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

Abstract

The invention is applicable to the technical field of genetic engineering, and provides an E2-1b + mi3 protein nanoparticle of bovine viral diarrhea virus as well as a preparation method and application of the E2-1b + mi3 protein nanoparticle, the E2-1b + mi3 protein nanoparticle is formed by coupling an E2-1b-st fusion protein and a mi3-sc fusion protein, the 1b 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 E2-1b-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 E2-1b + mi3 protein nano-particles, an effective immune protection effect can be generated, and the E2-1b + mi3 protein nano-particles have a good prevention and control application prospect on bovine viral diarrhea caused by BVDV 1b type viruses.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a bovine viral diarrhea virus E2-1b+mi3 protein nanoparticle 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 a bovine viral diarrhea virus E2-1b+mi3 protein nanoparticle, which aims to solve the problems raised in the background art.

[0006] To solve the above problems, the application is implemented as follows: a BVDV E2-1b+mi3 protein nanoparticle is composed of an E2-1b-st fusion protein and an mi3-sc fusion protein, and the BVDV type 1b E2 antigen 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 in the sequence table SEQ ID NO: 6; the amino acid sequence of the E2-1b-st fusion protein is shown in the sequence table SEQ ID NO: 2; and the amino acid sequence of the mi3-sc fusion protein is shown in the sequence table SEQ ID NO: 4.

[0007] Another object of the application is to provide a gene encoding the above-mentioned BVDV E2-1b+mi3 protein nanoparticle, including an E2-1b-st gene and an mi3-sc gene; the nucleotide sequence of the E2-1b-st gene is shown in the sequence table SEQ ID NO: 1; and the nucleotide sequence of the mi3-sc gene is shown in the sequence table SEQ ID NO: 3.

[0008] Another object of the application is to provide a recombinant expression vector containing the above-mentioned gene, specifically including an E2-1b-st expression vector and an mi3-sc expression vector; the E2-1b-st expression vector contains the E2-1b-st gene; and the mi3-sc expression vector contains the mi3-sc gene.

[0009] Another object of the application is to provide a preparation method of the above-mentioned E2-1b+mi3 protein nanoparticle, including the following steps: constructing an E2-1b-st expression vector of the BVDV E2-1b gene fused with a SpyTag003 tag, and constructing an mi3-sc expression vector of the mi3 gene fused with a SpyCatcher003; transforming the mi3-sc expression vector into a first host cell for expression to obtain the mi3-sc fusion protein; transfecting the E2-1b-st expression vector into a second host cell for expression to obtain the E2-1b-st fusion protein; mixing the E2-1b-st fusion protein with the mi3-sc fusion protein, and performing overnight standing connection to obtain the E2-1b+mi3 protein nanoparticle.

[0010] Further, the first host cell is an E. coli competent cell.

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

[0012] Further, the molar ratio of the E2-1b-st fusion protein to the mi3-sc fusion protein is (7-9):1.

[0013] Another object of the present application is to provide an application of the E2-1b+mi3 protein nanoparticle or the gene or the recombinant expression vector in the preparation of a medicine or vaccine for preventing and treating bovine viral diarrhea.

[0014] Another object of the present application is to provide a medicine or vaccine for preventing and treating bovine viral diarrhea, which comprises a pharmaceutically acceptable carrier and the E2-1b+mi3 protein nanoparticle.

[0015] The E2-1b+mi3 protein nanoparticle for bovine viral diarrhea virus provided by the present application can form a highly symmetrical, closed virus-like structure by introducing mi3, has excellent thermal stability and pH tolerance, can be made into a nanoparticle vaccine, 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 A schematic diagram of construction of the recombinant expression vector provided by the embodiment of the present application.

[0017] Figure 2 SDS-PAGE and Western Blot identification diagrams of the mi3-sc fusion protein (A) and the E2-1b-st fusion protein (B) provided by the embodiment of the present application.

[0018] Figure 3 An electron microscope diagram of the E2-1b+mi3 protein nanoparticle provided by the embodiment of the present application.

[0019] Figure 4 A serum IgG detection result diagram of a mouse immunized by the E2-1b+mi3 protein nanoparticle provided by the embodiment of the present application.

[0020] Figure 5 A spleen lymphocyte stimulation index detection result diagram of a mouse immunized by the E2-1b+mi3 protein nanoparticle provided by the embodiment of the present application.

[0021] Figure 6 A spleen cell supernatant cytokine IFN-γ expression level analysis result diagram of a mouse immunized by the E2-1b+mi3 protein nanoparticle provided by the embodiment of the present application.

[0022] Figure 7Figure 2 shows the results of the analysis of the expression level of the cytokine TNF-α in the supernatant of the spleen cells of the mice immunized with the E2-1b+mi3 protein nanoparticles according to the embodiments of the present application. DETAILED DESCRIPTION

[0023] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0024] 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 CHO cell lines optimized by process can reach several grams per liter. CHO cells are easy to suspend culture 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 small scale. Overall, CHO cells provide a reliable guarantee for large-scale supply of vaccines in terms of yield and scale-up production.

[0025] The mi3 protein nanoparticle is a dodecahedral protein nanocage (consisting of 60 subunits) with excellent particle uniformity and stability. By connecting the SpyCatcher003 sequence at its N-terminus, mi3 becomes a universal base that can spontaneously couple with any antigen carrying the SpyTag003 peptide after mixing. mi3-SpyCatcher003 (mi3-sc) is highly expressed in E. coli, and the prepared nanoparticles have excellent tolerance to the environment, and the performance of the coupled mi3 does not decrease after freeze-thaw and freeze-drying treatment. The mi3 platform shows compatibility and high coupling efficiency for different pathogenic antigens.

[0026] To study the mi3 protein nanoparticle vaccine capable of preventing BVDV, the present embodiment expresses the E2 antigen of BVDV type 1b fused with SpyTag003 in CHO cells. In addition, the mi3-SpyCatcher003 (mi3-sc) nanoparticle is expressed in the prokaryotic system of E. coli, and the purified E2-1b-SpyTag003 (E2-1b-st) is mixed with the mi3-SC at a certain ratio, and the specific covalent binding between SpyTag003 and SpyCatcher003 is used to construct the co-displayed recombinant nanoparticle. The structure can make the antigen display on the surface of the nanoparticle in a directional and regular manner, thereby simulating the natural virus antigen arrangement to the greatest extent, enhancing the activation of B cells and the production of neutralizing antibodies.

[0027] Specifically, in one embodiment of the present application, a BVDV E2-1b+mi3 protein nanoparticle is provided, which is composed of E2-1b-SpyTag003 (E2-1b-st) fusion protein and mi3-SpyChater003 (mi3-sc) fusion protein, and the E2 antigen of BVDV type 1b is displayed on the surface of the mi3 protein nanoparticle through an isopeptide bond (SpyCatcher003 / SpyTag003); the amino acid sequence of the mi3 protein nanoparticle is shown in SEQ ID NO: 6 of the sequence listing, and the nucleotide sequence is shown in SEQ ID NO: 5 of the sequence listing; the amino acid sequence of the E2-1b-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. The E2-1b-st fusion protein and the mi3-sc fusion protein are added with a signal peptide sequence at the N terminal and a His6 tag at the C terminal.

[0028] In another embodiment of the present application, a gene encoding the above-mentioned BVDV E2-1b+mi3 protein nanoparticle is also provided, which includes an E2-1b-st gene and a mi3-sc gene; the nucleotide sequence of the E2-1b-st gene is shown in SEQ ID NO: 1 of the sequence listing; and 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 application, a recombinant expression vector containing the above-mentioned gene is also provided, which specifically includes an E2-1b-st expression vector and a mi3-sc expression vector; the E2-1b-st expression vector contains the E2-1b-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 E2-1b+mi3 protein nanoparticles is also provided, which includes the following steps: S1. Construct the E2-1b-st expression vector of the BVDV E2-1b 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 E2-1b-st expression vector into the second host cell for expression to obtain the E2-1b-st fusion protein; S4. Mix the E2-1b-st fusion protein with the mi3-sc fusion protein and let it stand overnight to ligate, thus obtaining E2-1b+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 E2-1b-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 E2-1b+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 E2-1b+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-E2-1b) (GenBank ID: AGM75780.1) 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 the mammalian expression system to obtain the E2-1b-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 the prokaryotic expression system, it 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 the E2-1b-st fusion protein and the mi3-sc fusion protein, as detailed below: The E2-1b-st expression vector prepared in Example 1 above was introduced into CHO cells (electrocution cuvette 4 mm, cell number 1×10⁶) by 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, and after 7 days, the culture medium was replaced with fresh medium containing G418 to obtain a drug-resistant cell population. The supernatant of cells cultured for 4 days was collected and purified using affinity chromatography to obtain the E2-1b-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 E2-1b-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 E2-1b+mi3 protein nanoparticles, as detailed below: The E2-1b-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 E2-1b+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 E2-1b+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 three 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 E2-1b+mi3 group (E2-1b+mi3 protein nanoparticles prepared in Example 3) was 30 μg, combined with M903 adjuvant; the immunization dose of 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 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, the expression level of IgG in the E2-1b+mi3 group gradually increased with the increase of immunization time, reaching the highest value at 42 days. Furthermore, at 14 days of immunization, the IgG level in the E2-1b+mi3 group was significantly higher than that in the Dual vaccine group, and at 42 days, the difference between the E2-1b+mi3 and Dualvaccine groups was the most significant.

[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 E2-1b+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 E2-1b+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 E2-1b+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, at day 21, there was no statistically significant difference in IFN-γ expression levels between the E2-1b+mi3 and Dual vaccine groups, both slightly higher than the PBS group; at day 42, the E2-1b+mi3 group showed significantly higher IFN-γ expression than the Dual vaccine group. Furthermore, the E2-1b+mi3 group showed significantly higher TNF-α expression levels than both the PBS and Dual vaccine groups.

[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 E2-1b+mi3 protein nanoparticle, characterized in that, The E2-1b+mi3 protein nanoparticles are composed of E2-1b-st fusion protein and mi3-sc fusion protein coupled together, which display the bovine viral diarrhea virus type 1b E2 antigen 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 E2-1b-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 E2-1b+mi3 protein nanoparticles of bovine viral diarrhea virus as described in claim 1, characterized in that, It includes the E2-1b-st gene and the mi3-sc gene; the nucleotide sequence of the E2-1b-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 the E2-1b-st expression vector and the mi3-sc expression vector; the E2-1b-st expression vector contains the E2-1b-st gene; the mi3-sc expression vector contains the mi3-sc gene.

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

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

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

1.

8. The use of the E2-1b+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 E2-1b+mi3 protein nanoparticles as described in claim 1.

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