A subunit vaccine, its preparation method and application

Abstract

This invention discloses a subunit vaccine, its preparation method, and its application. The vaccine comprises an immunogenic rE2-2gD-Fc-M fusion protein and a pharmaceutically acceptable carrier. Using the rE2-2gD-Fc-M fusion protein as the antigen, the recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis exhibits high immunogenicity, long duration of immunity, low production cost, and a simple production process, offering numerous advantages such as safety, high efficiency, and low cost. Furthermore, a bovine polypeptide is incorporated as part of the vaccine composition, significantly enhancing antibody titer and antibody persistence. Even when the rE2-2gD-Fc-M fusion protein antigen content in the recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis is only 80 μg / mL, an antibody titer of over 1:512 can be achieved in the first week after a second immunization.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and to a subunit vaccine, its preparation method and application; particularly to a recombinant subunit fusion protein vaccine against bovine viral diarrhea virus and infectious bovine rhinotracheitis virus, and its preparation method. Background Technology

[0002] Bovine viral diarrhea virus (BVDV) belongs to the genus *swine fevervirus* in the family Flaviviridae, and is one of the pathogens causing bovine viral diarrhea (BVD). Currently, BVDV is widespread globally, with higher infection rates in livestock-developed regions. BVDV has a wide range of infective populations, including cattle, sheep, goats, deer, and camels. BVDV is a 12.3–12.5 kbp positive, single-stranded, enveloped RNA virus. Its encoded E2 protein is a viral structural protein composed of 370 amino acid residues with a molecular weight of 55–58 kDa, and can form homodimers independently.

[0003] Currently, there are two main methods for controlling the spread of BVDV: eliminating persistently infected animals and vaccination. Modified live or inactivated vaccines are primarily used in BVDV vaccination programs, but these vaccines each have safety risks or insufficient immune protection. Inactivated vaccines are safe for pregnant cows, but their immunity period is short; while attenuated vaccines have a longer immunity period, they pose safety risks to pregnant cows.

[0004] Infectious bovine rhinotracheitis (IBR) is a serious infectious disease caused by the infectious bovine rhinotracheitis virus (IBRV). It is widespread in cattle farms both domestically and internationally, causing significant economic losses to the livestock industry.

[0005] gD glycoprotein is located on the surface of IBRV envelope and infected cells. It is one of the main glycoproteins on the surface of IBRV and plays an important role in the process of viral adsorption and invasion of host cells. It can induce humoral immunity as well as cellular immunity. Furthermore, anti-gD monoclonal antibodies also have neutralizing activity and can neutralize the virus.

[0006] Currently, inactivated bivalent vaccines against BVDV and IBRV are available on the market. However, inactivated vaccines may lead to further viral mutations, increasing the risk of reduced vaccine efficacy. Furthermore, immunization with inactivated vaccines cannot distinguish between the titer produced by vaccine immunization and the titer produced by infection with the wild-type virus, thus failing to fundamentally eradicate these two diseases.

[0007] Subunit vaccines are composed of viral antigenic proteins, eliminating the risk of viral recombination and mutation, and can distinguish the titer produced by wild-type virus infection and vaccine immunization. Existing BVD-IBR bivalent subunit vaccines simply mix antigens and adjuvants in a certain ratio to improve immunogenicity, but this method has limited effectiveness. Therefore, based on these scientific questions, this study aimed to develop a BVD-IBR subunit vaccine. We successfully prepared a fusion protein using a mammalian cell expression system, expressing BVDV structural protein E2 and IBRV structural protein gD in tandem with bovine mutant Fc, achieving high-level stable expression of this protein in the CHO-K1 mammalian expression system. Through optimization of emulsification conditions and the specific peptides and their dosages, we successfully developed a BVDV-IBRV subunit vaccine and optimized the vaccine formulation to obtain a highly immunogenic combination. Summary of the Invention

[0008] In view of the deficiencies in the prior art, the present invention aims to provide a subunit vaccine, its preparation method, and its application; specifically, it provides the preparation and application of a fusion protein vaccine of bovine viral diarrhea virus E2 and bovine infectious rhinotracheitis virus gD; this vaccine can prevent infection with bovine viral diarrhea virus and bovine infectious rhinotracheitis virus, and has many advantages such as good immunogenicity, long duration of immunity, high efficiency, safety, and high cost-effectiveness; furthermore, the present invention screened a polypeptide SL9797 from multiple synthetic polypeptide sequences, and unexpectedly found through bovine immunization experiments that the addition of SL9797 can effectively improve the antibody titer and duration of antibody in the vaccine.

[0009] <First Aspect>

[0010] This invention provides a recombinant subunit fusion protein vaccine against bovine viral diarrhea virus (BVD) and infectious bovine rhinotracheitis virus (BVT). The vaccine comprises an immunogenic rE2-2gD-Fc-M fusion protein and a pharmaceutically acceptable vector. The rE2-2gD-Fc-M fusion protein has the amino acid sequence shown in SEQ ID NO. 7, or is a derived protein with immunogenicity, comprising substitutions, deletions, or additions of one or more amino acids as shown in SEQ ID NO. 7. One week after secondary immunization, the recombinant subunit fusion protein vaccine against BVD and BVT of this invention achieves an antibody titer of 1:512 or higher.

[0011] As one embodiment of the present invention, the rE2-2gD-Fc-M fusion protein antigen content in the recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis of the present invention is 40-200 μg / mL.

[0012] In the recombinant subunit fusion protein vaccine against bovine viral diarrhea virus (BVD) and infectious bovine rhinotracheitis virus (IVRV), the rE2-2gD-Fc-M fusion protein antigen content can be selected from 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, and 200 μg / mL. Even when the rE2-2gD-Fc-M fusion protein antigen content in the recombinant subunit fusion protein vaccine against BVD and IVRV is only 80 μg / mL, an antibody titer of 1:512 or higher can be achieved in the first week after secondary immunization.

[0013] In a preferred embodiment of the present invention, the rE2-2gD-Fc-M fusion protein antigen content in the recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis of the present invention is 120 μg / mL.

[0014] As one embodiment of the present invention, in the recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis, the pharmaceutically acceptable carrier includes an adjuvant.

[0015] The adjuvants include: (1) white oil, aluminum gel adjuvant, saponins, avrididine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; (3) polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives; and one or more of the following: RIBI adjuvant system, Blockco-polymer, SAF-M, monophospholipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-sensitive enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, Montanide ISA 206, 50V, and Gel adjuvant; preferably, Montanide ISA 50V.

[0016] In one embodiment of the present invention, the adjuvant content is 5%-60% V / V, preferably 20%-60% V / V, and more preferably 50% V / V.

[0017] As one embodiment of the present invention, the pharmaceutically acceptable carrier includes one or more of the following: drugs, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives.

[0018] In one embodiment of the present invention, the immunostimulant includes α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin-2 (IL2), as well as a polypeptide with immunomodulatory effects. The immunomodulatory polypeptides SL9795, SL9796, and SL9797 are selected, with amino acid sequences SEQ ID NO.15, SEQ ID NO.16, and SEQ ID NO.17, respectively. Preferably, a bovine polypeptide SL9797 is used.

[0019] In one embodiment of the present invention, the pharmaceutically acceptable carrier includes an immunostimulant, and the immunostimulant content in the recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis is 5 μg / mL to 20 μg / mL. Preferably, the immunostimulant content in the recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis is 10 μg / mL.

[0020] To prepare such a composition, methods known in the art can be used.

[0021] <Second aspect>

[0022] This invention provides a method for preparing a recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis, the method comprising the following steps:

[0023] S1. The gene encoding the rE2-Fc fusion protein was amplified and cloned into the expression vector to obtain a recombinant expression vector containing the rE2-Fc fusion protein gene.

[0024] Alternatively, the gene encoding the rgD-Fc fusion protein can be amplified and cloned into an expression vector to obtain a recombinant expression vector containing the rgD-Fc fusion protein gene;

[0025] Alternatively, the gene encoding the rE2-gD-Fc fusion protein can be amplified and cloned into an expression vector to obtain a recombinant expression vector containing the rE2-gD-Fc fusion protein gene;

[0026] Alternatively, the gene encoding the rgD-E2-Fc fusion protein can be amplified and cloned into an expression vector to obtain a recombinant expression vector containing the rgD-E2-Fc fusion protein gene;

[0027] Alternatively, the gene encoding the r2E2-gD-Fc fusion protein can be amplified and cloned into an expression vector to obtain a recombinant expression vector containing the r2E2-gD-Fc fusion protein gene;

[0028] Alternatively, the gene encoding the rE2-2gD-Fc fusion protein can be amplified and cloned into an expression vector to obtain a recombinant expression vector containing the rE2-2gD-Fc fusion protein gene;

[0029] Alternatively, the gene encoding the rE2-2gD-Fc-M fusion protein can be amplified and cloned into an expression vector to obtain a recombinant expression vector containing the rE2-2gD-Fc-M fusion protein gene;

[0030] S2. Transfect the host cell with the recombinant expression vector containing the fusion protein gene obtained in step S1;

[0031] S3. Cultivate the cells obtained in step S2 to express the fusion protein;

[0032] S4. Purify the fusion protein obtained in step S3, add a pharmaceutically acceptable vector, and obtain a recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis.

[0033] In some embodiments, the method includes: 1) cloning the gene encoding the fusion protein into a eukaryotic expression vector to obtain a recombinant plasmid containing the fusion protein encoding gene; 2) transfecting the recombinant plasmid containing the fusion protein encoding gene into a CHO cell line; 3) fermenting and culturing the cells described in 2), and purifying them to obtain the target protein; 4) purifying the fusion protein obtained in 3), adding different adjuvants, etc., to obtain the recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis.

[0034] In one embodiment of the present invention, in the method described in the present invention, the rE2-Fc, rgD-Fc, rE2-gD-Fc, rgD-E2-Fc, r2E2-gD-Fc, rE2-2gD-Fc, and rE2-2gD-Fc-M fusion proteins in step S1 are proteins with amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, or are derived proteins with amino acid sequences as shown above, including those with substitution, deletion, or addition of one or more amino acids and possessing immunogenicity.

[0035] As one embodiment of the present invention, the gene sequence of re2-fc is shown in SEQ ID NO.8;

[0036] As one embodiment of the present invention, the gene sequence of rgd-fc is shown in SEQ ID NO.9;

[0037] Alternatively, as one embodiment of the present invention, the gene sequence of re2-gd-fc is shown in SEQ ID NO.10;

[0038] Alternatively, as one embodiment of the present invention, the gene sequence of rgd-e2-fc is shown in SEQ ID NO.11;

[0039] Alternatively, as one embodiment of the present invention, the gene sequence of r2e2-gd-fc is shown in SEQ ID NO.12;

[0040] Alternatively, as one embodiment of the present invention, the gene sequence of re2-2gd-fc is shown in SEQ ID NO.13;

[0041] Alternatively, as one embodiment of the present invention, the gene sequence of re2-2gd-fc-M is shown in SEQ ID NO.14;

[0042] In one embodiment of the present invention, the expression vector in the method described herein is a mammalian cell expression vector. Preferably, the eukaryotic expression vector is pEE6.4, pEE12.4, pGL4.13, or pcDNA3.1; more preferably, the eukaryotic expression vector is pcDNA3.1.

[0043] In one embodiment of the present invention, the host cell in step S2 is a mammalian cell, including CHO cells and 293 cells, wherein CHO cells are preferred.

[0044] In one embodiment of the present invention, the fusion protein expressed in step S4 is a secretory soluble protein.

[0045] <Third aspect>

[0046] This invention provides a recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis, comprising rE2-2gD-Fc-M fusion protein obtained by fermentation and purification from a CHO cell line and a pharmaceutically acceptable vector; the CHO cell line is the Chinese hamster ovary cell SLPCB03 with the preservation number CCTCC NO:C2022315. In this invention, it is also referred to as Chinese hamster ovary cell 3F7-rE2-2gD-Fc-M.

[0047] <Fourth Aspect>

[0048] This invention provides the use of a recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis in the preparation of medicaments for the prevention and / or treatment of bovine viral diarrhea and infectious bovine rhinotracheitis.

[0049] The drugs for the prevention and / or treatment of bovine viral diarrhea and infectious bovine rhinotracheitis described in this invention are intended for use on cattle, sheep, deer, and camels.

[0050] In this invention, the Chinese hamster ovary cell SLPCB03 has been deposited with the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: C2022315 and deposit date of October 12, 2022.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1) The present invention uses rE2-2gD-Fc-M fusion protein as antigen to prepare a recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis. The vaccine has high immunogenicity, long duration of immunity, low production cost, and simple production process. It has many advantages such as safety, high efficiency and low cost.

[0053] 2) In this invention, even when the rE2-2gD-Fc-M fusion protein antigen content in the recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis is only 80 μg / mL, the antibody titer can reach more than 1:512 in the first week after secondary immunization.

[0054] 3) This invention uses a bovine polypeptide as part of the vaccine composition. After addition, it can significantly improve the antibody titer and duration of the vaccine. The optimal amount of polypeptide added is 10 μg / mL. Attached Figure Description

[0055] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0056] Figure 1 The image shows the SDS-PAGE results of the purified recombinant subunit fusion protein rE2-2gD-Fc-M. Detailed Implementation

[0057] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0058] Example 1: Construction of Recombinant Vector

[0059] 1.1 Synthesis of Gene Sequences. Using CHO as the host cell, the base sequences encoding the recombinant subunit fusion proteins rE2-Fc, gD-Fc, rE2-gD-Fc, rgD-E2-Fc, r2E2-gD-Fc, rE2-2gD-Fc, and rE2-2gD-Fc-M fusion proteins of this invention underwent codon optimization, as shown in SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14. The optimized base sequences were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0060] 1.2 Construction of recombinant expression plasmids pcDNA3.1-rE2-Fc, pcDNA3.1-rgD-Fc, pcDNA3.1-rE2-gD-Fc, pcDNA3.1-rgD-E2-Fc, pcDNA3.1-r2E2-gD-Fc, and pcDNA3.1-rE2-2gD-Fc.

[0061] (1) The sequences e2-fc, gd-fc, e2-gd-fc, gd-e2-fc, 2e2-gd-fc and e2-2gd-fc were synthesized by Genscript Biotech. The restriction endonuclease Nhe I site was introduced at the 5' end of the upstream of the sequence, and the Nhe I site sequence was GCTAGC. The restriction endonuclease Xho I site and a stop codon were introduced at the 3' end of the downstream of the sequence, and the Xho I site sequence was CTCGAG.

[0062] (2) The synthesized fragment was digested with restriction enzymes Nhe I and Xho I. The digested gene fragment was recovered and ligated into the pcDNA3.1 vector treated with the same restriction enzymes Nhe I and Xho I. The ligation product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing 100 μg / ml ampicillin. The plates were incubated at 37°C. When colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid LB agar containing 100 μg / ml ampicillin at 37°C. The plasmids were then extracted. Recombinant plasmids pcDNA3.1-E2-gD-Fc, pcDNA3.1-gD-E2-Fc, pcDNA3.1-2E2-gD-Fc, and pcDNA3.1-E2-2gD-Fc were obtained. The recombinant plasmids were confirmed to be consistent with the target sequence by sequencing.

[0063] 1.3 Construction of recombinant expression plasmid pcDNA3.1-rE2-2gD-Fc-M.

[0064] (1) The e2-2gd-fc-m sequence was synthesized by Genscript Biotech. The restriction endonuclease Nhe I site was introduced at the 5' end of the upstream of the sequence, and the Nhe I site sequence was GCTAGC. The restriction endonuclease Xho I site and a stop codon were introduced at the 3' end of the downstream of the sequence, and the Xho I site sequence was CTCGAG.

[0065] (2) The synthesized fragment was digested with restriction enzymes Nhe I and Xho I. The digested gene fragment was recovered and ligated into the pcDNA3.1 vector treated with the same restriction enzymes Nhe I and Xho I. The ligation product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing 100 μg / ml ampicillin. The plates were incubated at 37°C. When colonies were clearly visible, single colonies were picked and incubated in 3 ml of liquid LB agar containing 100 μg / ml ampicillin at 37°C. The plasmid was then extracted. The recombinant plasmid pcDNA3.1-E2-2gD-Fc-M was obtained. The recombinant plasmid was confirmed to be consistent with the target sequence by sequencing.

[0066] 1.3 Preparation of plasmids for transfection.

[0067] (1) Following the instructions of the endotoxin-free plasmid large-scale extraction kit (DP117, Tiangen Biotech (Beijing) Co., Ltd.), extract 2 ml of plasmids pcDNA3.1-E2-Fc, pcDNA3.1-gD-Fc, pcDNA3.1-E2-gD-Fc, pcDNA3.1-gD-E2-Fc, pcDNA3.1-2E2-gD-Fc, pcDNA3.1-E2-2gD-Fc, and pcDNA3.1-E2-gD-Fc-M.

[0068] (2) Take 1 μL of sample for agarose gel electrophoresis.

[0069] (3) The concentration of the extracted plasmids was detected by nanodrop, and the concentrations were as follows: pcDNA3.1-E2-Fc 823 ng / ul, pcDNA3.1-gD-Fc 796 ng / ul, pcDNA3.1-E2-gD-Fc 768 ng / ul, pcDNA3.1-gD-E2-Fc 793 ng / ul, pcDNA3.1-2E2-gD-Fc 806 ng / ul, pcDNA3.1-E2-2gD-Fc 772 ng / ul, and pcDNA3.1-E2-gD-Fc-M 736 ng / ul.

[0070] Example 2: Transient transfection of CHO-K1 cells for expression of fusion protein

[0071] (1) The cells used for transfection were CHO-K1 cells that had been domesticated to be cultured in suspension.

[0072] (2) One day before transfection (Day-1), CHO-K1 cells were divided into groups of 1×10⁻⁶ cells. 6 Live cells / mL were seeded into shake flasks containing 30 mL of Acti CHO serum-free medium (H310KJ, Shanghai Yuanpei Biotechnology Co., Ltd.).

[0073] (3) On the day of transfection (Day 0), the viable cell density and viability were measured. The viable cell density should reach 2 × 10⁻⁶. 6 -6×10 6 The cell viability should be higher than 95%, with 1 live cell / mL.

[0074] (4) Dilute 30 μg of recombinant plasmid with 1500 μL of dilution medium and mix thoroughly to form a dilution solution; dilute 40 μL of transfection reagent with 1960 μL of dilution medium and add the transfection reagent dilution solution to the plasmid dilution solution, vortex for 10 seconds and mix thoroughly; incubate at room temperature for 5 min.

[0075] (5) Add the complex to the cell culture medium and culture with shaking.

[0076] (6) On the 5th day after transfection, the expression supernatant was collected and SDS-PAGE electrophoresis was performed.

[0077] Example 3: Protein purification and its physicochemical analysis

[0078] (1) Harvest the fermentation supernatant, centrifuge at 8000 rpm for 30 min, and then filter it using a 0.45 μm filter membrane.

[0079] (2) Sample loading. Purification was performed using a 5 mL pre-packed protein A column at a flow rate of 2 mL / min.

[0080] (3) Washing. Buffer A (20mM PBS (pH 7.2)), 25mL, flow rate 2mL / min.

[0081] (4) Elution. Buffer B (25mM NaAc (pH 3.6)), 50mL, flow rate 2mL / min. Collect the elution peak.

[0082] (5) Equilibration. Buffer C (20mM PBS (pH 7.2)), 50mL volume, flow rate 2mL / min.

[0083] (6) Storage. 20% ethanol, 10 mL volume, 2 mL / min flow rate.

[0084] (7) The collected elution peak samples were subjected to SDS-PAGE electrophoresis. Some results are shown below. Figure 1 As shown, the concentration and purity of the recombinant protein were calculated using grayscale analysis.

[0085] (8) Purification Results. The purified antigen proteins were analyzed for protein concentration and purity. Gray-scale analysis was used, and samples were subjected to SDS-PAGE electrophoresis. The concentration and purity of each band in the samples were analyzed using a gel imaging system. The results are shown in Table 1. The expression levels and purified protein concentrations of these groups were all higher than 500 μg / mL.

[0086] (9) Sterility test. The test shall be conducted in accordance with the sterility test method in the appendix of the Pharmacopoeia of the People's Republic of China, and the result shall be sterile.

[0087] Table 1. Recombinant protein expression levels

[0088]

[0089] Example 4: Immunogenicity analysis of different antigen complex samples

[0090] 4.1 Preparation of Immunosamples. The purified recombinant proteins rE2-Fc and rgD-Fc were diluted to 200 μg / ml with PBS, mixed at a 1:1 ratio, and then emulsified with sterile Montanide ISA 50V adjuvant at a 1:1 volume ratio to prepare the immunization sample. rE2-gD-Fc, rgD-E2-Fc, r2E2-gD-Fc, rE2-2gD-Fc, and rE2-2gD-Fc-M were diluted to 400 μg / ml with PBS and emulsified with sterile Montanide ISA 50V adjuvant at a 1:1 volume ratio to prepare the immunization sample.

[0091] 4.2 Bovine Immunization Experiment.

[0092] Six groups of cattle were immunized using the prepared samples rE2-gD-Fc, rgD-E2-Fc, r2E2-gD-Fc, rE2-2gD-Fc, and rE2-2gD-Fc-M, along with PBS. Five cattle were immunized in each group. The immunization and blood collection procedures included: 1) approximately 5 ml of blood was collected before immunization; 2) Day 1: First immunization: 2 ml of blood was collected from each cow; 3) Day 21: Booster immunization: 2 ml of blood was collected from each cow; 4) Day 28: 10 ml of blood was collected from each cow, and antibody titer was detected by ELISA; 5) Day 35: 10 ml of blood was collected from each cow, and antibody titer was detected by ELISA.

[0093] 4.3 Antibody titer detection

[0094] 4.3.1 Immunotiter analysis of rE2-gD-Fc group, rgD-E2-Fc group, and 1:1 mixed group of rE-Fc and rgD-Fc

[0095] We designed an experiment to investigate the effect of the protein sequence of the fusion protein on immunogenicity in vivo. The antibody titers of the immune serum were detected using ELISA, and the results are shown in Table 2.

[0096] Table 2. Immunotiter analysis of rE2-gD-Fc group and rgD-E2-Fc group

[0097]

[0098]

[0099] The results of this experiment indicate that the order of the inserted proteins in the fusion protein affects its immunogenicity. On days 28 and 35 after immunization, the immunogenicity of the rE2-gD-Fc group against both E2 and gD was slightly higher than that of the rgD-E2-Fc group and the 1:1 mixture of rE-Fc and rgD-Fc, suggesting that the optimal fusion order for rE2-gD-Fc is selected.

[0100] 4.3.2 Immunotiter analysis of rE2-gD-Fc, r2E2-gD-Fc, and rE2-2gD-Fc groups

[0101] This experiment compared the effect of the protein ratio of the fusion protein on immunogenicity. The antibody titers of the immune serum were detected using the same ELISA assay as in 4.3.1, and the results are shown in Table 3.

[0102] Table 3. Immunotiter analysis of r2E2-gD-Fc and rE2-2gD-Fc groups.

[0103]

[0104]

[0105] These results indicate that the E2 and gD antibody titers of r2E2-gD-Fc and rE2-2gD-Fc are significantly higher than those of rE2-gD-Fc. On days 28 and 35 after immunization, the E2 antibody titer of r2E2-gD-Fc is not different from that of rE2-2gD-Fc, but the gD antibody titer is lower than that of rE2-2gD-Fc. These results suggest that the immunogenicity of the fusion protein is best when the E2 to gD ratio is 1:2.

[0106] 4.3.3 Immunotiter analysis between the E2-2gD-Fc-M group and the rE2-2gD-Fc group

[0107] This experiment compared the effect of the protein ratio of the fusion protein on immunogenicity. 4.3.1 The antibody titer of the immune serum was detected by ELISA, and the results are shown in Table 4.

[0108] Table 4. Analysis of the immunogenicity of rE2-2gD-Fc-M

[0109]

[0110] These results indicate that on days 28 and 35 after immunization, the E2 antibody titer of rE2-2gD-Fc was significantly lower than that of rE2-2gD-Fc-M, while the gD antibody titer showed no difference between the two. This suggests that the optimized Fc can enhance the immunogenicity of the E2 protein.

[0111] In summary, rE2-2gD-Fc-M exhibits the strongest immunogenicity among different recombinant antigens. Therefore, we will use rE2-2gD-Fc-M as the immunogenic antigen in the next phase of our research.

[0112] Example 5: Preparation of different vaccine complex samples

[0113] (1) Preparation of water-soluble complex adjuvant GEL vaccine samples.

[0114] The rE2-2gD-Fc-M antigen was diluted with PBS to 400 μg / mL and mixed with 20% (volume percentage) GEL adjuvant at a volume ratio of 1:1. The mixture was then incubated at 4°C for 12 h to obtain the vaccine sample V-rE2-2gD-Fc-M-GEL.

[0115] (2) Preparation of ISA206VG vaccine samples with water-in-oil-in-water adjuvant.

[0116] The rE2-2gD-Fc-M antigen was diluted to 400 μg / mL with PBS. The diluted antigen and ISA206VG adjuvant were heated to 37°C and then mixed at a volume ratio of 1:1. The mixture was then allowed to stand at 4°C for 12 h to obtain the vaccine sample V-rE2-2gD-Fc-M-206.

[0117] (3) Preparation of Montanide ISA50V vaccine samples with oil adjuvant.

[0118] The rE2-2gD-Fc-M antigen was diluted with PBS to 400 μg / mL and emulsified with Montanide ISA 50V adjuvant at a volume ratio of 1:1. After standing at 4°C for 12 h, the vaccine sample V-rE2-2gD-Fc-M-50V was obtained.

[0119] Example 6: Bovine Immunization Experiments with Different Vaccine Complex Samples

[0120] (1) 28 negative calves were selected. The requirements for the cattle are shown in Table 5 below.

[0121] Table 5: Animal Selection Requirements

[0122]

[0123] (2) The immunization procedure is shown in Table 6 below.

[0124] Table 6: Immunization Procedures

[0125]

[0126] (3) Antibody level detection. ELISA antibody detection was performed on the collected serum. The results showed that antibody levels were significantly increased after the second immunization in all vaccine groups, and the immunogenicity was good. The V-rE2-2gD-Fc-M-50V vaccine group was significantly better than the other two vaccine groups, indicating that the use of 50V adjuvant improved the immunogenicity of the vaccine better than water adjuvant and 206 adjuvant. Details are shown in Table 7 below.

[0127] Table 7: Results of antibody titer assays after immunization with vaccines using different adjuvants

[0128]

[0129]

[0130] Example 7: Optimization of emulsification conditions for the oil adjuvant Montanide ISA 50V

[0131] Based on the following optimization experiments, the optimal emulsification conditions were finally selected as a shearing speed of 12000 rpm, a shearing time of 12 min, and an oil-water ratio of 1:1.

[0132] (1) Optimize the shear force used in the emulsification process. 8000rpm, 10000rpm, 12000rpm and 14000rpm were used respectively. According to the uniformity and stability of the emulsified samples, the results showed that 12000rpm was better than 14000rpm, which was better than 10000rpm, which was better than 8000rpm.

[0133] (2) Optimize emulsification time. At 12000 rpm, shearing was performed for 10 min, 12 min and 14 min respectively. Based on the uniformity and stability of the emulsified samples, the results showed that 12 min was better than 10 min, which was better than 14 min.

[0134] (3) Optimize the ratio of 50V adjuvant to antigen buffer. The ratios of 50V adjuvant to antigen buffer were selected as 0.8:1, 1:1 and 1.2:1, respectively. The samples were sheared at 12000 rpm for 12 min. Based on the uniformity and stability of the emulsified samples, the results showed that 1:1 was better than 1.2:1, which was better than 0.8:1.

[0135] Example 8: Preparation of recombinant vaccines with different antigen contents

[0136] 8.1 Take an appropriate amount of adjuvant Montanide ISA 50V, autoclave at 121℃ for 30 minutes, and set aside.

[0137] 8.2 Take the protein sample rE2-2gD-Fc-M purified in the above examples and dilute it with sterile PBS to 160 μg / mL, 240 μg / mL, and 320 μg / mL, respectively, according to the measured concentration.

[0138] 8.3 Prepare an appropriate amount of adjuvant according to the ratio of oil:water = 1:1 (v:v), place it in a beaker, and immerse the shearing head in the adjuvant.

[0139] 8.4 Pre-emulsification. Begin shearing by slowly adding the aqueous phase to the adjuvant being sheared, ensuring thorough mixing.

[0140] 8.5 Emulsification. Shear at 12000 rpm for 12 minutes, moving the beaker along the stirring head to ensure uniform emulsification.

[0141] 8.6 Analysis. After the emulsion was prepared, it was left at room temperature overnight and then examined. 1 mL of the emulsion was centrifuged at 3000 rpm for 30 min. No aqueous phase precipitated at the bottom, indicating good emulsification.

[0142] 8.7 Vaccine samples V-rE2-2gD-Fc-M (80μg), V-rE2-2gD-Fc-M (120μg), and V-rE2-2gD-Fc-M (160μg) with antigen contents of 80μg / mL, 120μg / mL, and 160μg / mL were prepared.

[0143] Example 9: Bovine Immunization Experiments with Vaccine Samples of Different Antigen Content

[0144] (1) 32 negative cattle were selected, and the requirements for cattle are shown in Table 8.

[0145] Table 8: Animal Screening Requirements

[0146]

[0147]

[0148] (2) The immunization procedure is shown in Table 9 below.

[0149] Table 9: Immunization Procedures

[0150]

[0151] (3) Antibody level detection. ELISA antibody detection was performed on the collected serum. The results showed that antibodies in all vaccine groups significantly increased after the second immunization, indicating good immunogenicity. The 120 μg / mL antigen content in the vaccine group was superior to the other two vaccine groups; therefore, 120 μg / mL was considered the optimal antigen content. Details are shown in Table 10 below.

[0152] Table 10: Antibody titer test results of different immunization doses of V-rE2-2gD-Fc-M-50V

[0153]

[0154]

[0155] Example 10: Preparation of Recombinant Vaccines with Different Polypeptides

[0156] 10.1 Take an appropriate amount of adjuvant Montanide ISA50V, autoclave at 121°C for 30 minutes, and set aside.

[0157] 10.2 Take the protein sample rE2-2gD-Fc-M purified in the above examples, dilute it to 240 μg / mL with sterile PBS according to the measured concentration, divide it into three groups of samples, and add 10 μg / mL of each of the following peptides to each group: SL9795 (ANEIRANSHFIGITE TDL, SEQ ID No. 15), SL9796 (NYIRANSHFIGITE, SEQ ID No. 16), and SL9797 (FQNFTVSFWLRVPHVSASRLD, SEQ ID No. 17).

[0158] 10.3 Prepare an appropriate amount of adjuvant according to the ratio of oil:water = 1:1 (v:v), place it in a beaker, and immerse the shearing head in the adjuvant.

[0159] 10.4 Pre-emulsification. Begin shearing by slowly adding the aqueous phase to the adjuvant being sheared, ensuring thorough mixing.

[0160] 10.5 Emulsification. Shear at 12000 rpm for 12 minutes, moving the beaker along the stirring head to ensure uniform emulsification.

[0161] 10.6 Analysis. After the emulsion was prepared, it was left at room temperature overnight and then examined. 1 mL of the emulsion was taken and centrifuged at 3000 rpm for 30 min. No aqueous phase precipitated at the bottom, indicating good emulsification.

[0162] 10.7 Vaccine samples containing different peptides were prepared: V-rE2-2gD-Fc-M-50V-SL9795, V-rE2-2gD-Fc-M-50V-SL9796, and V-rE2-2gD-Fc-M-50V-SL9797.

[0163] Example 11: Bovine Immunization Experiments with Different Peptide Vaccine Samples

[0164] (1) 28 negative cattle were selected, and the requirements for cattle are shown in Table 11.

[0165] Table 11: Animal Screening Requirements

[0166]

[0167] (2) The immunization procedure is shown in Table 12 below.

[0168] Table 12: Immunization Procedures

[0169]

[0170] (3) Antibody level detection. ELISA antibody detection was performed on the collected serum. Peptide SL9795 reduced the immunogenicity of the vaccine, while SL9796 and SL9797 both enhanced the immunogenicity, with SL9797 showing a stronger enhancing effect. The results indicate that the effect of peptides on improving vaccine immunogenicity is uncertain; however, the addition of the currently screened peptide SL9797 significantly improved the immunogenicity of the vaccine. Details are shown in Table 13 below.

[0171] Table 13. Immunogenicity of recombinant vaccines with different peptides

[0172]

[0173]

[0174]

[0175] Example 12: Preparation of recombinant vaccines with different polypeptide contents

[0176] 12.1 Take an appropriate amount of adjuvant Montanide ISA50V, autoclave at 121 for 30 minutes, and set aside.

[0177] 12.2 Take the protein sample rE2-2gD-Fc-M purified in the above examples, dilute it to 240 μg / mL with sterile PBS according to the measured concentration, divide it into three groups of samples, and add peptides of 0 μg / mL, 10 μg / mL, 20 μg / mL and 40 μg / mL to each group respectively.

[0178] 12.3 Prepare an appropriate amount of adjuvant according to the ratio of oil:water = 1:1 (v:v), place it in a beaker, and immerse the shearing head in the adjuvant.

[0179] 12.4 Pre-emulsification. Begin shearing by slowly adding the aqueous phase to the adjuvant being sheared, ensuring thorough mixing.

[0180] 12.5 Emulsification. Shear at 12000 rpm for 12 minutes, moving the beaker along the stirring head to ensure uniform emulsification.

[0181] 12.6 Analysis. After the emulsion was prepared, it was left at room temperature overnight and then examined. 1 mL of the emulsion was centrifuged at 3000 rpm for 30 min. No aqueous phase precipitated at the bottom, indicating good emulsification.

[0182] 12.7 Vaccine samples containing different amounts of polypeptides were prepared: V-rE2-2gD-Fc-M-50V-SL9797 (0 μg / mL), V-rE2-2gD-Fc-M-50V-SL9797 (5 μg / mL), V-rE2-2gD-Fc-M-50V-SL9797 (10 μg / mL), and V-rE2-2gD-Fc-M-50V-SL9797 (20 μg / mL).

[0183] Example 13: Bovine Immunization Experiments with Vaccine Samples of Different Peptide Contents

[0184] (1) 35 negative cattle were selected, and the requirements for cattle are shown in Table 14.

[0185] Table 14: Animal Screening Requirements

[0186]

[0187] (2) The immunization procedure is shown in Table 15 below.

[0188] Table 15: Immunization Procedures

[0189]

[0190]

[0191] (4) Antibody level detection. ELISA antibody detection was performed on the collected serum. The results showed that the addition of peptides could improve the immunogenicity of the vaccine; most significantly, the vaccine group with added peptides at a concentration of 10 μg / mL showed the best enhanced immunogenicity, therefore, 10 μg / mL was considered the optimal peptide concentration. Details are shown in Table 16 below.

[0192] Table 16. Immunogenicity of recombinant vaccines with different peptide content

[0193]

[0194]

[0195] Example 14: Stability Analysis of Recombinant Vaccine

[0196] 14.1 The rE2-2gD-Fc-M-50V-SL9797 (10 μg / mL) vaccine was placed in a glass bottle and stored at 4℃, 20℃ and 37℃ respectively for stability studies.

[0197] 14.2 The criteria for determining the stability of the emulsion are: (1) the height of the precipitated layer of the sample is not greater than 5%; (2) it is kept in a sterile state; (3) after demulsification, the antigen content is not less than 80% compared with the original solution.

[0198] 14.3 The vaccine samples were tested and found to be stable after being stored at 4°C for 18 months, at 20°C for 3 months, and at 37°C for 1 month.

[0199] Example 13: Establishment of recombinant plasmid transfection of CHO-K1 cells and monoclonal screening.

[0200] 14.1 CHO-K1 cell transfection

[0201] (1) The cells used for transfection were CHO-K1 cells that had been domesticated to be cultured in suspension.

[0202] (2) One day before transfection (Day-1), CHO-K1 cells were divided into groups of 1×10⁻⁶ cells. 6 1 live cells / mL were seeded into a shake flask containing 30 mL of Acti CHO serum-free medium.

[0203] (3) On the day of transfection (Day 0), the viable cell density and viability were measured. The viable cell density should reach 2 × 10⁻⁶. 6 -6×10 6 The cell viability should be higher than 95%, with 1 live cell / mL.

[0204] (4) Dilute 30 μg of recombinant plasmid with 1500 μL of dilution medium and mix thoroughly to form a dilution solution; dilute 40 μL of transfection reagent with 1960 μL of dilution medium, add the transfection reagent dilution solution to the plasmid dilution solution, vortex for 10 seconds and mix thoroughly; incubate at room temperature for 1-5 min.

[0205] (5) Add the complex to the cell culture medium and culture with shaking.

[0206] (6) On the 3rd day after transfection, samples were taken and SDS-PAGE electrophoresis was performed.

[0207] 14.2 Cell line screening

[0208] (1) On the second day after transfection, add G418 solution with a final concentration of 600 μg / mL.

[0209] (2) On day 7 post-transfection, the viable cell density and viability were measured at a rate of 1×10⁻⁶ cells / day. 6 Live cells / mL were seeded into shake flasks of Acti CHO serum-free medium.

[0210] (3) Passage the cells every 3-4 days until cell viability recovers to over 90% and the viable cell density reaches 1×10⁻⁶. 6 live cells / mL.

[0211] (4) The limiting dilution method was used to screen single-clonal cell lines to obtain subclones with the highest recombinant protein expression in the supernatant.

[0212] (5) Three cell lines that highly expressed rE2-2gD-Fc-M were obtained through screening: 3F7, 4D3, and 7A10.

[0213] Example 15: CHO-K1 cell shake-flask fermentation

[0214] (1) The 3F7, 4D3, and 7A10 cell lines were respectively divided into 5×10⁻⁶ cells. 5 Live cells per ml were seeded into 125 mL shake flasks containing 30 mL of culture medium.

[0215] (2) Place the shake flask in a shaker at 37°C, 8% CO2, and 120 rpm for shaking culture.

[0216] (3) Cell viability was detected daily using trypan blue staining and counting. When cell viability was below 80%, the culture supernatant was harvested by centrifugation and further purification was carried out.

[0217] Example 16: Purification of the fusion protein

[0218] (1) Harvest the fermentation supernatant, centrifuge at 8000 rpm for 30 min, and then filter it using a 0.45 μm filter membrane.

[0219] (2) Sample loading. Purification was performed using a 5 mL pre-packed protein A column at a flow rate of 2 mL / min.

[0220] (3) Washing. Buffer A (20mM PBS (pH 7.2)), 25mL, flow rate 2mL / min.

[0221] (4) Elution. Buffer B (25mM NaAc (pH 3.6)), 50mL, flow rate 2mL / min. Collect the elution peak.

[0222] (5) Equilibration. Buffer C (20mM PBS (pH 7.2)), 50mL volume, flow rate 2mL / min.

[0223] (6) Storage. 20% ethanol, 10 mL volume, 2 mL / min flow rate.

[0224] (7) Purification results. Based on the purification collection volume, protein concentration, and culture volume, the purified protein content of the recombinant protein expressed per milliliter of culture volume was calculated, as shown in Table 17.

[0225] Table 17. Results of recombinant protein expression

[0226] Protein expression level (mg / ml) 1.3 1.12 0.96 Protein content after purification (mg / ml) 1.2 0.93 0.86 Protein purity (%) after purification 97 96 97

[0227] (6) The selected cell lines were cryopreserved under liquid nitrogen conditions.

[0228] (7) The 3F7 cell line has been deposited with the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO:C2022315.

[0229] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A recombinant subunit fusion protein vaccine against bovine viral diarrhea virus and infectious bovine rhinotracheitis virus, comprising rE2-2gD-Fc-M fusion protein and a pharmaceutically acceptable vector; wherein, The rE2-2gD-Fc-M fusion protein is a protein with the amino acid sequence shown in SEQ ID NO.

7.

2. The recombinant subunit fusion protein vaccine according to claim 1, characterized in that, The vaccine also includes one or more of the following: immunostimulants, antioxidants, surfactants, buffers, dispersants, and preservatives.

3. The recombinant subunit fusion protein vaccine according to claim 1, characterized in that, The vaccine also includes an immunostimulant; the immunostimulant includes at least one of α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, interleukin-2, and a polypeptide with immune-enhancing effects.

4. The recombinant subunit fusion protein vaccine according to claim 3, characterized in that, The amino acid sequence of the immune-enhancing polypeptide is shown in SEQ ID NO.

17.

5. The recombinant subunit fusion protein vaccine according to claim 1, characterized in that, The vaccine also includes adjuvants; the adjuvants include: (1) white oil, aluminum glue adjuvant, saponins, avrididine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; (3) polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives; and one or more of the following adjuvant systems: RIBI adjuvant system, Blockco-polymer, SAF-M, monophospholipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-sensitive enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, Montanide ISA 206, 50V, and Gel adjuvant.

6. The recombinant subunit fusion protein vaccine according to claim 1, characterized in that, The vaccine contains 40-200 μg / mL of rE2-2gD-Fc-M fusion protein antigen.

7. The recombinant subunit fusion protein vaccine according to claim 1, characterized in that, The vaccine contains 5%-60% v / v adjuvant and / or 5ug / mL-20ug / mL immunostimulant.

8. A recombinant subunit fusion protein vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis, comprising rE2-2gD-Fc-M fusion protein obtained by fermentation and purification of CHO cell line and a pharmaceutically acceptable vector; wherein the CHO cell line is Chinese hamster ovary cell SLPCB03 with accession number CCTCC NO:C2022315.

9. A method for preparing a recombinant subunit fusion protein vaccine according to any one of claims 1-7, characterized in that, The method includes the following steps: S1. The encoding gene of the rE2-2gD-Fc-M fusion protein was amplified and cloned into the expression vector to obtain a recombinant expression vector containing the rE2-2gD-Fc-M fusion protein gene. S2. Transfect the host cell with the recombinant expression vector containing the fusion protein gene obtained in step S1; S3. Cultivate the cells obtained in step S2 to express the fusion protein; S4. Purify the fusion protein obtained in step S3, add it to a pharmaceutically acceptable vector, and obtain the recombinant subunit fusion protein vaccine.

10. The use of a recombinant subunit fusion protein vaccine according to any one of claims 1-8 in the preparation of a medicament for the prevention of bovine viral diarrhea and infectious bovine rhinotracheitis.

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