3Ag-mi3 protein nanoparticles for preventing and treating BVD and IBR as well as preparation method and application of 3Ag-mi3 protein nanoparticles

Abstract

The invention is suitable for the technical field of gene engineering, and provides 3Ag-mi3 protein nanoparticles for preventing and treating BVD and IBR as well as a preparation method and application of the 3Ag-mi3 protein nanoparticles, the 3Ag-mi3 protein nanoparticles comprise E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein and gD-mi3 fusion protein; the amino acid sequences of the E2-1b-mi3 fusion protein, the E2-1d-mi3 fusion protein and the gD-mi3 fusion protein are respectively shown as SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 in a sequence table. After animals are immunized by the 3Ag-mi3 protein nanoparticles, an effective immune protection effect can be generated, and the 3Ag-mi3 protein nanoparticles have a good prevention and control application prospect on bovine viral diarrhea and bovine rhinotracheitis.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a 3Ag-mi3 protein nanoparticle for preventing and treating BVD and IBR, its preparation method and application. Background Technology

[0002] Bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis virus (IBRV) are pathogens that are prevalent in cattle herds worldwide. Their infection not only causes respiratory, reproductive, and digestive diseases such as bovine viral diarrhea (BVD) and bovine infectious rhinotracheitis (IBR), but also leads to decreased production performance and reproductive disorders in cattle herds, resulting in serious economic losses to the livestock industry.

[0003] BVDV belongs to the genus Menthaviridae in the family Flaviviridae. Based on genotype, it can be divided into BVDV-1, BVDV-2, and BVDV3. The main BVDV subtypes circulating in my country are BVDV 1a, BVDV 1b, and BVDV 1d. Its genome consists of approximately 12.3 kb of positive-sense single-stranded RNA, with the coding sequence as follows: Npro, capsid protein (C), Erns, E1, E2, p7, NS2 / NS3, NS4A, NS4B, NS5A, and NS5B. The E2 glycoprotein contains the major antigenic determinant, and its N-terminus is a major target of humoral immune responses and can induce neutralizing antibodies. The C-terminus of its extracellular domain has receptor binding and membrane fusion functions, which are key to mediating the binding of BVDV to host cell receptors (such as CD46 and LDL-R). In addition, recent studies have found that E2 can also bind to pattern recognition molecules involved in complement activation, making the virus more sensitive to complement lysis activity, thus indicating that E2 is an excellent candidate antigen for developing BVDV subunit vaccines.

[0004] While inactivated and attenuated vaccines widely used in clinical practice can alleviate viral outbreaks to some extent, their weak cross-protection, insufficient duration of immunity, and safety risks make them ineffective in controlling persistent infection and transmission, failing to fully meet the disease control needs of modern large-scale aquaculture systems. Therefore, developing novel vaccines with safe, efficient, and broad-spectrum protective effects has become a crucial direction for BVD and IBR prevention and control. In recent years, nanotechnology-based vaccine delivery systems, with their high-density antigen loading, strong immunogenicity, and excellent biocompatibility, have provided a new pathway for the development of multivalent vaccines. In-depth research on the immunizing effects of different nanoparticles on BVDV and IBRV vaccines, addressing the synergistic prevention and control needs, has significant theoretical and practical value. Summary of the Invention

[0005] The purpose of this invention is to provide a 3Ag-mi3 protein nanoparticle for preventing and treating BVD and IBR, aiming to solve the problems mentioned in the background art.

[0006] To address the aforementioned problems, this invention provides a 3Ag-mi3 protein nanoparticle for preventing and treating BVD and IBR, comprising an E2-1b-mi3 fusion protein, an E2-1d-mi3 fusion protein, and a gD-mi3 fusion protein; the amino acid sequences of the E2-1b-mi3 fusion protein, the E2-1d-mi3 fusion protein, and the gD-mi3 fusion protein are shown in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively.

[0007] Another object of the present invention is to provide a gene encoding the above-mentioned 3Ag-mi3 protein nanoparticles for preventing and treating BVD and IBR, said gene comprising the E2-1b-mi3 gene, the E2-1d-mi3 gene and the gD-mi3 gene; the nucleotide sequences of the E2-1b-mi3 gene, the E2-1d-mi3 gene and the gD-mi3 gene are shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, respectively.

[0008] Another object of the present invention is to provide a recombinant expression vector containing the above-mentioned genes, specifically including an E2-1b-mi3 expression vector, an E2-1d-mi3 expression vector, and a gD-mi3 expression vector; wherein the E2-1b-mi3 expression vector contains the E2-1b-mi3 gene; the E2-1d-mi3 expression vector contains the E2-1d-mi3 gene; and the gD-mi3 expression vector contains the gD-mi3 gene.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned 3Ag-mi3 protein nanoparticles, comprising the following steps: E2-1b-mi3 expression vector, E2-1d-mi3 expression vector and gD-mi3 expression vector were constructed using the genes with nucleotide sequences as shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, respectively. E2-1b-mi3 expression vector, E2-1d-mi3 expression vector and gD-mi3 expression vector were transformed into host cells for induced expression, and then the proteins were purified to obtain E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein and gD-mi3 fusion protein; The E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein, and gD-mi3 fusion protein were mixed to obtain 3Ag-mi3 protein nanoparticles.

[0010] Furthermore, the host cell is a CHO cell.

[0011] Furthermore, the molar ratio of the E2-1b-mi3 fusion protein, the E2-1d-mi3 fusion protein, and the gD-mi3 fusion protein is (0.5-1.5):(0.5-1.5):(0.5-1.5).

[0012] Another object of the present invention is to provide the use of the above-mentioned 3Ag-mi3 protein nanoparticles, or the above-mentioned gene, or the above-mentioned recombinant expression vector in the preparation of drugs or vaccines for the prevention and treatment of bovine viral diarrhea and / or bovine rhinotracheitis.

[0013] Another object of the present invention is to provide a drug or vaccine for the prevention and treatment of bovine viral diarrhea and / or bovine rhinotracheitis, comprising a pharmaceutically acceptable carrier and the aforementioned 3Ag-mi3 protein nanoparticles.

[0014] This invention provides a 3Ag-mi3 protein nanoparticle for the prevention and treatment of BVD and IBR. By introducing mi3, it can form a highly symmetrical and closed virus-like structure with excellent thermal stability and pH tolerance. This 3Ag-mi3 protein nanoparticle can be made into a nanoparticle vaccine that is non-toxic to mammals, poses no biosafety threat, and can induce a faster and stronger humoral immune response, including a more efficient germinal center response and the production of high titers of neutralizing antibodies. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the construction of the recombinant expression vector provided in an embodiment of the present invention.

[0016] Figure 2The images show SDS-PAGE and Western Blot diagrams of the expression and purification of E2-1b-mi3 fusion protein (A), E2-1d-mi3 fusion protein (B), and gD-mi3 fusion protein (C) provided in the embodiments of the present invention.

[0017] Figure 3 Electron micrographs of E2-1b-mi3 fusion protein (a), E2-1d-mi3 fusion protein (b), gD-mi3 fusion protein (c), and 3Ag-mi3 protein nanoparticles (d) provided in the embodiments of the present invention.

[0018] Figure 4 The image shows the serum IgG detection results of mice immunized with 3Ag-mi3 protein nanoparticles provided in this embodiment of the invention.

[0019] Figure 5 The image shows the results of spleen lymphocyte stimulation index detection in mice after immunization with 3Ag-mi3 protein nanoparticles provided in this embodiment of the invention.

[0020] Figure 6 The figure shows the results of the expression level analysis of the cytokine IL-6 in the serum cell supernatant of mice after immunization with the 3Ag-mi3 protein nanoparticles provided in the embodiments of the present invention.

[0021] Figure 7 The figure shows the results of the expression level analysis of the cytokine IFN-γ in the serum cell supernatant of mice after immunization with the 3Ag-mi3 protein nanoparticles provided in the embodiments of the present invention.

[0022] Figure 8 The figure shows the results of the analysis of the expression level of the cytokine TNF-α in the serum cell supernatant of mice after immunization with the 3Ag-mi3 protein nanoparticles provided in the embodiments of the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0024] The Chinese hamster ovary cells (CHO) used in this invention are one of the most widely used mammalian expression systems in vaccine antigen production. In recent years, many important vaccines (such as respiratory syncytial virus vaccines, shingles vaccines, and SARS-CoV-2 subunit vaccines) have used CHO cells to produce their recombinant protein antigens. CHO cells have excellent recombinant protein expression capabilities, enabling high-density cell culture in bioreactors to produce high concentrations of target proteins. Monoclonal antibody concentrations produced by optimized CHO cell lines can reach several grams per liter. CHO cells are easy to culture in suspension, allowing for large-scale production of vaccine antigens in large bioreactors while maintaining stable yields. In contrast, many other systems, while achieving high yields on a small scale, may face challenges when scaling up culture. Overall, CHO cells provide a reliable guarantee for large-scale vaccine supply in terms of yield and scale-up production.

[0025] mi3 nanoparticles are a man-made protein self-assembly platform composed of 60 subunits that can spontaneously form stable, symmetrical spherical structures with a diameter of approximately 25-30 nm within cells. In vaccine applications, mi3 nanoparticles can improve antigen stability in vivo and lymph node targeting efficiency, promote the uptake and activation of antigen-presenting cells, and effectively induce strong humoral and cellular immune responses. Their multivalent, repeating geometric arrangement can enhance B-cell receptor cross-linking, inducing higher titers of neutralizing antibodies and durable immune memory. As a novel nanovaccine platform, mi3 has broad application prospects in the development of vaccines for viral diseases, especially those requiring rapid induction of high-quality immune protection.

[0026] To investigate mi3 nanoparticle vaccines that can prevent BVDV and IBRV, this invention utilizes a CHO expression system and a mi3 self-assembled nanoparticle system. The E2 antigens of BVDV 1b and 1d types, and the gD antigen of IBRV, are expressed on the surface of mi3 nanoparticles via gene fusion. These three types of nanoparticles are then mixed in a specific ratio to prepare recombinant mi3 "cocktail" nanoparticles. This strategy preserves the structural integrity of multiple antigens and enhances antigen density through the multivalent structure of the nanoparticles, thereby improving immune activation efficiency. Further systemic immunological evaluations were conducted in BALB / c mice to analyze its induction capabilities in humoral immunity, cellular immunity, and immune memory, laying the foundation for subsequent vaccine optimization and application in cattle.

[0027] Specifically, in one embodiment of the present invention, a 3Ag-mi3 protein nanoparticle for preventing and treating BVD and IBR is provided, comprising an E2-1b-mi3 fusion protein, an E2-1d-mi3 fusion protein, and a gD-mi3 fusion protein; wherein, the E2-1b-mi3 fusion protein, the E2-1d-mi3 fusion protein, and the gD-mi3 fusion protein have a signal peptide sequence added to their N-terminus and a mi3 sequence and a His6 tag added to their C-terminus; the amino acid sequences of the above-mentioned E2-1b-mi3 fusion protein, the E2-1d-mi3 fusion protein, and the gD-mi3 fusion protein are shown in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively; the amino acid sequence of the above-mentioned mi3 sequence is shown in SEQ ID NO:8, and the nucleotide sequence is shown in SEQ ID NO:7.

[0028] In another embodiment of the present invention, a gene encoding the above-mentioned 3Ag-mi3 protein nanoparticles for preventing and treating BVD and IBR is also provided, the gene comprising the E2-1b-mi3 gene, the E2-1d-mi3 gene and the gD-mi3 gene; the nucleotide sequences of the E2-1b-mi3 gene, the E2-1d-mi3 gene and the gD-mi3 gene are shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, respectively.

[0029] In another embodiment of the present invention, a recombinant expression vector containing the above-mentioned genes is also provided, specifically including an E2-1b-mi3 expression vector, an E2-1d-mi3 expression vector, and a gD-mi3 expression vector; the E2-1b-mi3 expression vector contains the E2-1b-mi3 gene; the E2-1d-mi3 expression vector contains the E2-1d-mi3 gene; and the gD-mi3 expression vector contains the gD-mi3 gene. Preferably, the above-mentioned recombinant expression vector is a pcDNA3.1(+) plasmid containing the above-mentioned genes, and its promoter type is CMV promoter group.

[0030] In another embodiment of the present invention, a method for preparing the above-mentioned 3Ag-mi3 protein nanoparticles is also provided, comprising the following steps: S1. Construct E2-1b-mi3 expression vector, E2-1d-mi3 expression vector and gD-mi3 expression vector using the genes with nucleotide sequences as shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, respectively. S2. The E2-1b-mi3 expression vector, E2-1d-mi3 expression vector and gD-mi3 expression vector were transformed into host cells for induction expression, and then purified to obtain the E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein and gD-mi3 fusion protein. S3. Mix the E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein and gD-mi3 fusion protein to obtain 3Ag-mi3 protein nanoparticles.

[0031] Preferably, the host cell is a CHO cell; the molar ratio of the E2-1b-mi3 fusion protein, the E2-1d-mi3 fusion protein and the gD-mi3 fusion protein is (0.5-1.5):(0.5-1.5):(0.5-1.5), more preferably 1:1:1.

[0032] In another embodiment of the present invention, the use of the above-mentioned 3Ag-mi3 protein nanoparticles, or the above-mentioned gene, or the above-mentioned recombinant expression vector in the preparation of drugs or vaccines for the prevention and treatment of bovine viral diarrhea and / or bovine rhinotracheitis is also provided.

[0033] In another embodiment of the present invention, a drug or vaccine for preventing and treating bovine viral diarrhea and / or bovine rhinotracheitis is also provided, comprising a pharmaceutically acceptable carrier and the aforementioned 3Ag-mi3 protein nanoparticles.

[0034] The 3Ag-mi3 protein nanoparticles provided in this invention successfully combine advanced nanoparticle technology, a highly efficient mammalian cell expression system, and a key viral antigen to create a novel vaccine candidate with strong immunogenicity, high safety, and good stability. This provides a new solution for the effective prevention and control of bovine viral diarrhea and has significant practical application value and market prospects.

[0035] Example 1: This example provides a method for obtaining the target gene and constructing an expression vector, as detailed below: Gene sequences of bovine viral diarrhea virus (BVDV-E2-1b, GenBank ID: AGM75780.1), BVDV-E2-1d, GenBank ID: MF166858.1, and bovine rhinotracheitis virus (IBRV-gD, GenBank ID: QB159528.1) were selected and fused with mi3 sequences to construct gene sequences, which were then cloned into the mammalian expression vector pcDNA3.1(+). A signal peptide sequence was introduced at the N-terminus of the gene, and a mi3 sequence and a His6 tag were added at the C-terminus. Simultaneously, gene sequences were added to both ends of the constructed sequences. Hand III and EcoRI. Restriction site; all genes were codon-optimized according to mammalian expression systems to obtain E2-1b-mi3 expression vector, E2-1d-mi3 expression vector, and gD-mi3 expression vector; the gene mapping diagrams are shown below. Figure 1 As shown.

[0036] Example 2: This example provides a method for expressing and purifying E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein, and gD-mi3 fusion protein, as detailed below: The E2-1b-mi3 expression vector, E2-1d-mi3 expression vector, and gD-mi3 expression vector prepared in Example 1 were respectively introduced into CHO cells (electrocution cuvette 4 mm, cell number 1×10⁶) via electroporation. 7 After transfection, the cell culture density was 1×10⁶ cells / year. 6 After transfection for 48 h, G418 (final concentration 800 μg / mL) was added. After 7 days, the medium was replaced with fresh medium containing G418 to obtain a drug-resistant cell population. The supernatant from cells cultured for 4 days was collected and purified using affinity chromatography to obtain E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein, and gD-mi3 fusion protein. These proteins were identified by SDS-PAGE and Western blot. The primary antibody was rabbit-derived His (Abcam), and the secondary antibody was HRP-labeled goat anti-rabbit IgG. Results are shown below. Figure 2 As shown.

[0037] Example 3: This example provides a method for preparing and characterizing 3Ag-mi3 protein nanoparticles, as detailed below: The E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein, and gD-mi3 fusion protein prepared in Example 2 were mixed in a 1:1:1 molar ratio to obtain 3Ag-mi3 protein nanoparticle samples. The samples were dropped onto a carbon film copper mesh (300 mesh), allowed to stand at room temperature for 1 min, negatively stained with 2% phosphotungstic acid for 30 s, and after removing excess stain, allowed to air dry. The morphology of the E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein, gD-mi3 fusion protein, and 3Ag-mi3 protein nanoparticles was observed under an 80 kV transmission electron microscope. The results are as follows: Figure 3 As shown. DLS analysis: 300 μL of the 3Ag-mi3 protein nanoparticle suspension obtained in Example 3 above was taken and the average particle size (Z-Average) and polydispersity index (PDI) were measured in a Malvern Zetasizer Nano ZS instrument. Each sample was measured three times to analyze the diameter and uniformity of the nanoparticles.

[0038] Example 4: This example is a mouse immunization experiment, as detailed below: Forty 6-week-old SPF-grade female BALB / c mice were randomly divided into four groups of 10 mice each, with an average weight of 20 ± 2 g. Immunization was performed on days 0, 14, and 28. The total immunization dose for the 3Ag-mi3 group (3Ag-mi3 protein nanoparticles prepared in Example 3) and the 3Ag group (constructing E2-1b, E2-1d, and gD proteins directly using the BVDV-E2-1b, BVDV-E2-1d, and IBRV-gD genes according to the methods in Examples 1-2, without fusing the mi3 tag sequence, and then mixing the E2-1b, E2-1d, and gD proteins in a 1:1:1 molar ratio to obtain the 3Ag protein) was 30 μg, combined with M903 adjuvant; the total immunization dose for the commercially available vaccine group (Dualvaccine) was 100 μL; and the PBS control group received 100 μL of PBS. Blood samples were collected from the tail at 7, 14, 21, 28, 35 and 42 days after immunization.

[0039] Serum antibody levels were detected using ELISA at different time points (7 d, 14 d, 21 d, 28 d, 35 d, and 42 d) after mouse immunization. Serially diluted IgG standards and 1:100,000 diluted mouse serum samples were incubated in ELISA plates at 37°C for 1 h, washed, and HRP-labeled goat anti-mouse IgG secondary antibody was added, followed by incubation at 37°C for 1 h. After washing, 90 μL of TMB chromogenic solution was added, and the reaction was carried out at 37°C for 30 min. The reaction was terminated by adding 50 μL of 2 M H₂SO₄, and the absorbance was measured at 450 nm (OD450 nm). IgG antibody kinetic curves were plotted, and the results are shown below. Figure 4 As shown in the figure, on day 7 post-immunization, the serum IgG level in the 3Ag-mi3 group was significantly higher than that in the 3Ag group, and the IgG level increased with the extension of immunization time, reaching its highest level on day 42 post-immunization.

[0040] Splenic lymphocyte proliferation assay: On days 35 and 42 post-immunization, three mice from each group were randomly selected and sacrificed by dislocation. Splenic tissue was collected from the mice, and the cells were gently ground in spleen lymphocyte separation medium (Beijing Dakowei Biotechnology Co., Ltd.) to prepare a single-cell suspension. The cells were centrifuged at 800×g for 30 min to separate the cells into layers. The lymphocyte layer was aspirated and resuspended and washed with RPMI-1640 complete medium. Press 1×10 6Cells were seeded at a density of [number] cells / mL into 96-well plates, with 3Ag-mi3 protein nanoparticles (final concentration 5 μg / mL) added to each well. A commercially available vaccine group (dual vaccine) and a negative control group (PBS) were also established. After culturing at 37°C and 5% CO2 for 42 h, 10 μL of CCK-8 solution was added, and incubation continued at 37°C for another 4 h. OD values ​​were measured at 450 nm, and the lymphocyte proliferation index (SI) was calculated. SI = (OD value of experimental group - OD value of blank control group) / (OD value of negative control group - OD value of blank control group). Results are shown below. Figure 5 As shown, the SI in the PBS group remained at a low level at 35 days and 42 days without significant change (p>0.05); at 42 days, the stimulation index of the 3Ag-mi3 group was significantly higher than that at 35 days (p<0.001), indicating that the 3Ag-mi3 protein nanoparticles can more effectively promote T cell activation; this further verifies that the 3Ag-mi3 protein nanoparticles help enhance the T cell stimulation ability of antigens and improve cellular immune activity.

[0041] Analysis of Cytokine Levels in Mouse Serum Cell Supernatant: The secretion levels of cytokines in mouse serum cell supernatant were detected using a commercially available ELISA kit. First, mouse IL-6, IFN-γ, and TNF-α capture antibodies were coated onto 96-well ELISA plates and incubated overnight at 4°C, followed by blocking with 5% BSA for 1 h. Diluted serum was then added, and the plates were incubated at 37°C for 2 h, washed, and then biotin-labeled detection antibodies were added sequentially. The plates were incubated at 37°C for 30 min, and then washed to remove non-specific bindings. 90 μL of TMB chromogenic buffer was added, and the plates were reacted at 37°C in the dark for 15 min. Stop solution was then added, and the absorbance was measured at 450 nm. The concentrations of IFN-γ and TNF-α were calculated using a standard curve. Results are shown below. Figures 6-8 As shown, the expression levels of IL-6, IFN-γ, and TNF-α in the 3Ag-mi3 group were significantly higher than those in the PBS and Dual vaccine groups; the results indicate that the 3Ag-mi3 protein nanoparticles prepared in this embodiment of the invention have certain advantages in enhancing humoral and cellular immunity.

[0042] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A 3Ag-mi3 protein nanoparticle for preventing and treating BVD and IBR, characterized in that, The 3Ag-mi3 protein nanoparticles include E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein, and gD-mi3 fusion protein; the amino acid sequences of the E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein, and gD-mi3 fusion protein are shown in the sequence listing SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively.

2. A gene encoding 3Ag-mi3 protein nanoparticles for preventing and treating BVD and IBR as described in claim 1, characterized in that, The genes include the E2-1b-mi3 gene, the E2-1d-mi3 gene, and the gD-mi3 gene; the nucleotide sequences of the E2-1b-mi3 gene, the E2-1d-mi3 gene, and the gD-mi3 gene are shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, respectively.

3. A recombinant expression vector comprising the gene of claim 2, characterized in that, Specifically, it includes the E2-1b-mi3 expression vector, the E2-1d-mi3 expression vector, and the gD-mi3 expression vector; the E2-1b-mi3 expression vector contains the E2-1b-mi3 gene; the E2-1d-mi3 expression vector contains the E2-1d-mi3 gene; and the gD-mi3 expression vector contains the gD-mi3 gene.

4. A method for preparing 3Ag-mi3 protein nanoparticles as described in claim 1, characterized in that, Includes the following steps: E2-1b-mi3 expression vector, E2-1d-mi3 expression vector and gD-mi3 expression vector were constructed using the genes with nucleotide sequences as shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, respectively. E2-1b-mi3 expression vector, E2-1d-mi3 expression vector and gD-mi3 expression vector were transformed into host cells for induced expression, and then the proteins were purified to obtain E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein and gD-mi3 fusion protein; The E2-1b-mi3 fusion protein, E2-1d-mi3 fusion protein, and gD-mi3 fusion protein were mixed to obtain 3Ag-mi3 protein nanoparticles.

5. The method for preparing 3Ag-mi3 protein nanoparticles according to claim 4, characterized in that, The host cell is a CHO cell.

6. The method for preparing 3Ag-mi3 protein nanoparticles according to claim 4, characterized in that, The molar ratio of the E2-1b-mi3 fusion protein, the E2-1d-mi3 fusion protein, and the gD-mi3 fusion protein is (0.5-1.5):(0.5-1.5):(0.5-1.5).

7. The use of the 3Ag-mi3 protein nanoparticle as described in claim 1, or the gene as described in claim 2, or the recombinant expression vector as described in claim 3 in the preparation of a drug or vaccine for the prevention and treatment of bovine viral diarrhea and / or bovine rhinotracheitis.

8. A drug or vaccine for the prevention and treatment of bovine viral diarrhea and / or bovine rhinotracheitis, comprising a pharmaceutically acceptable carrier, characterized in that, It also includes the 3Ag-mi3 protein nanoparticles as described in claim 1.

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