A chimeric PEDV and TGEV spike protein vaccine, a bivalent subunit vaccine, its preparation method and application

Abstract

This invention discloses a chimeric spike protein of PEDV and TGEV, a bivalent subunit vaccine, its preparation method, and its applications, relating to the field of biotechnology. The amino acid sequence of the spike protein chimera is shown in SEQ ID NO.13. This invention disrupts the formation of the secondary structure of the TGEV S protein by mutating it, maintaining the pre-fusion conformation of the S protein. Furthermore, while maintaining the pre-fusion trimer conformation of TGEV S, the DO-NTD region of TGEV S is replaced with the DO-NTD region of PEDV S, thus obtaining a spike protein chimera. Immunization using this spike protein chimera can generate neutralizing antibodies against both PEDV and TGEV, providing simultaneous prevention and control of both PEDV and TGEV.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a chimeric spike protein of PEDV and TGEV, a bivalent subunit vaccine, its preparation method, and its application. Background Technology

[0002] Transmissible gastroenteritis virus (TGEV) is a member of the porcine enteric coronavirus (SeCoV) family. It spreads rapidly and can infect pigs of all ages, primarily causing severe diarrhea, vomiting, dehydration, and high mortality in piglets under two weeks of age. TGEV mainly infects the small intestine of pigs, causing villus atrophy, decreased villus height and crypt depth, and a reduction in the number of SIgA-positive cells and dendritic cells in the jejunum. Although the current incidence of TGEV is low and vaccination is effective, co-infection with other viruses has been detected in recent years. For example, co-infection with PEDV is relatively common, and recombination occurs, such as with the TGEVAHHF strain and the TGEV JS2012 strain. TGEV also poses a risk of cross-species transmission; therefore, prevention and control of TGEV cannot be relaxed.

[0003] Porcine enteric coronavirus (PEDV) is also a member of the alpha coronavirus family, belonging to the same genus as TGEV. Its clinical symptoms are similar to TGEV; it is an acute intestinal infectious disease that can infect pigs of all ages, primarily manifesting as a mortality rate of over 90% in piglets. The pathological changes of PEDV are mainly seen in the gastrointestinal tract, characterized by significant gastric distension, the presence of undigested curd in the stomach, marked atrophy of intestinal villi, thinning and near-transparency of the intestinal wall, pale yellow fluid accumulation in the intestine, significant vacuolation of intestinal cells, and severe atrophic enteritis in the jejunum. Since the outbreak of PEDV in 2010, highly virulent strains have gradually become dominant, and existing commercial vaccines cannot provide complete protection. Therefore, the development of a safe and effective PEDV vaccine is urgently needed for the prevention and control of PEDV.

[0004] The spike protein of coronaviruses plays a crucial role in viral infection of cells, marking the beginning of the virus's life cycle. The coronavirus S protein sequence contains approximately 1300 amino acids and is about 180 kDa in size. The S protein mainly comprises two subunits: S1, responsible for receptor binding, and S2, responsible for membrane fusion. During coronavirus invasion, the S protein must first recognize its receptor. This process is primarily related to the S1-CTD (binding protein receptor) and S1-NTD (binding sugar receptor) of the S protein. Another key function of the S protein is to utilize the S2 subunit to achieve membrane fusion; therefore, the S protein is considered an important protein for inducing the production of neutralizing antibodies.

[0005] Currently, traditional vaccines cannot effectively address illnesses caused by variant strains. Subunit vaccines, however, are considered to offer a faster response to emerging variants, requiring no whole virus and enabling rapid production by replacing corresponding protein sequences of circulating strains, while also providing high levels of effective antigens. Therefore, this invention aims to develop a novel subunit vaccine to provide strong technical support for the prevention and control of PEDV and TGEV. Summary of the Invention

[0006] The purpose of this invention is to provide a PEDV and TGEV spike protein chimera, a bivalent subunit vaccine, its preparation method, and its application, to solve the problems existing in the prior art. Immunization using this spike protein chimera can generate neutralizing antibodies against both PEDV and TGEV, thus providing simultaneous prevention and control of both PEDV and TGEV.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] The present invention provides a spike protein chimera, the amino acid sequence of which is shown in SEQ ID NO.15.

[0009] The present invention also provides a gene encoding the above-mentioned spike protein chimera.

[0010] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.16.

[0011] The present invention also provides a recombinant plasmid comprising the above-described encoding gene.

[0012] The present invention also provides a recombinant host cell comprising the above-described recombinant plasmid.

[0013] Furthermore, the recombinant host cell is a recombinant HEK293F cell.

[0014] The present invention also provides the application of the above-mentioned encoding gene, recombinant plasmid or recombinant host cell in the preparation of the above-mentioned spike protein chimera.

[0015] The present invention also provides the application of the above-described spike protein chimera in the preparation of a bivalent subunit vaccine.

[0016] The present invention also provides a method for preparing a bivalent subunit vaccine, comprising the step of mixing the above-mentioned spike protein chimera with a vaccine adjuvant evenly to prepare the bivalent subunit vaccine.

[0017] The present invention also provides a bivalent subunit vaccine, the active ingredient of which includes the above-mentioned spike protein chimera.

[0018] The present invention discloses the following technical effects:

[0019] This invention mutates glutamic acid at position 1139 and leucine at position 1140 of the TGEV S protein to proline, disrupting the formation of secondary structure and creating a rigid helix-loop-helix structure. This maintains the pre-fusion conformation of the S protein, improving stability and expression levels. Furthermore, while maintaining the pre-fusion trimer conformation of TGEV S, the TGEV SDO-NTD region is replaced with the PEDV SDO-NTD region. Eukaryotic expression using HEK-293F cells yields a highly pure spike protein chimera. The HEK-293F cell expression system offers advantages such as suspension culture, no need for serum to introduce exogenous viruses, simple production process, and good immunogenicity of the expressed protein.

[0020] This invention uses the spike protein chimera as an immunogen in animal experiments. Because it contains the core region of the TGEV S protein that induces the production of neutralizing antibodies—the RBD region—and the region of the PEDV S protein that binds to the sugar receptor—the D0-NTD region, it produces a certain degree of neutralizing antibodies against PEDV and TGEV, achieving "one injection for two protections" and having the effect of simultaneously preventing and controlling PEDV and TGEV. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 A schematic diagram illustrating the process of determining the 2P mutation site through sequence alignment with SARS-CoV-2;

[0023] Figure 2 Schematic diagram of the construction of TGEV and PEDV S proteins;

[0024] Figure 3 The results of PCR amplification of the TGEV S gene and the tag GCN4-8×His are shown.

[0025] Figure 4 The results of PCR amplification of the PEDV S gene and the tag GCN4-8×His are shown.

[0026] Figure 5 The images are of the TGEV S-2P recombinant plasmid (A) and the PEDV S-2P recombinant plasmid (B).

[0027] Figure 6The results of bacterial culture PCR identification of TGEV and PEDV S-2P recombinant plasmids;

[0028] Figure 7 The results are sequencing results for TGEV and PEDV S-2P; where A is the sequencing result for TGEV S-2P and B is the sequencing result for PEDV S-2P.

[0029] Figure 8 The purification results of TGEV and PEDV S-2P proteins are shown; where A is the purification peak diagram of TGEV S-2P protein.

[0030] B is the SDS-PAGE image of the target protein in collection tubes 19-42 in the purification peak diagram of TGEV S-2P protein; C is...

[0031] The purification peak diagram of PEDV S-2P protein; D is the SDS-PAGE diagram of the target protein in collection tubes 19-42 in the purification peak diagram of PEDV S-2P protein.

[0032] Figure 9 The results are SDS-PAGE and Western Blot identification results of TGEV and PEDV S-2P proteins; where A is the SDS-PAGE identification result of TGEV S protein; B is the SDS-PAGE identification result of PEDV S protein; C is the Western Blot identification result of TGEV S protein; and D is the Western Blot identification result of PEDV S protein.

[0033] Figure 10 This is a schematic diagram of the mouse immunization protocol;

[0034] Figure 11 The graph shows the results of indirect ELISA detection of IgG titers in mice immunized with TGEV and PEDV S-2P proteins; where A represents the detection results using TGEV S-2P protein as the antigen and B represents the detection results using PEDV S-2P protein as the antigen.

[0035] Figure 12 The results show the neutralizing titers and cross-protection of TGEV and PEDV S-2P protein-immunized mouse serum; where A represents the neutralizing antibody titer against TGEV and B represents the neutralizing antibody titer against PEDV.

[0036] Figure 13 A schematic diagram illustrating the construction of the spike protein chimera;

[0037] Figure 14The results are PCR amplification of the target gene of the spike protein chimera; among them, 1-2 are the D0-NTD fragment of PEDV S protein; 3-4 are the CTD-S2-GCN4-Strep-8×His fragment of TGEV S protein.

[0038] Figure 15 The results of PCR identification of spike protein chimera bacterial culture;

[0039] Figure 16 The figure shows the optimization results of the optimal expression time for the spike protein chimera;

[0040] Figure 17 The results show the purification of the spike protein chimera; where A is the purification peak diagram of TGEV S (replaced D0-NTD) protein; B is the SDS-PAGE diagram of the target protein in collection tubes 19-42 in the purification peak diagram of TGEV S (replaced D0-NTD) protein.

[0041] Figure 18 The results of SDS-PAGE (A) and Western Blot (B) identification of spike protein chimeras;

[0042] Figure 19 A diagram illustrating the timing of immunization and blood collection for weaned piglets;

[0043] Figure 20 The results show the monitoring results of neutralizing antibody levels for TGEV and PEDV; where A represents the result of neutralizing antibody titer detection against TGEV; B represents the result of neutralizing antibody titer detection against PEDV; TGEV S represents the TGEV S-2P proteome; PEDV S represents the PEDV S-2P proteome. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0046] Example 1

[0047] 1. Experimental Materials and Methods

[0048] 1.1 Experimental Materials

[0049] 1.1.1 Biomaterials

[0050] The Escherichia coli DH5α chemocompetent cells were obtained from Yibai Biotechnology Co., Ltd.

[0051] African green monkey kidney cells (Vero), porcine kidney cells (PK-15), and human embryonic kidney cell-F clone cells (HEK-293F) were provided by Professor Peng Guiqing's laboratory at Huazhong Agricultural University.

[0052] PEDV (CT P10 strain) was provided by Professor Peng Guiqing's laboratory at Huazhong Agricultural University, and TGEV (WH-1 strain) was donated by Professor He Qigai.

[0053] The full-length DNA fragments PEDV CT P10-S and TGEV WH-1-S (both optimized with mammalian cell-preferred codons) were synthesized by Nanjing Genscript Biotech Co., Ltd. The eukaryotic expression vector pCAGGS was purchased from AddGene.

[0054] The experimental animals, weaned piglets, came from Yingang Pig Farm in Hubei Province.

[0055] 1.1.2 Main Reagents and Consumables

[0056] Cell culture related reagents and consumables: Transfection reagent PEI40K was prepared and stored in our laboratory; trypsin powder and protein marker were purchased from Themo Fisher Scientific; 293 medium (serum-free) was purchased from Yonglian Biotechnology (Shanghai) Co., Ltd.; 4.5 g / L high glucose medium DMEM was purchased from Gibco; fetal bovine serum (FBS) was purchased from Wuhan Qingmu Biotechnology Co., Ltd.; penicillin-streptomycin antibiotics and ECL chemiluminescence staining solution were purchased from Beyotime Biotechnology Co., Ltd.; cell culture plates (96-well and 24-well plates), centrifuge tubes (15 and 50 mL) and cell culture flasks (T75 and T25) were purchased from Tiangen Biotech Co., Ltd.; 5 mL pipettes, 10 mL pipettes, 25 mL pipettes and cryopreservation tubes were purchased from Corning; dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich; serum-free cell cryopreservation solution was purchased from Xinsaimei Biotechnology Co., Ltd.

[0057] Molecular cloning related reagents and consumables: 2×Phanta Max Master Mix purchased from Nanjing Novizan Biotechnology Co., Ltd.; 2×MIX Taq enzyme purchased from Beijing Zhuangmeng Biotechnology Co., Ltd.; restriction endonucleases Xho I and EcoRI purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.; PCR tubes and disposable culture dishes purchased from Wuhan Houji Biotechnology Co., Ltd.; Goldview nucleic acid dye purchased from Xi'an Hete Biotechnology Co., Ltd.; DNA Markers (DL2000, DL5000 and DL15000) and high-purity low-electroosmotic agarose purchased from Wuhan Qingke Innovation Biotechnology Co., Ltd.; plasmid mini-extraction kit and gel extraction kit purchased from OMEGA; endotoxin-free large-scale plasmid extraction kit purchased from Tiangen Biotech Co., Ltd.; agar, imidazole, and ampicillin (Amp) purchased from Biosharp; Ni-TED Sepharose 6FF (His-Tag) was purchased from Sangon Biotech Co., Ltd.; 0.22μm filters, 0.45μm filters, and ultrafiltration tubes were purchased from Millipore; PVDF membranes and 30% acrylamide were purchased from Bio-Rad Laboratories; ELISA plates were purchased from Keqian Biotech Co., Ltd.; ELISA reagent was purchased from Beyotime Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled Anti-His antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd.; homologous recombination ligase was purchased from Beijing Jinsha Biotechnology Co., Ltd.; tryptone and yeast extract were purchased from OXOID.

[0058] Animal experiment-related reagents and consumables: MONTANIDE™ ISA 201VG adjuvant was purchased from Seppic Company, Shanghai, China;

[0059] Other reagents and consumables: EP tubes (2mL and 1.5mL) and pipette tips (yellow, blue, and white) were purchased from Wuhan Houji Biotechnology Co., Ltd.; sodium chloride (NaCl), disodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O), sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), anhydrous ethanol, methanol, glacial acetic acid, Tween-20, 84 disinfectant, medical alcohol, isopropanol, etc. were purchased from Sinopharm Reagent Co., Ltd.; primer synthesis and fragment sequencing were performed by Wuhan Qingke Innovation Biotechnology Co., Ltd.

[0060] 1.2 Experimental Methods

[0061] 1.2.1 Plasmid Construction

[0062] 1.2.1.1 Primer Design and Synthesis

[0063] Based on existing research on SARS-CoV-2 S protein and PEDV S protein, using the full-length DNA fragments of TGEV WH-1 strain S and PEDV CT P10 strain S as templates, a chimera of spike protein displaying the PEDVSD0-NTD region with the TGEV S CTD-S2 region as the backbone was designed. Two proline mutations (TGEV S: E1139, L1140; PEDV S: S1076, L1077) were designed to improve its expression level and stability. To enable the target protein to be secreted and expressed in HE K-293F cells, the Kozak sequence (GCCACC) was added to the upstream primer, the S protein's own signal peptide was used, and a trimer tag GCN4 (SEQ ID NO.1) was added to the N-terminus of the S protein, followed by a Strep-8×His tag sequence (SEQ ID NO.2) to facilitate protein purification.

[0064] The primers involved in this invention are shown in Table 1.

[0065] Table 1. Primer Design

[0066]

[0067] 1.2.1.2 PCR Amplification

[0068] Table 2 PCR amplification system

[0069]

[0070] The PCR amplification system is shown in Table 2. The reaction conditions are as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 60℃ annealing for 15 s; 72℃ extension for 15 s to 3 min (depending on the size of different amplified fragments, 30 s / 1 kb); 72℃ extension for 10 min; 16℃ for 10 min; 35 cycles for steps two to four. After the process, the product is stored at 4℃.

[0071] 1.2.1.3 Restriction endonuclease digestion

[0072] The restriction endonucleases used for the pCAGGS vector were EcoR I and Xho I. The double digestion system is shown in Table 3.

[0073] Table 3 Double enzyme digestion system

[0074]

[0075] Water bath at 37℃ for 30 minutes.

[0076] 1.2.1.4 Electrophoretic detection of PCR products or enzyme digestion products

[0077] After PCR or enzyme digestion, electrophoresis was performed on a 1% agarose gel, with DL5000 DNA Marker added as a control. The electrophoresis was carried out at 120V for 30 minutes. The PCR products or enzyme digestion products were analyzed by electrophoresis, and the band size was observed and photographed using an agarose gel imaging system. The products were then recovered to obtain the target DNA fragment.

[0078] 1.2.1.5 Ligation of the target fragment to the enzyme digestion vector

[0079] Table 4 Recombination Reaction System

[0080]

[0081] The recombination reaction system is shown in Table 4. When inserting multiple fragments, the optimal amount (ng) for each fragment is 0.02 × the number of base pairs of the fragment. The fragment is then diluted according to the desired fragment recovery concentration and added to the appropriate volume of the recombination reaction system. All components are then gently mixed and reacted at 50°C for 30 min.

[0082] 1.2.1.6 Transformation of ligation products or positive plasmids

[0083] (1) Plate preparation: Add 200 μL of ampicillin antibiotic to 200 mL of LB solid medium at a ratio of 1:1000;

[0084] (2) Add 10 μL of recombinant product or 1 μL of positive plasmid to DH5α competent cells after they have been thawed on ice;

[0085] (3) Ice bath for 30 min, 42℃ water bath heat shock for 45 s, quickly transfer centrifuge tubes to ice, ice bath for 2 min;

[0086] (4) Add 800 μL of antibiotic-free LB liquid culture medium to each tube, gently shake in a shaker at 37°C for 1 hour to allow the bacteria to recover;

[0087] (5) Plate preparation: After centrifugation at 5000r / min for 5min, resuspend the bacterial cells in 100μL of antibiotic-free LB liquid medium, spread them evenly on plates, and incubate the plates at 37℃ for 16h.

[0088] (6) Prepare LB liquid culture medium containing ampicillin antibiotic at a ratio of 1:1000, mix well, add 500 μL to a sterile EP tube, use tweezers to pick up a single colony with a white pipette tip and gently transfer it into the EP tube, and incubate in a shaker at 37°C.

[0089] 1.2.1.7 Bacterial culture PCR identification

[0090] The bacterial culture, which has been incubated at 37°C for 5 hours in a shaker, is removed and visibly turbid; this indicates that bacterial culture PCR identification can be performed. The bacterial culture PCR system is shown in Table 5.

[0091] Table 5 Bacterial PCR System

[0092]

[0093] Reaction conditions: 94℃ pre-denaturation for 10 min; 94℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 15 s, 30 cycles; 72℃ complete extension for 3 min; 16℃ for 2 min; and finally, storage at 4℃. After bacterial PCR, agarose gel electrophoresis analysis was performed. Correctly identified bacterial cultures were sent to Qingke Biotechnology or Hexex Genetic Sequencing.

[0094] 1.2.1.8 Extraction of positive plasmids

[0095] Plasmid mini-extraction was performed using the PlasmidMini Kit for purification.

[0096] 1.2.2 Expression and purification of recombinant protein in HEK-293F cells

[0097] 1.2.2.1 Endotoxin Removal and Plasmid Extraction

[0098] Endotoxin-free plasmid extraction was performed using the EndoFree Maxi Plasmid Kit.

[0099] 1.2.2.2 Transfection of HEK-293F cells

[0100] (1) Take 10 μL of HEK-293F cells in the growth period, mix with 10 μL of trypan blue staining, and add to a cell counting chamber for counting.

[0101] (2) Based on cell density, HEK-293F cells were diluted with culture medium to a density of 2×10⁻⁶. 6 The sample size is 1 / mL, and the total volume is 600mL.

[0102] (3) Add 12 mL of culture medium and 600 μg of recombinant plasmid to a 50 mL centrifuge tube, vortex for 30 s, then add 2.4 mL of PEI 40k transfection reagent in the dark, vortex again for 30 s, and let the mixture stand for 30 min.

[0103] (4) Add the mixture to the cells while shaking the culture flask, place the cells in a 37℃ 8% CO2 shaker, let stand for 6 minutes, shake for 30 seconds, and repeat 4 times.

[0104] (5) Culture the cells in a shaker at 37°C and 8% CO2 in the dark for about 5-6 days.

[0105] 1.2.2.3 Purification and Detection of Recombinant Proteins

[0106] (1) Take the culture flask containing HEK-293F cells transfected for 5-6 days from the shaker, aliquot it into centrifuge bottles, balance it, centrifuge at 2000 r / min at 4℃ for 30 min, pour the supernatant into a clean centrifuge bottle, balance it, centrifuge at 8000 r / min at 4℃ for 30 min.

[0107] (2) Collect the supernatant, filter it with a 0.45μm filter, and then load the supernatant onto the packing material using a peristaltic pump so that the target protein binds to the corresponding packing material.

[0108] (3) Clean the FPLC protein purification instrument with 30 mL Binding Buffer.

[0109] (4) Connect the column to the purification system and elute the target protein with Wash Buffer and Elution Buffer. The elution program is as follows: 100% Wash Buffer, 1 mL / min for 40 min; 10% Elution Buffer, 1 mL / min for 10 mL; 10%-40% Elution Buffer, 1 mL / min for 60 mL; 100% Elution Buffer, 1 mL / min for 30 mL. At the same time, prepare 50 2 mL EP tubes for sample collection, and elute 2 mL / tube.

[0110] (5) After the procedure is completed, connect the column to the peristaltic pump, wash with ddH2O for 50 mL, then wash with 1mol / LNaOH for 50 mL, and finally wash with ddH2O for 50 mL. Store the column at 4℃.

[0111] (6) Cap the EP tube containing the collected protein sample, label it with the serial number, and temporarily store it at 4°C.

[0112] The collected protein samples were analyzed by SDS-PAGE and Western blot.

[0113] 1.2.2.4 Preservation of the target protein

[0114] After SDS-PAGE and Western blot analysis, the target protein with good purity was collected, concentrated to 500 μL by centrifugation at 4000 r / min at 4℃ using a concentration tube, replaced twice with PBS (containing 10% glycerol), and concentrated to 500 μL by centrifugation at 4000 r / min at 4℃ using a concentration tube. After concentration measurement, the protein was aliquoted, flash-frozen in liquid nitrogen, and stored at -80℃.

[0115] 1.2.3 Animal Immunization

[0116] 1.2.3.1 Mouse Immunization

[0117] (1) According to the needs of animal experiments, the corresponding number of 6-week-old BALB / c mice was purchased from the Experimental Animal Center of Huazhong Agricultural University and randomly assigned, with 4 mice in each group.

[0118] (2) Each group of mice was placed in different cages. Before the experiment, the mice were allowed to adapt to the environment for 3 days, and then protein was injected intramuscularly for the first immunization. The immunization dose was 20 μg of protein per mouse (diluted with normal saline), and it was mixed evenly with QuickAntibody-Mouse3W adjuvant at a ratio of 1:1. The MOCK group was injected intramuscularly with normal saline, which was recorded as day 0.

[0119] (3) On the 14th day, the mice were immunized for the second time. On the 21st day after immunization, the blood of the mice was collected to measure the IgG titer and neutralizing antibody.

[0120] (4) During the experiment, the mice were fed with mouse food and provided with additional drinking water every day. At the same time, the cage bedding was changed every day.

[0121] During the above animal experiments, the relevant regulations on animal ethics and welfare were followed, and the experiments were carried out according to the protocol approved by the Scientific Ethics Committee of Huazhong Agricultural University (Experimental Animal License Number: SYXK(E)2020-0084; Ethics Number: HZAUMO-2025-0079).

[0122] 1.2.3.2 Immunization of weaned piglets

[0123] (1) According to the needs of animal experiments, the corresponding number of weaned piglets (negative for PEDV, TGEV antigen and antibody detection) was purchased from Hubei Yingang Pig Farm and randomly divided into 5 groups, with 5 pigs in each group.

[0124] (2) After adjusting the antigen protein concentration to 100 μg / mL, it was mixed with MONTANIDETM ISA 201VG adjuvant in a water bath at 31 °C for 20 - 30 min and then emulsified by mixing at a ratio of 1:1 to make the final concentration of the antigen protein 50 μg / mL, that is, each pig was inoculated once with 2 mL, containing 100 μg of antigen protein. The MOCK group was injected intramuscularly with PBS (emulsified with adjuvant) in the neck.

[0125] (3) On day 0 and day 14, the piglets were immunized for the first and second times respectively. After immunization, the blood of the piglets was collected every 7 days to separate serum for measuring the neutralizing antibody titers against TGEV and PEDV.

[0126] (4) During the experiment, the piglets were fed with feed and provided with additional drinking water every day. At the same time, the feeding environment was cleaned every day.

[0127] During the above animal experiments, the relevant regulations on the ethics and welfare of experimental animals were followed, and the experiments were carried out in accordance with the protocol approved by the Scientific Ethics Committee of Huazhong Agricultural University (Experimental Animal License Number: SYXK(E)2020-0084; Ethics Number: HZAUSW-2025-0027).

[0128] 1.2.4 Indirect ELISA

[0129] The IgG titer against the S protein in the immune sera of each protein was detected by indirect ELISA, and the specific operation steps were as follows:

[0130] (1) Coating: Add 100 μL / well of His-tagged S protein with a concentration of 10 μg / mL to the ELISA plate and coat it overnight at 4°C.

[0131] (2) Blocking: Discard the coating solution, add 200 μL of PBST to each well and wash the plate 3 times, 5 minutes each time. Pat dry, then add 100 μL / well of 2% BSA blocking solution and block at room temperature for 1 h.

[0132] (3) Primary antibody dilution: Add 396 μL of blocking solution to the first tube of each sample, and add 360 μL of blocking solution to the remaining tubes. After vortexing and centrifuging the serum, take 4 μL and add it to the first tube. After vortexing and centrifuging the first tube, take 40 μL and add it to the second tube, and dilute sequentially to the last tube.

[0133] (4) Primary antibody incubation: Discard the blocking solution, add 200 μL of PBST to each well and wash the plate 3 times, 5 minutes each time. After patting dry, add 100 μL of the diluted mouse serum after immunization with each protein to each well and incubate at 37°C for 1 h.

[0134] (5) Secondary antibody incubation: Discard the primary antibody, add 200 μL of PBST to each well and wash the plate 3 times, 5 minutes each time. Then add 100 μL of HRP-labeled Anti-His mouse antibody to each well and incubate at 37°C in the dark for 1 h.

[0135] (6) Discard the secondary antibody, add 200 μL of PBST to each well and wash the plate 3 times, 5 minutes each time. Add 50 μL of TMB chromogenic solution to each well, terminate the reaction after 7 minutes with ELISA termination solution, and finally read the OD 630nm and OD 450nm absorbance value.

[0136] 1.2.5 Virus neutralization experiment

[0137] 1.2.5.1 Virus TCID 50 Determination

[0138] Determination of TGEV virus TCID 50 Determination:

[0139] (1) Prepare cells in good growth condition, digest them with trypsin after they have grown to about 80%, and then plate them into 96-well plates.

[0140] (2) After 12 hours, the virus was serially diluted 10-fold in a sterile 1.5 mL EP tube with maintenance solution. The amount of virus suspension in each well was 100 μL. Each dilution was replicated in 8 wells. Cell control wells were also prepared. The plate was shaken well and then transferred to a cell culture incubator at 37°C and 5% CO2.

[0141] (3) After 48 hours, the 96-well plate was removed and the cells were observed under a microscope day by day, and the results were recorded.

[0142] (4) Calculate the virus TCID using the Reed and Muench methods. 50 .

[0143] PEDV virus TCID 50 Measurement:

[0144] (1) Prepare cells in good growth condition, digest them with trypsin after they have grown to about 90%, and then plate them into 96-well plates.

[0145] (2) After 24 hours, the virus was serially diluted 10-fold in a sterile 1.5 mL EP tube with serum-free culture medium. The volume of virus suspension in each well was 100 μL. Each dilution was replicated in 8 wells. Cell control wells were also prepared. The tubes were shaken to mix well.

[0146] (3) Take out the 96-well plate, discard the original culture medium, add 100 μL PBS to each well, wash twice, and then add 100 μL of the corresponding virus suspension to each well. Transfer the 96-well plate to a cell culture incubator at 37°C and 5% CO2 and incubate for 1 hour.

[0147] (4) After incubation, discard the liquid in the 96-well plate, add 100 μL of culture medium containing 10 μg / mL trypsin and without serum to wash once, then add 100 μL of culture medium containing 10 μg / mL trypsin and without serum, and put it back into the 37℃ incubator for incubation.

[0148] (5) After 24 hours, the 96-well plate was removed and the cells were observed under a microscope day by day, and the results were recorded.

[0149] (6) Calculate the virus TCID using the Reed-Muench method 50 .

[0150] 1.2.5.2 Virus neutralization experiment of TGEV

[0151] (1) Lay the cells required for the determination of neutralizing antibodies into a 96-well plate one day in advance.

[0152] (2) After the serum to be tested is inactivated at 56℃ for 30 min, it is continuously diluted 2-fold with maintenance solution.

[0153] (3) Measure the TCID 50 TGEV virus solution diluted to 100 TCID 50 / 50μL of virus solution.

[0154] (4) Take 250 μL of serially diluted serum and mix it with 250 μL of virus solution, and incubate at 37°C for 1 h.

[0155] (5) Inoculate the mixture into a monolayer of PK-15 cells cultured in a 96-well plate, 100 μL / well, and make 4 replicates for each dilution.

[0156] (6) Assume 100TCID 50 / 100μL, 10TCID 50 / 100μL, 1TCID 50 / 100μL, 0.1TCID 50 A 100 μL virus control and a column of cell controls were prepared, with 8 wells in each control.

[0157] (7) After placing the 96-well plate in a 37℃ 5% CO2 incubator for 48 hours, observe the cytopathic effect (CPE) and record the number of wells with cytopathic effects. Observe for 48-72 hours.

[0158] (8) The serum neutralization titer was calculated using the Reed-Muench method based on the number of cytopathic wells.

[0159] 1.2.5.3 PEDV Virus Neutralization Experiment

[0160] (1) Lay the cells required for the determination of neutralizing antibodies into a 96-well plate one day in advance.

[0161] (2) After the serum to be tested is inactivated at 56°C for 30 min, it is continuously diluted 2-fold with a culture medium that does not contain serum or trypsin.

[0162] (3) Measure the TCID 50 The PEDV virus solution was diluted to 100 TCID using serum-free and trypsin-free medium. 50 / 50μL of virus solution.

[0163] (4) Take 250 μL of serially diluted serum and mix it with 250 μL of virus solution, and incubate at 37°C for 1 h.

[0164] (5) Discard the original culture medium from the 96-well plate and wash it twice with PBS.

[0165] (6) Inoculate the mixture into a monolayer of Vero cells cultured in a 96-well plate, 100 μL / well, and perform 4 replicates for each dilution.

[0166] (7) Assume 100TCID 50 / 100μL, 10TCID 50 / 100μL, 1TCID 50 / 100μL, 0.1TCID 50 A 100 μL virus control and a column of cell controls were prepared, with 8 wells in each control.

[0167] (8) Place the 96-well plate in a 37℃ 5% CO2 incubator for 1 h, aspirate the inoculum, wash once with culture medium containing 10 μg / mL trypsin and no serum, add 100 μL of DMEM containing 10 μg / mL trypsin to each well, observe cytopathic effect (CPE) daily and record the number of wells with cytopathic effect, and observe for 24-72 h.

[0168] (9) The serum neutralization titer was calculated using the Reed-Muench method based on the number of cytopathic pores.

[0169] 1.2.6 Biological Software and Databases

[0170] Biological software includes: SnapGene, PyMOL, GraphPad Prism, etc.

[0171] Online analysis software includes: ABCpred, BepiPred2.0, BcePred, ESPript3server, multalin, Swiss Model, etc.

[0172] Databases: PDB (https: / / www.rcsb.org / ), NCBI (https: / / www.ncbi.nlm.nih.gov / ), IEDB (https: / / www.iedb.org), etc.

[0173] 1.2.7 Statistical Analysis

[0174] All statistical analyses were performed using GraphPad Prism 8.0.2 software. Data were evaluated using ANOVA and t-tests. A p-value > 0.05 was considered not significant (ns), p-value < 0.05 was considered statistically significant (*), p-value < 0.01 was considered statistically significant (**), p-value < 0.001 was considered highly significant (***), and p-value < 0.0001 was considered extremely significant (****).

[0175] 2. Experimental Results

[0176] 2.1 Expression of the full length of TGEV and PEDV S-2P

[0177] 2.1.1 Identification of mutation sites by comparing with SARS-CoV-2-S protein sequences

[0178] To improve the stability and expression levels of TGEV and PEDV S proteins, a pre-fusion conformation of the S protein was designed, referencing the SARS-COV-2 strategy of enhancing stability through proline mutation. To determine the proline mutation sites in TGEV and PEDV S proteins, the S protein sequences of wild-type SARS-COV-2, TGEV WH-1, and PEDV CT P10 strains were downloaded from NCBI. Amino acid sequence alignment of these three strains was performed using the online websites mutalin and ESPript3server. It was found that the proline mutation sites corresponding to amino acids 986-987 in the PEDV S protein and SARS-COV-2 S protein were serine at position 1076 and leucine at position 1077, respectively. Similarly, the proline mutation sites corresponding to amino acids 986-987 in the TGEV S protein and SARS-COV-2 S protein were glutamate at position 1139 and leucine at position 1140, respectively. Figure 1 Mutating these sites to proline disrupts the formation of secondary structures, creating a rigid helix-loop-helix structure that maintains the S protein in its pre-fusion conformation.

[0179] 2.1.2 Amplification of the full-length gene fragments of TGEV and PEDV S

[0180] To obtain TGEV and PEDV S proteins, in addition to secretory expression using their own signal peptides, a GCN4 sequence was added to the C-terminus of the S protein sequence to form a trimer structure, and then a Strep-8×His tag was introduced at the posterior end for purification. Figure 2 Based on the target protein construction strategy, using the cDNA sequences of TGEV S and PEDV S as templates, relevant primers were designed, and the fragments TGEV-S-1, TGEV-S-2, PEDV-S-1, PEDV-S-2, and GCN4-8×His were amplified by PCR. The gel electrophoresis results showed that the amplified target gene fragments appeared at 3458bp, 780bp, 3269bp, 762bp, and 211bp, respectively, which were consistent with the expected size. The target gene fragments TGEV-S-1, TGEV-S-2, PEDV-S-1, PEDV-S-2, and GCN4-8×His were successfully amplified. Figures 3-4 ).

[0181] 2.1.3 Identification of full-length recombinant plasmids of TGEV and PEDV S

[0182] After gel extraction of the obtained target gene, it was analyzed according to the plasmid map ( Figure 5 The target gene was ligated into the restriction enzyme vector pCAGGS and transformed. After incubation at 37℃ for 16 hours, single colonies were picked for bacterial PCR identification. Agarose gel electrophoresis showed that the target bands appeared at 780bp and 762bp, respectively. The identified fragments were TGEV-S-2 and PEDV-S-2, consistent with the expected sizes. Positive bacterial cultures containing the TGEV and PEDV S-2P recombinant plasmids were successfully obtained. Figure 6 The positive bacterial culture was sent to a co-testing gene sequencing site for sequencing. The results showed that the 2P mutation of TGEV and PEDV S was successfully performed, and the correctly sequenced positive plasmid was obtained. Figure 7 ).

[0183] 2.1.4 Large-scale expression and purification of TGEV and PEDV S proteins in HEK-293F cells

[0184] TGEV S-2P and PEDV S-2P plasmids were transfected into 600 mL of healthy, growing HEK-293F cells for high-level expression. Supernatants were collected on days 5-6 post-transfection and purified using Ni-TED packing material. Samples from different collection tubes were then analyzed by SDS-PAGE. Figure 8 Based on the results, protein solutions from collection tubes with high purity were selected for concentration.

[0185] 2.1.5 SDS-PAGE and Western Blot were used to identify protein expression.

[0186] Take 16 μL of the concentrated protein solution for SDS-PAGE, stain with Coomassie Brilliant Blue, and then destain. Take another 16 μL for Western blotting, incubate with 6×His-HRP antibody, and then develop. Based on the gel image and development results (… Figure 9 The target bands were all located at around 180 kDa, consistent with the expected size of the target protein, and the TGEV S-2P and PEDV S-2P proteins were successfully purified.

[0187] 2.1.6 Comparison of the immunogenicity of TGEV and PEDV S protein

[0188] 2.1.6.1 TGEV and PEDV S protein immunization strategy

[0189] Purified TGEV S-2P and PEDV S-2P proteins were used to immunize mice. Six-week-old BALB / c mice were purchased from the Experimental Animal Center of Huazhong Agricultural University, and four mice were randomly selected as a group. Each mouse in the experimental group was immunized with 20 μg of protein in the thigh muscle, while each mouse in the control group was immunized with physiological saline. The first and second immunizations were performed on day 0 and day 14, respectively, and blood was collected on day 21. Figure 10 The whole blood was placed in a 37°C water bath for 1 hour, and the precipitated serum was separated, aliquoted, and stored at -80°C.

[0190] 2.1.6.2 Detection of Specific Antibody IgG for TGEV and PEDV S Protein

[0191] Serums immunized with TGEV S-2P and PEDV S-2P proteins, respectively, were subjected to indirect ELISA to determine the titer of nonspecific IgG in the serum. TGEV S-2P and PEDV S-2P proteins were used as antigens for detection, with immune serum as the primary antibody and goat anti-mouse IgG as the secondary antibody. Results are as follows: Figure 11 As shown, serum immunized against TGEV S-2P protein produced IgG titers against TGEV S-2P protein up to 1:1,000,000, while serum immunized against PEDV S-2P protein produced IgG titers against TGEV S-2P protein at 1:10,000. Similarly, serum immunized against PEDV S-2P protein produced IgG titers against PEDV S-2P protein up to 1:1,000,000, while serum immunized against TGEV S-2P protein produced IgG titers against PEDV S-2P protein at 1:10,000. Therefore, the IgG titers produced by immunization against TGEV S-2P and PEDV S-2P are similar, and the two exhibit weak cross-protection at the level of specific antibody IgG.

[0192] 2.1.6.3 Detection of neutralizing antibodies and cross-protection against TGEV and PEDV S proteins

[0193] Serum from mice immunized with TGEV S-2P and PEDV S-2P proteins was used to determine the neutralizing titers against TGEV WH-1 and PEDVCT P10 strains, respectively, and to determine whether there was cross-protection between the two strains. Results are as follows: Figure 12As shown, serum obtained after immunizing mice with TGEV S-2P protein produced high levels of neutralizing antibodies, with an average neutralizing titer of 1:7491, while serum obtained after immunizing mice with PEDV S-2P protein produced low levels of neutralizing antibodies, with an average neutralizing titer of 1:123. Therefore, the neutralizing effect of immunization with TGEV S-2P protein is significantly better than that of immunization with PEDV S-2P protein, and there is no cross-protection between the two.

[0194] 2.2 Construction, expression and evaluation of spike protein chimeras

[0195] 2.2.1 Construction strategy of spike protein chimera

[0196] Based on existing research, it has been confirmed that the immune response of the coronavirus S protein maintaining its pre-fusion trimeric structure is superior to that of the monomeric S1 protein and CTD protein. Furthermore, the TGEV S protein exhibits better immune response than the PEDV S protein of the same species. This is presumably because the TGEV S protein backbone better displays the various domains of its S1 structure. Additionally, the highly variable region of the PEDV S protein, concentrated in the D0-NTD region in recent years, possesses potential for vaccine design. Therefore, the D0-NTD domain of PEDV S was replaced on the TGEV S protein to display the PEDV S0-NTD domain, with the aim of obtaining an immune spike protein chimera that can provide better protection against PEDV or simultaneously generate immune protection against both TGEV and PEDV. See the detailed construction diagram below. Figure 13 .

[0197] The spike protein chimera of the present invention is composed of the CTD to S2 regions of the extracellular domain of the TGEV S protein as a backbone, and the D0-NTD region of the PEDV S protein is intercalated to form it. It is named TGEV S (replacing D0-NTD), and its amino acid sequence is shown in SEQ ID NO. 15. After mammalian codon optimization, the nucleotide sequence of the coding gene for the spike protein chimera TGEV S (replacing D0-NTD) is shown in SEQ ID NO. 16.

[0198] 2.2.2 Amplification of spike protein chimeric gene fragments

[0199] Based on the spike protein chimera construction strategy, a corresponding plasmid map was designed, and specific amplification primers with homologous arms were synthesized. The target fragment was amplified using PCR. According to the agarose gel electrophoresis results, the sizes of the amplified fragments matched the target fragment size, successfully amplifying the desired target gene. Figure 14 ).

[0200] 2.2.3 PCR identification of spike protein chimera bacterial culture

[0201] After gel recovery of the obtained target gene, homologous recombination was performed with the enzyme-digested vector pCAGGS according to the plasmid map. The recombination product was then added to competent DH5α cells for transformation. After 16 hours of culture, single colonies were picked and cultured in LB broth containing ampicillin for 4 hours on a shaker. Colony PCR was then performed. Agarose gel electrophoresis confirmed that the target band appeared at 780 bp, consistent with the size of the identified TGEV-S-2 fragment, successfully obtaining positive bacterial cultures for each spike protein chimera. Figure 15 ).

[0202] 2.2.4 Exploring the expression time of spike protein chimeras

[0203] The recombinant plasmid of the spike protein chimera TGEV S (replacing D0-NTD) was transfected into 50 mL of healthy HEK-293F cells for low-level expression. Cell supernatants were collected from day 2 to day 7, and cell status was observed. Microscopically, cell density continued to increase from day 2 to day 4, but the proliferation rate gradually slowed down, reaching its peak on day 5. On day 6, cell status began to deteriorate significantly, with a marked increase in cell debris. On day 7, a thickened ring of dead cells appeared on the flask wall. Western blotting was performed on the cell supernatants from day 2 to day 7. The results showed that the content of the spike protein chimera increased daily, reaching its highest level on day 6, and significant protein degradation began to occur on day 7, with a decrease in content. Figure 16 Based on the above results, the sixth day is the optimal time for sample collection.

[0204] 2.2.5 Large-scale expression and purification of spike protein chimeras

[0205] The recombinant plasmid of the spike protein chimera TGEV S (replacing DO-NTD) was transfected into 600 mL of well-grown HEK-293F cells for high-level expression. Samples were collected at the optimal expression time, and the protein in the cell supernatant was bound to Ni-TED packing material. After purification, samples from collection tubes showing peaks were subjected to SDS-PAGE identification. Protein solutions from collection tubes with higher purity were selected and concentrated to obtain the corresponding protein. Figure 17 ).

[0206] 2.2.6 SDS-PAGE and Western Blot were used to identify protein expression.

[0207] Based on the SDS-PAGE gel image, proteins from tubes 26-42 are typically collected and concentrated. After replacement with PBS and 10% glycerol, the concentrate is brought to 500 μL. 16 μL samples are then prepared and subjected to SDS-PAGE and Western blotting to identify the target protein. Figure 18As shown, the target bands appeared at around 180 kDa, and the size was consistent, indicating that the spike protein chimera was successfully purified.

[0208] 2.3.1 Immunogenicity evaluation of candidate subunit vaccines in weaned piglets

[0209] 2.3.1.1 Immunization Strategy for Candidate Subunit Vaccines

[0210] To compare the immunogenicity of the spike protein chimera, TGEV S (replaced D0-NTD) protein was selected as the experimental group, TGEV S-2P and PEDV S-2P were used as positive control groups, and PBS was used as the negative control group. A subunit vaccine of 2 mL / head was prepared by fully emulsifying the antigen dose (100 μg / head) with adjuvant and administering it to weaned piglets. Five piglets were immunized in each group, receiving the first and second immunizations on days 0 and 14. Blood samples were collected every 7 days to determine the neutralizing titer. Figure 19 ).

[0211] 2.3.1.2 Neutralizing antibody detection of candidate subunit vaccines

[0212] The weekly serum samples were analyzed for neutralizing titers against TGEV and PEDV to assess antibody titers and cross-protection. Results are as follows: Figure 20 As shown, in the positive control group, the TGEV S-2P protein group had the highest antibody level on day 21, with an average of 1:25927, while the PEDV S-2P protein group had the highest antibody level on day 35, with an average of 1:158. In the experimental groups, the TGEV S (replaced D0-NTD) group produced neutralizing antibody levels close to those of the TGEV S group, with the highest average neutralizing antibody titer on day 21, reaching 1:12129. It also produced neutralizing antibodies against PEDV, with the highest average neutralizing antibody titer on day 28, reaching 1:57.

[0213] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A spike protein chimera, characterized in that, The amino acid sequence is shown as SEQ ID NO.

15.

2. A coding gene of the Spike protein chimera according to claim 1.

3. The genetic code according to claim 2, wherein, The nucleotide sequence of the coding gene is shown as SEQ ID NO.

16.

4. A recombinant plasmid, characterized by comprising the nucleotide sequence of SEQ ID NO:

1. comprising the coding gene according to claim 2 or 3.

5. A recombinant host cell, characterized in that, comprising the recombinant plasmid according to claim 4.

6. The recombinant host cell of claim 5, wherein, The recombinant host cell is a recombinant HEK293F cell.

7. Use of the coding gene according to claim 2 or 3, the recombinant plasmid according to claim 4, or the recombinant host cell according to claim 5 or 6 in the preparation of the Spike protein chimera according to claim 1.

8. Use of the Spike protein chimera according to claim 1 in the preparation of a bivalent subunit vaccine.

9. A method for the preparation of a diphtheria-tetanus subunit vaccine, characterized in that, comprising the step of mixing the Spike protein chimera according to claim 1 with a vaccine adjuvant uniformly to obtain the bivalent subunit vaccine.

10. A dvalent subunit vaccine, characterized in that, The active ingredient comprises the Spike protein chimera according to claim 1.

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