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

Abstract

The invention is applicable to the technical field of genetic engineering, and provides an E2-1d-mi3 fusion protein of bovine viral diarrhea virus and a preparation method and application thereof, the E2-1d-mi3 fusion protein is formed by fusing a mi3 tag sequence with a 1d type E2 genome of the bovine viral diarrhea 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 E2-1d-mi3 fusion protein is as shown in SEQ ID NO: 2 in a sequence table. After animals are immunized by the E2-1d-mi3 fusion protein, an effective immune protection effect can be generated, and the E2-1d-mi3 fusion protein has a good prevention and control application prospect on bovine viral diarrhea caused by BVDV 1d type viruses.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to an E2-1d-mi3 fusion protein of bovine viral diarrhea virus and a preparation method and application thereof. BACKGROUND

[0002] Bovine viral diarrhea (BVD) is an acute and persistent infectious disease caused by bovine viral diarrhea virus (BVDV), which mainly infects cattle, but also can infect sheep, pigs and other odd-toed animals; the virus can cause gastrointestinal, respiratory and reproductive system diseases in infected animals, and is accompanied by immunosuppression, increasing the risk of secondary infection, and thus reducing the reproduction and growth efficiency of animals and increasing the mortality of young animals; the prevention and control of BVDV is a persistent challenge for the dairy cattle breeding industry.

[0003] 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, and the main subtypes of BVDV prevalent in China are BVDV 1a, BVDV 1b and BVDV 1d, etc. The genome 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, 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] At present, although there are some BVDV vaccines on the market (such as inactivated vaccines, attenuated vaccines, etc.), there are still problems such as safety, insufficient immune efficacy, etc. Therefore, it is of great significance to develop a safe, efficient and suitable for industrial production BVDV nanoparticle vaccine for effectively controlling the spread of bovine viral diarrhea. SUMMARY

[0005] The purpose of the present application is to provide an E2-1d-mi3 fusion protein of bovine viral diarrhea virus, which aims to solve the problems raised in the background art.

[0006] To solve the above problems, the application is implemented as follows: an E2-1d-mi3 fusion protein of bovine viral diarrhea virus is assembled by fusing a mi3 tag sequence with a 1d type E2 gene of bovine viral diarrhea 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 E2-1d-mi3 fusion protein is shown in the sequence table SEQ ID NO:2.

[0007] Another object of the application is to provide a gene encoding the E2-1d-mi3 fusion protein of bovine viral diarrhea virus, and the nucleotide sequence of the gene is shown in the sequence table SEQ ID NO:1.

[0008] Another object of the application is to provide a recombinant expression vector comprising the gene.

[0009] Another object of the application is to provide a host cell comprising at least one of the E2-1d-mi3 fusion protein, the gene and the recombinant expression vector.

[0010] Another object of the application is to provide a preparation method of the E2-1d-mi3 fusion protein, comprising the following steps: constructing a recombinant expression vector by using the gene with the 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 E2-1d-mi3 fusion protein.

[0011] Further, the host cell is a CHO cell.

[0012] Another object of the application is to provide an application of the E2-1d-mi3 fusion protein, the gene, the recombinant expression vector or the host cell in preparing a medicine or vaccine for preventing and treating bovine viral diarrhea.

[0013] Another object of the application is to provide a medicine or vaccine for preventing and treating bovine viral diarrhea, comprising a pharmaceutically acceptable carrier and the E2-1d-mi3 fusion protein.

[0014] Further, the vaccine is a nanoparticle vaccine.

[0015] The application provides a bovine viral diarrhea virus E2-1d-mi3 fusion protein, by introducing mi3, a highly symmetrical, closed virus-like structure can be formed, which has excellent thermal stability and pH tolerance; the E2-1d-mi3 fusion protein can be made into a nanoparticle vaccine, which is non-toxic to mammals, has no biological safety threat, and can induce a more rapid and strong humoral immune response, including a more efficient germinal center reaction and high-titer neutralizing antibody production. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The construction schematic diagram of the recombinant expression vector provided for the embodiment of the application.

[0017] Figure 2 The SDS-PAGE graph (A) and Western Blot identification graph (B) of E2-1d-mi3 fusion protein expression and purification provided for the embodiment of the application.

[0018] Figure 3 The electron microscope graph of the E2-1d-mi3 fusion protein nanoparticle provided for the embodiment of the application.

[0019] Figure 4 The serum IgG detection result graph of the E2-1d-mi3 fusion protein nanoparticle after immunizing mice provided for the embodiment of the application.

[0020] Figure 5 The spleen lymphocyte stimulation index detection result graph of the E2-1d-mi3 fusion protein nanoparticle after immunizing mice provided for the embodiment of the application.

[0021] Figure 6 The expression level analysis result graph of the cytokine IL-6 in the spleen cell supernatant of the E2-1d-mi3 fusion protein nanoparticle after immunizing mice provided for the embodiment of the application.

[0022] Figure 7 The expression level analysis result graph of the cytokine IFN-γ in the spleen cell supernatant of the E2-1d-mi3 fusion protein nanoparticle after immunizing mice provided for the embodiment of the application.

[0023] Figure 8 The expression level analysis result graph of the cytokine TNF-α in the spleen cell supernatant of the E2-1d-mi3 fusion protein nanoparticle after immunizing mice provided for the embodiment of the application. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the application clearer, further detailed description will be made to the application in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the application, and are not used to limit the application.

[0025] The Chinese hamster ovary (CHO) cells used in the embodiments of the present application are one of the most widely used mammalian expression systems for vaccine antigen production. In recent years, a variety of important vaccines (such as respiratory syncytial virus vaccine, herpes zoster vaccine, and new coronavirus subunit vaccine) have used CHO cells to produce their recombinant protein antigens. CHO cells have excellent recombinant protein expression capacity and can achieve high cell density culture in bioreactors, thereby producing high concentrations of target proteins. The concentration of monoclonal antibodies produced by a CHO cell line optimized through a process can reach several grams per liter. CHO cells are easy to culture in suspension and can be scaled up to produce vaccine antigens in large bioreactors while maintaining stable yields. In contrast, many other systems may face challenges when scaled up, although they have higher yields at a small scale. Overall, CHO cells provide a reliable guarantee for large-scale supply of vaccines in terms of yield and scale-up production.

[0026] Mi3 nanoparticles are an artificially designed protein self-assembly platform composed of 60 subunits that can spontaneously form stable, symmetrical spherical structures in cells, with a diameter of about 25-30 nm. In vaccine applications, mi3 nanoparticles can improve the stability and lymph node targeting efficiency of antigens in vivo, promote the uptake and activation of antigen-presenting cells, and effectively induce strong humoral and cellular immune responses. Their multivalent and repetitive geometric arrangement can enhance B cell receptor crosslinking, induce higher titers of neutralizing antibodies and long-lasting immune memory. As a new type of nano-vaccine platform, mi3 has broad application prospects in the development of vaccines for viral diseases, especially those that require rapid induction of high-quality immune protection.

[0027] To study the mi3 fusion nanoparticle vaccine that can prevent BVDV, the embodiments of the present application use a CHO expression system and mi3 self-assembly nanoparticle system to display the E2 antigen of BVDV type 1d on the surface of mi3 nanoparticles through gene fusion expression. This strategy preserves the structural integrity of multiple antigens and also enhances antigen density through the multivalent structure of nanoparticles, thereby improving immune activation efficiency. Further immunological evaluation in BALB / c mice analyzes its ability to induce humoral immunity, cellular immunity, and immune memory, laying a foundation for subsequent vaccine optimization and application in cattle.

[0028] Specifically, in one embodiment of the present invention, an E2-1d-mi3 fusion protein of bovine viral diarrhea virus is provided, which is formed by fusing a mi3 tag sequence with the 1d type E2 genome of bovine viral diarrhea 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 E2-1d-mi3 fusion protein is shown in SEQ ID NO:2.

[0029] In another embodiment of the present invention, a gene encoding the E2-1d-mi3 fusion protein of the above-mentioned bovine viral diarrhea virus is also provided, the nucleotide sequence of which is shown in the sequence listing SEQ ID NO:1.

[0030] In another embodiment of the present invention, a recombinant expression vector comprising the above-described genes is also provided.

[0031] 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.

[0032] In another embodiment of the present invention, a host cell is also provided, comprising at least one of the above-described E2-1d-mi3 fusion protein, the above-described gene, and the above-described recombinant expression vector.

[0033] In another embodiment of the present invention, a method for preparing the above-mentioned E2-1d-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 E2-1d-mi3 fusion protein. Preferably, the host cells are CHO cells.

[0034] In another embodiment of the present invention, the use of the above-mentioned E2-1d-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 preventing and treating bovine viral diarrhea is also provided.

[0035] In another embodiment of the invention, a drug or vaccine for preventing bovine viral diarrhea is also provided, comprising a pharmaceutically acceptable carrier and the aforementioned E2-1d-mi3 fusion protein. Preferably, the vaccine is a nanoparticle vaccine.

[0036] The E2-1d-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 viral diarrhea and has significant practical application value and market prospects.

[0037] Example 1: This example provides a method for obtaining the target gene and constructing an expression vector, as detailed below: The bovine viral diarrhea virus (BVDV)-E2-1d (GenBank ID: MF166858.1) gene sequence was selected and fused with the mi3 tag sequence (nucleotide sequence shown in SEQ ID NO:3 in the sequence listing) to construct the gene named E2-1d-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.

[0038] Example 2: This example provides a method for expressing and purifying E2-1d-mi3 fusion protein nanoparticles, as detailed below: The recombinant expression vector E2-1d-mi3 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 Cells were transfected (cell / mL). G418 (final concentration 800 μg / mL) was added 48 h later, and the medium was replaced with fresh medium containing G418 7 days later. The supernatant from cells cultured for 4 days was collected and purified using affinity chromatography to obtain an E2-1d-mi3 fusion protein nanoparticle suspension. 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.

[0039] Example 3: This example describes the identification and characterization experiments of the E2-1d-mi3 fusion protein nanoparticles, as detailed below: Take 2 mL of the E2-1d-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 E2-1d-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.

[0040] 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 E2-1d-mi3 group (containing the E2-1d-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 an 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.

[0041] 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, the IgG level in the E2-1d-mi3 group gradually increased with the extension of immunization time. Between 21 and 28 days post-immunization, the serum IgG content in the E2-1d-mi3 group was significantly higher than that in the Dual vaccine group and the PBS group. These results indicate that the E2-1d-mi3 fusion protein nanoparticles provided in this embodiment of the invention can effectively enhance humoral immune responses.

[0042] 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 E2-1d-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 E2-1d-mi3 group was slightly higher than that in the PBS group, and at 42 days, the SI in the E2-1d-mi3 group was higher than that at 35 days, indicating that the E2-1d-mi3 fusion protein nanoparticles produced the same immunostimulatory capacity as the antigen group.

[0043] 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 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. Figure 6-8 As shown, the expression levels of IL-6, IFN-γ, and TNF-α in the E2-1d-mi3 group were significantly higher than those in the E2-1d, PBS, and Dual vaccine groups; the results indicate that the E2-1d-mi3 fusion protein nanoparticles prepared in this embodiment of the invention have certain advantages in enhancing humoral and cellular immunity.

[0044] 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. An E2-1d-mi3 fusion protein of bovine viral diarrhea virus, characterized in that, The E2-1d-mi3 fusion protein is formed by fusing a mi3 tag sequence with the 1d type E2 genome of bovine viral diarrhea virus; the amino acid sequence of the mi3 tag sequence is shown in SEQ ID NO:4 of the sequence listing; the amino acid sequence of the E2-1d-mi3 fusion protein is shown in SEQ ID NO:2 of the sequence listing.

2. A gene encoding the E2-1d-mi3 fusion protein of bovine viral diarrhea virus as described in claim 1, characterized in that, The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO:

1.

3. A recombinant expression vector, characterized in that, It contains the gene described in claim 2.

4. A host cell, characterized in that, It comprises at least one of the E2-1d-mi3 fusion protein of claim 1, the gene of claim 2, and the recombinant expression vector of claim 3.

5. The host cell according to claim 4, characterized in that, The host cell is a CHO cell.

6. A method for preparing the E2-1d-mi3 fusion protein as described in claim 1, characterized in that, 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 E2-1d-mi3 fusion protein.

7. The method for preparing the E2-1d-mi3 fusion protein according to claim 6, characterized in that, The host cell is a CHO cell.

8. The use of the E2-1d-mi3 fusion protein as described in claim 1, or the gene as described in claim 2, or the recombinant expression vector as described in claim 3, or the host cell as described in claim 4 in the preparation of a drug or vaccine for preventing and treating 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 fusion protein as described in claim 1.

10. The drug or vaccine for preventing and treating bovine viral diarrhea according to claim 9, characterized in that, The vaccine in question is a nanoparticle vaccine.

Images