GD-mi3 fusion protein of infectious bovine rhinotracheitis virus as well as preparation method and application of gD-mi3 fusion protein

Abstract

The invention is applicable to the technical field of genetic engineering, and provides a gD-mi3 fusion protein of infectious bovine rhinotracheitis virus and a preparation method and application thereof, the gD-mi3 fusion protein is formed by fusing a mi3 tag sequence with a gD genome of the infectious bovine rhinotracheitis virus; the amino acid sequence of the mi3 tag sequence is as shown in a sequence table SEQ ID NO: 4; the amino acid sequence of the gD-mi3 fusion protein is shown as SEQ ID NO: 2 in a sequence table. After animals are immunized by the gD-mi3 fusion protein, an effective immune protection effect can be generated, and the gD-mi3 fusion protein has a good prevention and control application prospect on infectious bovine rhinotracheitis caused by the IBRV virus.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a gD-mi3 fusion protein of bovine infectious rhinotracheitis virus, its preparation method and application. Background Technology

[0002] Bovine Infectious Rhinotracheitis (IBR), also known as red nose disease, is a contagious disease caused by bovine infectious rhinotracheitis virus (IBRV), and is classified as a Class II animal disease in my country. IBRV, also known as bovine herpesvirus-1 (BoHV-1), is highly contagious and can be transmitted through droplets, artificial insemination, and direct contact. Calf infection rates and mortality are high, often accompanied by neurological symptoms. Furthermore, BoHV-1 can remain latent in cattle and be carried for extended periods, easily activating and shedding the virus under stress, becoming a persistent source of infection and increasing the difficulty of prevention and control. The widespread transmission and latent nature of this disease seriously threaten the healthy development of the cattle industry, causing severe economic losses.

[0003] While traditional vaccines can control the epidemic to some extent, they are less effective at blocking latent infection and reactivation of the virus. Therefore, developing new and highly effective vaccines is of great significance for the prevention and control of IBRV. Summary of the Invention

[0004] The purpose of this invention is to provide a gD-mi3 fusion protein of bovine infectious rhinotracheitis virus, which aims to solve the problems mentioned in the background art.

[0005] To address the aforementioned problems, the present invention provides a gD-mi3 fusion protein of bovine infectious rhinotracheitis virus, which is formed by fusing a mi3 tag sequence with the gD genome of bovine infectious rhinotracheitis virus; the amino acid sequence of the mi3 tag sequence is shown in SEQ ID NO:4; and the amino acid sequence of the gD-mi3 fusion protein is shown in SEQ ID NO:2.

[0006] Another object of the present invention is to provide a gene encoding the gD-mi3 fusion protein of the above-mentioned bovine infectious rhinotracheitis virus, the nucleotide sequence of which is shown in the sequence listing SEQ ID NO:1.

[0007] Another object of the present invention is to provide a recombinant expression vector comprising the above-described genes.

[0008] Another object of the present invention is to provide a host cell comprising at least one of the above-described gD-mi3 fusion protein, the above-described gene, and the above-described recombinant expression vector.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned gD-mi3 fusion protein, which includes the following steps: A recombinant expression vector was constructed using the gene whose nucleotide sequence is shown in SEQ ID NO:1 of the sequence listing. The recombinant expression vector was transformed into host cells for induced expression, and then the protein was purified to obtain the gD-mi3 fusion protein.

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

[0011] Another object of the present invention is to provide the use of the above-mentioned gD-mi3 fusion protein, or the above-mentioned gene, or the above-mentioned recombinant expression vector, or the above-mentioned host cell in the preparation of drugs or vaccines for the prevention and treatment of bovine infectious rhinotracheitis.

[0012] Another object of the present invention is to provide a drug or vaccine for the prevention and treatment of bovine infectious rhinotracheitis, comprising a pharmaceutically acceptable carrier and the above-described gD-mi3 fusion protein.

[0013] Furthermore, the vaccine is a nanoparticle vaccine.

[0014] This invention provides a gD-mi3 fusion protein of bovine infectious rhinotracheitis virus. By introducing mi3, a highly symmetrical and closed virus-like structure can be formed, exhibiting excellent thermal stability and pH tolerance. This gD-mi3 fusion protein can be formulated into nanoparticle vaccines that are non-toxic to mammals, pose 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 2 The SDS-PAGE image (A) and Western Blot identification image (B) of the gD-mi3 fusion protein expression and purification provided in the embodiments of the present invention.

[0017] Figure 3 Electron micrograph of gD-mi3 fusion protein nanoparticles provided in an embodiment of the present invention.

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

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

[0020] Figure 6 The figure shows the results of the expression level analysis of IFN-γ cytokine in the spleen cell supernatant of mice after immunization with gD-mi3 fusion protein nanoparticles provided in this embodiment of the invention.

[0021] Figure 7 The figure shows the results of the expression level analysis of the cytokine TNF-α in the spleen cell supernatant of mice after immunization with gD-mi3 fusion protein nanoparticles provided in the embodiments of the present invention. Detailed Implementation

[0022] 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.

[0023] 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.

[0024] 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.

[0025] To investigate mi3 fusion nanoparticle vaccines that can prevent IBRV, this invention utilizes a CHO expression system and a mi3 self-assembled nanoparticle system. The IBRV gD antigen is expressed on the surface of mi3 nanoparticles via gene fusion. 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 evaluation was 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.

[0026] Specifically, in one embodiment of the present invention, a gD-mi3 fusion protein of bovine infectious rhinotracheitis virus is provided, which is formed by fusing a mi3 tag sequence with the gD genome of bovine infectious rhinotracheitis virus, with a signal peptide sequence added at the N-terminus and a His6 tag added at the C-terminus; wherein, the amino acid sequence of the above-mentioned mi3 tag sequence is shown in SEQ ID NO:4, and the nucleotide sequence is shown in SEQ ID NO:3; the amino acid sequence of the above-mentioned gD-mi3 fusion protein is shown in SEQ ID NO:2.

[0027] In another embodiment of the present invention, a gene encoding the gD-mi3 fusion protein of the above-mentioned bovine infectious rhinotracheitis virus is also provided, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0028] In another embodiment of the present invention, a recombinant expression vector comprising the above-described genes is also provided.

[0029] Preferably, the recombinant expression vector can be normally expressed in CHO cells; more preferably, the recombinant expression vector is a pcDNA3.1(+) plasmid containing the above-mentioned gene, and its promoter type is CMV promoter group.

[0030] In another embodiment of the present invention, a host cell is also provided, comprising at least one of the above-described gD-mi3 fusion protein, the above-described gene, and the above-described recombinant expression vector.

[0031] In another embodiment of the present invention, a method for preparing the above-mentioned gD-mi3 fusion protein is also provided, which includes the following steps: S1. Construct a recombinant expression vector using the gene whose nucleotide sequence is shown in SEQ ID NO:1 of the sequence listing; S2. The recombinant expression vector is transformed into host cells for induced expression, followed by protein purification to obtain the gD-mi3 fusion protein. Preferably, the host cells are CHO cells.

[0032] In another embodiment of the present invention, the use of the above-mentioned gD-mi3 fusion protein, or the above-mentioned gene, or the above-mentioned recombinant expression vector, or the above-mentioned host cell in the preparation of a drug or vaccine for the prevention and treatment of bovine infectious rhinotracheitis is also provided.

[0033] In another embodiment of the invention, a drug or vaccine for preventing and treating bovine infectious rhinotracheitis is also provided, comprising a pharmaceutically acceptable carrier and the aforementioned gD-mi3 fusion protein. Preferably, the vaccine is a nanoparticle vaccine.

[0034] The gD-mi3 fusion protein provided in this invention successfully combines 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 infectious rhinotracheitis 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: The bovine infectious rhinotracheitis virus (IBRV)-gD gene sequence (GenBank ID: QB159528.1) was selected and fused with the mi3 tag sequence (nucleotide sequence shown in SEQ ID NO:3 in the sequence listing) to construct a gene named gD-mi3. This gene was then cloned into the pcDNA3.1(+) expression vector. A signal peptide sequence was introduced at the N-terminus of the gene, and a mi3 tag sequence and a His6 tag were added at the C-terminus. Simultaneously, additional tags were added to both ends of the constructed sequence. Hand III and EcoR I. Restriction site; all genes were codon-optimized according to mammalian expression systems to obtain recombinant expression vectors; gene construction map as shown below. Figure 1 As shown.

[0036] Example 2: This example provides a method for expressing and purifying gD-mi3 fusion protein nanoparticles, as detailed below: The recombinant expression vector gD-mi3 prepared in Example 1 was introduced into CHO cells via electroporation (electroclavicle cup size: 4 mm, cell number: 1 × 10⁻⁶). 7 After transfection, the cell culture density was 1×10⁶ cells / year. 6Cells were transfected (cell / mL), and G418 (final concentration 800 μg / mL) was added 48 h later. After 7 days, the medium was replaced with fresh medium containing G418. The supernatant from cells cultured for 4 days was collected and purified using affinity chromatography to obtain a suspension of gD-mi3 fusion protein nanoparticles. The suspension was 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 describes the identification and characterization experiments of gD-mi3 fusion protein nanoparticles, as detailed below: Take 2 mL of the gD-mi3 fusion protein nanoparticle sample obtained in Example 2 above. Drop the sample onto a carbon film copper mesh (300 mesh), let it stand at room temperature for 1 min, negatively stain with 2% phosphotungstic acid for 30 s, remove excess stain, and allow it to air dry. Observe the morphology of the nanoparticles under an 80 kV transmission electron microscope. The results are as follows: Figure 3 As shown. DLS analysis: 300 μL of the gD-mi3 fusion protein nanoparticle suspension obtained in Example 2 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 three groups of 10 mice each, with an average weight of 20 ± 2 g. Immunization was performed on days 0, 14, and 28. The gD-mi3 group (containing gD-mi3 fusion protein nanoparticles prepared in Example 2) received a total immunization dose of 30 μg, combined with M903 adjuvant; the commercially available dual vaccine group received a total immunization dose of 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.

[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 mouse serum samples diluted 1:100,000 were incubated in ELISA plates at 37°C for 1 h, followed by washing. HRP-labeled goat anti-mouse IgG secondary antibody was added, and the plates were incubated 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. Results are shown below. Figure 4 As shown, with the extension of immunization time, the IgG level in the gD-mi3 group gradually increased. From 21 to 28 days after immunization, the serum IgG content in the gD-mi3 group was significantly higher than that in the Dual vaccine group and the PBS group. The results indicate that the gD-mi3 fusion protein nanoparticles provided in this embodiment of the invention can effectively enhance humoral immune response.

[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 6 Cells were seeded at a density of [number] cells / mL into 96-well plates, with gD-mi3 fusion 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, at 35 days post-immunization, the SI in the gD-mi3 group was higher than that in the PBS group, and at 42 days, the SI in the gD-mi3 group was higher than that at 35 days. The results indicate that the gD-mi3 fusion protein nanoparticles produced the same immunostimulatory capacity as the antigen group.

[0041] Analysis of Cytokine Levels in Mouse Spleen Cell Supernatant: The secretion levels of cytokines in mouse spleen 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 and Figure 7 As shown, the IFN-γ level in the gD-mi3 group was higher than that in the PBS and Dual vaccine groups, and the TNF-α expression level in the gD-mi3 group was significantly higher than that in the PBS and Dual vaccine groups. These results indicate that the gD-mi3 fusion protein nanoparticles provided in this 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 gD-mi3 fusion protein of Infectious Bovine Rhinotracheitis virus, characterized in that, The gD-mi3 fusion protein is assembled by fusing a mi3 tag sequence with a gD gene of bovine infectious rhinotracheitis virus; the amino acid sequence of the mi3 tag sequence is shown in the sequence table SEQ ID NO: 4; and the amino acid sequence of the gD-mi3 fusion protein is shown in the sequence table SEQ ID NO:

2.

2. A gene encoding the gD-mi3 fusion protein of the infectious bovine rhinotracheitis virus according to claim 1, characterized by, The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO:

1.

3. A recombinant expression vector, characterized in that, The gene of claim 2.

4. A host cell, characterized in that, At least one of the gD-mi3 fusion protein of claim 1, the gene of claim 2, and the recombinant expression vector of claim 3.

5. The host cell of claim 4, wherein, The host cell is a CHO cell.

6. A method of producing the gD-mi3 fusion protein of claim 1, wherein, The method comprises the following steps: Constructing a recombinant expression vector by using a gene with a nucleotide sequence shown in the sequence table SEQ ID NO: 1; Transforming the recombinant expression vector into a host cell for induced expression, and then performing protein purification to obtain the gD-mi3 fusion protein.

7. The method of claim 6, wherein the gD-mi3 fusion protein is prepared by, The host cell is a CHO cell.

8. Use of the gD-mi3 fusion protein of claim 1, or the gene of claim 2, or the recombinant expression vector of claim 3, or the host cell of claim 4 in the preparation of a medicine or vaccine for preventing and treating bovine infectious rhinotracheitis.

9. A medicament or vaccine for the prevention or treatment of contagious bovine rhinotracheitis, comprising a pharmaceutically acceptable carrier, characterized in that, The gD-mi3 fusion protein of claim 1.

10. The medicament or vaccine for the prevention of contagious bovine rhinotracheitis according to claim 9, characterized in that, The vaccine is a nanoparticle vaccine.

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