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-st protein, E2-1d-st protein, gD-st protein and mi3-sc protein; the E2-1b-st protein, the E2-1d-st protein and the gD-st protein are coupled to the surface of the same mi3-sc protein through an isopeptide bond to form 3Ag + mi3 protein nanoparticles; the amino acid sequences of the E2-1b-st protein, the E2-1d-st protein, the gD-st protein and the mi3-sc protein are respectively shown as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 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] The application belongs to the technical field of genetic engineering, and particularly relates to 3Ag+mi3 protein nanoparticles for preventing and treating BVD and IBR, a preparation method and application thereof. BACKGROUND

[0002] Bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis virus (IBRV) are widely prevalent pathogens in global cattle herds. Infection with the viruses not only causes respiratory tract, reproductive tract and digestive tract diseases such as bovine viral diarrhea (BVD) and bovine infectious rhinotracheitis (IBR) in animals, but also causes production performance decline and reproductive disorders in cattle herds, and further causes serious economic losses to the animal husbandry.

[0003] Among them, BVDV belongs to the Pestivirus genus of the Flaviviridae family, and can be divided into BVDV-1, BVDV-2 and BVDV3 types according to the genotype. The main subtypes of BVDV prevalent in China are BVDV 1a, BVDV 1b and BVDV 1d, etc. The genome of BVDV is composed of a positive single-stranded RNA of about 12.3 kb, and the coding order is Npro, capsid protein (C), Erns, E1, E2, p7, NS2 / NS3, NS4A, NS4B, NS5A and NS5B. The E2 glycoprotein contains the main antigenic determinant, the N-terminal of which is the main target of the humoral immune response and can induce neutralizing antibodies; the C-terminal of the extracellular domain has receptor binding and membrane fusion functions, which is the key to mediating the binding of BVDV to host cell receptors (such as CD46 and LDL-R); in addition, the latest research 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 a good candidate antigen for developing BVDV subunit vaccine.

[0004] The inactivated vaccine and attenuated vaccine currently widely used in clinical practice can alleviate the virus epidemic to a certain extent, but due to their weak cross-protection, insufficient immune persistence and safety risks, it is difficult to effectively control the persistent infection and transmission of the virus, and it cannot fully meet the demand of modern large-scale breeding system for disease prevention and control. Therefore, the development of new vaccines with safety, high efficiency and broad-spectrum protection effect has become an important direction for the prevention and control of BVD and IBR. In recent years, the vaccine delivery system based on nanotechnology has the characteristics of high-density loading of antigens, strong immunogenicity and excellent biocompatibility, which provides a new path for the development of multi-union multi-valent vaccine. In view of the cooperative prevention and control demand of BVDV and IBRV, it has important theoretical significance and practical application value to further study the immune effect of different nanoparticles on BVDV and IBRV vaccine. SUMMARY

[0005] The purpose of the present application is to provide a 3Ag+mi3 protein nanoparticle for preventing and treating BVD and IBR, which aims to solve the problems raised in the background art.

[0006] In order to solve the above problems, the present application is realized as follows: a 3Ag+mi3 protein nanoparticle for preventing and treating BVD and IBR, comprising E2-1b-st protein, E2-1d-st protein, gD-st protein and mi3-sc protein; the E2-1b-st protein, E2-1d-st protein and gD-st protein are coupled to the surface of the same mi3-sc protein through isopeptide bond to form the 3Ag+mi3 protein nanoparticle; the amino acid sequences of the E2-1b-st protein, E2-1d-st protein, gD-st protein and mi3-sc protein are shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8 respectively.

[0007] Another purpose of the present application is to provide a gene encoding the 3Ag+mi3 protein nanoparticle for preventing and treating BVD and IBR described above, comprising E2-1b-st gene, E2-1d-st gene, gD-st gene and mi3-sc gene; the nucleotide sequences of the E2-1b-st gene, E2-1d-st gene, gD-st gene and mi3-sc gene are shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7 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-st expression vector, an E2-1d-st expression vector, a gD-st expression vector, and a mi3-sc expression vector; wherein the E2-1b-st expression vector contains the E2-1b-st gene; the E2-1d-st expression vector contains the E2-1d-st gene; the gD-st expression vector contains the gD-st gene; and the mi3-sc expression vector contains the mi3-sc gene.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned 3Ag+mi3 protein nanoparticles, which includes the following steps: E2-1b-st expression vector, E2-1d-st expression vector, gD-st expression vector and mi3-sc expression vector were constructed using the genes with nucleotide sequences as shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, respectively. The mi3-sc expression vector was transformed into the first host cell for induced expression, and then the protein was purified to obtain the mi3-sc protein. The E2-1b-st expression vector, E2-1d-st expression vector and gD-st expression vector were transformed into second host cells for induced expression, and then the proteins were purified to obtain E2-1b-st protein, E2-1d-st protein and gD-st protein. E2-1b-st protein, E2-1d-st protein and gD-st protein were mixed to obtain 3Ag protein nanoparticles; 3Ag protein nanoparticles were mixed with mi3-sc protein and ligated overnight to obtain 3Ag+mi3 protein nanoparticles.

[0010] Furthermore, the first host cell is a competent Escherichia coli cell.

[0011] Furthermore, the second host cell is a CHO cell.

[0012] Furthermore, the molar ratio of the E2-1b-st protein, E2-1d-st protein, and gD-st protein is (0.5-1.5):(0.5-1.5):(0.5-1.5).

[0013] Furthermore, the molar ratio of the Ag protein nanoparticles to the mi3-sc protein is (6-8):1.

[0014] 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.

[0015] 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.

[0016] 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 uniform 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

[0017] Figure 1 This is a schematic diagram illustrating the construction of the recombinant expression vector provided in an embodiment of the present invention.

[0018] Figure 2 SDS-PAGE images of the expression and purification of E2-1b-st, E2-1d-st, and gD-st proteins provided in the embodiments of the present invention.

[0019] Figure 3 Electron micrograph of 3Ag+mi3 protein nanoparticles provided in an embodiment of the present invention.

[0020] 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.

[0021] Figure 5 The graph 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.

[0022] Figure 6 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.

[0023] Figure 7 The figure shows the results of the analysis of the expression level of TNF-α cytokine in the serum cell supernatant of mice after immunization with 3Ag+mi3 protein nanoparticles provided in the embodiments of the present invention. Detailed Implementation

[0024] 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.

[0025] 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.

[0026] mi3 protein nanoparticles are dodecahedral protein nanocages (composed of 60 subunits) exhibiting excellent particle uniformity and stability. By linking the SpyCatcher003 sequence to its N-terminus, mi3 becomes a universal locator, spontaneously coupling with any antigen carrying the SpyTag003 peptide after mixing. mi3-SpyCatcher (mi3-sc) is highly expressed in *E. coli*, and the prepared nanoparticles exhibit excellent environmental tolerance. The mi3 nanoparticles obtained through coupling do not lose performance after freeze-thaw and lyophilization treatments. The mi3 platform demonstrates compatibility and high coupling efficiency with various pathogen antigens.

[0027] To investigate mi3 nanoparticle vaccines that can prevent BVDV and IBRV, this invention expresses BVDV 1b and 1d E2 antigens fused with SpyTag003 and IBRV gD antigen in CHO cells. Furthermore, mi3-SpyCatcher003 (mi3-sc) nanoparticles are expressed in an E. coli prokaryotic system. Purified E2-1b-SpyTag003 (E2-1b-st), E2-1d-SpyTag003 (E2-1d-st), and gD-SpyTag003 (gD-st) are mixed with mi3-SpyCatcher003 (mi3-sc) in a specific ratio. Through the specific covalent binding between SpyTag003 and SpyCatcher003, recombinant nanoparticles exhibiting all three antigens are constructed. This structure allows antigens to be displayed on the surface of nanoparticles in a directional and regular manner, thereby maximally mimicking the antigen arrangement of natural viruses and enhancing the activation of B cells and the production of neutralizing antibodies.

[0028] Specifically, in one embodiment of the present invention, a 3Ag+mi3 protein nanoparticle for preventing and treating BVD and IBR is provided, comprising E2-1b-st protein (E2-1b fused with SpyTag003 sequence), E2-1d-st protein (E2-1d fused with SpyTag003 sequence), gD-st protein (gD fused with SpyTag003 sequence), and mi3-sc protein (mi3 sequence fused with SpyCatcher003); the E2-1b-st protein, E2-1d-st protein, and gD-st protein are coupled to the surface of the same mi3-sc protein via isopeptide bonds to form the 3Ag+mi3 protein nanoparticle; the amino acid sequences of the E2-1b-st protein, E2-1d-st protein, gD-st protein, and mi3-sc protein are shown in the sequence listing SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively, with a signal peptide sequence added at the N-terminus and a His6 tag added at the C-terminus. The amino acid sequence of the mi3 sequence is shown in SEQ ID NO:10, and the nucleotide sequence is shown in SEQ ID NO:9.

[0029] 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, including the E2-1b-st gene, the E2-1d-st gene, the gD-st gene, and the mi3-sc gene; the nucleotide sequences of the E2-1b-st gene, the E2-1d-st gene, the gD-st gene, and the mi3-sc gene are shown in the sequence listings SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, respectively.

[0030] In another embodiment of the present invention, a recombinant expression vector containing the above-mentioned genes is also provided, specifically including an E2-1b-st expression vector, an E2-1d-st expression vector, a gD-st expression vector, and a mi3-sc expression vector; the E2-1b-st expression vector contains the E2-1b-st gene; the E2-1d-st expression vector contains the E2-1d-st gene; the gD-st expression vector contains the gD-st gene; and the mi3-sc expression vector contains the mi3-sc gene. Preferably, the above-mentioned recombinant expression vector is a pET28a(+) or pcDNA3.1(+) plasmid containing the above-mentioned genes, and its promoter type is a T7 promoter or a CMV promoter.

[0031] 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. Using the genes with nucleotide sequences as shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, construct E2-1b-st expression vector, E2-1d-st expression vector, gD-st expression vector, and mi3-sc expression vector, respectively. S2. The mi3-sc expression vector was transformed into the first host cell for induced expression, and then the protein was purified to obtain the mi3-sc protein; S3. The E2-1b-st expression vector, E2-1d-st expression vector and gD-st expression vector were transformed into second host cells for expression, and then the proteins were purified to obtain E2-1b-st protein, E2-1d-st protein and gD-st protein; S4. Mix E2-1b-st protein, E2-1d-st protein and gD-st protein to obtain 3Ag protein nanoparticles. S5. Mix the 3Ag protein nanoparticles with the mi3-sc protein and ligate them overnight to obtain 3Ag+mi3 protein nanoparticles.

[0032] Preferably, the first host cell is a competent Escherichia coli cell, and the second host cell is a CHO cell; the molar ratio of E2-1b-st protein, E2-1d-st protein and gD-st protein is (0.5-1.5):(0.5-1.5):(0.5-1.5), more preferably 1:1:1; the molar ratio of Ag protein nanoparticles to mi3-sc protein is (6-8):1, more preferably 7:1.

[0033] 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.

[0034] 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.

[0035] Example 1: This example provides a method for obtaining the target gene and constructing an expression vector, as detailed below: The 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 the SpyTag003 sequence 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 His6 tag was added at the C-terminus; additionally, gene sequences were added to both ends of the constructed sequences. Bam HI and Eco RI restriction sites; all genes were codon-optimized according to mammalian expression systems to obtain E2-1b-st, E2-1d-st, and gD-st expression vectors; additionally, the mi3 sequence was fused with the SpyChater003 sequence, and a His6 tag was added to the C-terminus. After codon optimization according to prokaryotic expression systems, the vector was constructed into pET28a(+) to obtain the mi3-sc expression vector; the map of the above gene construction is shown below. Figure 1 As shown.

[0036] Example 2: This example provides a method for expressing and purifying E2-1b-st, E2-1d-st, gD-st, and mi3-sc proteins, as detailed below: The E2-1b-st expression vector, E2-1d-st expression vector, and gD-st 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-st, E2-1d-st, and gD-st proteins, which were then 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. The results are as follows: Figure 2 As shown; in addition, following a similar method, the mi3-sc expression vector prepared in Example 1 was transformed into E. coli BL21(DE3) competent cells for induced expression to obtain the mi3-sc protein. Example 3: This example provides a method for preparing and characterizing 3Ag+mi3 protein nanoparticles, as detailed below: The E2-1b-st, E2-1d-st, and gD-st proteins prepared in Example 2 were mixed in a 1:1:1 molar ratio. Under aseptic conditions, the three proteins were mixed by pipetting to obtain a 3Ag protein mixture (hereinafter referred to as 3Ag). Separately, 3Ag was mixed with the mi3-sc protein prepared in Example 2 in a 7:1 molar ratio and incubated overnight at room temperature. Free protein was removed by ultrafiltration (PBs replaced 3-5 times the volume), yielding a concentrated solution, which is the 3Ag+mi3 protein nanoparticle. Large particle impurities were removed by filtration through a 0.22 μm filter membrane to obtain the sample. The sample was dropped onto a carbon membrane 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 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.

[0037] 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 (3Ag protein nanoparticles prepared in Example 3) was 30 μg, combined with M903 adjuvant; the immunization dose for the commercially available dual vaccine group was 100 μL, and the PBS control group received 100 μL of PBS. Blood was collected from the tail at days 7, 14, 21, 28, 35, and 42 after immunization.

[0038] 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, at day 42, the IgG level in the 3Ag+mi3 group was significantly increased (p<0.001), significantly higher than that in the PBS group, indicating that the mi3 nanoparticle strategy and multi-antigen synergistic strategy have certain advantages in enhancing antibody production.

[0039] 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 added to each well (final concentration 5 μg / mL). 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, on day 42, the SI value of the 3Ag+mi3 group was close to 3.5, which was significantly higher than that of the PBS group (p<0.01), indicating that the mi3 display strategy can stimulate T cell response in the early stage and maintain a strong secondary response capability.

[0040] 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 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. Figure 6-7 As shown, on day 21, the serum IFN-γ and TNF-α expression in all immunization groups increased. On day 42, the serum IFN-γ and TNF-α levels in the 3Ag+mi3 group were significantly higher than those in the PBS group, indicating that the mi3 display strategy and multi-antigen combination may activate cellular immune responses in the later stages of immunization.

[0041] 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-st protein, E2-1d-st protein, gD-st protein, and mi3-sc protein; the E2-1b-st protein, E2-1d-st protein, and gD-st protein are coupled to the surface of the same mi3-sc protein via isopeptide bonds to form the 3Ag+mi3 protein nanoparticles; the amino acid sequences of the E2-1b-st protein, E2-1d-st protein, gD-st protein, and mi3-sc protein are shown in the sequence listing SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, 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-st gene, the E2-1d-st gene, the gD-st gene, and the mi3-sc gene; the nucleotide sequences of the E2-1b-st gene, the E2-1d-st gene, the gD-st gene, and the mi3-sc gene are shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, respectively.

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

4. A method for preparing 3Ag+mi3 protein nanoparticles as described in claim 1, characterized in that, Includes the following steps: E2-1b-st expression vector, E2-1d-st expression vector, gD-st expression vector and mi3-sc expression vector were constructed using the genes with nucleotide sequences as shown in the sequence listing SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, respectively. The mi3-sc expression vector was transformed into the first host cell for induced expression, and then the protein was purified to obtain the mi3-sc protein. The E2-1b-st expression vector, E2-1d-st expression vector and gD-st expression vector were transformed into second host cells for induced expression, and then the proteins were purified to obtain E2-1b-st protein, E2-1d-st protein and gD-st protein. E2-1b-st protein, E2-1d-st protein and gD-st protein were mixed to obtain 3Ag protein nanoparticles; 3Ag protein nanoparticles were mixed with mi3-sc protein and ligated overnight to obtain 3Ag+mi3 protein nanoparticles.

5. The method for preparing 3Ag+mi3 protein nanoparticles according to claim 4, characterized in that, The first host cell is a competent Escherichia coli cell.

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

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

8. The method for preparing 3Ag+mi3 protein nanoparticles according to claim 4 or 7, characterized in that, The molar ratio of the Ag protein nanoparticles to the mi3-sc protein is (6-8):

1.

9. 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.

10. 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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