DIV1 recombinant antigen, gene encoding same and use thereof, and yolk antibody and method for preparing same

Abstract

The application relates to the technical field of molecular immunology, and discloses a DIV1 recombinant antigen, a coding gene and application thereof, and egg yolk antibody and a preparation method thereof. The DIV1 recombinant antigen provided by the application can stimulate the body to produce specific immune response against Decapoda iridovirus 1 by the specific amino acid sequence shown in SEQ ID No. 1, and can be used for preparing egg yolk antibody against the DIV1 and related immune additives, and has high specificity and inhibition capacity against the DIV1, thereby providing a feasible, easily large-scale popularization and application immunological solution for the prevention and treatment of DIV1 virus infection in Macrobrachium rosenbergii culture.

Description

Technical Field

[0001] This application relates to the field of molecular immunology technology, specifically to a DIV1 recombinant antigen and its encoding gene and its applications, as well as egg yolk antibodies and their preparation methods. Background Technology

[0002] The giant freshwater prawn (Macrobrachium rosenbergii), a globally important freshwater aquaculture species, is highly favored by farmers and consumers due to its short growth cycle, rich protein content, and high market value. However, the sustainable development of this industry faces severe challenges, primarily due to the widespread transmission of decapodiridescentvirus 1 (DIV1). DIV1 belongs to the Iridoviridae family and has a double-stranded DNA structure. Its infection mechanism involves invading key organs of shrimp, such as the hepatopancreas, hematopoietic tissue, and gills, leading to cell necrosis, immune system collapse, and metabolic disorders. In high-density aquaculture, the virus can spread rapidly through water, feed, or contact, triggering mass mortality events and causing significant economic losses.

[0003] Currently, the prevention and control system against DIV1 has significant shortcomings. On the one hand, there are no approved vaccines or chemotherapeutic drugs, and aquaculture practices mainly rely on passive prevention strategies, such as screening for virus-free shrimp larvae, strengthening the disinfection of the water environment and tools, and reducing stocking density. However, these measures can only delay the spread of the virus and cannot completely block the source of infection. Moreover, their effectiveness is limited by cost and technology in actual operation. On the other hand, although some literature mentions using traditional Chinese medicines such as aloe-emodin to enhance the antiviral ability of shrimp through intramuscular injection, this method has exposed many problems in large-scale production: the injection process is time-consuming and labor-intensive, causing severe stress and mechanical damage to the shrimp, increasing the risk of secondary infections, and it is difficult to achieve uniform drug administration, resulting in unstable protective effects. Therefore, the aquaculture industry urgently needs a safe, convenient, and large-scale antiviral solution, especially the development of formulations that can be taken orally or by soaking to simplify the application process and reduce interference with shrimp.

[0004] Egg yolk immunoglobulin (IgY), a polyclonal antibody synthesized by avian B lymphocytes in response to antigen stimulation and enriched in egg yolk, exhibits unique advantages: its production requires no animal slaughter, resulting in low cost; it possesses good thermal stability and acid tolerance, making it suitable for oral administration; and it carries no risk of cross-reactivity associated with mammalian antibodies, ensuring high safety. In the field of aquatic disease control, IgY has been proven to effectively neutralize pathogens, but the development of specific IgY targeting DIV1 faces a core obstacle—the lack of efficient, mass-producible DIV1 antigens. Natural DIV1 antigens are extracted from infected tissues, resulting in extremely low yields, insufficient purity, and significant batch-to-batch variations, failing to meet antibody production demands. Simultaneously, existing technologies have failed to provide recombinant antigens with well-defined structures and strong immunogenicity, hindering the development of DIV1-specific egg yolk antibodies and severely restricting the practical application of IgY in giant freshwater prawn (Macrobrachium rosenbergii) farming. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a DIV1 recombinant antigen and its encoding gene, enabling the efficient production of a structurally well-defined DIV1 recombinant antigen, supporting the development of anti-DIV1 egg yolk antibodies and immune additive products, and providing a safe and convenient antiviral solution for shrimp farming such as giant freshwater prawns.

[0006] Another objective of this application is to provide egg yolk antibodies prepared based on the above-mentioned DIV1 recombinant antigen and its encoding gene, as well as the preparation method thereof and the immune additive products, which have better neutralizing ability and can effectively inhibit DIV1 infection of shrimp such as Macrobrachium rosenbergii.

[0007] In order to solve the above-mentioned technical problems or at least partially solve the above-mentioned technical problems, this application provides a method for solving the above-mentioned technical problems or at least partially solving the above-mentioned technical problems. As a first aspect of this application, a DIV1 recombinant antigen is provided, which has the amino acid sequence shown in SEQ ID No. 1.

[0008] As a second aspect of this application, a gene encoding the DIV1 recombinant antigen described in this application is provided.

[0009] Optionally, the gene has the nucleotide sequence shown in SEQ ID No. 2.

[0010] As a third aspect of this application, the use of the recombinant antigen or gene described in this application in the preparation of anti-DIV1 egg yolk antibodies or immune additives is provided.

[0011] As a fourth aspect of this application, an anti-DIV1 egg yolk antibody is provided, which is obtained by immunizing laying hens with the recombinant antigen described in this application and then isolating it from the egg yolk.

[0012] As a fifth aspect of this application, a method for preparing an anti-DIV1 egg yolk antibody is provided, comprising: The gene encoding the DIV1 recombinant antigen described in this application is inserted into the multiple cloning site of a vector using genetic engineering techniques to obtain a recombinant vector; The recombinant vector was transformed into the expression host for induced expression, and the inclusion bodies were collected for protein purification to obtain the DIV1 recombinant antigen. The DIV1 recombinant antigen was mixed with an immune adjuvant and then used to immunize laying hens. High-immune eggs were collected based on the titer after immunization. The yolks of hyperimmune eggs were collected, lipids were removed, and yolk antibodies were isolated and purified.

[0013] Optionally, the immune adjuvant includes Tween adjuvant, which includes white oil, Span, and aluminum stearate.

[0014] As a sixth aspect of this application, an egg yolk antibody powder immune additive is provided, which is obtained by spray drying coated egg liquid; the coated egg liquid includes a protectant and egg liquid obtained after immunizing laying hens with the recombinant antigen described in this application as an immunogen.

[0015] As a seventh aspect of this application, the use of the egg yolk antibody or the egg yolk antibody powder immunomodulator described in this application in the preparation of products for the treatment and / or prevention of DIV1 infection is provided.

[0016] As an eighth aspect of this application, a product for treating and / or preventing DIV1 infection is provided, comprising the egg yolk antibody and / or the egg yolk antibody powder immune additive described in this application.

[0017] The DIV1 recombinant antigen provided in this application, through its specific amino acid sequence shown in SEQ ID No. 1, can serve as a highly efficient immunogen, stimulating the body to produce a specific immune response against Decapoda iridovirus 1. Therefore, this recombinant antigen can be used to prepare anti-DIV1 egg yolk antibodies and related immunomodulatory additives, exhibiting high specificity and inhibitory ability against DIV1. This provides a feasible and easily scalable immunological solution for the prevention and control of DIV1 virus infection in giant freshwater prawn farming. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0019] Figure 1The image shows the SDS-PAGE electrophoresis identification (A) and Western blot verification (B) of the DIV1 recombinant antigen; M: Marker; 1 and 2 of A and B: total protein before and after induction by pET-28a-DIV1 / Rosetta transformant; Figure 2 The image shows the SDS-PAGE electrophoresis analysis of the purified and renatured DIV1 recombinant antigen; M, Marker; 1, Rosetta empty bacteria induction product; 2, pET-28a-DIV1 / Rosetta transformed bacteria induction product; 3, DIV1 purified by nickel column. Figure 3 The figure shows the change in titer of anti-DIV1 yolk antibodies in egg liquid after immunization. Figure 4 The image shows IgY at different purification stages. DIV1 Purification results; M, 180kDa Marker; 1, IgY extraction solution; 2, IgY after one ammonium sulfate precipitation; 3, IgY after two ammonium sulfate precipitations; 20μg protein was loaded into each lane. Figure 5 The image shows IgY. DIV1 Specificity identification; M, 180kDa Marker; 1, Total protein after induction of Rosetta empty bacteria without pET-28a(+); 2, Total protein after induction of Rosetta carrying pET-28a(+) empty vector; 3, Total protein after induction of Rosetta carrying pET-28a-DIV1; Figure 6 The image shows the indirect immunofluorescence detection of IgY in Sf9 cells infected with DIV1. DIV1 Specificity; cell nuclei stained with DAPI show blue fluorescence; FITC shows green fluorescence; Merge is a laser confocal technique; (Bar=50μm); Figure 7 The image shows the pathological effect of Sf9 cells after DIV1 inoculation; A, normal Sf9 cells; B, positive control inoculated only with DIV1; C, ordinary egg solution control; D, E, and F, anti-DIV1IgY diluted 1:2500, 1:5000, and 1:7500, respectively; arrows point to cells exhibiting pathological effects; (Bar=50μm); Figure 8 The image shows feeding with IgY. DIV1 The effect of powdering on the survival of DIV1-infected giant freshwater prawns; different letters in the figure indicate significant differences between groups (p<0.05, n=3). Figure 9The image shows the changes in hematopoietic tissue of shrimp after injection of DIV1 virus in each treatment group; where A is the normal giant freshwater shrimp group; B is the positive (virus challenge only) control group; C is the ordinary egg powder group; D, E and F are the groups containing 0.1%, 0.3% and 0.6% specific egg yolk antibodies, respectively; the red arrows point to the hematopoietic tissue. Figure 10 The image shows feeding IgY after a DIV1 attack. DIV1 The effect on the survival of giant freshwater prawns; different letters in the figure indicate significant differences between groups (p<0.05, n=3). Figure 11 The image shows the SDS-PAGE electrophoresis identification (A) and Western blot verification (B) of the recombinant DIV1-168L protein; M, Marker: 1 and 2 in Figures A and B, total protein before and after induction by pET-28a-DIV1-168L / Rosetta transformant; Figure 12 The image shows the SDS-PAGE electrophoresis analysis of the purified and renatured DIV1-168L recombinant protein; M, Marker; 1, Rosetta empty bacteria induction product; 2, pET-28a-DIV1-168L / Rosetta transformant induction product; 3, DIV1-168L purified by nickel column. Figure 13 The image shows IgY. DIV1-168L Specific identification; A, IgY DIV1-168L Western blot B, as the primary antibody, reacts with IgY of the pre-incubated antigen. DIV1-168L Western blot as primary antibody; M, 180 kDa Marker; 1, Total protein after induction with pET-28a(+)-DIV1-168L Rosetta empty bacteria; 2, Total protein after induction with Rosetta carrying pET-28a(+) empty vector; 3, Total protein after induction with pET-28a(+) Rosetta empty bacteria. Figure 14 The image shows the indirect immunofluorescence detection of IgY in Sf9 cells infected with DIV1. DIV1-168L The specificity of the cells; the cell nuclei stained with DAPI show blue fluorescence; FITC shows green fluorescence; Merge is a laser confocal technique (Bar=50μm); Figure 15 The image shows IgY. DIV1-168L Neutralizing effect of DIV1 on Sf9 cells; A, normal Sf9 cells; B, positive control with only DIV1 inoculated; C, ordinary egg solution control; D, E and F, IgYDIV1-168L diluted 1:2500, 1:5000 and 1:7500 respectively; arrows point to cells showing pathological effects; (Bar=50μm). Detailed Implementation

[0020] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this application. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0022] In the first aspect of this application, a recombinant DIV1 antigen with the amino acid sequence shown in SEQ ID No. 1 is provided. This recombinant antigen can be used as an immunogen to prepare anti-DIV1 egg yolk antibodies, thereby providing a feasible and easily applicable large-scale DIV1 prevention and control strategy for aquaculture industries such as giant freshwater prawns. The preparation of this recombinant antigen can be achieved in various ways. For example, conventional genetic engineering techniques can be used to insert a gene fragment encoding a DIV1 virus-related protein into an expression vector, express it in suitable host cells, and then collect and purify the expression product to obtain the DIV1 recombinant antigen. Alternatively, a peptide with a specific amino acid sequence can be directly synthesized through chemical synthesis, and then modified or conjugated to enhance its immunogenicity, thereby obtaining the DIV1 recombinant antigen.

[0023] In a second aspect of this application, a gene encoding a DIV1 recombinant antigen is proposed. Its implementation may include, but is not limited to: one approach is to reverse translate the amino acid sequence of the DIV1 recombinant antigen (e.g., SEQ ID No. 1) and optimize its design by incorporating the codon preferences of the target expression host (e.g., E. coli, yeast, or insect cells), and then obtain it through gene synthesis technology; another approach is to screen and extract fragments encoding target antigen epitopes from known DIV1 viral genome sequences and perform necessary modifications (e.g., truncation, addition of tag sequences, or codon optimization) to construct the gene encoding the recombinant antigen.

[0024] In some embodiments of this application, the gene has the nucleotide sequence shown in SEQ ID No. 2. The nucleotide sequence is optimized for expression in *E. coli* by appropriately modifying the codons based on the *Macrobrachium rosenbergii* iridovirus sequence, without altering the translated protein.

[0025] In a third aspect of this application, to further verify the egg yolk antibody (IgY) prepared via the recombinant antigen of this application... DIV1 To determine the specificity of the antibody, this application performed Western blot and indirect immunofluorescence experiments. Western blot results showed that purified IgY specifically recognized the DIV1 recombinant antigen, and excess antigen blocked antibody binding, indicating that the preparation of the recombinant antigen was successful. Indirect immunofluorescence results showed that purified IgY specifically recognized Sf9 cells infected with DIV1, while no fluorescence signal was observed in the normal group and the non-specific control group, further confirming the antibody's specificity and reaffirming that DIV1 can infect Sf9 cells. Simultaneously, this application used the Sf9 cell line to detect IgY... DIV1 The results showed that its neutralizing capacity reached 1:10864, ​​which could effectively inhibit DIV1 infection in Sf9 cells.

[0026] Furthermore, this application utilizes spray drying and the addition of trehalose as a protective agent to spray-dry high-titer egg liquid into egg yolk antibody powder. ELISA testing confirmed that spray drying has minimal impact on IgY activity. The drying process allows for the preparation of egg yolk powder from high-titer whole egg liquid, enabling long-term storage and demonstrating good application results in animal oral administration experiments.

[0027] Based on the above-mentioned superior effects, this application provides the use of the recombinant antigen or the gene described in this application in the preparation of anti-DIV1 egg yolk antibodies or immune additives.

[0028] In a fourth aspect of this application, an anti-DIV1 egg yolk antibody is provided, which is isolated from the egg yolk after immunizing laying hens with the recombinant antigen described in this application as an immunogen. The immunization process typically includes primary immunization and multiple booster immunizations. The immunization route can be intramuscular injection, subcutaneous injection, or intraperitoneal injection, and immune adjuvants can be used to enhance the immunization effect. For example, oil emulsion adjuvants or aluminum hydroxide adjuvants can be used.

[0029] This application uses the DIV1 recombinant antigen with the amino acid sequence shown in SEQ ID No. 1 as an immunogen, which ensures that the generated egg yolk antibodies have high specificity and effectiveness against the DIV1 virus, thereby avoiding non-specific immune responses. Furthermore, obtaining antibodies from egg yolks is not only simple, but the resulting egg yolk antibodies also have good stability and are easy to prepare into oral or soaking forms of immune additives, directly meeting the practical needs of preventing and treating DIV1 infection in aquaculture.

[0030] In a fifth aspect of this application, a method for preparing an anti-DIV1 egg yolk antibody is provided, comprising: The gene encoding the DIV1 recombinant antigen described in this application is inserted into the multiple cloning site of a vector using genetic engineering techniques to obtain a recombinant vector; The recombinant vector was transformed into the expression host for induced expression, and the inclusion bodies were collected for protein purification to obtain the DIV1 recombinant antigen. The DIV1 recombinant antigen was mixed with an immune adjuvant and then used to immunize laying hens. Hyperimmune eggs were collected at an appropriate time after the last immunization. The yolks of hyperimmune eggs were collected, lipids were removed, and yolk antibodies were isolated and purified.

[0031] Common vectors include plasmids, bacteriophages, and viral vectors. Multiple cloning sites are regions on a vector containing multiple restriction endonuclease recognition sites, facilitating the targeted insertion of the target gene.

[0032] Transformation refers to the process of introducing recombinant DNA molecules constructed in vitro into recipient cells. This invention achieves this through a heat shock method. The expression host is a prokaryotic cell (such as Escherichia coli or Bacillus subtilis). By adding inducers (such as IPTG or lactose) and optimizing culture conditions, the host cell expresses the target protein in large quantities, and high-purity DIV1 recombinant antigen is obtained through affinity chromatography.

[0033] The DIV1 recombinant antigen, mixed with an immune adjuvant, is used to immunize laying hens to enhance the body's immune response to the recombinant antigen and increase the titer of anti-DIV1 yolk antibodies. The immune adjuvant is a non-specific immune enhancer that can enhance the immunogenicity of the antigen, prolong its duration of action in the body, and promote the activation and proliferation of immune cells. Common immune adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, and oil emulsion adjuvant. Eggs laid by laying hens are collected when their antibody levels reach peak levels after multiple immunizations. At this time, the yolk antibody content in the eggs is highest and the activity is strongest, making it the optimal time to prepare highly effective yolk antibodies.

[0034] After collecting the yolks from hyperimmune eggs, lipids need to be removed. Methods for lipid removal include polyethylene glycol (PEG) precipitation, dextran sulfate precipitation, or organic solvent extraction. Subsequently, yolk antibodies are extracted from the lipid-removed yolks using separation and purification techniques. Separation and purification methods can employ salting out (e.g., ammonium sulfate precipitation), affinity chromatography, ion exchange chromatography, or gel filtration chromatography to obtain high-purity, highly active anti-DIV1 yolk antibodies.

[0035] In some embodiments of this application, the immune adjuvant includes Tween adjuvant, which includes white oil, Span, and aluminum stearate. In some embodiments of this application, Tween accounts for 3-5% of the volume of the egg yolk antibody; Tween, as a nonionic surfactant, mainly functions to reduce the surface tension of the liquid, which helps to uniformly disperse the DIV1 recombinant antigen and the oil phase adjuvant, prevents stratification or aggregation, and ensures that the immunogenic components can be fully exposed and recognized by the immune system. In some embodiments of this application, Tween accounts for 3-5% of the volume of the egg yolk antibody. The adjuvant generally refers to substances that can non-specifically enhance the body's immune response to antigens in vaccines, including white oil, Span, and aluminum stearate; its mechanism of action is diverse, including forming an antigen reservoir, activating immune cells, and inducing cytokine secretion. In some embodiments of this application, the adjuvant specifically includes 92% white oil, 6% Span 80, and 2% aluminum stearate.

[0036] In the adjuvants of this application, white oil is a highly refined mineral oil that primarily serves as the oil phase matrix in the oil-emulsion adjuvant, used to encapsulate the DIV1 recombinant antigen to form a water-in-oil or oil-in-water emulsion. Its function is to provide sustained release of the antigen, prolonging its duration of action in the body, thereby continuously stimulating the immune system and enhancing the immune response. Span is a fatty acid sorbitan ester, belonging to the nonionic surfactant class. In this application, it is used in conjunction with Tween as an emulsifier to stabilize the oil-water emulsion. Span can reduce the tension at the oil-water interface, promoting emulsion formation and stability, and preventing emulsion breakage or separation. Aluminum stearate is a metal soap that primarily serves as a stabilizer and immunostimulant in the adjuvant. It can increase the viscosity of the emulsion, improve its stability, and form a precipitate with the DIV1 recombinant antigen, prolonging the antigen release time. Simultaneously, aluminum stearate itself also has a certain immunostimulatory effect, activating immune cells and enhancing the immune response.

[0037] This application addresses the problem of inappropriate adjuvant selection in the preparation of anti-DIV1 egg yolk antibodies by specifically defining the composition of the immune adjuvant, namely, a combination of Tween, white oil, Span, and aluminum stearate. Tween, as a surfactant, helps emulsify the mixture of DIV1 recombinant antigen and adjuvant, improving uniformity and dispersibility, and preventing stratification or aggregation. White oil provides a sustained-release base, Span enhances emulsion stability, and aluminum stearate strengthens the adjuvant's adhesion and immunostimulatory effects. These components work synergistically to promote the sustained release and enhanced immunogenicity of the DIV1 recombinant antigen, thereby optimizing the immune response and ensuring a highly efficient, safe, and stable immunization process.

[0038] In a sixth aspect of this application, an egg yolk antibody powder immune additive is provided, which is obtained by spray drying coated egg liquid; the coated egg liquid includes a protectant, the protectant being more preferably trehalose, and egg liquid obtained after immunizing laying hens with the recombinant antigen described in this application as an immunogen.

[0039] The coated egg liquid is a liquid precursor used to prepare egg yolk antibody powder. Its core function is to uniformly mix the active ingredient (i.e., egg yolk antibody against DIV1) with protective ingredients (such as trehalose) to form a stable system, providing a guarantee for the subsequent drying process. This egg liquid is obtained by immunizing laying hens with the DIV1 recombinant antigen (having the amino acid sequence shown in SEQ ID No. 1). It can be the whole egg liquid produced by immunized hens or the yolk liquid after preliminary separation, which is rich in specific egg yolk antibodies against the DIV1 recombinant antigen. Trehalose is a natural non-reducing disaccharide, which serves as an excellent protective agent in this application. Its main function is to protect the structure and function of the egg yolk antibody during spray drying. During the drying process, trehalose can form a glassy matrix around the antibody molecules, replacing water molecules to bind to the antibody protein, thereby preventing protein denaturation, aggregation, and inactivation. In some embodiments of this application, the egg liquid coating method includes: weighing whole eggs with a potency of 1:8000 or higher into a container, mixing the egg liquid on ice, weighing 12% of the egg liquid mass of trehalose, dissolving it in deionized water at 70°C with 20% of the trehalose mass of trehalose, slowly pouring it into the egg liquid, and stirring thoroughly to coat the egg liquid.

[0040] Spray drying is a highly efficient drying technology that atomizes liquid coated egg liquid into tiny droplets and exposes them to hot air, rapidly evaporating moisture to obtain a powdered product. The process is optimized based on the characteristics of the egg liquid and the requirement for a powder moisture content below 5%. In some embodiments of this application, the inlet and outlet air temperatures are adjusted to 180°C and 80°C respectively, and the peristaltic speed is adjusted to 19 rpm to ensure that antibody activity is not impaired.

[0041] The egg yolk antibody powder of this application not only greatly improves the stability of the product and extends its shelf life, but also significantly reduces storage and transportation costs. At the same time, it facilitates precise measurement and uniform feeding in practical applications such as aquaculture, thereby overcoming the limitations of liquid egg yolk antibodies in terms of stability, storage and large-scale application, and providing a more efficient, convenient and economical form of immune additive for the prevention and control of DIV1 infection.

[0042] In the seventh aspect of this application, the egg yolk antibody powder immunomodulator of this application was used for in vivo and in vitro detection experiments. In vitro experiments showed that IgY diluted 5000 times... DIV1 The powder can significantly protect Sf9 cells from DIV1 infection, and the cell state is close to that of normal cells that have not been challenged.

[0043] In vivo experiments showed that adding 0.3% and 0.6% doses of IgY... DIV1The powder can effectively prevent the occurrence and control of disease outbreaks of decapod iridovirus in giant freshwater prawns, with a maximum immunization rate of 54%. It significantly reduces the DIV1 load in infected prawns, alleviates tissue damage, strengthens the prawns' own immunity, and improves the survival rate of giant freshwater prawns.

[0044] Based on the above-mentioned excellent effects, this application provides the use of the egg yolk antibody or the egg yolk antibody powder immune additive described in this application in the preparation of products for treating and / or preventing DIV1 infection.

[0045] The preparation of products for treating and / or preventing DIV1 infection refers to the use of egg yolk antibodies or egg yolk antibody powder as an immune additive as an active ingredient in the production or manufacture of formulations that enhance animal immunity and resist DIV1 virus infection. These formulations can be prepared in various forms; for example, they can be mixed with feed to form oral additives for animal consumption; or dissolved in water to form soaking agents for absorption by farmed animals; or they can be formulated as sprays for environmental spraying to reduce the risk of virus transmission. These formulation forms aim to transform egg yolk antibodies or egg yolk antibody powder into easily applicable and highly effective products to combat DIV1 infection. The application referred to in this application involves using the aforementioned egg yolk antibodies or egg yolk antibody powder as the core active ingredient, combined with other excipients (such as carriers, stabilizers, palatability enhancers, etc.) to prepare the final immune formulation product. Alternatively, they can be added as a functional component to existing feed or water treatment agents to impart anti-DIV1 immune function, thereby achieving the purpose of treating and / or preventing DIV1.

[0046] In an eighth aspect of this application, a product for treating and / or preventing DIV1 infection is provided, comprising the egg yolk antibody and / or the egg yolk antibody powder immune additive described in this application. The product may contain only egg yolk antibody, or only egg yolk antibody powder immune additive, or, depending on the actual application requirements, a mixture of egg yolk antibody and egg yolk antibody powder immune additive in a specific ratio.

[0047] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all purchased from commercial channels. In the specific embodiments of this application, unless otherwise specified, the experimental environment and parameter conditions of each group in the tests are consistent.

[0048] The following provides further details regarding a DIV1 recombinant antigen, its encoding gene, its applications, and an egg yolk antibody and its preparation method.

[0049] Example 1: Preparation of Egg Yolk Antibodies (1) Sequence The iridovirus sequence (KY681039.1) of giant freshwater prawn published on NCBI was sent to Nanjing Genscript Biotech Co., Ltd. The codons were optimized to make it suitable for expression in E. coli without changing the translated protein. The protein was truncated to improve its immunogenicity. The sequence was inserted into the commercial pET-28a(+) vector through the NdeI / XhoI restriction site to construct a recombinant plasmid named pET-28a-DIV1.

[0050] The amino acid sequence of the recombinant antigen is as follows (SEQ ID No. 1): The coding sequence of the recombinant antigen is as follows (SEQ ID No. 2): (2) pET-28a-DIV1 plasmid transformation The synthesized 4 μg lyophilized plasmid was centrifuged at 12,000 r / min for 1 min, and 200 μL of TE buffer was added and vortexed to mix. 2 μL of pET-28a-DIV1 and 2 μL of pET-28a(+) empty vector were respectively transformed into *E. rosetta* bacteria and analyzed using 50 μg / mL Kansin buffer. + Positive colonies were selected using LB solid plates.

[0051] (3) Induced expression The cultured bacterial culture was transferred to a 50 mL centrifuge tube and centrifuged at 5,000 r / min for 10 min at 4 °C. The supernatant was removed, and the bacterial sludge was washed twice with PBS and stored at -80 °C. The pET-28a-DIV1 / Rosetta cells were autoclaved and inclusion bodies were collected. After washing, the inclusion bodies were resuspended in denaturing buffer, and the supernatant was collected by centrifugation to obtain the denatured protein. The target protein was then purified by Ni column purification, and the purified recombinant antigen was obtained by dialysis and renaturation.

[0052] (4) Immunization of laying hens Calculate the required protein volume (0.5 mg per bird) based on the number of immunized chickens. Add 4% of the protein volume of Tween 80 and an equal volume of homemade adjuvant (adjuvant: protein = 1:1). Vortex for 30 minutes to fully emulsify the protein, and centrifuge at low speed for 1 minute until no separation occurs.

[0053] Five laying hens were injected with the vaccine at three points: the left and right pectoral muscles and the subcutaneous tissue of the neck. Each hen received 0.5 mg of protein, and the injection volume per hen should not exceed 1 mL. A second immunization was administered 10 days after the first immunization. The antibody titer in the egg liquid was then measured using an indirect ELISA method. A booster immunization was administered when the titer dropped below 1:8000.

[0054] (4) Extraction and purification of anti-DIV1 IgY Eggs with an efficacy titer of 1:16000 or higher were collected. After removing the egg white, anti-DIV1 IgY was extracted from the eggs using a water dilution-salting-out method. IgY was extracted from ordinary eggs using the same method and reserved for subsequent negative control.

[0055] Example 2: Preparation of Egg Yolk Antibody Powder (1) Egg coating: Beat all whole eggs with a potency of 1:8000 or higher into a beaker and weigh them. Use a stirrer to mix the egg mixture on ice. Weigh 12% of the egg mixture mass of trehalose, dissolve it in 20% of the trehalose mass of deionized water in a 70°C water bath, and slowly pour it into the egg mixture. Stir on ice for 10 minutes to fully coat the egg mixture. (2) Operation of spray drying equipment: Connect the vacuum pump and spray chamber device to the power supply, install the powder collection bottle, feed pipe and peristaltic pump, adjust the inlet and outlet air temperatures to 180℃ and 80℃ respectively, connect the peristaltic pump power supply, put the pump pipe into deionized water, adjust the peristaltic speed to 19 rpm, and observe that mist gas is sprayed out in the spray chamber. (3) Loading the egg liquid: Transfer the pump tube into the egg liquid. The egg liquid should be diluted with an appropriate amount of deionized water in advance to prevent clogging of the pump head. Spray dry the egg liquid. Every 15 minutes, tap the spray chamber with a hammer to facilitate the entry of egg powder into the collection bottle. (4) Turn off the instrument: After all the egg liquid has been sprayed and dried, transfer the pump tube to deionized water. After observing that all the egg liquid in the pump tube has been sprayed, continue to add water for 5 minutes to thoroughly clean the pump tube. Remove the pump tube from the water. After all the water in the pump tube has been sprayed, turn off the peristaltic pump and vacuum pump. Open the top cover of the spray chamber and then close the spray chamber. Sweep out the residual egg powder in the spray chamber. Disconnect the tube connecting the spray chamber and the collection bottle and wash it thoroughly with water to prevent insect infestation. (5) Egg powder preservation: Transfer the egg powder in the collection bottle and spray chamber into the sealing film, place it in a desiccator and store at room temperature for subsequent functional evaluation.

[0056] Example 3: Characterization and Validation of Recombinant Antigens (1) Validation of recombinant antigen-induced expression pET-28a-DIV1 and pET-28a(+) plasmids were transformed into E. coli Rosetta competent cells for protein expression induction, and identified by SDS-PAGE electrophoresis. The results showed that after induction, pET-28a-DIV1 exhibited a thick band at approximately 50 kDa. Figure 1The area indicated by the arrow in lane A2 (48.47 kDa) matches the expected size. Western blot analysis of the induced protein using a His-tag antibody showed a distinct band at approximately 50 kDa for both the total pET-28a-DIV1 protein and the sonicated precipitate. Figure 1 (The area indicated by the arrow in lane B) No obvious bands were observed in the total protein before and after induction with pET-28a(+) empty vector and before induction with pET-28a-DIV1, indicating that the protein at around 50 kDa is the target protein.

[0057] (2) Purification of DIV1 recombinant antigen After large-scale induction, the bacterial cells were high-pressure disrupted and centrifuged to collect inclusion body proteins. After one ultrasonic wash with 3M urea inclusion body washing buffer, the main band at around 50 kDa was obvious, but there were still many impurities. After two ultrasonic washes, the impurities were reduced significantly, but some faint bands still existed. The washed inclusion bodies were denatured and purified by Ni column. The refolded target protein was free of other impurities. Figure 2 This meets the requirements for producing immunogens.

[0058] (3) The variation pattern of anti-DIV1 egg yolk antibody titer Egg IgY was plotted based on indirect ELISA detection results. DIV1 The pattern of valence change, the results are as follows Figure 3 After the first immunization, IgY DIV1 There was no significant change in titer. A second immunization was administered 10 days after the first immunization, using IgY. DIV1 The titer rose to 1:4000, failing to reach the expected 1:8000. The adjuvant-to-protein ratio was adjusted from 1:1 to 2:1, and a third immunization was administered. Post-immunization, IgY... DIV1 The titer gradually increased, reaching a peak of 1:32,000, and then gradually decreased after 3 days, dropping below 1:8,000. A fourth immunization was then administered, and after 10 days, the titer rose to 1:64,000, remaining there for two weeks before gradually decreasing again. Thirteen weeks after immunization, the titer fell below 1:8,000. A fifth immunization was then administered, and five days later, the titer increased to 1:64,000, remaining there for two weeks before gradually decreasing again. After nine weeks, the titer dropped to 1:16,000. Since the chickens stopped laying eggs, no further testing was conducted.

[0059] (4) Isolation and purification of anti-DIV1 egg yolk antibody The fat in the egg yolk was removed by acidifying the water, and then precipitated twice using ammonium sulfate precipitation to obtain IgY. DIV1 The antibody was purified to obtain pure IgY after dialysis to remove ammonium sulfate. DIV1The protein concentration was determined after different purification steps; disulfide bonds were opened in the presence of β-mercaptoethanol, and SDS-PAGE analysis was performed. The results are as follows: Figure 4 As shown, the egg yolk extract diluted with acidified water contained a large amount of impurities. After salting out, the number of impurities decreased significantly. The heavy chain showed a thickened band at 65 kDa, and the light chain at 25 kDa, consistent with the expected size. IgY was successfully isolated and purified. DIV1 Antibodies are stored at -80°C for subsequent specific testing.

[0060] (5) Neutralizing titer of anti-DIV1 egg yolk antibody against DIV1 The titer of anti-DIV1 egg yolk antibodies was detected using a neutralization assay. The results are shown in Table 1. The egg yolk antibody (DIV1 IgY) prepared by immunizing with recombinant DIV1 antigen exhibited significant neutralizing activity, with a neutralizing titer of 10. -4.036 (1:10864), meaning that the anti-DIV1 egg yolk antibody at a dilution of 1:10864 can protect 50% of cells from DIV1 damage and prevent cellular pathological effects.

[0061] Table 1. Neutralization titer determination of egg yolk antibodies prepared after immunization with recombinant DIV1 protein. Note: The number of lesion wells refers to the number of wells in a 96-well plate where Sf9 cells showed signs of fusion and enlargement.

[0062] Distance ratio = (Positive percentage above 50% – 50) / (Positive percentage above 50% – Positive percentage below 50%) = (53.3 - 50) / (53.3 - 25) = 0.12; ND 50 The logarithm of the dilution is equal to the logarithm of the dilution with a lesion rate higher than 50% plus the distance ratio multiplied by the difference between the logarithms of the dilutions. lgND 50 = -4 + 0.12 × (-0.3) = -4 + (-0.036) = -4.036; ND 50 = 10 -4.036 = 1:10,864; (6) Specificity of anti-DIV1 egg yolk antibody Purified DIV1 recombinant antigen was used as the antigen, IgY DIV1 HRP-labeled goat anti-chicken antibody was used as the primary antibody and as the secondary antibody in Western blot analysis. The results are as follows: Figure 5 As shown, no target band was observed after pre-incubation with excess antigen and antibody. Figure 5A), the unincubated sample showed a distinct band at 50 kDa (A). Figure 5 Arrow B indicates that excess antigen can bind to the antibody, preventing it from binding to the antigen on the PVDF membrane and thus not displaying a band. Antibodies without pre-incubated antigen can bind to the antigen on the membrane, thus displaying a band, indicating that IgY... DIV1 The antibodies obtained from immunizing chickens with egg yolks are correct because they can specifically recognize proteins expressed in prokaryotes.

[0063] Sf9 cells were seeded on a slide. IgY extracted from immunized eggs was used as the primary antibody, and FITC-labeled goat anti-chicken IgY was used as the secondary antibody. Indirect immunofluorescence experiments were performed on Sf9 cells 12 h after challenge. A control group without challenge but incubated with a specific primary antibody was also included, as well as a control group challenged and incubated with a non-inventory-specific protein antibody IgY. PirA The negative control group, the results are as follows Figure 6 As shown, cell nuclei exhibited blue fluorescence after DAPI staining, and IgY was used to further fluoresce. DIV1 Sf9 cells infected with DIV1 showed green fluorescence after incubation with IgY. DIV1 Incubation of healthy Sf9 cells showed no fluorescence, and incubation of DIV1-infected Sf9 cells with non-specific IgY also showed no fluorescence, indicating that specific IgY can recognize and bind to native DIV1 viral particles.

[0064] Example 4: Evaluation of the control effect of anti-DIV1 IgY on iridovirus disease in giant freshwater prawns The egg yolk antibody (IgY) used in this embodiment DIV1 The sample is the anti-DIV1 egg yolk antibody powder prepared by spray drying in Example 2.

[0065] (1) The effect of anti-DIV1 yolk antibody on neutralizing DIV1 200 TCID 50 DIV1 virus solution and IgY at different dilutions DIV1 After a 1-hour pre-incubation, the cells were seeded onto SF9 cells, with normal SF9 cells serving as a control. Results after 3 days are shown below. Figure 7As shown, compared with normal SF9 cells (A), cells inoculated only with DIV1 virus solution showed enlargement (B). Cells inoculated with egg solution made from 1:2500 diluted ordinary egg powder and DIV1 solution also showed enlargement (C), indicating that ordinary egg powder did not neutralize DIV1 and could still cause cytopathic effects. Cells inoculated with 1:2500 and 1:5000 diluted DIV1 egg yolk antibody solutions were uniform in size, with no significant cell enlargement observed (D, E), indicating that the added specific egg yolk antibody neutralized DIV1 virus particles, preventing or minimizing viral invasion. Abnormally enlarged cells appeared in the group inoculated with 1:7500 diluted DIV1 egg yolk antibody solution (F), possibly due to excessively high dilution, which failed to completely neutralize the virus.

[0066] (2) The preventive effect of anti-DIV1 egg yolk antibody on iridovirus disease in giant freshwater shrimp To evaluate the preventive effect of anti-DIV1 egg yolk antibody against iridovirus disease in giant freshwater prawns, different doses of egg powder were added to the feed for 16 days, followed by intramuscular injection to challenge the giant freshwater prawns with the virus. The results were recorded and analyzed to evaluate the preventive effect.

[0067] With 3.3×10 3 The shrimp were challenged with intramuscular injection of a single dose of virus. Daily mortality was recorded for the negative control (healthy shrimp), positive control (injected with virus), 0.6% ordinary egg powder treatment group, and 0.1%, 0.3%, and 0.6% egg yolk antibody treatment groups. Data was collected up to day 14 post-challenge. Survival curves were plotted using the average of the three replicates. The results are as follows: Figure 8As shown, under the experimental culture conditions, 100% of the giant freshwater prawns in the negative control group survived. No shrimp deaths were observed in any of the experimental groups within 1-2 days. On day 3 after infection, shrimp deaths occurred in all groups. The 0.3% and 0.6% specific treatment groups had significantly fewer shrimp deaths than the positive control and the ordinary egg powder group. Shrimp continued to die on days 4 and 5. Subsequently, only a very small number of shrimp died in the 0.3% and 0.6% specific treatment groups, while the other groups continued to experience intermittent mortality. On day 14 post-infection, the mortality rates of the 0.3% and 0.6% specific treatment groups were significantly lower than those of the other groups (p < 0.05), at 50 ± 4.08% and 35 ± 2.36%, respectively. The 0.1% specific treatment group had no significant effect, with a mortality rate of 65 ± 4.08%, which was not significantly different from the 0.6% ordinary egg powder group (p > 0.05). Compared with the positive control group, the immunoprotective efficacy of the 0.6% ordinary egg powder group and the groups supplemented with 0.1%, 0.3%, and 0.6% specific egg yolk antibodies was 7%, 13%, 33%, and 53%, respectively. Survival curves were plotted using Graph Pad Prism, showing that adding more than 0.3%, preferably 0.3-0.6% specific egg yolk antibodies (e.g., 0.4%, 0.5%) to the feed had a significant preventive effect, with 0.6% showing the most significant effect.

[0068] (3) The therapeutic effect of anti-DIV1 egg yolk antibody on the outbreak of iridovirus disease in giant freshwater shrimp after DIV1 infection. To evaluate the therapeutic effect of anti-DIV1 egg yolk antibody on giant freshwater prawns infected with iridovirus, different doses of egg powder were added to the feed and the prawns were fed to the feed after intramuscular injection for 14 days. The therapeutic effect was recorded and analyzed.

[0069] A. Symptoms of hematopoietic tissue in diseased giant freshwater shrimp 3 days after intramuscular injection of DIV1. With 3.3×10 3 After infecting healthy giant freshwater prawns with intramuscular injection of a dose per tail, they were fed with regular feed, 0.6% regular egg powder feed, and 0.1%, 0.3%, and 0.6% IgY, respectively. DIV1 Feed samples were taken on day 3, and photographs were taken to record the lesions in the hematopoietic tissue. The results were as follows: Figure 9 As shown, the hematopoietic tissue of normal giant freshwater prawns is pale yellow and transparent (A), while after being challenged with DIV1, it becomes opaque and milky white (B, C), consistent with the symptoms of giant freshwater prawn iridovirus reported in the literature. The milky whiteness of the groups fed with 0.1% and 0.3% doses of specific egg yolk antibody was reduced compared with the positive control group (D, E). The hematopoietic tissue of the group fed with 0.6% dose of specific egg yolk antibody was almost identical to that of the normal group (A), indicating that the specific egg yolk antibody played an antiviral role, with the 0.6% dose showing the best effect.

[0070] B. Relative immune protection Mortality of giant freshwater prawns in each group was recorded daily after challenge treatment, up to day 14 post-challenge. On day 3, one prawn from each parallel sample was counted as a survivor. Survival curves were plotted using GraphPad Prism based on the results, as shown below. Figure 10 As shown, in the laboratory culture environment, 100% of the shrimp in the negative control group survived. Shrimp in all challenged groups died on day 3, with a significant number of deaths occurring between days 3 and 5, indicating a mortality outbreak. Shrimp continued to die thereafter. At 14 days post-challenge, the mortality rate in the 0.6% specific treatment group was significantly lower than other groups (p < 0.05), at 30 ± 4.08%. The mortality rates in the 0.1% and 0.3% treatment groups were comparable, both significantly higher than the positive control group and the ordinary egg powder group (p < 0.05), at 45 ± 4.71% and 45 ± 2.36%, respectively. There was no significant difference between the positive control group and the ordinary egg powder group. Compared with the positive control group, the immunoprotective efficacy of the 0.6% ordinary egg powder group and the groups supplemented with 0.1%, 0.3%, and 0.6% specific egg yolk antibodies was 8%, 31%, 31%, and 54%, respectively. The results showed that adding more than 0.1% of specific egg yolk antibodies (preferably 0.3-0.6% specific egg yolk antibodies, such as 0.4% or 0.5%) to the feed could effectively inhibit the virus and reduce the mortality of giant freshwater prawns, with the 0.6% dose showing the most significant therapeutic effect.

[0071] Comparative Example 1: During the research and development process of this application, we attempted to use CQIV-168L protein as an antigen to prepare egg yolk antibodies based on existing literature reports such as "Identification and characterization of an envelope protein 168L in Cherax quadricarinatus iridovirus (CQIV)" and "Decapod iridescent virus 1 (DIV1) 168L can targetcuticle protein 8 from Litopenaeus vanname". However, the neutralizing effect of the egg yolk antibodies obtained was poor and they were not suitable as antigens for preparing egg yolk antibodies.

[0072] CQIV, or the late-expressed membrane protein of DIV1 virus, is a component of the viral envelope and can bind to and interact with host gill epidermal cells to invade the host. Therefore, existing literature reports the preparation of antibodies against this protein, which have potential anti-invasion functions. The protein has a molecular weight of approximately 57 kDa. Based on epitope prediction, a new sequence was formed with a molecular weight of approximately 30.72 kDa. The specific preparation process and related verification experiments in this application are as follows: (1) Sequence The iridovirus sequence (PQ724921.1) of giant freshwater prawn published on NCBI was sent to Nanjing Genscript Biotech Co., Ltd. Based on the DIV1-168L sequence (XTR93210.1), multiple antigenic epitopes were screened and screening tags were added, resulting in a significant shortening of the amino acid sequence. Codons were slightly optimized to make them suitable for expression in E. coli without altering the translated protein. The sequence was then inserted into the pET-28a(+) vector via NcoI / XhoI restriction sites to construct a recombinant plasmid named pET-28a-DIV1-168L. The relevant preparation process can be found in the aforementioned examples.

[0073] The amino acid sequence of the DIV1-168L recombinant antigen is as follows (SEQ ID No. 3): MDGVIRAISTQSSDIHAIVDGAHGVIENDVLTQQQKIFSAIQQEAKSIVSGLNLAQVNVTNLVSSRLTCNKINYIYKHIDGPFILKDVKLDQIQDIFSSCIQNNKVVQDISQLSSWVIVGIVAVMIGLPIVGGVAIGKTAL KFIFPLVLIAAITFIVLYFVKTSGFKVVPYSSPCNSELLTVECKAVYWEKYIHKAPQDRDAYVDDKLWYQYNAKDIFLRYQPTRVESVEYVPGNVWITILKIYKWMLYVGVLAIIIGILGTGITFYLDRKKSELEHHHHHH The coding sequence of the DIV1-168L recombinant antigen is as follows (SEQ ID No. 4): ATGGATGGAGTAATAAGGGCTATTTCAACACAAAGCTCTGATATCCATGCAATAGTTGACGGCGCACATGGTGTGATTGAAAACGATGTTTTGACCCAGCAACAGAAAATCTTCTCTGCGATCCAGCAAGAAGCGAAGTCCATCGTTTCCGGTCTGAATCTGGCTCAGGTTAATGTCACCAACCTGGTGAGCTCGCGCTTAACGTG CAATAAGATCAACTATATCTACAAGCACATTGATGGTCCGTTTATCCTGAAGGACGTTAAGTTGGACCAGATTCAAGACATCTTCAGCTCTTGTATCCAAAACAACAAAGTTGTTCAGGACATCAGCCAGCTGAGTAGCTGGGTCATTGTGGGCATCGTGGCGGTGATGATTGGCCTTCCGATTGTCGGCGGTGTGGCCATCGGCA AAACTGCGCTGAAGTTTATTTTCCCGCTCGTGCTGATTGCGGCGATTACCTTCATCGTTTTGTACTTCGTGAAAACCAGCGGTTTTAAGGTGGTGCCATATTCGTCCCCGTGCAACAGCGAACTGCTGACCGTTGAGTGCAAAGCTGTTTACTGGGAGAAATACATCCACAAAGCTCCGCAGGACCGCGATGCATATGTTGATGAT AAACTGTGGTATCAGTATAACGCCAAGGACATCTTCCTGCGTTACCAACCGACGCGTGTTGAGAGCGTAGAATACGTACCGGGTAATGTGTGGATTACCATTCTCAAGATCTACAAGTGGATGCTGTACGTGGGTGTCCTGGCGATCATTATCGGCATTTTGGGAACCGGTATTACCTTTTATCTGGATCGTAAAAAAAGCGAGTAA (2) Validation of DIV1-168L recombinant protein induced expression pET-28a-DIV1-168L and pET-28a(+) plasmids were transformed into E. coli Rosetta competent cells for protein expression induction, and identified by SDS-PAGE electrophoresis. The results showed that pET-28a-DIV1-168L exhibited a thick band at approximately 33 kDa after induction. Figure 11 The area indicated by the arrow in lane A2 (31.8 kDa) matches the expected size. Western blot analysis of the induced protein using a His-tag antibody showed a distinct band at approximately 33 kDa in both the total pET-28a-DIV1-168L protein and the sonicated precipitate. Figure 11 (The area indicated by the arrow in lane B) No obvious bands were observed in the total protein before and after induction with pET-28a(+) empty vector and before induction with pET-28a-DIV1-168L, indicating that the protein at around 33 kDa is the target protein.

[0074] (3) Purification of DIV1-168L recombinant protein After large-scale induction, the bacterial cells were high-pressure disrupted and centrifuged to collect inclusion body proteins. After one ultrasonic wash with 3M urea inclusion body washing buffer, the main band at approximately 33 kDa was clear, but there were still many impurities. After two ultrasonic washes, the impurities were significantly reduced, but some faint bands remained. The washed inclusion bodies were denatured and purified by Ni column chromatography. The refolded target protein was free of other impurities. Figure 12 This meets the requirements for producing immunogens.

[0075] (4) Specificity of anti-DIV1-168L egg yolk antibody Purified DIV1-168L recombinant protein was used as the antigen, IgY DIV1-168L HRP-labeled goat anti-chicken antibody was used as the primary antibody and as the secondary antibody in Western blot analysis. The results are as follows: Figure 13 As shown, IgY DIV1-168L As a primary antibody, it can bind to the antigen on the membrane, thereby enabling the display of the target band ( Figure 13 (indicated by arrow A), the antibody indicates IgY. DIV1-168L It can specifically recognize proteins expressed in prokaryotes; no target band was observed after pre-incubation with excessive antigen and antibody. Figure 13 B) indicates that excess antigen can bind to the antibody, preventing the antibody from binding to the antigen on the PVDF membrane and thus not showing a band. The experimental results show that the egg yolk antibody obtained by immunizing chickens is correct.

[0076] Sf9 cells were seeded on slides. IgY extracted from egg liquid with a titer of 1:32000 was diluted 1:300 with 0.1% skim milk powder as the primary antibody. FITC-labeled goat anti-chicken IgY was used as the secondary antibody. Indirect immunofluorescence assays were performed on Sf9 cells 12 h after challenge. Separate groups were established: one group incubated with the specific primary antibody without challenge, and the other group incubated with the non-specific IgY. PirA Group, results as follows Figure 14 As shown, cell nuclei exhibited blue fluorescence after DAPI staining, and IgY was used to further fluoresce.DIV1-168L Incubation of healthy Sf9 cells showed no fluorescence, and incubation of DIV1-infected Sf9 cells with nonspecific IgY also did not produce fluorescence. DIV1-168L Sf9 cells infected with DIV1 exhibited weak green fluorescence after incubation, with a fluorescence intensity much weaker than that of IgY in the embodiments of this application. DIV1 (combined) Figure 6 (Comparison), indicating specific IgY DIV1-168L Although it can recognize and bind to natural DIV1 virus particles, the binding force is significantly weaker than that of IgY in the embodiments of this application. DIV1 This proves that its ability to neutralize viruses is very weak.

[0077] (5) The effect of anti-DIV1-168L egg yolk antibody in neutralizing DIV1 200 TCID 50 DIV1 virus solution and IgY at different dilutions DIV1-168L After a 1-hour pre-incubation, the cells were seeded onto Sf9 cells, with normal Sf9 cells serving as a control. Results after 3 days are as follows: Figure 15 As shown, A represents normal Sf9 cells; compared to normal Sf9 cells, Sf9 cells infected with only DIV1 virus solution showed enlarged lesions (B), and cells infected with DIV1 virus solution after being inoculated with 1:2500 diluted ordinary egg solution also showed enlarged lesions (C), indicating that ordinary egg powder has no neutralizing effect on DIV1 and can still cause cytopathic effects. Cells inoculated with 1:2500 and 1:5000 diluted IgY... DIV1-168L Sf9 cells infected with DIV1 viral fluid still showed cytopathic effects, but the cytopathic cells were slightly smaller than those infected with DIV1 viral fluid alone. Furthermore, as the antibody level decreased, the number and size of the cytopathic cells increased (D, E, F), indicating that the added IgY... DIV1-168L It can bind to DIV1 viral particles in Sf9 cells, but its ability to neutralize the virus is very weak, and it cannot effectively prevent viral replication or protect cells. Therefore, it is not used for subsequent in vivo challenge protection.

[0078] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the appended claims and their equivalents.

[0079] The present application and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present application. Actual applications are not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present application, such designs should fall within the protection scope of this application.

Claims

1. A DIV1 recombinant antigen, characterized in that, It has the amino acid sequence shown in SEQ ID No.

1.

2. The gene encoding the DIV1 recombinant antigen as described in claim 1.

3. The gene according to claim 2, characterized in that, It has the nucleotide sequence shown in SEQ ID No.

2.

4. The use of the recombinant antigen of claim 1 or the gene of any one of claims 2-3 in the preparation of anti-DIV1 egg yolk antibodies or immune additives.

5. An egg yolk antibody against DIV1, characterized in that, It is obtained by immunizing laying hens with the recombinant antigen described in claim 1 and then isolating it from the egg yolk.

6. A method for preparing an anti-DIV1 egg yolk antibody, characterized in that, include: The gene encoding the DIV1 recombinant antigen of claim 1 is inserted into the multiple cloning site of the vector using genetic engineering technology to obtain the recombinant vector; The recombinant vector was transformed into the expression host for induced expression, and the inclusion bodies were collected for protein purification to obtain the DIV1 recombinant antigen. The DIV1 recombinant antigen was mixed with an immune adjuvant and then used to immunize laying hens. High-immune eggs were collected based on the titer after immunization. The yolks of hyperimmune eggs were collected, lipids were removed, and yolk antibodies were isolated and purified.

7. The preparation method according to claim 6, characterized in that, The immune adjuvant includes Tween adjuvant, which includes white oil, Span, and aluminum stearate.

8. An egg yolk antibody powder immune additive, characterized in that, It is produced by spray drying coated egg liquid; the coated egg liquid includes a protectant and egg liquid obtained after immunizing laying hens with the recombinant antigen of claim 1 as an immunogen.

9. The use of the egg yolk antibody of claim 5 or the egg yolk antibody powder immune additive of claim 8 in the preparation of products for the treatment and / or prevention of DIV1 infection.

10. A product for treating and / or preventing DIV1 infection, characterized in that, It includes the egg yolk antibody as described in claim 5 and / or the egg yolk antibody powder immune additive as described in claim 8.

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