A preparation method and application of a recombinant pig delta coronavirus (PDCoV) S1 protein and subunit vaccine expressed by CHO cells

Abstract

This invention discloses a method for preparing and applying a recombinant porcine deltacoronavirus (PDCoV) S1 protein and subunit vaccine expressed in CHO cells, belonging to the field of animal vaccines and veterinary biological products technology. Its purpose is to provide a method for large-scale industrial production of a recombinant subunit vaccine against porcine deltacoronavirus. The recombinant subunit vaccine preparation method provided by this invention includes the following steps: 1. Cloning a eukaryotic expression vector containing the PDCoV S1 protein encoding gene; 2. Transfecting CHO cells, and obtaining a CHO cell line that stably and efficiently expresses the PDCoV S1 protein in suspension through selection, screening, and domestication; 3. Thoroughly mixing the PDCoV S1 protein with ISA 201 VG to obtain the recombinant subunit vaccine. Animal experiments on piglets show that high levels of neutralizing antibodies are produced 21 days after immunization.

Description

Technical Field

[0001] This invention belongs to the field of animal vaccines and veterinary biological products technology, specifically relating to a method for preparing and applying a recombinant porcine deltacoronavirus (PDCoV) S1 protein and subunit vaccine expressed in CHO cells. Background Technology

[0002] Porcine deltacoronavirus (PDCoV) is a newly emerging enteropathogenic coronavirus that has become widespread in many countries around the world (including China, South Korea, and Thailand) since its initial discovery in the United States in 2012. The virus primarily infects piglets aged 7–21 days, causing severe watery diarrhea, vomiting, dehydration, and even death, with a mortality rate reaching 30%–50%, resulting in significant economic losses to the global pig farming industry.

[0003] Currently, there is still a lack of highly effective and safe commercial vaccines for the prevention and control of PDCoV. Although some inactivated or attenuated vaccines are in the research and development or trial stage, they generally have the following problems: (1) Inactivated vaccines rely on high-titer virus culture, while PDCoV has low in vitro proliferation efficiency and unstable titer, resulting in limited antigen production and large batch-to-batch differences; (2) Attenuated vaccines have the risk of virulence reversion or recombination, and biosafety is difficult to guarantee; (3) Most existing vaccines use whole virus antigens, which contain non-protective or even immunosuppressive components, which are prone to causing adverse reactions and are difficult to distinguish between naturally infected and immunized animals (DIVA strategy is lacking).

[0004] In recent years, subunit vaccines based on the viral surface spike (S) protein have become an important direction in coronavirus vaccine development due to their advantages such as high safety, well-defined composition, and ease of quality control. Studies have shown that the S1 subunit of PDCoV contains a receptor-binding domain (RBD), a key antigenic region for inducing neutralizing antibodies. However, efficiently expressing and correctly folding the PDCoV S1 protein remains a technical bottleneck in achieving its immunogenicity.

[0005] Although prokaryotic systems (such as E. coli) have high expression levels, they cannot perform eukaryotic-specific post-translational modifications (such as glycosylation), resulting in protein conformational distortion and significantly reduced immunogenicity. Insect cells (baculovirus system) can achieve partial glycosylation, but the glycan structure differs from that of mammals, which may affect the presentation of antigenic epitopes. CHO (Chinese hamster ovary) cells, as a mainstream mammalian expression platform, possess complete protein folding, processing, and humanized glycosylation capabilities, and have been widely used in the production of human and veterinary recombinant protein vaccines (such as foot-and-mouth disease VP1 vaccine, pseudorabies gE / gI subunit vaccine, etc.).

[0006] Nevertheless, achieving high-yield, stable, and functional expression of the PDCoV S1 gene in CHO cells remains challenging: issues such as codon mismatch in natural viral gene usage in CHO cells, poor mRNA stability, and low protein secretion efficiency severely restrict its industrial application. Furthermore, optimizing culture processes and purification strategies to obtain high-purity, highly active S1 antigen, and combining it with suitable adjuvants to elicit potent mucosal and humoral immunity, is also crucial for the success of subunit vaccines.

[0007] Therefore, there is an urgent need to develop a safe and efficient PDCoV recombinant S1 subunit vaccine based on the CHO cell expression system to meet clinical prevention and control needs. Summary of the Invention

[0008] To achieve the above objectives, the present invention first provides a recombinant PDCoV S1 protein expressed by CHO cells. The S1 protein is encoded by a PDCoV S1 gene optimized for CHO cell codon preference and is prepared by a CHO cell expression system. Its amino acid sequence is shown in SEQ ID NO:2. It can induce animals to produce neutralizing antibodies against porcine deltacoronavirus.

[0009] The present invention also proposes a nucleic acid molecule that has been optimized for CHO cell codon preference and encodes the recombinant PDCoV S1 protein of claim 1, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0010] An expression vector, wherein the expression vector is a eukaryotic expression vector, comprising the nucleic acid molecule of claim 2.

[0011] A recombinant CHO cell line comprising the expression vector and stably expressing the recombinant S1 protein at an expression level of not less than 2 g / L.

[0012] This invention also proposes a subunit vaccine of recombinant PDCoV S1 protein, the vaccine comprising the following components: an aqueous phase containing the recombinant PDCoV S1 protein of claim 1 at a content of 100 μg / dose; an oil phase containing ISA 201 VG adjuvant; and an aqueous phase to oil phase mass ratio of 1:1.

[0013] In addition, a method for preparing a PDCoV subunit vaccine was proposed, including the following steps: (1) Construct a eukaryotic expression vector containing the porcine deltacoronavirus (PDCoV) S1 gene optimized for CHO cell codon preference; (2) The vector was transfected into CHO cells, and a single-clonal cell line that stably expressed PDCoV S1 protein was obtained by screening. (3) The cell line was cultured in suspension in serum-free medium and PDCoVS1 protein was induced to be expressed by cooling. The cell culture supernatant was harvested and PDCoVS1 protein was purified from the supernatant. (4) The PDCoV S1 protein is mixed and emulsified with adjuvant ISA 201 VG to prepare the subunit vaccine.

[0014] Furthermore, the eukaryotic expression vector in step (1) is pCI-neo; in step (2), electroporation transfection is used, and a single clone cell line is obtained by selective screening combined with limiting dilution method, with an S1 protein expression level of 2 g / L.

[0015] Furthermore, the initial inoculation density in step (3) is 0.8–1.2 × 10⁻⁶. 6 cells / mL, when the cell density reaches 10–12 × 10 6 When the cell / mL concentration was reduced to 32–34℃ for induction expression, the target recombinant PDCoV S1 protein was purified by nickel affinity chromatography in step (3) by eluting the target recombinant PDCoV S1 protein with a 150–250 mM imidazole gradient.

[0016] Furthermore, in step (4), the mass ratio of PDCoV S1 protein to ISA 201 VG is 1:1, and each dose of vaccine contains 100 μg of PDCoV S1 protein.

[0017] Finally, this invention also proposes the use of a subunit vaccine of recombinant PDCoV S1 protein in the preparation of a drug for preventing porcine deltacoronavirus infection, wherein the drug is an injectable preparation for intramuscular injection into healthy piglets aged 3 days or older with maternal PDCoV neutralizing antibody titers not exceeding 1:4, at a dose of 2 mL per piglet. 21 days post-immunization, serum neutralizing antibody titers reach 1:96 to 1:256, and significantly reduce the incidence of diarrhea, intestinal PDCoV viral load, and intestinal tissue pathological damage after challenge; the maternal PDCoV neutralizing antibody titer in the piglets is not higher than 1:4.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Strong antigenic immunogenicity: This invention optimizes the CHO cell codon preference of the PDCoV S1 gene, which can effectively induce piglets to produce high levels of PDCoV-specific neutralizing antibodies (titer reaches 1:96–1:256 21 days after immunization), which is significantly better than the prokaryotic expression product.

[0019] 2. The production process is stable and suitable for industrial scale-up. By using a selective screening system combined with limiting dilution, high-yield and stable monoclonal CHO cell lines are obtained, with S1 protein expression levels exceeding 2 g / L under serum-free suspension culture conditions. Combined with cooling induction and precise feeding processes, high batch-to-batch consistency is achieved, meeting GMP production requirements.

[0020] 3. Highly efficient purification process and high product purity. A single-step nickel affinity chromatography step can efficiently purify the target protein from the culture supernatant with a purity ≥90%, high recovery rate, avoiding activity loss caused by multi-step purification, and containing no endotoxins or host protein residues, ensuring good safety.

[0021] 4. Optimized vaccine formulation and excellent immunization effect. The purified S1 protein was emulsified with ISA 201 VG adjuvant at a 1:1 ratio to form an emulsion with uniform particle size and good stability. This not only enhances humoral immunity but also promotes the balanced Th1 / Th2 response. In the piglet challenge experiment, it significantly reduced the incidence of diarrhea, enterovirus load and pathological damage, and provided good clinical protection.

[0022] 5. High safety and wide applicability to a wide age range. The vaccine does not contain live virus or viral nucleic acid, and there is no risk of shedding, recombinant transmission, or virulence reversion; it is suitable for piglets older than 3 days, and can effectively immunize even in the presence of maternal antibodies (neutralizing antibodies ≤1:4), filling the gap in early immunization.

[0023] In summary, the PDCoV subunit vaccine provided by this invention has high safety, strong immunogenicity, good process adaptability, and clear clinical protective efficacy, providing a novel and reliable technical means for the scientific prevention and control of PDCoV, and has significant economic value and social benefits. Attached Figure Description

[0024] Figure 1 The results are for double enzyme digestion identification of recombinant plasmids, including: 1. pCI-neo-PDCoV-S1-1; 2. pCI-neo-PDCoV-S1-2; 3. pCI-neo-PDCoV-S1-3; Figure 2 The results of PDCoV-S1 sequence optimization are as follows: 1. PDCoV-S1 sequence expression was not optimized; 2. PDCoV-S1 sequence expression was optimized. Figure 3 The following are the SDS-PAGE analysis results of recombinant S1 protein: 1. Before cooling, 2. 2 days after cooling, 3. 3 days after cooling, 4. 4 days after cooling, 5. 5 days after cooling, 6. 6 days after cooling, 7. 7 days after cooling, 8. 8 days after cooling, 9. 9 days after cooling. Figure 4The following are the results of Western blot analysis of recombinant S1 protein: 1. Before cooling; 2. 2 days after cooling; 3. 4 days after cooling; 4. 5 days after cooling; 5. 6 days after cooling; 6. 7 days after cooling; 7. 9 days after cooling. Figure 5 SDS-PAGE analysis results of recombinant S1 protein purified by nickel affinity chromatography: 1. Before loading CHO-PDCoV, 2. elution with 50 μM imidazole, 3. elution with 100 imidazole, 4. elution with 100 imidazole, 5. elution with 200 imidazole, 6. elution with 200 imidazole, 7. elution with 300 imidazole, 8. elution with 300 imidazole, 9. elution with 500 imidazole. Figure 6 The results of dialysis analysis of recombinant S1 protein purification are as follows: A: SDS-PAGE analysis results of recombinant S1 protein after dialysis purification; B: Purity analysis results of recombinant S1 protein after dialysis purification. Figure 7 The results show the diarrhea symptoms of piglets aged 3-5 days after immunization and challenge. A represents subunit vaccine immunization, B represents commercial vaccine immunization, C represents blank adjuvant control, and D represents healthy control. Detailed Implementation

[0025] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments. Obviously, the embodiments described below are only a part of the embodiments, and all other embodiments obtained by those skilled in the art without creative effort are still within the scope of protection of the present invention.

[0027] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0028] Example 1: Construction and validation of recombinant expression vector pCI-neo-PDCoV-S1 This embodiment aims to construct a eukaryotic expression vector for PDCoV S1 protein that is suitable for efficient expression in CHO cells.

[0029] 1. Gene design and synthesis Based on the S1 gene sequence of the circulating strain of porcine deltacoronavirus (PDCoV) published in NCBI GenBank (accession number KX361344.1), and considering the codon usage preferences of CHO cells, the S1 gene was optimized to remove potential splicing sites and unstable elements, resulting in the synthesis of the PDCoV-S1 gene sequence. The optimized S1 gene is approximately 1500 bp in length, as shown in SEQ ID NO:1, and encodes 500 amino acids, as shown in SEQ ID NO:2. This sequence was synthesized by Shanghai Sangon Biotech Co., Ltd., and cloned into the pUC57 vector as an intermediate vector.

[0030] 2. Vector digestion and ligation The synthesized S1 gene and pCI-neo vector were double-digested with HindIII and EcoRI, respectively, and reacted at 37°C for 2 hours. The digestion products were separated by agarose gel electrophoresis and then purified using a gel recovery kit.

[0031] Results: After double enzyme digestion and purification, PDCoV-S1 and pCI-neo vector were ligated to construct the recombinant plasmid pCI-neo-PDCoV-S1. The plasmid was identified by double enzyme digestion with HindIII and EcoRI, and the results are as follows: Figure 1 Three single colonies were selected after transformation with recombinant plasmids. Plasmids were extracted and enzyme digestion was performed to identify the gene fragments, which were approximately 1,000 and 8,000 in size, respectively.

[0032] 3. Connection and Transformation The S1 fragment was mixed with the linearized pCI-neo vector at a molar ratio of 3:1, and T4 DNA ligase was added and ligated overnight at 16°C. The ligation product was transformed into E. coli DH5α competent cells, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated at 37°C for 16 hours.

[0033] 4. Screening and sequencing verification of positive clones Single colonies were selected and amplified, plasmids were extracted, and identified by HindIII / EcoRI double digestion. Clones with correct restriction enzyme patterns were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequencing results showed that the inserted fragment sequence was completely consistent with the design. The PDCoV-S1 nucleotide sequence is shown in SEQ ID NO:1, and the PDCoV-S1 amino acid sequence is shown in SEQ ID NO:2. The verified recombinant plasmid was named pCI-neo-PDCoV-S1 and cultured in glycerol at -80℃ for later use.

[0034] Example 2: Establishment of a CHO cell line stably expressing recombinant PDCoV-S1 protein In this embodiment, a high-yield and stable CHO cell line was obtained through electroporation transfection and stepwise scale-up screening.

[0035] 1. Plasmid extraction The positive bacterial culture verified in Example 1 was inoculated at a ratio of 1:1000 into LB liquid medium containing 100 μg / ml ampicillin and cultured overnight at 37°C and 220 r / min. Plasmids were extracted according to the instructions of the endotoxin-free plasmid extraction kit, and the plasmid mass concentration was determined. The plasmids were stored at -80°C.

[0036] 2. Electroporation 6.25 × 10⁶ CHO cells in the logarithmic growth phase were collected. 6 After centrifugation, cells were resuspended in 1 ml of CHO-ATG medium. 0.8 ml of the cell suspension was mixed with 50 μg of plasmid (filtered through a 0.22 μm filter) in an EP tube and transferred to a pre-chilled electroporation cuvette for electrotransfection. After electrotransfection, 0.6 ml of the liquid from the cuvette was transferred to a T-25 cm⁻¹ container containing 5 ml of CHO-ATG medium. 2 Incubate statically in the culture flask for 24 hours.

[0037] 3. Initial screening and amplification of cell lines 24 hours after electroporation, take an appropriate amount of T-25cm 2 Cells in culture flasks were cultured at a concentration of 0.5 × 10⁻⁶. 4 Seeds were seeded at a density of 200 μl / well in 96-well plates and incubated statically. When cell confluence reached 80%, cells were passaged at a 1:4 ratio, and the supernatant was collected for preliminary analysis using SDS-PAGE. Positive wells with high expression levels were transferred to 24-well plates, and when cell confluence reached 80%, they were transferred to T-25cm plates. 2 Culture flasks, when the viable cell density is higher than 0.5 × 10⁻⁶ 6 When the concentration is increased to 100 cells / ml, the concentration can be further increased to T-75cm. 2 Cultured in culture flasks, the viable cell density is also higher than 0.5 × 10⁻⁶. 6 When the number of cells / mL reaches a certain level, transfer the culture to a 125 mL shake flask.

[0038] 4. Monoclonal screening To further improve stability, a limiting dilution method (0.5 cells / well) was used for single-clone screening. Combined with ELISA or Western blot to detect the S1 protein expression level as 2 g / L, a high-yield single-clone cell line was finally obtained.

[0039] 5. The original PDCoV-S1 sequence without optimization was used to establish a control cell line through the same electroporation transfection and screening process to evaluate the effect of the optimized sequence on protein expression. SDS-PAGE analysis showed that the optimized sequence significantly increased the expression of PDCoV-S1 protein. Figure 2 ).

[0040] Example 3 Expression and purification of recombinant PDCoV-S1 protein This embodiment establishes a protein expression and purification process suitable for industrial production.

[0041] 1. Optimization of expression conditions The CHO-PDCoV-S1 cell line screened in Example 2 was expanded and cultured at 1×10⁻⁶ cells / year. 6 Cells / ml were seeded in serum-free medium and cultured at 37°C, 130 rpm, and 5% CO2. When the cell density reached 10–12 × 10⁶ cells / ml, the culture was continued. 6 Expression was performed at cell counts / ml using a cooling method. The culture conditions were 130 rpm, 33°C, and a CO2 incubator. Sugar was added daily during the cooling culture, and feed was added every other day. Expression continued for 9 days. When cell viability fell below 50%, the cell supernatant was collected by centrifugation and filtered through a 0.22 μm filter for sterilization, yielding a clear expression filter. SDS-PAGE and Western blot analysis showed the following results: Figure 3 and Figure 4 As shown.

[0042] 2. Nickel affinity chromatography purification The target protein was purified using nickel affinity chromatography. After loading the filtered protein expression solution onto the PBS buffer containing 50 mM imidazole, non-specifically bound proteins were removed. Eluent was then applied with PBS buffers containing 100 mM, 150 mM, 200 mM, and 300 mM imidazole, respectively. Results showed that after elution with solutions containing different concentrations of imidazole, SDS-PAGE (…)… Figure 5 The results showed that the PDCoV-S1 protein was mainly found in the elution buffer containing 200 mM imidazole, indicating that 200 mM imidazole could effectively elute the target protein.

[0043] The 200 mM imidazole eluent was collected and dialyzed. After dialyzing, the protein concentration was measured to be 2.4 mg / mL using a micro-ultraviolet spectrophotometer. SDS-PAGE analysis confirmed the protein concentration. Figure 6 A) Protein purity was analyzed using ImageJ software, and the protein purity was 90.51% (A). Figure 6 B).

[0044] Example 4: Preparation of a porcine deltacoronavirus subunit vaccine 1. Aqueous phase preparation: The purified PDCoV-S1 protein was diluted with sterile PBS, 100ug per dose.

[0045] 2. Emulsification and packaging: The aqueous phase and adjuvant ISA201 VG were mixed at a mass ratio of 1:1 and emulsified for 5 minutes using a high-speed emulsifier under ice bath conditions to form an emulsion. After passing the test, the emulsion was quantitatively dispensed, capped, and labeled.

[0046] One batch of vaccine was prepared according to the above method, with batch number 20250401.

[0047] 3. Quality Inspection (1) Appearance: It is a milky white, uniform emulsion.

[0048] (2) Dosage form: water-in-oil-in-water. Take a clean pipette, draw a small amount of vaccine and drop it into cold water. It will diffuse in a cloud-like manner.

[0049] (3) Stability: Take 10.0 ml of vaccine and add it to a centrifuge tube. Centrifuge at 3000 r / min for 15 minutes. The aqueous phase liquid precipitated at the bottom of the centrifuge tube should not exceed 0.5 ml.

[0050] (4) Viscosity was determined according to the appendix of the current Chinese Pharmacopoeia and met the requirements.

[0051] (5) The dosage was checked in accordance with the current appendix of the Chinese Pharmacopoeia and met the requirements.

[0052] (6) Sterility test: The test shall be conducted in accordance with the appendix of the current Chinese Pharmacopoeia, and sterile growth shall be observed.

[0053] (7) Mycoplasma test: The test was conducted according to the appendix of the current Chinese Veterinary Pharmacopoeia. No obvious color change was found in the small vials and tubes of culture. No "fried egg" mycoplasma colonies were found on the solid culture medium of the transplanted liquid culture.

[0054] (8) The test for exogenous viruses was conducted in accordance with the current appendix of the Chinese Veterinary Pharmacopoeia, and no exogenous virus contamination was found.

[0055] Example 5: Immunization test and evaluation of neutralizing antibodies in piglets 1. Animal experiments Healthy piglets aged 3-5 days born to sows with PDCoV neutralizing antibody levels not exceeding 1:4 were randomly divided into two groups of 5 piglets each. The experimental group received an intramuscular injection of 2 ml of PDCoV subunit vaccine per piglet in the neck; the control group received 2 ml of commercially available PDCoV vaccine per piglet, each dose containing 100 μg of antigen.

[0056] 2. Sampling and Testing Serum was collected from the anterior vena cava before immunization and 21 days after immunization. The serum was separated and the PDCoV neutralizing antibody in the serum was measured using the virus neutralization test (VNT).

[0057] The methods for detecting PDCoV neutralizing antibodies are as follows: Serum treatment: Inactivate the serum to be tested by water bath at 56℃ for 30 minutes; Cell preparation: LLCPK15 cells were seeded in 96-well microcell culture plates and the experiment was carried out when the cells grew into a monolayer. Serum dilution: The serum to be tested was serially diluted in EP tubes with serum-free DMEM medium containing 7.5 μg / ml trypsin, from 1:4 to 1:512. Virus neutralization: The pre-measured TCID virus was neutralized in serum-free DMEM medium containing 7.5 μg / ml trypsin. 50 The PDCoVDK01 strain of virus was diluted to 200 TCID. 50 / 100µl. Mix equal volumes of the diluted virus solution with serum of different dilutions and incubate at 37°C in a 5% CO2 incubator for 1 hour; Inoculation of serum-virus mixture: Wash the LlcPK15 cells that have grown into a monolayer in a 96-well microcell culture plate twice with serum-free DMEM culture medium. Inoculate the neutralized serum-virus mixtures into the cell wells at 100 µl per well, with 4 wells inoculated for each dilution. Control settings: Serum toxicity control (appropriately diluted serum was mixed with an equal volume of serum-free DMEM medium containing 7.5 μg / ml trypsin, 100 μl per well for each sample), and virus control (100 TCID). 50 50µl, 10TCID 50 / 50µl, 1TCID 50 / 50µl and 0.1TCID 50 Four virus controls with different viral loads were inoculated into four wells of the above 96-well microcell culture plate, 50 μl per well, and then 50 μl of serum-free DMEM medium containing 7.5 μg / ml trypsin was added to each well. A cell control was also prepared (four cell control wells were set up in the above 96-well microcell culture plate, and 100 μl of serum-free DMEM medium containing 7.5 μg / ml trypsin was added to each well). Adsorption: The above 96-well microcell culture plate was placed in an incubator at 37°C with 5% CO2 for 1 hour for adsorption, and the inoculum in all test wells was discarded; the cells were washed twice with serum-free DMEM medium, and 100 μl of serum-free DMEM medium containing 7.5 μg / ml trypsin was added to each well, and the plate was placed in an incubator at 37°C with 5% CO2 for further culture; Results Interpretation: Observe and record cytopathic effect (CPE) daily for 4 consecutive days. Cells in both the serum toxicity control wells and the normal cell control wells should be normal. The virus control should contain 100 TCID50. 50 and 10TCID 50 All wells should show cytopathic effects and 1.0 TCID. 50 There should be 0-2 wells showing cytopathic effects and 0.1 TCID. 50 No cytopathic effects should be observed in any of the wells. The titer of the PDCoV neutralizing antibody in the tested serum should be calculated using the Reed-Muench method.

[0058] 3. Results As shown in Table 1, all piglets had pre-immunization antibody titers <1:4. Twenty-one days after immunization, the geometric mean titer (GMT) of neutralizing antibodies in the experimental group was 1:152, with an individual range of 1:96–1:256. The GMT in the control group was 1:96, with an individual range of 1:64–1:128. The antibody levels in the experimental group were significantly higher than those in the control group (p < 0.05).

[0059] Table 1. Detection results of PDCoV neutralizing antibodies Example 6: Virus Challenge Protection Test This embodiment evaluates the protective effect against viral load and viral load inhibition ability of the porcine deltacoronavirus subunit vaccine prepared in Example 4 in piglets.

[0060] 1. Laboratory animals and grouping Twenty healthy piglets aged 3-5 days were selected, all from PDCoV neutralizing antibody-negative sows (serum neutralizing antibody titer less than 1:4). They were confirmed by PCR and ELISA to be free of PDCoV and other common diarrhea pathogens (such as PEDV, TGEV, and rotavirus).

[0061] They were randomly divided into 4 groups, with 5 animals in each group: Group A (subunit vaccine immunization and challenge): PDCoV subunit vaccine (batch number 20250401) was injected intramuscularly into the neck, 2 mL / head; Group B (immunization and challenge with commercially available vaccines): intramuscular injection of commercially available PDCoV inactivated vaccine in the neck, 2 mL / head; the commercially available vaccine is an inactivated complete PDCoV virus particle, containing all structural proteins and non-structural proteins of the virus, such as S, M, N, and E.

[0062] Group C (blank adjuvant control, challenge): 2 mL / head of an equal volume of ISA 201 VG adjuvant emulsion (without antigen) was injected intramuscularly into the neck. Group D (healthy controls, no challenge): 2 mL / head of PBS was injected intramuscularly into the neck, without challenge. All piglets were vaccinated once and kept in an isolated negative pressure animal room with free access to food and water.

[0063] 2. Virus attack plan Challenge the virus 21 days after immunization (i.e., 24-26 days old).

[0064] Challenge strain: Using recently isolated PDCoV DK01 strain tissue virus, via ID 50 The toxicity was determined to be 10. 3.5 ID 50 / mL.

[0065] Method of infection control: oral gavage, total dose of 10 mL (containing 100 ID50). 50 (Virus). Group D will not attack the virus, while the other three groups will attack the virus simultaneously.

[0066] 3. Observation indicators (1) Measurement of piglet body temperature Temperature was measured for 10 consecutive days before and after the virus challenge, with the piglets' body temperature measured and recorded at 9:00 AM each day.

[0067] (2) Observation of clinical symptoms in piglets after viral challenge After the virus challenge, observe the piglets daily for their mental state, appetite, and coat condition; check for vomiting, diarrhea, dehydration, and death. Observe continuously for 10 days and record details.

[0068] (3) Determination of viral load in anal swabs after challenge Sample collection and processing: Anal swab collection method: Fresh fecal swabs were collected daily after the challenge for 10 consecutive days. The fecal samples were diluted with 1 ml of PBS, and the samples were repeatedly frozen and thawed 3 times. They were then stored in a refrigerator below -70°C for later use.

[0069] Detection of viral load in anal swabs by real-time quantitative RT-PCR The repeatedly freeze-thawed anal swabs were removed from a freezer below -70°C, and the diluted fecal solution was centrifuged at 12000 rpm for 3 minutes. The supernatant was then used for total RNA extraction. Subsequently, the viral load in the anal swabs was detected using quantitative real-time RT-PCR.

[0070] 4. Results 4.1 Results of body temperature monitoring in piglets after viral challenge Twenty-one days after immunization of 3-5 day-old piglets, PDCoV DK01 strain tissue virus was administered orally to the immunized piglets. The specific results of body temperature monitoring are shown in Tables 1 and 2. The body temperature of all piglets fluctuated within the normal physiological range after challenge with the virus, and there were no obvious abnormalities. Occasionally, some piglets showed signs of depression, decreased appetite, and rough coat after challenge with the virus.

[0071] Table 2. Body temperature and routine clinical manifestations of 3-5 day old piglets after immunization and challenge. Table 3. Body temperature and routine clinical manifestations of 3-5 day old piglets after immunization and challenge. Note: "-" indicates that the piglet's spirit, appetite and coat are relatively normal, "a" indicates that the piglet is depressed, "b" indicates that the piglet's appetite is reduced, and "c" indicates that the piglet's coat is rough.

[0072] 4.2 Observation results of clinical symptoms of diarrhea in piglets after viral challenge After viral challenge, the clinical symptoms of diarrhea in piglets were closely observed daily, and photos were taken and recorded. The statistical results of diarrhea-related clinical symptoms are shown in Table 3 below. The diarrhea symptoms are as follows: Figure 7 As shown.

[0073] Table 4. Statistical results of diarrhea-related clinical symptoms in 3-5 day old piglets after immunization and challenge. Note: " / " indicates the absence of clinical symptoms such as diarrhea, vomiting, and dehydration.

[0074] Based on the statistical results of diarrhea-related clinical symptoms in Table 3 and Figure 7 The results of the study on diarrhea in piglets showed that in Group A (subunit vaccine immunization, challenge), all piglets did not show diarrhea symptoms within 1-10 days after challenge, demonstrating complete protection; in Group B (commercial inactivated vaccine immunization, challenge), some piglets showed symptoms, such as piglet No. 07, which showed diarrhea symptoms on the 3rd-4th day after challenge, but the overall morbidity and severity of symptoms were significantly lower than in Group C; in Group C (blank adjuvant control, challenge), all 5 piglets showed diarrhea symptoms accompanied by vomiting and dehydration, and the symptoms lasted for a long time (3-7 days); in Group D (healthy control, no challenge), all 5 piglets were normal.

[0075] 4.3 Results of anal swab excretion in piglets after viral challenge Anal swabs were collected daily for 10 consecutive days after challenge. After sample processing, the supernatant was used for total RNA extraction. Viral shedding in the anal swabs was detected by quantitative real-time RT-PCR. Specific statistical results are shown in Table 4. In Group A, only piglet No. 03 showed a very low viral load on days 3-4; the remaining piglets showed no viral load throughout the process, indicating rapid viral clearance. In Group B, only piglet No. 07 showed a viral load on days 3-4, with a significantly lower peak and duration of viral load compared to Group C. In Group C, all piglets showed high viral loads from days 1-8, with a long viral duration, indicating no effective protection was acquired. In Group D, no virus was detected throughout the process, verifying the initial health status of the experimental animals.

[0076] Table 5. Results of viral load detection in anal swabs of piglets aged 3-5 days after immunization and challenge. Conclusion: The porcine deltacoronavirus subunit vaccine of this invention can effectively induce a protective immune response, significantly reduce clinical symptoms and viral replication levels after challenge, and its protective efficacy is superior to existing commercial vaccines. Therefore, the subunit vaccine's precise targeting of the S1 protein-induced immune response provides a more efficient new option for PDCoV prevention and control.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant PDCoV S1 protein expressed in CHO cells, characterized in that, The S1 protein is encoded by the PDCoV S1 gene optimized for CHO cell codon preference and prepared by a CHO cell expression system. Its amino acid sequence is shown in SEQ ID NO:

2. It can induce animals to produce neutralizing antibodies against porcine deltacoronavirus.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule is optimized for CHO cell codon preference and encodes the recombinant PDCoV S1 protein of claim 1, the nucleotide sequence of which is shown in SEQ ID NO:

1.

3. An expression carrier, characterized in that, The expression vector is a eukaryotic expression vector containing the nucleic acid molecule described in claim 2.

4. A recombinant CHO cell line, characterized in that, The cell line comprises the expression vector of claim 3 and stably expresses the recombinant S1 protein of claim 1 at an expression level of not less than 2 g / L.

5. A subunit vaccine for recombinant PDCoV S1 protein, characterized in that, The vaccine comprises the following components: an aqueous phase containing the recombinant PDCoV S1 protein of claim 1 at a content of 100 μg / dose; an oil phase containing ISA 201 VG adjuvant; and an aqueous phase to oil phase mass ratio of 1:

1.

6. A method for preparing a recombinant PDCoV subunit vaccine, characterized in that, Includes the following steps: (1) Construct a eukaryotic expression vector containing the porcine deltacoronavirus (PDCoV) S1 gene optimized for CHO cell codon preference; (2) The vector was transfected into CHO cells, and a single-clonal cell line that stably expressed PDCoV S1 protein was obtained by screening. (3) The cell line was cultured in suspension in serum-free medium and PDCoV S1 protein was induced to be expressed by cooling. The cell culture supernatant was harvested and the PDCoV S1 protein was purified from the supernatant. (4) The PDCoV S1 protein is mixed and emulsified with adjuvant ISA 201 VG to prepare the subunit vaccine.

7. The preparation method according to claim 6, characterized in that, The eukaryotic expression vector in step (1) is pCI-neo; in step (2), electroporation transfection is used, and a single clone cell line is obtained by selective medium screening combined with limiting dilution method, and the expression level of S1 protein is 2 g / L.

8. The preparation method according to claim 6, characterized in that, The initial inoculation density in step (3) is 0.8–1.2 × 10⁻⁶. 6 cells / mL, when the cell density reaches 10–12 × 10 6 When the cell / mL concentration was reduced to 32–34℃ for induction expression, the target recombinant PDCoV S1 protein was purified by nickel affinity chromatography in step (3) by eluting the target recombinant PDCoV S1 protein with a 150–250 mM imidazole gradient.

9. The preparation method according to claim 6, characterized in that, The mass ratio of PDCoV S1 protein to ISA 201VG in step (4) is 1:1, and each dose of vaccine contains 100 μg of PDCoV S1 protein.

10. The use of the recombinant PDCoV S1 protein subunit vaccine of claim 5 in the preparation of a medicament for the prevention of porcine deltacoronavirus infection, characterized in that, The drug is an injectable preparation used for intramuscular injection into healthy piglets aged 3 days or older with a maternal PDCoV neutralizing antibody titer not exceeding 1:

4. The dosage is 1 mL per piglet.

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