing TW-type IBV egg yolk antibodies, an indirect ELISA detection method, and their antiviral applications. The preparation method includes the following steps: TW-type IBV is inactivated with β-propiolactone, then emulsified with white oil at a volume ratio of oil phase to water phase of 3:2 to obtain an immunogen; the immunogen is administered to adult laying hens via intramuscular injection, with the following immunization schedule: one primary immunization, a first booster immunization 14 days later, and a second booster immunization 14 days later; hyperimmune eggs are collected two weeks after each immunization; egg yolk antibodies are extracted from hyperimmune eggs using the caprylic acid method. This invention utilizes inactivated virus immunization to prepare specific egg yolk antibodies, establishes an indirect ELISA detection system based on prokaryotic expression of recombinant S1 protein, and simultaneously verifies the anti-TW-type IBV activity of the antibody, enabling accurate detection of strain-specific antibodies and providing practical technical support for the biocontrol of this disease.

Description

Technical Field

[0001] This invention relates to the field of immunology, specifically to the preparation of TW-type IBV egg yolk antibodies, an indirect ELISA detection method, and their antiviral applications. Background Technology

[0002] Infectious bronchitis (IB) is an acute and highly contagious disease of chickens caused by the avian infectious bronchitis virus (IBV). It can occur at any age of chicken, but it is most infectious to chicks under 4 weeks old. It is characterized by high morbidity, high mortality, and extremely high infectivity. After infection, secondary infections such as mycoplasma and Escherichia coli are common, leading to various clinical types such as respiratory, renal, proventricular, and reproductive types. This results in serious consequences such as damage to tracheal cilia, decreased egg production, and increased mortality, causing huge economic losses to the poultry industry.

[0003] Currently, the main diagnostic methods for IBV include clinical diagnosis and laboratory diagnosis. Clinical diagnosis is rapid but highly subjective and prone to misdiagnosis. In laboratory diagnosis, etiological diagnosis (virus isolation) is accurate but time-consuming and not suitable for large-scale testing; serological diagnoses such as virus neutralization test (VNT), hemagglutination inhibition test (HI), and enzyme-linked immunosorbent assay (ELISA) each have limitations: VNT is sensitive but expensive and cannot distinguish between vaccine antibodies and infection antibodies; ELISA is simple to operate and has high throughput, but generally can only detect serotype-specific antibodies and cannot distinguish serotypes; molecular biological diagnoses (PCR, qPCR, RT-PCR) are sensitive and specific, but require specialized equipment and reagents, and are relatively expensive.

[0004] Immunoglobulin of yolk (IgY) is a type of antibody produced in birds after their immune system is activated by external antigens. As the immune system is activated, B cells in the bursa of Fabricius differentiate into plasma cells and produce corresponding antibodies that enter the bloodstream. These antibodies accumulate in the yolk through receptor action. While structurally and functionally similar to mammalian IgG, IgY has a unique molecular structure with no hinge region and more constant heavy chain domains, resulting in greater structural stability. It also exhibits excellent resistance to acids and alkalis, high temperatures, and has a long shelf life, demonstrating superior physicochemical properties.

[0005] Compared to traditional antibiotics and mammalian antibodies, IgY leaves no drug residues, does not induce drug resistance, has no toxic side effects, and does not trigger cross-immune reactions. It aligns with the trend of banning antibiotics in livestock and poultry farming and is a highly promising antibiotic alternative. In terms of preparation, IgY can be extracted simply by collecting hyperimmune eggs from immunized laying hens, eliminating the need for blood collection, thus protecting animal welfare. It also boasts high yield, a short preparation cycle, low production costs, and supports oral administration, offering significant advantages in emergency disease control.

[0006] Furthermore, IgY has a wide range of applications, possessing multiple biological activities such as antibacterial, antiviral, antiparasitic, and anti-allergic properties, and its application prospects in the emergency prevention and control of avian viral diseases are broad. Currently, the use of specific IgY to control avian infectious diseases has become a research hotspot in the veterinary field. Therefore, conducting research on the preparation of TW-type IBV-specific egg yolk antibodies, and simultaneously establishing a supporting rapid ELISA detection method, is of significant practical importance and application value for improving the IBV diagnostic system, developing highly effective control agents, achieving precise prevention and control of this disease, and reducing losses from poultry diseases. Summary of the Invention

[0007] To address the shortcomings of existing technologies such as the lack of specific detection methods for TW-type IBV, the scarcity of dedicated prevention and control agents, the crude antibody extraction process, and the unclear control efficacy, this invention provides a method for preparing TW-type IBV egg yolk antibodies, an indirect ELISA detection method, and their antiviral application. The invention involves preparing an immunogen by inactivating the virus and immunizing laying hens to generate specific egg yolk antibodies. Simultaneously, an indirect ELISA detection method is established by expressing the S1 recombinant protein in prokaryotes. The antiviral efficacy of this egg yolk antibody against TW-type IBV is verified, thereby achieving accurate detection of TW-type IBV antibodies and biocontrol of this disease.

[0008] To achieve the above objectives, the technical solution specifically adopted by the present invention is as follows: A method for preparing TW-type IBV yolk antibody includes the following preparation steps: S1. Antigen preparation: TW type avian infectious bronchitis virus was selected and inactivated with β-propiolactone at 4℃ for 36h. After inactivation, it was placed at 37℃ for 4h to allow β-propiolactone to fully decompose. The inactivated virus was emulsified with white oil adjuvant at a ratio of oil phase:water phase = 3:2 to prepare the immunogen. S2. Animal immunization: Select healthy adult laying hens and immunize them by intramuscular injection in the pectoral muscle. The immunization program is one primary immunization and two booster immunizations, with an interval of 14 days between each immunization. S3. Collection of hyperimmune eggs: Collect hyperimmune eggs 14 days after the end of each immunization; S4. Egg yolk antibody extraction: Egg yolk antibodies were extracted using an optimized caprylic acid method. The extraction system had a pH of 5.2 and a caprylic acid concentration of 2.5%, yielding purified egg yolk antibodies with a protein concentration of up to 10.88 mg / mL. Furthermore, the TW-type IBV egg yolk antibody levels stabilized in the second week after the third immunization, and the neutralizing titer in the chicken embryo neutralization test was no less than 1:27.

[0009] This invention also provides an indirect ELISA detection method for TW-type IBV egg yolk antibodies, using TW-type IBVS1 recombinant protein as the coating antigen at a concentration of 1 µg / mL, a 1:40 dilution of the egg yolk antibody to be tested, 5% skim milk as the blocking buffer for 2 hours, rabbit anti-chicken IgG-HRP at a dilution of 1:5000 for 30 minutes, and a color development time of 8 minutes; wherein, the preparation method of the S1 recombinant protein includes: (1) Construct a pET-32a(+) recombinant plasmid containing the S1 gene and transform it into competent Escherichia coli cells; (2) Induction of expression: IPTG final concentration 0.5mM, induced culture at 16℃ and 150r / min for 16h to obtain S1 protein expressed in inclusion body form, with a protein size of 75kDa; (3) Protein refolding: The inclusion bodies were refolded by a gradient dialysis method of 6M, 4M, 2M, 1M and 0M urea to obtain purified refolded S1 protein with a protein concentration of 116 μg / mL.

[0010] After detecting 12 negative egg yolk antibodies using the established indirect ELISA method, the cut-off value was determined to be 0.24. The sensitivity of this method was 1:1280 dilution, and the coefficients of variation for inter-assay and intra-assay repeatability were both less than 20%.

[0011] The TW-type IBV yolk antibody prepared by this invention can be used to prepare anti-TW-type IBV drugs. The yolk antibody can delay the onset of disease in chicken flocks, reduce viral shedding in pharyngeal and cloacal swabs, and reduce mortality in infected chickens. The anti-TW-type IBV drug achieves at least one of the following effects: delaying onset of disease, reducing viral shedding, reducing mortality, alleviating tracheal ciliary damage, alleviating lung tissue damage, alleviating kidney tissue damage, reducing viral load in the larynx and trachea, reducing viral load in kidney tissue, and reducing viral load in lung tissue.

[0012] Compared with the prior art, the present invention has the following beneficial effects: 1) Reliable antigen expression and purification refolding methods: For the first time, an optimized prokaryotic expression and inclusion body refolding process was established for TW-type IBVS1 protein, obtaining immunoreactive recombinant S1 protein, providing a high-quality coated antigen for the establishment of subsequent detection methods.

[0013] 2) The egg yolk antibody preparation process is efficient and safe: β-propiolactone is used to inactivate the virus, which is thorough and completely decomposed, avoiding the residual toxicity that may be caused by formaldehyde inactivation; the optimized oil emulsion immunogen and pectoral muscle injection immunization program can efficiently induce laying hens to produce high-titer egg yolk antibodies; the modified caprylic acid method (pH 5.2, caprylic acid concentration 2.5%) enables the large-scale, high-purity, and high-concentration extraction of egg yolk antibodies, with a protein concentration of 10.88 mg / mL.

[0014] 3) The indirect ELISA detection method is sensitive and specific: For the first time, a method for detecting the titer of TW-type IBV egg yolk antibodies based on recombinant S1 protein was established. The sensitivity reached 1:1280, the cut-off value was 0.24, and the coefficient of variation was less than 20%. It can accurately monitor changes in antibody levels after immunization and guide the timing of immunization and the application of egg yolk antibodies.

[0015] 4) Significant antiviral effect: The TW type IBV yolk antibody prepared in this invention showed high neutralization titer (>1:128) in chicken embryo neutralization test; in SPF chickens, it can significantly delay the onset of disease, reduce viral shedding, reduce mortality, reduce damage to tracheal cilia and lung and kidney tissues, and reduce tissue viral load, showing excellent antiviral therapeutic effect.

[0016] 5) Broad application prospects: This invention provides a new technical means for the prevention and control of TW-type IBV. The prepared egg yolk antibody can be used as a biological agent for emergency prevention and treatment, replacing or reducing the use of antibiotics, which meets the requirements of animal welfare and food safety. Attached Figure Description

[0017] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 For the structural and hydrophobic analysis of the S1 protein; In the diagram: A, signal peptide detection; B, hydrophilicity detection.

[0018] Figure 2 Construct a map of the S1 protein particle.

[0019] Figure 3 This is the result of PCR amplification of the S1 sequence; In the figure: M, Marker; 1-8, 8 S1 amplified samples.

[0020] Figure 4 The results are for S1pET-32a(+) double digestion. In the figure: M, Marker; 1-6, 6 double-digested samples.

[0021] Figure 5 PCR results for bacterial culture In the figure: M, Marker; 1-8, PCR bands of 8 positive colonies.

[0022] Figure 6 These are the results of gene sequencing.

[0023] Figure 7 To identify S1 protein expression using SDS-PAGE; In the figure: M, Marker; 1, before induction; 2, after induction; 3, supernatant after crushing; 4, precipitate after crushing.

[0024] Figure 8 To identify the purification efficiency of S1 protein using SDS-PAGE; In the diagram: M, Marker; 1, Flow-through solution; 2, First wash; 3, Second wash; 4, Third wash; 5, First elution; 6, Second elution; 7, Third elution.

[0025] Figure 9 The refolding effect of S1 protein was identified by SDS-PAGE (A) and Western blot (B). In the figure: M, Marker; 1-4, four protein samples after S1 protein renaturation.

[0026] Figure 10 To establish a standard curve for protein concentration determination, the formula obtained from the standard curve is: y = 0.6903x + 0.0262, R² = 0.9964.

[0027] Figure 11 Optimize color development time.

[0028] Figure 12 For the stability test of the immunogen; In the diagram: A, state after centrifugation; B, state after standing at room temperature.

[0029] Figure 13 For the safety testing of immunogens; In the figure: A: blank control; B: immunogen.

[0030] Figure 14 Optimization of caprylic acid concentration for egg yolk antibody extraction method; In the figure: A, protein purity; B, state after centrifugation. M, marker; 1-8, caprylic acid concentrations of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, and 4.5%.

[0031] Figure 15 For safety inspection In the figure: A, blank control; B, egg yolk antibody.

[0032] Figure 16 For the detection of egg yolk antibody titer.

[0033] Figure 17 This is for the detection of yolk antibody neutralization capacity.

[0034] Figure 18 Clinical symptom observation after IBV challenge; In the figure: A, clinical symptom score; B, mortality rate.

[0035] Figure 19 The tracheociliary score is given after IBV challenge.

[0036] Figure 20 Observation of lesions in the larynx, trachea, kidneys, and lungs after IBV challenge. In the figure: A, lesion observation on day 5 post-infection; B, lesion observation on day 10 post-infection.

[0037] Figure 21 Histopathological changes (HE staining) of the larynx, trachea, kidneys, and lungs after IBV challenge. In the figure: A, histopathological changes on day 5 post-infection; B, histopathological changes on day 10 post-infection.

[0038] Figure 22 For testing viral shedding after IBV infection; In the image: A, the viral shedding from a throat swab after IBV infection; B, the viral shedding from a cloacal swab after IBV infection.

[0039] Figure 23 The viral load in the trachea, lungs, and kidneys after IBV challenge.

[0040] In the diagram: A, viral load in the trachea; B, viral load in the lungs; C, viral load in the kidneys. Detailed Implementation

[0041] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. Example

[0042] 1.1 Test Materials SPF chicken embryos were purchased from Shandong Haotai Experimental Animal Breeding Co., Ltd.; TW type IBV strain and pET-32a(+) plasmid were preserved by the Animal Science and Veterinary Medicine Laboratory of Tianjin Agricultural College; TW type IBV egg yolk antibody positive standard was prepared by our laboratory in preliminary experiments.

[0043] 1.2 Preparation of the main solution (1) Preparation of ethanol solution: Dilute anhydrous ethanol to 75%.

[0044] (2) LB liquid culture medium: 8g LB broth to a final volume of 400mL.

[0045] (3) LB solid medium: 8g LB nutrient agar to a final volume of 400mL.

[0046] (4) Coomassie brilliant blue staining solution: 0.5g R-250 + 45mL methanol + 10mL glacial acetic acid + 45mL distilled water.

[0047] (5) Inclusion body washing solution I: 25 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, 1% Triton-X100, pH 8.0.

[0048] (6) Inclusion body washing solution II: 2M urea, 10mM MTT, 25mM Tris-HCl, 1mM EDTA, 100mM NaCl, 0.1% Triton-X100, pH 8.0.

[0049] (7) Inclusion body lysis buffer: 8M urea, 50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0. (8) Washing buffer: 8M urea, 50mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH 8.0.

[0050] (9) Eluent: 8M urea, 50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH 8.0.

[0051] (10) Inclusion body refolding solution: 6M, 4M, 2M, 1M, 0M urea gradient, each containing 10mM reduced glutathione and 1mM oxidized glutathione, adjusted to volume with 1×PBS, pH 8.0.

[0052] 1.3 Test Methods 1.3.1 Structural and hydrophobicity analysis of S1 protein Download the GenBank TW IBV sequence, analyze the S1 protein structure and remove the signal peptide using the TMHMM website; perform protein hydrophobicity analysis using the Expasy website.

[0053] 1.3.2 Plasmid Construction The extracted S1 gene sequence and the pET-32a(+) vector sequence were used to construct a plasmid map using GeneiousPrime software.

[0054] 1.3.3 Primer Design and Synthesis Primers for amplification and detection were designed using Primer 5.0 software and synthesized by Qingke Biotechnology. The primer sequences are shown in Table 1.

[0055] Table 1 Primer sequences for constructing recombinant plasmids

[0056] 1.3.4 IBV Reproduction Select 9-10 day old SPF chicken embryos, sterilize them, punch holes in the air cell and allantoic cavity, inoculate each embryo with 200 μL IBV of protovirus, and culture at 37℃. Remove dead embryos within 24 hours, culture for 72 hours, then incubate overnight at 4℃, aseptically collect allantoic fluid, filter through a 0.45 μm filter membrane, and store at -80℃.

[0057] 1.3.5 Total RNA Extraction The Xavier Total RNA Extraction Kit was used, with the following specific operating parameters: Allantoic fluid stored at -80℃ was taken out, thawed at 4℃, and centrifuged at 12000 r / min at 4℃ for 10 min; 200 μL of supernatant was aspirated, 10 μL of Proteinase K and 500 μL of Buffer RL1 pre-containing 40 μL DTT were added, and the mixture was incubated in a water bath at 56℃ for 10 min; subsequent steps of centrifugation, genomic removal, binding, multiple washing, and two elutions were performed to extract total viral RNA, thereby improving the RNA recovery rate.

[0058] 1.3.6 Reverse transcription According to the instructions for the reverse transcription kit provided by Qingke Biotechnology Co., Ltd., the total RNA extracted in the previous step was reverse transcribed. The specific steps are as follows: (1) Thaw the total RNA and the reagents in the kit at 4°C.

[0059] (2) Add the following system (10µL) to an RNase-free and sterile PCR centrifuge tube on ice, as shown in Table 2.

[0060] Table 2 Reverse Transcription System I

[0061] (3) After repeatedly blowing and mixing the above system, centrifuge, incubate at 42℃ for 2 min, and incubate at 60℃ for 1 min.

[0062] (4) After incubation, add the following mixture (10 µL), as shown in Table 3: Table 3 Reverse Transcription System II

[0063] (5) After mixing the above system by pipetting, briefly centrifuge, incubate at 50°C for 15 min, incubate at 85°C for 5 s, and then store the cDNA obtained by reverse transcription at -20°C.

[0064] 1.3.7 Amplification of the S1 sequence According to the KODOne™ PCRMasterMix-Blue instructions provided by Toyobo (Shanghai) Biotechnology Co., Ltd., the S1 gene fragment was amplified by PCR according to the following reaction system (Table 4) and reaction procedure (Table 5).

[0065] Table 4 PCR reaction system (50µL)

[0066] Table 5 PCR amplification reaction procedure

[0067] The amplified products were identified by 1% agarose gel electrophoresis.

[0068] 1.3.8 Gel recovery of PCR products The agarose gel DNA recovery kit was used, and the specific operating parameters were as follows: 500 μL of equilibration buffer BL was added to the CA2 adsorption column, and the column was centrifuged at 12000 rpm for 1 min at room temperature to complete column equilibration; a single target DNA gel band was cut and weighed, and sol was added at a ratio of 100 μL LPN sol per 0.1 g of gel. The column was heated in a 50°C metal bath and repeatedly turned until the gel block was completely melted; the sol solution was transferred to the equilibrated adsorption column, incubated at room temperature for 2 min, and centrifuged at 12000 rpm for 2 min at room temperature; 600 μL of LPW rinsing buffer was added sequentially for washing twice, centrifuged at 12000 rpm for 1 min each time; after washing, the column was centrifuged for 2 min to completely remove alcohol; the adsorption column was placed in a sterile centrifuge tube, and 35 μL of EB elution buffer preheated to 65°C was added dropwise to the adsorption membrane. After standing at room temperature for 5 min, the column was centrifuged to elute, and the nucleic acid was collected and the nucleic acid concentration was determined using a spectrophotometer.

[0069] 1.3.9 Vector double enzyme digestion The pET-32a(+) vector was double-digested with XhoI and BamHI, and incubated at 37°C for 15 min. The digestion system is shown in Table 6. Table 6 Double enzyme digestion system

[0070] After mixing the above system thoroughly, incubate it in a 37°C water bath for 15 minutes. The enzyme digestion products are identified by 1% agarose gel electrophoresis.

[0071] 1.3.10 Gel recovery of double enzyme digestion products The steps are as described in 1.3.8.

[0072] 1.3.11 Homologous Recombination According to the instructions of the homologous recombination kit provided by Mona Biotechnology Co., Ltd., the gel-recovered product of PCR-amplified S1 gene fragment was homologously recombinated with the double-digested vector pET-32a(+). The homologous recombination system (10 μL) is as follows (Table 7).

[0073] Table 7 Homologous Recombination System

[0074] After mixing the prepared system thoroughly, place it in a PCR instrument and incubate at 50°C for 15 minutes. Then store at -20°C.

[0075] 1.3.12 Conversion Follow the steps outlined in the instructions for DH5a competent cells provided by Qingke Biotechnology Co., Ltd.: Take 100µL LDH5α competent cells, add 5µL of homologous recombinant product, and incubate on ice for 30min; heat shock at 42℃ for 55s, then incubate on ice for 2min; add 700µL of antibiotic-free LB, and revive at 37℃ and 200r / min for 1h; spread on ampicillin-resistant plates and incubate overnight at 37℃.

[0076] 1.3.13 Bacterial PCR and Sequencing Eight single colonies were picked and placed in 700 µL of ampicillin-resistant LB agar, and incubated at 37 °C and 200 rpm for 4 h. PCR verification was performed using the bacterial culture as a template (primers 3 and 4; system and procedure are shown in Tables 10 and 11). Positive strains were sent for sequencing.

[0077] Table 10 Bacterial PCR System

[0078] Table 11 PCR amplification reaction procedure for bacterial culture

[0079] 1.3.14 Plasmid Extraction Plasmid extraction was performed according to the instructions of the endotoxin-free plasmid small-scale extraction kit provided by Tiangen Biotech Co., Ltd.

[0080] Take 100 µL of the correctly sequenced bacterial culture and inoculate it into 50 mL of ampicillin LB. After overnight incubation, collect the bacterial cells by centrifugation at 12,000 rpm for 10 min. Resuspend the cells in 4 mL P1, lyse with 4 mL P2 by inversion 8 times, neutralize with 5.6 mL P3 by inversion 8 times, and centrifuge at 12,000 rpm for 10 min. Pass the supernatant through a CP4 column (pre-equilibrated with 500 µL BL by centrifugation at 12,000 rpm for 1 min), centrifuge at 12,000 rpm for 1 min; wash twice with 600 µL LPW, centrifuging at 12,000 rpm for 1 min each time; centrifuge for 2 min. Add 150 µL LEB (preheated to 65 °C) to the center of the membrane, incubate at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min; repeat the elution once. Measure the concentration using a spectrophotometer and store at -20 °C.

[0081] 1.3.15 Conversion Refer to the instructions for BL21(DE3) competent cells provided by Qingke Biotechnology Co., Ltd., and follow the steps in 1.3.12.

[0082] 1.3.16S1 protein induction The following day, a single transformed colony was picked and incubated overnight at 37°C and 220 rpm in 50 mL of ampicillin LB. 100 μL of the inoculum was then transferred to 100 mL of ampicillin LB and incubated at 37°C and 220 rpm until OD (digesterone) was reached. 600 =0.6, add IPTG for induction (take 1 mL of bacterial culture as a control before induction). After induction, centrifuge the bacterial culture at 12,000 r / min, 4℃ for 20 min, and wash twice with PBS (take 1 mL of PBS as a control after induction before centrifugation). Resuspend the precipitate in 10 mL PBS, add 1 mM PMSF, and sonicate on ice (200 W, sonicate for 2 s, pause for 4 s, total 15 min). Centrifuge at 12,000 r / min for 20 min at 4℃, and take 50 μL each of the pre-induction, post-induction, and lysed supernatant and lysed precipitate to prepare protein samples. Heat at 100℃ for 10 min and store at -80℃.

[0083] 1.3.17 SDS-PAGE analysis of S1 protein The prepared pre-induction control, post-induction control, lysed supernatant, and lysed precipitate protein samples were analyzed by SDS-PAGE to determine the expression of S1 protein. The upper and lower gels were prepared according to the instructions of the one-step PAGE color gel ultra-rapid preparation kit provided by Wuhan Sewell Biotechnology Co., Ltd., as detailed in Table 12.

[0084] Table 12 Protein Gel Preparation

[0085] The steps are as follows: Load 8 μL of protein sample into each well, electrophoresis at 240V for 30 min, Coomassie brilliant blue staining for 25 min, and microwave heating with water for 10 min to decolorize.

[0086] 1.3.1 Purification and refolding of 8S1 protein S1 protein was expressed in inclusion body form and purified using NiNTA affinity chromatography followed by renaturation via urea gradient dialysis. 2 mL of NiNTABeads packing material was used to pack a column, and column equilibration was achieved using 3 column volumes of lysis buffer. After amplification and induction of the bacterial cells, the precipitate was ultrasonically disrupted, and then washed sequentially with Wash Buffer I and Wash Buffer II (12000 rpm, room temperature, 10 min each time). The precipitate was resuspended in lysis buffer and incubated overnight at 4°C. The supernatant was collected by centrifugation. Samples were loaded onto the column and incubated at 4°C for 3–5 h, and the flow-through was collected. The sample was washed three times with 4 column volumes of washing buffer (≥10 min each time), followed by elution three times with 1 column volume of elution buffer (≥20 min each time). The elutions were collected for SDS-PAGE analysis to assess the purification effect. After purification, the packing material was washed and stored. The dialysis bag was pretreated by boiling in water for 10 minutes. The purified protein was placed in the dialysis bag and sequentially placed in a 6M, 4M, 2M, 1M, and 0M urea gradient refolding solution containing reduced glutathione / oxidized glutathione (GSH / GSSG). Dialysis was performed stepwise at 4°C for 12 hours per step.

[0087] 1.3.19 SDS-PAGE and Western blot detection of purified and refolded S1 protein After purification and refolding, the S1 protein was analyzed by SDS-PAGE (same as 1.3.17) and then Western blot was performed for identification. The PVDF membrane was activated with methanol and transferred at 1.3A, 25V for 10 min; it was then blocked with 5% skim milk at room temperature for 2 h. The primary antibody was HisTag mouse monoclonal antibody (1:1000), incubated at room temperature for 1 h followed by overnight incubation at 4℃; the secondary antibody was HRP-labeled goat anti-mouse IgG (1:1000), incubated at room temperature for 1 h; after each antibody incubation, the membrane was washed three times with 1×TBST for 5 min each time. Finally, the membrane was developed using a DAB chromogenic kit for 10 min to complete the specificity identification.

[0088] 1.3.20S1 protein concentration determination The concentration of S1 protein after renaturation was determined using the Beyotime Bradford Protein Assay Kit. Using 20 mg / mL standard protein as the starting material, it was serially diluted with PBS to 1.5 mg / mL, 1 mg / mL, 0.75 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0 mg / mL. 10 μL of each standard protein was mixed with 300 μL of LG250 staining solution, and the absorbance was measured to plot a standard curve. 10 μL of the S1 protein to be tested was then analyzed using the same method, and the absorbance was measured. The protein concentration was calculated by substituting the absorbance into the standard curve.

[0089] 1.3.21 Main experimental steps of the indirect ELISA method Coating: Dilute S1 protein with coating buffer, 100 μL per well, and incubate overnight at 4°C.

[0090] Wash plate: Wash with 360μL PBST for 2 min, then pat dry.

[0091] Sealing: 360 μL of sealing solution per well, incubate at 37°C for 2 hours. Wash the plate 3 times.

[0092] Primary antibody: 100 μL of egg yolk antibody per well, 37°C for 1 hour. Wash the plate 3 times.

[0093] Secondary antibody: 100 μL rabbit anti-chicken IgG-HRP per well, incubated at 37°C for 1 hour. Wash the plate 3 times.

[0094] Color development: 100 μL of TMB per well, 37℃ for 10 min.

[0095] Termination: 50 μL of stop solution per well.

[0096] Reading: OD measured within 15 minutes after color development is terminated. 450nm value.

[0097] 1.3.22 Optimization of Conditions for Indirect ELISA Method 1.3.22.1 Optimization of Coating Concentration and Antibody Dilution Optimization was performed using a checkerboard method: S1 protein dilution gradients were 8 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL, 100 μL per well, coated overnight at 4°C, with 3 replicates per concentration; positive and negative egg yolk antibody dilution gradients were 1:10, 1:20, 1:40, 1:80, and 1:160, with 3 replicates per dilution; secondary antibody dilution ratio was 1:10000, 100 μL per well. The P / N ratio (positive and negative OD values) was used to determine the optimal concentration. 450nm The ratio is used to select the optimal condition.

[0098] 1.3.22.2 Optimization of Sealing Fluid Type and Sealing Time Optimal antigen and antibody concentrations were fixed, and the checkerboard method was used for optimization: blocking solutions were set at 1% BSA, 3% BSA, 5% BSA, and 5% skim milk; blocking times were set at 60 min, 120 min, and 180 min. Positive and negative controls were set up, and the optimal blocking conditions were screened based on the P / N ratio.

[0099] 1.3.22.3 Optimization of Secondary Antibody Dilution and Incubation Time With fixed optimal preconditions, the secondary antibody dilution gradients were: 1:5000, 1:10000, 1:15000, 1:20000, 1:25000, and 1:30000; the incubation times were set to 30 min, 60 min, and 90 min, and the optimal reaction parameters were determined based on the P / N ratio.

[0100] 1.3.22.4 Optimization of color development time With the above optimal conditions fixed, the color development time gradient is: 6 min, 8 min, 10 min, 12 min, 14 min, 16 min, 18 min. The optimal color development time is determined based on the P / N value.

[0101] 1.3.23 Interpretation of Results from Indirect ELISA Methods The established indirect ELISA method was used to detect OD antibodies in 12 negative egg yolk samples. 450 nm value, calculate the average (X) and OD of 12 negative egg yolk antibodies. 450nm The standard deviation (SD) of the values ​​is used to calculate the critical value according to the formula cut-off = X + 3SD, which serves as the basis for determining the positive or negative status of the sample.

[0102] 1.3.24 Indirect ELISA Method Test 1.3.24.1 Sensitivity test of indirect ELISA method Two positive egg yolk antibodies were taken and serially diluted with PBST: 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, and 1:2560. OD values ​​were then measured. 450 nm, with the first dilution below the cut-off value as the lowest limit of detection.

[0103] 1.3.24.2 Repeatability Test of Indirect ELISA Method Intra-batch replication: Take one sample each of positive, weakly positive, and negative egg yolk antibodies, and perform repeated testing 12 times on the same ELISA plate; Inter-batch replication: Take three positive and three negative samples, and test them on days 1, 3, and 5, respectively. Calculate the coefficient of variation using the formula CV = SD / X × 100%, where CV is the coefficient of variation; SD is the standard deviation; and X is the mean. The coefficient of variation should be ≤20%.

[0104] 1.4 Test Results 1.4.1 S1 protein structure and hydrophobicity analysis The amino acid sequence of TW-type IBV was obtained from GenBank; analysis using the TMHMM website confirmed that the S1 protein contains a signal peptide, which needs to be cleaved to adapt it for prokaryotic expression; analysis using the Expasy website showed that the protein is a hydrophilic protein, providing a theoretical basis for the purification method.

[0105] 1.4.2 Plasmid Construction Using GeneiousPrime software, the S1 gene sequence without the signal peptide was combined with the pET-32a(+) vector to construct a recombinant plasmid. Figure 2 ).

[0106] 1.4.3 PCR amplification of the S1 sequence PCR amplification was performed using IBV reverse transcribed cDNA as a template, and a specific band of 1563 bp was observed by 1% agarose gel electrophoresis. Figure 3 The concentration of the recovered adhesive product was 48 ng / mL.

[0107] 1.4.4 pET-32a (+) double enzyme digestion The vector was double-digested with XhoI and BamHI, and electrophoresis showed the target band at 5897 bp. Figure 4 The concentration of the enzyme digestion product recovered from the gel was 58 μg / mL.

[0108] 1.4.5 Bacterial PCR The recombinant product was transformed into DH5α competent cells, and a specific band appeared at 1000 bp on PCR electrophoresis of the bacterial culture, as expected. Figure 5 ).

[0109] 1.4.6 Gene sequencing of positive colonies Positive colonies were screened and sent for testing. Sequencing results confirmed that the S1 gene was successfully ligated into the pET-32a(+) vector. Figure 6 ).

[0110] 1.4.7 Prokaryotic expression of S1 protein Following the method in 1.3.16, 0.5 mM / L IPTG was added, and the protein was induced at 16℃ and 150 r / min for 12–16 h. Samples were prepared before induction, after induction, from the lysed supernatant, and from the precipitated protein. SDS-PAGE showed the target protein to be approximately 75 kDa. Figure 7 ).

[0111] 1.4.8S1 protein purification Experiments showed that the S1 protein was expressed as inclusion bodies; the imidazole concentration settings for the purification system were: 10 mM for lysis, 20 mM for washing (3 washes), and 250 mM for elution (3 elutions); the flow-through, washing, and elution buffers were collected separately for sample preparation and analysis. Electrophoresis showed that the purified protein band was single. Figure 8 ).

[0112] 1.4.9S1 protein refolding Refolding was performed using a gradient dialysis with 6M, 4M, 2M, 1M, and 0M urea; SDS-PAGE and Western blot analysis showed that the refolded protein bands were single, indicating good refolding performance. Figure 9 ).

[0113] 1.4.10 Protein Concentration Determination The Bradford method was used to serially dilute the 20 mg / mL standard protein in PBS, with absorbance values ​​corresponding to the gradient concentrations being 1.074, 0.705, 0.555, 0.328, 0.217, 0.127, and 0.068, respectively; the standard curve showed R² = 0.9964, indicating high reliability. Figure 10 The concentration of S1 protein after renaturation was measured to be 116 μg / mL.

[0114] 1.4.11 Optimization of S1 protein coating concentration and yolk antibody dilution Following the checkerboard method optimization described in 1.3.22.1, the highest P / N value of 5.141 was achieved when the S1 protein coating concentration was 1 μg / mL and the egg yolk antibody dilution was 1:40; among which, the positive OD... 450 nm=1.589, negative OD 450 nm=0.460, this condition was determined to be the optimal reaction condition for ELISA (Tables 13-15). Table 13 IBV-positive egg yolk antibody OD 450nm value

[0115] Table 14 IBV-negative egg yolk antibody OD 450nm value

[0116] Table 15 IBV-positive egg yolk antibody OD 450nm With negative egg yolk antibody OD 450nm Ratio (P / N)

[0117] 1.4.12 Optimization of sealing fluid type and sealing time Based on the optimized S1 protein coating concentration and yolk antibody dilution, the blocking solution type and blocking time were further optimized. The results showed that the highest P / N value (5.596) was achieved when the blocking solution was 5% skim milk and the blocking time was 120 min. At this time, the OD of the positive yolk antibody was [missing value]. 450 =1.638, negative OD 450 =0.301 (Tables 16-18). Therefore, the optimal conditions for this indirect ELISA method are 5% skim milk as the blocking solution and 120 min as the blocking time.

[0118] Table 16 IBV-positive egg yolk antibody OD 450nm value

[0119] Table 17 IBV-negative egg yolk antibody OD 450nm value

[0120] Table 18 IBV-positive egg yolk antibody OD 450nm With negative egg yolk antibody OD 450nm Ratio (P / N)

[0121] 1.4.13 Optimization of dilution and incubation time for rabbit anti-chicken IgG-HRP Based on the optimized coating concentration, antibody dilution, and blocking conditions, the dilution of the secondary antibody (rabbit anti-chicken IgG-HRP) and the incubation time were further optimized. The results showed that the P / N value was highest (8.009) when the secondary antibody dilution was 1:5000 and the incubation time was 30 min. At this point, the positive OD... 450 =1.386, negative OD 450 =0.173 (Tables 19-21). Therefore, a dilution of anti-chicken IgG-HRP of 1:5000 and an incubation time of 30 min are considered the optimal conditions for this indirect ELISA method.

[0122] Table 19 IBV-positive egg yolk antibody OD 450nm value

[0123] Table 20 IBV-negative egg yolk antibody OD 450nm value

[0124] Table 21 IBV-positive egg yolk antibody OD 450nm With negative egg yolk antibody OD 450nm Ratio (P / N)

[0125] 1.4.14 Optimization of color development time for TMB single-component colorimetric solution Based on the optimization of the aforementioned conditions, the TMB development time was further optimized. The results showed that the P / N value was highest (10.378) at a development time of 8 min, and the P / N ratio decreased at times lower or higher than 8 min (Table 22). Figure 11 Therefore, a color development time of 8 minutes is considered the optimal condition for this indirect ELISA method.

[0126] Table 22 IBV-positive egg yolk antibody OD 450nm With negative egg yolk antibody OD 450nm Ratio (P / N)

[0127] 1.4.15 Interpretation of results from indirect ELISA methods Twelve negative egg yolk antibodies were collected and tested using the optimized indirect ELISA method. After the colorimetric reaction was terminated, the OD values ​​of each well were read. 450 Values ​​(Table 23). The average OD values ​​of the 12 negative samples were calculated. 450 The value (X) was 0.181, and the standard deviation (SD) was 0.018. According to the formula cut-off = X + 3SD, the cut-off value was calculated to be 0.24, which was used as the standard for determining the positivity or positivity of egg yolk antibodies in this indirect ELISA method.

[0128] Table 23 IBV-negative egg yolk antibody OD 450nm value

[0129] 1.4.16 Sensitivity Test of Indirect ELISA Method Based on the established ELISA method and cut-off criteria, two positive egg yolk antibodies were randomly selected, serially diluted using PBST, and tested. The first dilution below the cut-off value was used as the minimum detection threshold (Table 24). OD values ​​of the two positive samples at a 1:1280 dilution were... 450 The nm values ​​were 0.216 and 0.225, respectively, both lower than the critical value of 0.24, thus determining the minimum detection limit of this method to be 1:1280.

[0130] Table 24 Sensitivity Tests for Indirect ELISA Method

[0131] 1.4.17 Repeatability Test of Indirect ELISA Method (1) Intra-assay repeatability test: Based on the established indirect ELISA method, two positive and one negative egg yolk antibodies were randomly selected, and each sample from the same batch of ELISA plates was tested 12 times. The standard deviations were calculated to be 0.072, 0.047, and 0.026, respectively, and the mean values ​​were 1.225, 0.962, and 0.197, respectively. The coefficients of variation were 5.90%, 4.90%, and 13.2%, respectively, all below 20%, indicating that the method has good intra-assay repeatability (Table 25).

[0132] Table 25 Intra-batch repeatability test using the indirect ELISA method

[0133] (2) Inter-batch repeatability test: Three positive and three negative egg yolk antibodies were randomly selected and tested using three batches of ELISA plates coated on days 1, 3, and 5, respectively. The mean values ​​of the six samples were calculated to be 1.284, 0.951, 1.148, 0.214, 0.193, and 0.209, respectively, with standard deviations of 0.048, 0.045, 0.033, 0.006, 0.004, and 0.010, respectively. The coefficients of variation were 3.70%, 4.70%, 2.90%, 2.80%, 2.10%, and 4.90%, respectively, all below 20%, indicating good inter-batch repeatability of the method (Table 26).

[0134] Table 26. Inter-batch repeatability test using the indirect ELISA method.

[0135] In summary, this embodiment establishes an indirect ELISA method for detecting TW type egg yolk antibodies by expressing and purifying the refolded S1 protein in prokaryotes and using the refolded S1 protein as a coating antigen. This provides data support for the expression and purification of this genotype IBVS1 protein and the establishment of an indirect ELISA method. Example

[0136] 2.1 Biomaterials SPF chicken embryos were purchased from Shandong Haotai; the TW type IBV strain was preserved by the Tianjin Agricultural College laboratory; the TW type IBV yolk antibody positive standard was prepared by our laboratory for preliminary testing; 17-day-old SPF chicks were purchased from Boehringer Ingelheim Viton Biotechnology Co., Ltd. in Beijing.

[0137] 2.2 Preparation of the main solution (1) Preparation of ethanol solution: Dilute anhydrous ethanol to 75% and 20% by multiples.

[0138] (2) Coomassie brilliant blue staining solution, PBST same as 1.2.

[0139] (3) Preparation of acidified water: Weigh 2.05g of sodium acetate with an M / V of 0.41% into 500mL of pure water, heat at 45℃ and use immediately.

[0140] 2.3 Test Methods 2.3.1EID 50 Measurement The bred IBV was serially diluted 10-fold (10³~10⁻⁶) with PBS. 5 Five 9-day-old SPF chicken embryos were inoculated at 100 μL per embryo for each dilution, with PBS injected as a negative control. Incubation was carried out at 37°C. Dead embryos were discarded after 24 hours. Observations were conducted every 24 hours thereafter, and typical lesions (developmental delay, dwarfism, curling, etc.) and dead embryos were counted after 144 hours. The number of positive embryos at each dilution was recorded, and the EID was calculated using the Reed-Muench method. 50 : Distance ratio = (Positive percentage with a positive rate higher than 50% - 50%) / (Positive percentage with a positive rate higher than 50% - Positive percentage with a positive rate lower than 50%) × Correction factor logEID 50 = Logarithm of viral dilution (infection rate above 50%) + corresponding distance ratio × logarithm of dilution factor 2.3.2 Preparation of Immunogen To prepare the oil-emulsion immunogen after inactivating IBV virus: 10 mL of IBV was thawed at low temperature, and 0.05% β-propiolactone was added for inactivation at 4℃ for 36 h. The inactivating agent was then decomposed by placing the mixture at 37℃ for 4 h. For the oil phase, sterile white oil and Span 80 were stirred at a ratio of 94:6 for 1 h. For the aqueous phase, the inactivated virus solution and Tween 80 were stirred at a ratio of 96:4 for 1 h. After mixing and stirring the oil and aqueous phases for 1 h, the mixture was emulsified at 12000 rpm for 3 min, with a 3-min interval, and this emulsification was repeated 3 times to complete the immunogen preparation. 2.3.3 Immunogen testing 2.3.3.1 Physical Inspection The physical state of the prepared IBV immunogen emulsion was observed by slowly adding 100 μL of the emulsion to distilled water using a pipette and observing whether diffusion occurred. If no diffusion occurred, the emulsion was a water-in-oil emulsion.

[0141] 2.3.3.2 Stability Test After the prepared IBV immunogen emulsion was placed at room temperature for 24 hours, it was observed whether there was any layering or uneven emulsion. In addition, the emulsion was centrifuged at 3,000 r / min at room temperature for 15 minutes to observe whether there was any layering.

[0142] 2.3.3.3 Safety Inspection The prepared IBV immunogen emulsion and sterile PBS were respectively coated onto the surface of LB solid medium without resistance and placed in an incubator at 37°C. After 24 hours, the presence of colonies was observed.

[0143] 2.3.4 Immunization The prepared IBV immunogen emulsion was used to immunize adult laying hens in the chicken farm. The specific immunization procedure is shown in Table 27.

[0144] Table 27 Immunization Schedule

[0145] 2.3.5 General steps for extracting egg yolk antibodies Egg yolk antibodies were extracted using the caprylic acid method: Egg white was removed and egg yolk was collected. The yolk was diluted with acidified water at a volume ratio of 3:1 (v / v), stirred for 10 min, and the pH was adjusted before continued stirring for 1 h. The mixture was then centrifuged at 12000 rpm and 4 °C for 30 min, and the supernatant was collected. Caprylic acid was added to the supernatant and stirred for 1 h. The mixture was then centrifuged again under the same conditions for 30 min, and the lower layer was collected. Finally, the mixture was sterilized using a 0.22 μm filter membrane and stored at 4 °C.

[0146] 2.3.6 Optimization of Egg Yolk Antibody Extraction Conditions pH and octanoic acid concentration were used as optimization indicators. pH optimization: pH gradients of 4.0, 4.6, 5.2, 5.8, 6.4, and 7.0 were set. After treatment, 2% octanoic acid was added, followed by centrifugation and sample preparation. Protein purity was analyzed by SDS-PAGE. Octanoic acid concentration optimization: The optimal pH was fixed, and octanoic acid concentration gradients of 0.5% to 4.5% were set. The remaining steps were the same. The optimal extraction conditions were screened by combining physical conditions and SDS-PAGE.

[0147] 2.3.7 Egg yolk antibody test Based on the optimized conditions for extracting egg yolk antibodies, the extracted egg yolk antibodies were subjected to physical testing, protein concentration testing, and safety testing.

[0148] 2.3.7.1 Physical Inspection Observe the color and state of the extracted egg yolk antibodies.

[0149] 2.3.7.2 Protein Concentration Determination Same as the method and steps in 1.3.20.

[0150] 2.3.7.3 Safety Inspection Same as the method and steps in 2.3.3.3.

[0151] 2.3.8 Detection of egg yolk antibody titer The virus neutralizing capacity of egg yolk antibodies was detected using the chicken embryo method: Egg yolk antibodies were extracted from hyperimmune eggs and serially diluted 2-fold with PBS (2...0 ~2 8 Dilute the virus to 200 EID. 50 / 100μL. Take an equal volume (100μL each) of antibody and virus solution, mix well, and incubate at 37℃ for 1 hour. Inoculate the mixture into 9-day-old SPF chicken embryos, with 5 replicates per dilution; set up a virus positive control group and a PBS negative control group. Non-specific dead embryos within 24 hours are removed, and the survival of chicken embryos is observed every 24 hours; after 144 hours, necropsy is performed, and the appearance of typical lesions such as poor development and curling or death during the period is used to determine the positive result. The number of positive chicken embryos in each group is counted.

[0152] 2.3.10 Evaluation of the efficacy of egg yolk antibodies Using 21-day-old SPF chicks as experimental subjects, TW-type IBV egg yolk antibodies were injected before and after challenge. The in vivo protective effect of the egg yolk antibodies was comprehensively evaluated by clinical symptom scores, tracheal ciliary scores, virus shedding detection, tissue lesion observation, and tissue viral load.

[0153] 2.3.10.1 Experimental Grouping Seventeen-day-old SPF chickens were divided into five groups: PC (Positive control), T3 (Treatment 3 injection), T1 (Treatment 3 injection), P1 (Preventive 1 injection), and NC (Negative control), with 16 chickens in each group. After grouping, the five groups were placed in isolation acclimatization facilities for 3 days.

[0154] 2.3.10.2 Virus challenge and egg yolk antibody injection Treatment methods for each group: P1 group received an intramuscular injection of 1 mL of egg yolk antibody at 20 days of age; PC, T3, T1, and P1 groups received 100 μL of yolk antibody via nasal drops or eye drops at 21 days of age. 5.8 EID 50 IBV virus dosage; T3 group was injected with 1 mL of egg yolk antibody at 22, 23 and 24 days of age; T1 group was injected with 1 mL of egg yolk antibody at 22 days of age; NC group was injected with 100 μL PBS at 21 days of age.

[0155] 2.3.10.3 Clinical Symptom Observation Clinical scores were assessed daily for 14 consecutive days following viral challenge: 0 points for normal symptoms; 1 point for mild respiratory symptoms; 2 points for worsening symptoms and lethargy; 3 points for severe illness with diarrhea; and 4 points for death. Two chickens were randomly necropsyed on days 5 and 10 after challenge to observe lesions in the larynx, trachea, kidneys, and lungs. Tracheal, larynx, kidney, and lung tissues were collected for ciliary scoring and viral load detection.

[0156] 2.3.10.4 Monitoring of Detoxification Pharyngeal and cloacal swabs were collected on days 1, 3, 5, 7, 9, 11, and 14 post-infection and placed in 0.5 mL PBS. After vortexing, the mixture was centrifuged at 12000 rpm for 5 min at room temperature. 200 μL of the supernatant was collected to extract RNA, which was then reverse transcribed. Viral shedding dynamics within 14 days were detected by qPCR. The total RNA extraction and reverse transcription procedures were the same as those in 1.3.5 and 1.3.6.

[0157] 2.3.10.5 Tracheal Ciliary Scoring The trachea was divided into three segments: upper, middle, and lower. A 1.5 mm tracheal ring was prepared for each segment and placed in a 96-well plate containing DMEM culture medium. The ciliary movement was observed and scored using an inverted microscope: 0 points for all movement, 1 point for 25% inactivity, 2 points for 50% inactivity, 3 points for 75% inactivity, and 4 points for no movement at all.

[0158] 2.3.10.6 Organize viral load monitoring 20 mg of tissue samples were collected from the larynx, kidney, and lung, respectively. The samples were then ground in Buffer RL1 containing DTT for 1 min. After centrifugation at 12000 rpm for 5 min at room temperature, RNA was extracted and reverse transcribed according to method 1.3.6. Viral load in the tissues was detected by qPCR on days 5 and 10. 2.3.10.7 Primer Design Primers were designed using Primer 5.0 software based on the IBV whole genome sequence on GeneBank and synthesized by Qingke Biotechnology Co., Ltd. The primer sequences are shown in Table 28.

[0159] Table 28 Primers for IBV Quantitative Detection

[0160] 2.3.10.8 qPCR The cDNA obtained by reverse transcription in steps 2.3.10.4 and 2.3.10.6 was used as a template for qPCR, with two replicates for each sample. qPCR was performed according to the Beijing ArtiCanATMSYBRqPCRMix procedure, and the specific reaction volume (20 μL) and reaction program are shown in Tables 29 and 30.

[0161] Table 29 qPCR reaction system (20 μL)

[0162] Table 30 qPCR amplification reaction procedure

[0163] 2.3.11 Statistical Analysis of Data GraphPadPrism9 software was used for statistical analysis of the experimental data, and One-Way ANOVA was used for significance analysis of differences.

[0164] 2.4 Test Results 2.4.1EID 50 Measurement According to the calculation method in 2.3.1, the toxicity of IBV after reproduction is 10. 5.8 EID 50 / 100μL.

[0165] 2.4.2 Physical testing of immunogens Physical testing mainly involves observing the dosage form and appearance of the immunogen emulsion. According to the results, the emulsion does not diffuse when the second drop is placed in water, proving that the dosage form of the emulsion is water-in-oil. In addition, the emulsion appears to be a milky white oil emulsion.

[0166] 2.4.3 Stability Test The stability test was conducted to verify the stability of the prepared emulsion after 24 hours at room temperature or after centrifugation at 3,000 rpm for 15 minutes at room temperature. The results showed no stratification or emulsion heterogeneity under either test method, indicating that the prepared immunogen meets the stability requirements (e.g., ...). Figure 12 ).

[0167] 2.4.4 Safety Inspection Safety testing is primarily to ensure the immunogen is used under sterile conditions. The results showed that when the immunogen and PBS were spread onto the surface of antibiotic-free LB solid medium and placed in a 37°C incubator for 24 hours, no colonies were observed (e.g., ...). Figure 13 ).

[0168] 2.4.5 Optimization of Egg Yolk Antibody Conditions (1) Following the general steps of extracting egg yolk antibodies using the caprylic acid method in section 2.3.5, the pH was adjusted to 4.0, 4.6, 5.2, 5.8, 6.4, and 7.0. The physical state of the egg yolk antibodies after centrifugation was observed, and the protein purity of the extracted egg yolk antibodies at different pH values ​​was analyzed using SDS-PAGE. The results showed that esters were difficult to separate at pH values ​​below 5.2 after centrifugation, while the bands were relatively simple and the physical state was clearer and more transparent at pH 5.2.

[0169] (2) The optimal pH was fixed, and an octanoic acid concentration gradient of 0.5% to 4.5% was set for antibody extraction. The results showed that the lipid separation effect was poor when the octanoic acid concentration was below 2.5%; the solution was clear and the protein electrophoresis bands were single when the concentration was ≥2.5%. Considering that high concentrations of octanoic acid would reduce antibody titer, an octanoic acid concentration of 2.5% was adopted as the optimal condition for extracting egg yolk antibodies.

[0170] 2.4.6 Physical test for egg yolk antibodies Egg yolk antibodies were extracted using the optimized caprylic acid method. The physical state of the extracted egg yolk antibodies was observed, and the results showed that the extracted egg yolk antibodies were mainly in the form of a pale yellow liquid.

[0171] 2.4.7 Determination of Egg Yolk Antibody Concentration The extracted egg yolk antibody was analyzed for protein concentration using the Bradford protein concentration assay kit provided by Beyotime, specifically following step 2.3.10. The results showed that the protein concentration of the extracted egg yolk antibody was 10.88 mg / mL.

[0172] 2.4.8 Safety testing of egg yolk antibodies To verify the safety of the extracted egg yolk antibody, PBS and the egg yolk antibody were spread onto LB solid medium without antibiotics and incubated at 37°C for 24 hours. No colonies were observed to grow (e.g., ...). Figure 15 ).

[0173] 2.4.9 Egg yolk antibody titer detection Antibodies were extracted from hyperimmune egg yolk using an optimized caprylic acid method, and antibody titers were measured every two weeks using an established indirect ELISA method. Results showed that antibody levels were significantly higher in the control group from 14 to 21 days post-immunization; antibody levels continued to rise from 14 to 42 days, and stabilized after 35 days. Figure 16 ).

[0174] 2.4.10 Detection of egg yolk antibody neutralization The neutralizing ability of egg yolk antibodies against TW type IBV was verified using the chicken embryo method. Results showed that the antibody dilution ratio was 1:2. 6 At that time, only one chicken embryo showed typical IBV lesions; the dilution was 1:2. 7 At that time, two chicken embryos showed lesions; the dilution ratio was less than 1:2. 6 At that time, the chicken embryos showed no lesions. Figure 17 Therefore, this indicates that the neutralizing titer of the prepared egg yolk antibody is 1:2. 7 above.

[0175] 2.4.11 Evaluation of the efficacy of egg yolk antibodies Following the grouping method in 2.3.10.1 and the treatment method in 2.3.10.2, clinical symptom scoring and mortality statistics were conducted for 14 consecutive days after challenge in the PC, T3, T1, P1, and NC groups. Autopsies were performed on days 5 and 10 after challenge to assess the degree of tracheal ciliary damage and observe lesions in the larynx, trachea, kidneys, and lungs. Viral load in organs of each group was detected using qPCR, and viral shedding dynamics were monitored at days 1, 3, 5, 7, 9, 11, and 14 post-challenge.

[0176] 2.4.11.1 Clinical Symptom Observation Following the grouping method in 2.3.10.1 and the IBV challenge and yolk antibody injection methods in 2.3.10.2, clinical symptom scores and mortality were recorded daily for the PC, T3, T1, P1, and NC groups after IBV infection for a total of 14 days (e.g., ...). Figure 18 ).

[0177] Regarding clinical symptoms, comparative analysis revealed that the PC group developed typical IBV clinical symptoms from day 4 onwards, with clinical symptom scores continuously increasing, peaking on day 8 and then gradually decreasing; while the NC group showed no obvious clinical symptoms throughout the course of infection. Compared with the PC group, the clinical symptom scores of the P1, T1, and T3 groups were significantly lower, with a significantly delayed peak and a significantly lower peak value. Among them, the T3 group showed a highly significant decrease in clinical symptom scores from day 4 to day 10 after infection (P<0.001), demonstrating the most outstanding relief effect; the T1 and P1 groups also showed highly significant symptom relief from day 4 to day 8 after infection (P<0.001), with a significant reduction in symptom severity. These results indicate that yolk antibody intervention can effectively alleviate clinical symptoms induced by IBV infection, significantly reduce symptom severity, and delay disease progression.

[0178] Regarding survival rates, the PC group had the lowest survival rate, with only 60% survival on day 14 after challenge; the T3 group had the highest survival rate, with no deaths throughout the process and a survival rate of 100%; the P1 and T1 groups had survival rates of 90% and 80% respectively, both significantly higher than the PC group. In summary, yolk antibody intervention can not only significantly alleviate clinical symptoms after IBV infection and delay the onset of disease, but also effectively improve the survival rate of infected chickens, with the T3 group showing the best protective effect.

[0179] 2.4.11.2 Tracheal Ciliary Score Tracheal rings were prepared according to method 2.3.10.6. Tracheal cilia damage was assessed on days 5 and 10 after IBV infection to evaluate the degree of ciliary lesions in each group. Results showed that the model PC group had the highest ciliary scores on days 5 and 10 after challenge, indicating severe IBV infection-induced tracheal cilia damage. Compared to the PC group, the ciliary scores in groups P1, T1, and T3 were significantly lower on day 5. P<0.001), and on day 10, the scores of each antibody treatment group were still significantly lower than those of the PC group ( P <0.001), with the T3 group having the lowest score and the mildest ciliary damage; the blank NC group consistently had a low score and no obvious ciliary damage. In summary, egg yolk antibodies can significantly alleviate IBV-induced tracheal ciliary damage, with the T3 group showing the best protective effect ( Figure 19 ).

[0180] 2.4.11.3 Observation of lesions during necropsy Regarding the observation of lesions from larynx and trachea necropsy, in the PC group, symptoms of mucus secretion and petechiae were observed on days 5 and 10 post-IBV infection, both typical lesions caused by IBV. No obvious lesions of the larynx and trachea were found in the P1, T1, T3, and NC groups on days 5 and 10 post-infection. In conclusion, yolk antibodies can reduce the pathological damage of IBV to the larynx and trachea.

[0181] Regarding the observation of lesions from renal tissue necropsy, in the PC group, renal enlargement and typical "mottled kidney" lesions were observed on days 5 and 10 post-IBV infection. In the egg yolk antibody injection groups, except for the T1 group which showed renal enlargement on day 5 post-infection, no significant lesions were observed in any of the other groups at any time point. In conclusion, egg yolk antibodies can inhibit the occurrence of IBV lesions in renal tissue.

[0182] Regarding the observation of lesions from lung tissue autopsy, typical lesions of hemorrhage and congestion were observed in the PC group on day 5 post-IBV infection. In the egg yolk antibody injection groups, except for the T1 group which showed mild pulmonary congestion on day 5 post-infection, no obvious lesions were observed in any group at any time point. In conclusion, egg yolk antibodies can inhibit the occurrence of lesions in lung tissue caused by IBV (such as...). Figure 20 ).

[0183] 2.4.11.4 Histopathological observation Pathological changes in tracheal tissue: In the NC group, the tracheal mucosal epithelium was intact, the cilia were neatly arranged, and there was no inflammatory cell infiltration. Significant lesions appeared in the PC group on day 5 after infection, manifested as tracheal mucosal epithelial shedding, extensive cilia loss, and extensive inflammatory cell infiltration in the lamina propria and submucosa; the lesions worsened further on day 10, with severe damage to the mucosal structure. In the P1, T1, and T3 groups, the tracheal mucosal structure was basically normal on days 5 and 10 after infection, with intact epithelium, regular cilia arrangement, and only very slight inflammatory cell infiltration, without obvious pathological damage.

[0184] Kidney histopathological changes: In the NC group, the renal tubular structure was clear, the glomeruli were normal in morphology, and there was no interstitial congestion or edema. In the PC group, degeneration and necrosis of renal tubular epithelial cells were observed as early as day 5, with dilation of some renal tubular lumens, interstitial congestion and edema accompanied by a large number of inflammatory cell infiltrations; the lesions were more severe on day 10, with extensive renal tubular necrosis, interstitial fibrosis, and a large number of lymphocyte infiltrations. In the P1, T1, and T3 groups, the renal histological structure was close to normal at both time points, with intact renal tubular epithelial cell morphology and no obvious degeneration, necrosis, or interstitial inflammatory infiltration.

[0185] Pathological changes in lung tissue: In the NC group, the alveolar structure was intact, the alveolar walls were thin, and there was no congestion, edema, or inflammatory exudate. In the PC group, on day 5, alveolar wall congestion and thickening were observed, with red blood cells and inflammatory exudate in the alveolar cavities, and pulmonary congestion and consolidation in some areas; on day 10, the lesions further aggravated, with extensive alveolar wall necrosis and fusion, accompanied by a large number of inflammatory cell infiltrations. In the P1, T1, and T3 groups, the lung tissue structure was basically normal on days 5 and 10 after viral challenge, with no significant thickening of the alveolar walls, and no congestion, edema, or inflammatory exudate.

[0186] In summary, the PC group showed typical histopathological damage to the trachea, kidneys, and lungs after IBV challenge, while no significant pathological changes were observed in any of the egg yolk antibody intervention groups (P1, T1, T3). This suggests that egg yolk antibodies can effectively block the histopathological damage induced by IBV infection and have a good protective effect on the trachea, kidneys, and lungs. Figure 21 ).

[0187] 2.4.11.5 Detoxification Status Pharyngeal and cloacal swabs were collected at 1, 3, 5, 7, 9, 11, and 14 days post-infection. Virus shedding levels were detected using qPCR. Figure 21 Pharyngeal swab results showed that viral load in the PC group initially increased and then decreased, peaking at 7–9 days. Viral shedding decreased in all antibody intervention groups, with the T3 group showing significant inhibition from day 3, exhibiting extremely low viral load from 5–14 days (P<0.001). The P1 and T1 groups showed even lower peak viral loads and a faster decline. Cloacal swab results showed that viral load rapidly increased in the PC group from day 1, maintaining a high viral load from 3–9 days. The T3 group showed extremely significantly lower viral loads than the PC group from day 3 (P<0.001). The peak viral loads in the P1 and T1 groups were suppressed and declined earlier. No virus was detected in the NC group. In conclusion, egg yolk antibodies significantly inhibited viral shedding, with the T3 group showing the best effect. Figure 22 ).

[0188] 2.4.11.6 Organ viral load According to the methods in 2.3.10.6 and 2.3.10.7, the viral load in the larynx, trachea, lungs, and kidneys was measured on days 5 and 10 after infection. Figure 22 , Figure 23The results showed that viral load in all organs of the PC group was at a high level. Compared with the PC group, the viral load in the trachea, lungs, and kidneys of the P1, T1, and T3 groups was significantly reduced (P<0.001), and the viral load in the T3 group was the lowest at all time points. In conclusion, egg yolk antibodies can significantly inhibit IBV replication in tissues, with the T3 group showing the best inhibitory effect.

[0189] In summary, this embodiment successfully prepared egg yolk antibodies against TW-type IBV. The tests showed that the egg yolk antibodies have the ability to neutralize the virus and have a good protective effect against IBV infection.

[0190] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for the production of a TW-type IBV egg yolk antibody, characterized in that, The method comprises the following steps: S1, emulsifying TW type IBV after inactivated by β-propiolactone with white oil according to the ratio of oil phase to water phase volume ratio of 3:2 to obtain an immunogen; S2, immunizing adult hens by intramuscular injection with the immunogen, and the immunization procedure is as follows: primary immunization once, the first booster immunization after 14 days, and the second booster immunization after another 14 days; S3, collecting high immune eggs after two weeks of each immunization; S4, extracting yolk antibodies in the high immune eggs by caprylic acid method.

2. The production method according to claim 1, wherein The inactivation condition in step S1 is that β-propiolactone is inactivated at 4℃ for 36h, and then is placed at 37℃ for 4h to decompose β-propiolactone.

3. The production method according to claim 1, wherein The condition of the caprylic acid method extraction in step S4 is that the pH is 5.2, and the caprylic acid concentration is 2.5%.

4. An indirect ELISA method for detecting TW type IBV egg yolk antibodies, characterized by, The recombinant S1 protein is used as a coating antigen, the coating concentration is 1µg / mL, the dilution multiple of the tested yolk antibody is 1:40, the blocking liquid is 5% skimmed milk, the blocking time is 2h, the secondary antibody is rabbit anti-chicken IgG-HRP, the dilution is 1:5000, the incubation time is 30min, and the color development time is 8min.

5. The indirect ELISA method according to claim 4, wherein The recombinant S1 protein is obtained by the following method: homologous recombination of the S1 gene of TW type IBV with the prokaryotic expression vector pET-32a(+), transformation of Escherichia coli, prokaryotic expression under the condition of IPTG final concentration of 0.5mM and 16℃ induction for 16h, the expression product exists in the form of inclusion body, the size is 75KDa, and the recombinant protein is obtained after urea gradient renaturation.

6. The indirect ELISA method as claimed in claim 4, wherein, The cut-off value of the method is 0.24, the sensitivity is 1:1280, and the variation coefficients of intra-batch repeatability and inter-batch repeatability are both less than 20%.

7. The TW type IBV yolk antibody prepared by the method of any one of claims 1-3 is applied to the preparation of an anti-TW type IBV medicine.

8. Use according to claim 7, wherein the compound is ###0002### The anti-TW type IBV medicine is used to achieve at least one of the following effects: delaying the onset time, reducing virus shedding, reducing the mortality rate, reducing tracheal cilia damage, reducing lung tissue damage, reducing kidney tissue damage, reducing the virus load in the laryngeal trachea, reducing the virus load in the kidney tissue, and reducing the virus load in the lung tissue.

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