E2-1d + mi3 protein nanoparticles of bovine viral diarrhea virus as well as preparation method and application of E2-1d + mi3 protein nanoparticles

Abstract

The invention is applicable to the technical field of genetic engineering, and provides an E2-1d + mi3 protein nanoparticle of bovine viral diarrhea virus as well as a preparation method and application of the E2-1d + mi3 protein nanoparticle, the E2-1d + mi3 protein nanoparticle is formed by coupling an E2-1d-st fusion protein and a mi3-sc fusion protein, the 1d type E2 antigen of the bovine viral diarrhea virus is displayed on the surface of the mi3 protein nanoparticle through an isopeptide bond; the amino acid sequence of the mi3 protein nanoparticle is shown as SEQ ID NO: 6 in a sequence table; the amino acid sequence of the E2-1d-st fusion protein is as shown in SEQ ID NO: 2 in a sequence table; the amino acid sequence of the mi3-sc fusion protein is shown as SEQ ID NO: 4 in a sequence table. After animals are immunized by the E2-1d + mi3 protein nano-particles, an effective immune protection effect can be generated, and the E2-1d + mi3 protein nano-particles have a good prevention and control application prospect on bovine viral diarrhea caused by BVDV 1d type viruses.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to E2-1d+mi3 protein nanoparticles of bovine viral diarrhea virus, their preparation method and application. Background Technology

[0002] Bovine viral diarrhea (BVD) is an acute, persistent infectious disease caused by bovine viral diarrhea virus (BVDV). It primarily infects cattle, but can also infect cloven-hoofed animals such as sheep and pigs. The virus can cause gastrointestinal, respiratory, and reproductive system diseases in infected animals, accompanied by immunosuppression, increasing the risk of secondary infections, thereby reducing the reproductive and growth efficiency of animals and increasing the mortality rate of young animals. The prevention and control of BVDV is an ongoing challenge for the dairy farming 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 1d, 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] Currently, although some BVDV vaccines are available on the market (such as inactivated vaccines and attenuated vaccines), problems such as insufficient safety and immunogenicity still exist. Therefore, developing a safe, efficient, and industrially viable BVDV nanoparticle vaccine is of great significance for effectively controlling the spread of bovine viral diarrhea. Summary of the Invention

[0005] The purpose of this invention is to provide E2-1d+mi3 protein nanoparticles of bovine viral diarrhea virus, aiming to solve the problems mentioned in the background art.

[0006] To address the aforementioned problems, this invention provides a bovine viral diarrhea virus (BVM) E2-1d+mi3 protein nanoparticle, composed of an E2-1d-st fusion protein and a mi3-sc fusion protein coupled together. The BVM displays the 1d-type E2 antigen of BVM on the surface of the mi3 protein nanoparticle via isopeptide bonds. The amino acid sequence of the mi3 protein nanoparticle is shown in SEQ ID NO:6; the amino acid sequence of the E2-1d-st fusion protein is shown in SEQ ID NO:2; and the amino acid sequence of the mi3-sc fusion protein is shown in SEQ ID NO:4.

[0007] Another object of the present invention is to provide a gene encoding E2-1d+mi3 protein nanoparticles of the above-mentioned bovine viral diarrhea virus, comprising the E2-1d-st gene and the mi3-sc gene; the nucleotide sequence of the E2-1d-st gene is shown in SEQ ID NO:1 of the sequence listing; the nucleotide sequence of the mi3-sc gene is shown in SEQ ID NO:3 of the sequence listing.

[0008] Another object of the present invention is to provide a recombinant expression vector containing the above-mentioned genes, specifically including an E2-1d-st expression vector and a mi3-sc expression vector; the E2-1d-st expression vector contains the E2-1d-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 E2-1d+mi3 protein nanoparticles, which includes the following steps: Construct the E2-1d-st expression vector of the BVDV E2-1d gene fused with the SpyTag003 tag, and construct the mi3-sc expression vector of the mi3 gene fused with SpyCatcher003. The mi3-sc expression vector was transformed into the first host cell for expression to obtain the mi3-sc fusion protein; The E2-1d-st expression vector was transfected into a second host cell for expression, resulting in the E2-1d-st fusion protein. The E2-1d-st fusion protein was mixed with the mi3-sc fusion protein and allowed to stand overnight for ligation to obtain E2-1d+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 E2-1d-st fusion protein to the mi3-sc fusion protein is (7-9):1.

[0013] Another object of the present invention is to provide the application of the above-mentioned E2-1d+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.

[0014] Another object of the present invention is to provide a drug or vaccine for the prevention and treatment of bovine viral diarrhea, comprising a pharmaceutically acceptable carrier and the aforementioned E2-1d+mi3 protein nanoparticles.

[0015] This invention provides E2-1d+mi3 protein nanoparticles of bovine viral diarrhea virus. By introducing mi3, these nanoparticles can form a highly symmetrical, closed virus-like structure with excellent thermal stability and pH tolerance. These E2-1d+mi3 protein nanoparticles can be used to make 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

[0016] Figure 1 This is a schematic diagram illustrating the construction of the recombinant expression vector provided in an embodiment of the present invention.

[0017] Figure 2 The images show SDS-PAGE and Western Blot diagrams of the mi3-sc fusion protein (A) and E2-1d-st fusion protein (B) expressed and purified according to embodiments of the present invention.

[0018] Figure 3 Electron micrograph of E2-1d+mi3 protein nanoparticles provided in an embodiment of the present invention.

[0019] Figure 4 The image shows the serum IgG detection results of mice immunized with E2-1d+mi3 protein nanoparticles provided in this embodiment of the invention.

[0020] Figure 5 The image shows the results of spleen lymphocyte stimulation index detection in mice after immunization with E2-1d+mi3 protein nanoparticles provided in this embodiment of the invention.

[0021] Figure 6 The figure shows the results of the expression level analysis of IFN-γ cytokine in the supernatant of spleen cells after mice were immunized with E2-1d+mi3 protein nanoparticles provided in the embodiments of the present invention.

[0022] Figure 7The figure shows the results of the analysis of the expression level of TNF-α cytokine in the spleen cell supernatant of mice after immunization with E2-1d+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 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-SpyCatcher003 (mi3-sc) is highly expressed in E. coli, and the prepared nanoparticles exhibit excellent environmental tolerance. The mi3-coupled nanoparticles retain their performance after freeze-thaw and lyophilization treatments. The mi3 platform demonstrates compatibility and high coupling efficiency with various pathogen antigens.

[0026] To investigate a mi3 protein nanoparticle vaccine capable of preventing BVDV, this invention expresses the BVDV 1d type E2 antigen fused with SpyTag003 in CHO cells. Furthermore, mi3-SpyCatcher003 (mi3-sc) nanoparticles are expressed in an E. coli prokaryotic system, and purified E2-1d-SpyTag003 (E2-1d-st) is mixed with mi3-SC in a specific ratio. Utilizing the specific covalent binding between SpyTag003 and SpyCatcher003, recombinant nanoparticles are constructed to co-display the antigen. This structure allows the antigen to be displayed on the nanoparticle surface in a directional and regular manner, thereby maximally mimicking the antigen arrangement of the natural virus and enhancing B cell activation and the production of neutralizing antibodies.

[0027] Specifically, in one embodiment of the present invention, a bovine viral diarrhea virus (BVM) E2-1d+mi3 protein nanoparticle is provided, which is composed of an E2-1d-SpyTag003 (E2-1d-st) fusion protein and a mi3-SpyChater003 (mi3-sc) fusion protein coupled together. The BVM 1d-type E2 antigen is displayed on the surface of the mi3 protein nanoparticle via a heteropeptide bond (SpyCatcher003 / SpyTag003). The amino acid sequence of the mi3 protein nanoparticle is shown in SEQ ID NO:6, and the nucleotide sequence is shown in SEQ ID NO:5. The amino acid sequence of the E2-1d-st fusion protein is shown in SEQ ID NO:2, and the amino acid sequence of the mi3-sc fusion protein is shown in SEQ ID NO:4. A signal peptide sequence is added to the N-terminus of both the E2-1d-st and mi3-sc fusion proteins, and a His6 tag is added to the C-terminus.

[0028] In another embodiment of the present invention, a gene encoding E2-1d+mi3 protein nanoparticles of the above-mentioned bovine viral diarrhea virus is also provided, including the E2-1d-st gene and the mi3-sc gene; the nucleotide sequence of the E2-1d-st gene is shown in SEQ ID NO:1 of the sequence listing; the nucleotide sequence of the mi3-sc gene is shown in SEQ ID NO:3 of the sequence listing.

[0029] In another embodiment of the present invention, a recombinant expression vector containing the above-mentioned genes is also provided, specifically including an E2-1d-st expression vector and a mi3-sc expression vector; the E2-1d-st expression vector contains the E2-1d-st gene; and the mi3-sc expression vector contains the mi3-sc gene.

[0030] In another embodiment of the present invention, a method for preparing the above-mentioned E2-1d+mi3 protein nanoparticles is also provided, which includes the following steps: S1. Construct the E2-1d-st expression vector of the BVDV E2-1d gene fused with the SpyTag003 tag, and construct the mi3-sc expression vector of the mi3 gene fused with SpyCatcher003. S2. The mi3-sc expression vector was transformed into the first host cell for expression to obtain the mi3-sc fusion protein; S3. Transfect the E2-1d-st expression vector into the second host cell for expression to obtain the E2-1d-st fusion protein; S4. Mix the E2-1d-st fusion protein with the mi3-sc fusion protein and let it stand overnight to ligate, thus obtaining E2-1d+mi3 protein nanoparticles.

[0031] Preferably, the first host cell is Escherichia coli competent cells; the second host cell is CHO cells; and the molar ratio of E2-1d-st fusion protein to mi3-sc fusion protein is (7-9):1, more preferably 8:1.

[0032] In another embodiment of the present invention, the application of the above-mentioned E2-1d+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 is also provided.

[0033] In another embodiment of the present invention, a drug or vaccine for preventing and treating bovine viral diarrhea is also provided, comprising a pharmaceutically acceptable carrier and the aforementioned E2-1d+mi3 protein nanoparticles.

[0034] Example 1: This example provides a method for obtaining the target gene and constructing an expression vector, as detailed below: The gene sequence of bovine viral diarrhea virus (BVDV-E2-1d) (GenBank ID: MF166858.1) was selected, fused with the SpyTag003 sequence, and cloned into the pcDNA3.1(+) expression vector. A signal peptide sequence was introduced at the N-terminus of the gene, and a His6 tag was added at the C-terminus. Simultaneously, additives were added to both ends of the constructed sequence. BamH I and EcoR I. Restriction site; all genes were codon-optimized according to mammalian expression systems to obtain the E2-1d-st expression vector. 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 gene construction map is shown below. Figure 1 As shown.

[0035] Example 2: This example provides a method for expressing and purifying E2-1d-st fusion protein and mi3-sc fusion protein, as detailed below: The E2-1d-st expression vector prepared in Example 1 was introduced into CHO cells via electroporation (electroclavicle cuvette 4 mm, cell number 1×10⁶). 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, and the medium was replaced with fresh medium containing G418 after 7 days to obtain a drug-resistant cell population. The supernatant of cells cultured for 4 days was collected and purified by affinity chromatography to obtain the E2-1d-st fusion protein. Following the same process, the mi3-sc expression vector prepared in Example 1 was transformed into *E. coli* BL21(DE3) competent cells for expression to obtain the mi3-sc fusion protein. The E2-1d-st and mi3-sc fusion 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. The results are as follows: Figure 2 As shown.

[0036] Example 3: This example provides a method for preparing and characterizing E2-1d+mi3 protein nanoparticles, as detailed below: The E2-1d-st fusion protein prepared in Example 2 above was mixed with the mi3-SC fusion protein at a molar ratio of 8:1. Under aseptic conditions, the mixture was pipetted and incubated overnight at room temperature. Free antigen was removed by ultrafiltration, and the concentrated solution yielded E2-1d+mi3 protein nanoparticles. Large particle impurities were then 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, and negatively stained with 2% phosphotungstic acid for 30 s. After removing excess stain, the nanoparticles were air-dried. The morphology of the 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 E2-1d+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 via subcutaneous multi-site injection at 0, 14, and 28 days. The total immunization dose of the E2-1d+mi3 group (E2-1d+mi3 protein nanoparticles prepared in Example 3) was 30 μg, combined with M903 adjuvant; the commercially available dual vaccine group received 100 μL of immunization; and the PBS control group received 100 μL of PBS. Blood was collected from the tail at 7, 14, 21, 28, 35, and 42 days 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, serum IgG levels in the E2-1d+mi3 group gradually increased from 7 to 42 days post-immunization. At 14 days, the serum IgG level in the E2-1d+mi3 group was significantly higher than that in the commercially available dual vaccine group; at 42 days, the difference between the two groups widened and was much higher than that in the PBS group. These results indicate that E2-1d+mi3 promotes the production of serum IgG in mice.

[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 E2-1d+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, on day 42, the SI value of the E2-1d+mi3 group was close to 3.5, which was significantly higher than that of the PBS group (p<0.01). The results indicate that the E2-1d+mi3 protein nanoparticles can stimulate T cell responses in the early stage and maintain a strong immune response.

[0040] Analysis of Cytokine Levels in Mouse Spleen Cell Supernatant: The secretion levels of cytokines in the supernatant of mouse spleen cells at different time points (21 days and 42 days) after immunization were detected using a commercially available ELISA kit. First, mouse IFN-γ and TNF-α capture antibodies were coated onto 96-well ELISA plates, incubated overnight at 4°C, and then blocked with 5% BSA for 1 h. Diluted serum was then added, and the plates were incubated at 37°C for 2 h, followed by washing. Biotin-labeled detection antibodies were then added sequentially, and the plates were incubated at 37°C for 30 min. The plates were 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 expression levels of IFN-γ and TNF-α in the E2-1d+mi3 group were significantly higher than those in the PBS and Dual vaccine groups. These results indicate that the E2-1d+mi3 protein nanoparticles provided in this invention have certain advantages in enhancing humoral and cellular immunity.

[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 bovine viral diarrhea virus E2-1d+mi3 protein nanoparticle, characterized in that, The E2-1d+mi3 protein nanoparticles are composed of E2-1d-st fusion protein and mi3-sc fusion protein coupled together, which display the 1d type E2 antigen of bovine viral diarrhea virus on the surface of the mi3 protein nanoparticles through isopeptide bonds; the amino acid sequence of the mi3 protein nanoparticles is shown in SEQ ID NO:6 of the sequence listing; the amino acid sequence of the E2-1d-st fusion protein is shown in SEQ ID NO:2 of the sequence listing; and the amino acid sequence of the mi3-sc fusion protein is shown in SEQ ID NO:4 of the sequence listing.

2. A gene encoding E2-1d+mi3 protein nanoparticles of bovine viral diarrhea virus as described in claim 1, characterized in that, It includes the E2-1d-st gene and the mi3-sc gene; the nucleotide sequence of the E2-1d-st gene is shown in SEQ ID NO:1 of the sequence listing; the nucleotide sequence of the mi3-sc gene is shown in SEQ ID NO:3 of the sequence listing.

3. A recombinant expression vector comprising the gene of claim 2, characterized in that, It includes an E2-1d-st expression vector and a mi3-sc expression vector; the E2-1d-st expression vector contains the E2-1d-st gene; the mi3-sc expression vector contains the mi3-sc gene.

4. A method for preparing E2-1d+mi3 protein nanoparticles as described in claim 1, characterized in that, Includes the following steps: Construct the E2-1d-st expression vector of the BVDV E2-1d gene fused with the SpyTag003 tag, and construct the mi3-sc expression vector of the mi3 gene fused with SpyCatcher003. The mi3-sc expression vector was transformed into the first host cell for expression to obtain the mi3-sc fusion protein; The E2-1d-st expression vector was transfected into a second host cell for expression, resulting in the E2-1d-st fusion protein. The E2-1d-st fusion protein was mixed with the mi3-sc fusion protein and allowed to stand overnight for ligation to obtain E2-1d+mi3 protein nanoparticles.

5. The method for preparing E2-1d+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 E2-1d+mi3 protein nanoparticles according to claim 4, characterized in that, The second host cell is a CHO cell.

7. The method for preparing E2-1d+mi3 protein nanoparticles according to claim 4, characterized in that, The molar ratio of the E2-1d-st fusion protein to the mi3-sc fusion protein is (7-9):

1.

8. The use of the E2-1d+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.

9. A drug or vaccine for the prevention and treatment of bovine viral diarrhea, comprising a pharmaceutically acceptable carrier, characterized in that, It also includes the E2-1d+mi3 protein nanoparticles as described in claim 1.

Images