PEDV (porcine epidemic diarrhea virus) and TGEV (transmissible gastroenteritis virus) spike protein chimera, bigeminy subunit vaccine as well as preparation method and application of bigeminy subunit vaccine

Abstract

The invention relates to the technical field of biology, in particular to a PEDV (porcine epidemic diarrhea virus) and TGEV (transmissible gastroenteritis virus) spike protein chimera, a bivalent subunit vaccine as well as a preparation method and application of the bivalent subunit vaccine. The amino acid sequence of the spike protein chimera is as shown in SEQ ID NO. 13. According to the invention, the TGEV S protein is subjected to mutation treatment, so that the formation of a secondary structure is destroyed, the S protein keeps the conformation before fusion, and a TGEV S D0-NTD region is further replaced by a PEDV S D0-NTD region on the basis that the TGEV S keeps the trimer conformation before fusion, so that the spike protein chimera is obtained. When the spike protein chimera is used for immunization, a neutralizing antibody aiming at the PEDV and the TGEV at the same time can be generated, and the spike protein chimera has the effect of preventing and controlling the PEDV and the TGEV at the same time.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a PEDV and TGEV spike protein chimera, a two-subunit vaccine, and a preparation method and application thereof. Background Art

[0002] Porcine transmissible gastroenteritis virus (TGEV) is a member of the porcine enteric coronavirus (SeCoV). It spreads rapidly and can infect pigs of different ages, mainly causing severe diarrhea, vomiting, dehydration and high mortality in piglets under two weeks of age. TGEV mainly infects the small intestine of pigs, causing intestinal villus atrophy, decreased villus height and crypt depth, and a decrease in the number of SIgA-positive cells and dendritic cells in the jejunum. Although the current incidence of TGEV is low and the vaccine prevention and control effect is good, mixed infection with other viruses has been detected in recent years. For example, mixed infection of TGEV with PEDV is more common, and there is a phenomenon of recombination, such as the TGEVAHHF strain and the TGEV JS2012 strain. At the same time, TGEV also has the risk of cross-species transmission. Therefore, the prevention and control of TGEV cannot be relaxed.

[0003] PEDV is also a member of the porcine enteric coronavirus family, belonging to the same alphacoronavirus family as TGEV. Its clinical symptoms are similar to those of TGEV. It is an acute enteric infectious disease that can infect pigs of all ages, primarily resulting in a mortality rate exceeding 90% in piglets. PEDV pathological changes are primarily seen in the gastrointestinal tract, characterized by marked stomach dilation, the presence of undigested curds in the stomach, marked atrophy of the intestinal villi, thinning of the intestinal wall to near transparency, yellowish fluid accumulation in the intestine, and significant enterocyte vacuolation. Severe atrophic enteritis may also be seen in the jejunum. Since the PEDV outbreak in 2010, highly virulent strains have gradually become prevalent, and existing commercial vaccines do not provide complete protection. Therefore, the development of a safe and effective PEDV vaccine is urgently needed for PEDV prevention and control.

[0004] The coronavirus's spike protein plays a key role in the process of viral infection of cells, and the exercise of this role marks the beginning of the virus's life cycle. The coronavirus's S protein sequence contains approximately 1,300 amino acids and is approximately 180 kDa in size. The S protein mainly consists of two subunits, one is S1, which is responsible for receptor binding, and the other is S2, which is responsible for membrane fusion. During the coronavirus invasion process, the S protein must first recognize the receptor. The realization of this process is mainly related to the S protein's S1-CTD (protein receptor binding) and S1-NTD (sugar receptor binding). Another key function of the S protein is to use the S2 subunit to achieve membrane fusion. Therefore, the S protein is considered to be an important protein for inducing the production of neutralizing antibodies.

[0005] Currently, traditional vaccines are unable to promptly respond to outbreaks caused by variant strains. Subunit vaccines are believed to be more responsive to the emergence of variants. They do not require the entire virus; instead, they can be rapidly produced by replacing protein sequences corresponding to prevalent strains, resulting in high levels of effective antigens. Therefore, the present invention proposes to develop a novel subunit vaccine, thereby providing powerful technical support for the prevention and control of PEDV and TGEV. Summary of the Invention

[0006] The present invention aims to provide a PEDV and TGEV spike protein chimera, a bivalent subunit vaccine, and its preparation and use to address the aforementioned problems of the prior art. Immunization with this spike protein chimera can produce neutralizing antibodies against both PEDV and TGEV, effectively preventing and controlling both PEDV and TGEV.

[0007] To achieve the above object, the present invention provides the following solutions:

[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 as SEQ ID NO.16.

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

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

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

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

[0015] The present invention also provides the use of the above-mentioned spike protein chimera in the preparation of a two-subunit vaccine.

[0016] The present invention also provides a method for preparing a bivalent subunit vaccine, comprising the steps of uniformly mixing the above-mentioned spike protein chimera with a vaccine adjuvant 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] The present invention mutates the 1139th glutamic acid (Glutamic) and the 1140th leucine (Leucine) of the TGEV S protein to proline (Proline), respectively, thereby destroying the formation of the secondary structure and forming a rigid helix-loop-helix structure, so that the S protein maintains the pre-fusion conformation, thereby improving the stability and expression level. Further, on the basis of maintaining the pre-fusion trimer conformation of TGEV S, the TGEV SD0-NTD region is replaced with the PEDV SD0-NTD region, and HEK-293F cells are used for eukaryotic expression to obtain a spike protein chimera with higher purity. Among them, the HEK-293F cell expression system has the advantages of suspension culture, no need for serum and no introduction of exogenous viruses, and a simple production process. The expressed protein has good immunogenicity and the like.

[0020] The present invention used the spike protein chimera as an immunogen for animal experiments. Because it contained the core region of TGEV S protein that induces the production of neutralizing antibodies - the RBD region, and the region of PEDV S protein that binds to sugar receptors - the D0-NTD region, it produced a certain degree of neutralizing antibodies against PEDV and TGEV, achieving "one shot, two protections" and having the effect of simultaneously preventing and controlling PEDV and TGEV. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Schematic diagram for determining the 2P mutation site by alignment with the SARS-COV-2 sequence;

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

[0024] Figure 3 This is the PCR amplification result of TGEV S gene and tag GCN4-8×His;

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

[0026] Figure 5 The maps of TGEV S-2P recombinant plasmid (A) and PEDV S-2P recombinant plasmid (B);

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

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

[0029] Figure 8 The purification results of TGEV and PEDV S-2P proteins are shown in Figure 1. A is the purified peak of TGEV S-2P protein.

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

[0031] Purification peak of PEDV S-2P protein; D is the SDS-PAGE image of the target protein in the 19th-42nd collection tube corresponding to the purification peak of PEDV S-2P protein;

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

[0033] Figure 10 Schematic diagram of the mouse immunization scheme;

[0034] Figure 11 The results of indirect ELISA testing of IgG titers in mice immunized with TGEV and PEDV S-2P proteins are shown in Figure 1. A shows the results of the test using TGEV S-2P protein as an antigen; B shows the results of the test using PEDV S-2P protein as an antigen.

[0035] Figure 12 The results of the neutralization titer and cross-protection test of the serum of mice immunized with TGEV and PEDV S-2P proteins are shown in Table 1. A is the neutralizing antibody titer test result against TGEV; B is the neutralizing antibody titer test result against PEDV.

[0036] Figure 13 Schematic diagram for the construction of spike protein chimeras;

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

[0038] Figure 15 This is the result of PCR identification of the spike protein chimera bacterial solution;

[0039] Figure 16 This is the result diagram of the optimization of the optimal expression time of the spike protein chimera;

[0040] Figure 17 The purification results of the spike protein chimera; A is the purification peak of TGEV S (replacing D0-NTD) protein; B is the SDS-PAGE image of the target protein in the 19th to 42nd collection tubes corresponding to the purification peak of GEV S (replacing D0-NTD) protein;

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

[0042] Figure 19 This is a schematic diagram of the immunization and blood collection time for weaned piglets;

[0043] Figure 20 The monitoring results of the neutralizing antibody levels of TGEV and PEDV are shown. A is the neutralizing antibody titer test result for TGEV; B is the neutralizing antibody titer test result for PEDV; TGEV S represents the TGEV S-2P protein group; PEDV S represents the PEDV S-2P protein group. DETAILED DESCRIPTION

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

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0046] Example 1

[0047] 1. Materials and Methods

[0048] 1.1 Experimental Materials

[0049] 1.1.1 Biomaterials

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

[0051] African green monkey kidney cells (Vero), porcine kidney cells (PK-15), and human embryonic kidney cells-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 a gift from Professor He Qigai.

[0053] The full-length DNA fragments of PEDV CT P10-S and TGEV WH-1-S (both codon-optimized for mammalian cell preference) were synthesized by Nanjing GenScript Biotech Co., Ltd. The eukaryotic expression vector pCAGGS was purchased from Addgene.

[0054] The experimental animals, weaned piglets, were from Hubei Yingang Pig Farm.

[0055] 1.1.2 Main reagents and consumables

[0056] Cell culture-related reagents and consumables: PEI40K transfection reagent was prepared and stored in our laboratory; trypsin powder and protein marker were purchased from Thermo Fisher Scientific; 293 culture medium (serum-free) was purchased from Yonglian Biotechnology (Shanghai) Co., Ltd.; 4.5 g / L high-glucose DMEM was purchased from Gibco; fetal bovine serum (FBS) was purchased from Wuhan Qingmu Biotechnology Co., Ltd.; penicillin-streptomycin antibiotics and ECL chemiluminescence colorimetric solution were purchased from Shanghai 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 Biochemical Technology 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; and serum-free cell freezing medium was purchased from Xinsaimei Biotechnology Co., Ltd.

[0057] Molecular cloning-related reagents and consumables: 2×Phanta Max Master Mix was purchased from Nanjing Novozymes Biotechnology Co., Ltd.; 2×MIX Taq enzyme was purchased from Beijing Zhuangmeng Biotechnology Co., Ltd.; restriction endonucleases Xho I and EcoR I were purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.; PCR tubes and disposable culture dishes were purchased from Wuhan Houji Biological Co., Ltd.; nucleic acid dye Goldview was purchased from Xi'an Het Biotechnology Co., Ltd.; DNAMarker (DL2000, DL5000, and DL15000) and high-purity low-electrosmotic agarose were purchased from Wuhan Qingke Innovation Biotechnology Co., Ltd.; plasmid miniprep kit and gel recovery kit were purchased from OMEGA; endotoxin-free large-scale plasmid extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.; agar, imidazole, and ampicillin (Amp) were purchased from Biosharp; Ni-TED Sepharose 6FF (His-Tag) was purchased from Sangon Biotechnology Co., Ltd.; 0.22 μm filter, 0.45 μm filter, and ultrafiltration tube were purchased from Millipore; PVDF membrane and 30% acrylamide were purchased from Bio-Rad; ELISA plate was purchased from Keqian Company; ELISA colorimetric solution was purchased from Shanghai 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 (TRYPTONE) and yeast extract (YEAST EXTRACT) were purchased from OXOID Company.

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

[0059] Other reagents and consumables: EP tubes (2 mL and 1.5 mL) and pipette tips (yellow, blue, and white) were purchased from Wuhan Houji Biological 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 Company; primer synthesis and fragment sequencing were completed 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 the existing research on SARS-COV-2S protein and PEDV S protein, the full-length DNA fragment of TGEV WH-1 strain S and the full-length DNA fragment of PEDV CT P10 strain S were used as templates to design a spike protein chimera with the TGEV S CTD-S2 region as the skeleton to display the PEDVSD0-NTD region. The present invention designed two proline mutations (TGEV S: E1139, L1140; PEDV S: S1076, L1077) to improve its expression level and stability. In order to enable the target protein to be secreted and expressed in HE K-293F cells, when designing the primers, a 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, and a Strep-8×His tag sequence (SEQID NO.2) was added thereafter to facilitate protein purification.

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

[0065] Table 1 Related 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 were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s; annealing at 60°C for 15 s; extension at 72°C for 15 s to 3 min (depending on the size of the amplified fragment, 30 s / 1 kb), extension at 72°C for 10 min; and 16°C for 10 min. Steps 2 to 4 were repeated 35 times, followed by storage at 4°C.

[0071] 1.2.1.3 Restriction enzyme digestion

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

[0073] Table 3 Double enzyme digestion system

[0074]

[0075] Incubate in a 37°C water bath for 30 minutes.

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

[0077] After PCR or enzyme digestion, perform electrophoresis on a 1% agarose gel with a DL5000 DNA marker as a control and separate at 120 V for 30 minutes. Analyze the PCR or enzyme digestion products by electrophoresis. Observe the band sizes using an agarose gel imager and take photos for preservation. Product recovery is then performed to obtain the desired DNA fragment.

[0078] 1.2.1.5 Ligation of target fragment and enzyme-digested 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 of each fragment (ng) = 0.02 × the number of base pairs in the fragment. Dilute the fragment according to the recovered concentration of the target fragment and add the appropriate volume to the recombination reaction system. Gently mix all components and react at 50°C for 30 minutes.

[0082] 1.2.1.6 Transformation of ligation products or positive plasmids

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

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

[0085] (3) Ice bath for 30 min, heat shock in a 42°C water bath for 45 s, quickly transfer the centrifuge tube to ice, and ice bath for 2 min;

[0086] (4) Add 800 μL of antibiotic-free LB liquid medium to each tube and incubate at 37°C with gentle shaking for 1 h to allow the bacteria to recover;

[0087] (5) Plate: After centrifugation at 5000 rpm for 5 min, resuspend the bacteria in 100 μL of antibiotic-free LB liquid medium, spread evenly on the plate, and incubate the plate at 37°C for 16 h.

[0088] (6) Prepare LB liquid culture medium containing ampicillin antibiotics at a ratio of 1:1000, mix well, and add 500 μL to a sterilized EP tube. Use tweezers to pick up a white pipette tip and lightly dip a single colony into the EP tube. Culture in a shaking incubator at 37°C.

[0089] 1.2.1.7 PCR identification of bacterial fluid

[0090] After incubating at 37°C for 5 hours on a shaker, remove the bacterial solution. If it becomes turbid to the naked eye, PCR identification of the bacterial solution can be performed. The bacterial solution PCR system is shown in Table 5.

[0091] Table 5 Bacterial liquid PCR system

[0092]

[0093] Reaction conditions: 94°C pre-denaturation for 10 minutes; 30 cycles of 94°C denaturation for 15 seconds, 60°C annealing for 15 seconds, and 72°C extension for 15 seconds; 72°C full extension for 3 minutes; 16°C for 2 minutes; and storage at 4°C. After PCR, analyze the bacterial solution by agarose gel electrophoresis. Select the correct bacterial solution and send it to Qingke Biotechnology or Hesen Gene for sequencing.

[0094] 1.2.1.8 Extraction of positive plasmid

[0095] Plasmids were purified using the Plasmid Mini Kit.

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

[0097] 1.2.2.1 Endotoxin removal and plasmid extraction

[0098] EndoFree Maxi Plasmid Kit was used for endotoxin-free plasmid extraction.

[0099] 1.2.2.2 Transfection of HEK-293F cells

[0100] (1) Take 10 μL of HEK-293F cells in the growth phase and mix with 10 μL of trypan blue stain, then add to the cell counting plate for counting.

[0101] (2) According to the cell density, HEK-293F cells were diluted with culture medium to a density of 2×10 6 / mL, 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 and vortex for 30 seconds. Then, add 2.4 mL of PEI 40k transfection reagent in the dark, vortex again for 30 seconds, and let the mixture stand for 30 minutes.

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

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

[0105] 1.2.2.3 Purification and detection of recombinant proteins

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

[0107] (2) Collect the supernatant, filter it with a 0.45 μm filter, and then load the supernatant with a peristaltic pump to allow the target protein to bind to the corresponding filler.

[0108] (3) Wash the FPLC protein purifier with 30 mL of Binding Buffer.

[0109] (4) Connect the column to the purifier 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; and 100% Elution Buffer, 1 mL / min for 30 mL. At the same time, prepare 50 2 mL EP tubes for sample collection, eluting 2 mL per tube.

[0110] (5) After the program is completed, connect the column to a peristaltic pump and wash it with 50 mL of ddH2O, then 50 mL of 1 mol / L NaOH, and finally 50 mL of ddH2O. Store the column at 4°C.

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

[0112] The collected protein samples were subjected to SDS-PAGE and Western-blot detection.

[0113] 1.2.2.4 Preservation of target protein

[0114] After SDS-PAGE and Western-blot detection, the target protein with good purity was collected and concentrated to 500 μL by centrifugation at 4000 r / min at 4°C using a concentrator tube, replaced twice with PBS (containing 10% glycerol), and continued to be concentrated to 500 μL by centrifugation at 4000 r / min at 4°C using a concentrator tube. After measuring the concentration, the aliquots were packaged, quick-frozen in liquid nitrogen, and stored at -80°C.

[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 they were immunized for the first time by intramuscular injection of protein. The immunization dose was 20 μg of protein per mouse (diluted with normal saline), and it was mixed with QuickAntibody-Mouse3W adjuvant at a ratio of 1:1. The MOCK group was intramuscularly injected with normal saline, which was recorded as day 0.

[0119] (3) The mice were immunized for the second time on the 14th day, and the blood of the mice was collected on the 21st day after immunization 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 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: 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 and 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 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 intramuscularly injected with PBS (emulsified with adjuvant) in the neck.

[0125] (3) The piglets were immunized for the first and second times on day 0 and day 14 respectively. After immunization, the blood of the piglets was collected every 7 days to separate serum for measuring the neutralizing antibody titer 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 according to 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, and then add 100 μL / well of 2% BSA blocking solution and block at room temperature for 1 hour.

[0132] (3) Primary antibody dilution: Add 396 μL of blocking solution to the first tube of each sample, and 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, pat dry, and then add 100 μL of the diluted mouse serum after immunization with each protein to each well and incubate at 37°C for 1 hour.

[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, and then add 100 μL of HRP-labeled Anti-His murine antibody to each well and incubate at 37°C in the dark for 1 hour.

[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 grow to about 80%, and plate them into a 96-well plate.

[0140] (2) After 12 hours, dilute the virus 10-fold with maintenance solution in a sterile 1.5 mL EP tube. The volume of virus suspension in each well is 100 μL. Repeat 8 wells for each dilution, and prepare cell control wells. Shake evenly and steadily move the 96-well plate to a cell culture incubator at 37°C and 5% CO2.

[0141] (3) After 48 hours, the 96-well plate was removed and the cell pathological changes were observed daily under a microscope and the results were recorded.

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

[0143] PEDV virus TCID 50 Determination:

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

[0145] (2) After 24 hours, dilute the virus 10-fold in a sterile 1.5 mL EP tube with serum-free culture medium. The volume of virus suspension in each well is 100 μL. Repeat 8 wells for each dilution, and prepare a cell control well. Shake evenly.

[0146] (3) Remove the 96-well plate, discard the original culture medium, add 100 μL of PBS to each well, wash twice, then add 100 μL of the corresponding virus suspension to each well, and steadily move the 96-well plate to a cell culture incubator at 37°C and 5% CO2 for incubation 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 return to the 37°C incubator for culture.

[0148] (5) After 24 hours, the 96-well plate was removed and the cell pathological changes were observed daily under a microscope and the results were recorded.

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

[0150] 1.2.5.2 TGEV virus neutralization experiment

[0151] (1) One day in advance, prepare the cells required for neutralizing antibody determination in a 96-well plate.

[0152] (2) After the serum to be tested is inactivated at 56°C for 30 min, it is serially diluted 2-fold using the maintenance solution.

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

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

[0155] (5) The mixed solution was inoculated into the PK-15 cell monolayer cultured in a 96-well plate at 100 μL / well, and 4 replicate wells were made for each dilution.

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

[0157] (7) Place the 96-well plate in a 37°C 5% CO2 incubator for 48 hours and then observe the cytopathic effect (CPE). Record the number of wells with lesions and observe for 48-72 hours.

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

[0159] 1.2.5.3 PEDV virus neutralization experiment

[0160] (1) One day in advance, prepare the cells required for neutralizing antibody determination in a 96-well plate.

[0161] (2) After the serum to be tested is inactivated at 56°C for 30 min, it is serially diluted 2-fold using serum-free and trypsin-free culture medium.

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

[0163] (4) Mix 250 μL of serially diluted serum 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) The mixed solution was inoculated into a Vero cell monolayer cultured in a 96-well plate at 100 μL / well, with 4 replicate wells for each dilution.

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

[0167] (8) Place the 96-well plate in a 37°C 5% CO2 incubator and incubate for 1 hour. Aspirate the inoculum and wash once with serum-free culture medium containing 10 μg / mL trypsin. Add 100 μL of DMEM containing 10 μg / mL trypsin to each well. Observe the cytopathic effect (CPE) daily and record the number of wells with lesions for 24-72 hours.

[0168] (9) Calculate the serum neutralization titer based on the number of cytopathic wells using the Reed-Muench method.

[0169] 1.2.6 Biological Software and Databases

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

[0171] Online analysis software: 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 analysis of variance and t-test. P values ​​> 0.05 were considered nonsignificant. ns ), P value < 0.05 was considered statistically significant ( * ), P value < 0.01 was considered statistically significant ( ** ), P value < 0.001 indicates a very significant statistical difference ( *** ), P value < 0.0001 indicates extremely significant statistical difference ( **** ).

[0175] 2. Experimental Results

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

[0177] 2.1.1 Comparison with SARS-COV-2-S protein sequence to determine mutation sites

[0178] In order to improve the stability and expression level of TGEV and PEDV S proteins, the pre-fusion conformation of S proteins was designed with reference to the strategy of improving stability through proline mutation of SARS-COV-2. To determine the proline mutation sites of TGEV and PEDV S proteins, the S protein sequences of SARS-COV-2 Wuhan-Hu-1 strain, TGEV WH-1 strain, and PEDV CT P10 strain were first downloaded from NCBI. The amino acid sequences of these three strains were compared using the online websites multalidin and ESPript3server. It was found that the proline mutation sites corresponding to the 986-987 amino acids of the PEDV S protein and the SARS-COV-2 S protein were serine at position 1076 and leucine at position 1077, respectively. The proline mutation sites corresponding to the 986-987 amino acids of the TGEV S protein and the SARS-COV-2 S protein were glutamic acid at position 1139 and leucine at position 1140, respectively. Figure 1 After these sites were mutated to proline, the secondary structure was destroyed, and a rigid helix-loop-helix structure was formed, allowing the S protein to maintain its pre-fusion conformation.

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

[0180] In order to obtain TGEV and PEDV S proteins, in addition to using their own signal peptides for secretory expression, a GCN4 sequence was added to the C-terminus of the S protein sequence to form a trimer structure, and a Strep-8×His tag was introduced at the rear end for purification ( Figure 2). According to the target protein construction strategy, the cDNA sequences of TGEV S and PEDV S were used as templates, and relevant primers were designed. The fragments were amplified by PCR: TGEV-S-1, TGEV-S-2, PEDV-S-1, PEDV-S-2, GCN4-8×His. According to the gel results, the amplified target genes showed target bands at 3458bp, 780bp, 3269bp, 762bp, and 211bp, respectively, which were in line with the expected size. The target gene fragments TGEV-S-1, TGEV-S-2, PEDV-S-1, PEDV-S-2, GCN4-8×His ( Figure 3-Figure 4 ).

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

[0182] After the target gene was recovered by gel, the plasmid map ( Figure 5 ), the target gene was connected to the enzyme-cut vector pCAGGS for transformation, and after culturing in a 37°C incubator for 16 hours, a single colony was picked for bacterial liquid PCR identification. According to the agarose gel results, the products showed target bands at 780bp and 762bp, respectively. The identified fragments were TGEV-S-2 and PEDV-S-2, which were consistent with the expected sizes. The positive bacterial liquid containing TGEV and PEDV S-2P recombinant plasmids was successfully obtained ( Figure 6 The positive bacterial solution was sent to the combined gene for sequencing. The results showed that TGEV and PEDV S were successfully mutated by 2P, and the positive plasmid with correct sequencing 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 HEK-293F cells in good condition and in the growth phase for large-scale expression. The supernatants were collected on the 5th to 6th day after transfection and purified using Ni-TED filler. Samples from different collection tubes were taken for SDS-PAGE identification ( Figure 8 ), and based on the results, select the protein solution in the collection tube with high purity for concentration.

[0185] 2.1.5 Identification of protein expression by SDS-PAGE and Western Blot

[0186] 16 μL of the concentrated protein solution was taken for sample preparation and subjected to SDS-PAGE. The sample was stained with Coomassie Brilliant Blue and then destained. Another 16 μL of sample was taken for Western Blot and incubated with 6×His-HRP antibody and then developed. According to the gel image and development results ( Figure 9 ) all showed the target bands at around 180 KDa, which was consistent with the expected target protein size. TGEV S-2P protein and PEDV S-2P protein were successfully purified.

[0187] 2.1.6 Comparison of immune effects of TGEV and PEDV S proteins

[0188] 2.1.6.1 TGEV and PEDV S protein immunization strategies

[0189] The purified TGEV S-2P protein and PEDV S-2P protein were used to immunize mice. Six-week-old BALB / c mice were purchased from the Experimental Animal Center of Huazhong Agricultural University. Four mice were randomly selected as a group. Each mouse in the experimental group was immunized with 20 μg protein in the thigh muscle, and each mouse in the control group was immunized with normal saline. The first and second immunizations were performed on day 0 and day 14, respectively. 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 and stored at -80°C after packaging.

[0190] 2.1.6.2 Detection of IgG-specific antibodies to TGEV and PEDV S proteins

[0191] The serum obtained by immunizing TGEV S-2P and PEDV S-2P proteins was subjected to indirect ELISA to determine the nonspecific IgG titer in the serum. TGEV S-2P protein and PEDV S-2P protein were coated as antigens for detection, with immune serum as primary antibody and goat anti-mouse IgG as secondary antibody. The results are shown in Figure 2. Figure 11 As shown in the results, the IgG titer against TGEV S-2P protein produced by sera immunized with TGEV S-2P protein reached 1:1000000, while the IgG titer against TGEV S-2P protein produced by sera immunized with PEDV S-2P protein was 1:10000; the IgG titer against PEDV S-2P protein produced by sera immunized with PEDV S-2P protein reached 1:1000000, while the IgG titer against PEDV S-2P protein produced by sera immunized with TGEV S-2P protein was 1:10000. Therefore, the IgG titers produced by immunization with TGEV S-2P and PEDV S-2P were similar, and the two showed weak cross-protection at the level of specific antibody IgG.

[0192] 2.1.6.3 Neutralizing Antibodies and Cross-Protection Detection of TGEV and PEDV S Proteins

[0193] The neutralization titers of the sera from mice immunized with TGEV S-2P and PEDV S-2P proteins were determined against TGEV WH-1 strain and PEDV CT P10 strain, and the cross-protection between the two was determined. Figure 12 As shown, the serum obtained from mice immunized with TGEV S-2P protein produced a high level of neutralizing antibodies, with an average neutralization titer of 1:7491, while the serum obtained from mice immunized with PEDV S-2P protein produced a lower level of neutralizing antibodies, with an average neutralization titer of 1:123. Therefore, the neutralization effect produced by immunization with TGEV S-2P protein is significantly better than that produced by immunization with PEDV S-2P protein, and there is no neutralization 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 chimeras

[0196] Based on existing research, it has been confirmed that the immune effect of the coronavirus S protein maintaining the pre-fusion trimer structure is better than that of the monomeric S1 protein and CTD protein. Moreover, the immune effect of the TGEV S protein is better than that of the PEDV S protein of the same species. It is speculated that this may be because the TGEV S protein skeleton can better display the various domains of its S1, and in recent years, the highly variable regions of the PEDV S protein are concentrated in the D0-NTD region, which has the potential for designing vaccines. Therefore, the D0-NTD domain of PEDV S is replaced with the TGEV S protein to display the PEDV SD0-NTD domain, in order to obtain an immune spike protein chimera that can provide better protection against PEDV or simultaneously produce immune protection against TGEV and PEDV. See the specific construction diagram for details. Figure 13 .

[0197] The spike protein chimera of the present invention is composed of the CTD to S2 regions of the TGEV S protein extracellular domain as the backbone, and the chimeric display of the D0-NTD region of the PEDV S protein. 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 gene encoding the spike protein chimera TGEV S (replacing D0-NTD) is shown in SEQ ID NO. 16.

[0198] 2.2.2 Amplification of the Spike Protein Chimera Gene Fragment

[0199] According to the spike protein chimera construction strategy, the corresponding plasmid map was designed, and specific amplification primers with homology arms were synthesized. The target fragment was amplified by PCR. According to the agarose gel results, the size of each amplified fragment was consistent with the target fragment size, and the desired target gene was successfully amplified ( Figure 14 ).

[0200] 2.2.3 PCR identification of spike protein chimeras

[0201] After gel recovery, the target gene was homologously recombined with the vector pCAGGS obtained by enzyme digestion according to the plasmid map. The recombinant product was then added to the competent DH5α for transformation. After culturing for 16 hours, a single colony was picked and added to LB containing ampicillin antibiotics. After culturing on a shaker for 4 hours, bacterial liquid PCR was performed. The products were identified by agarose gel, and the target band appeared at 780 bp, which was consistent with the size of the identified TGEV-S-2 fragment. The positive bacterial liquid of each spike protein chimera was successfully obtained ( Figure 15 ).

[0202] 2.2.4 Exploring the Expression Timing of Spike Protein Chimeras

[0203] The recombinant plasmid of the spike protein chimera TGEV S (replacing D0-NTD) was transfected into 50mL HEK-293F cells in good growth condition for small-scale expression. The cell supernatants on days 2-7 were collected and the cell status was observed. Microscopically, the cell density continued to increase from day 2 to day 4, the proliferation rate gradually slowed down, and reached a peak on day 5; on day 6, the cell state began to deteriorate significantly, and the cell debris increased significantly; on day 7, a significantly thickened circle of dead cells appeared on the flask wall. The cell supernatants from days 2-7 were prepared and tested by Western Blot. The results showed that the content of the spike protein chimera protein increased day by day, reaching the highest on the sixth day. On the seventh day, obvious protein degradation began to occur, and the content decreased ( Figure 16 ). Based on the above results, the sixth day is the best time to collect samples.

[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 D0-NTD) was transfected into 600 mL of HEK-293F cells in good growth condition for large-scale expression. Samples were collected at the optimal expression time, and the protein in the cell supernatant was bound to the filler Ni-TED. After purification, the sample in the collection tube where the peak appeared was taken for SDS-PAGE identification, and the protein solution in the collection tube with higher purity was concentrated to obtain the corresponding protein ( Figure 17 ).

[0206] 2.2.6 Identification of protein expression by SDS-PAGE and Western Blot

[0207] According to the SDS-PAGE gel image, the proteins in the collection tubes 26-42 are generally concentrated and replaced with PBS + 10% glycerol, and then concentrated to 500 μL. After taking 16 μL of each sample, SDS-PAGE and Western Blot are performed to identify the target protein. Figure 18 As shown, the target bands appeared at around 180 KDa, which were consistent in size, indicating that the spike protein chimera was successfully purified.

[0208] 2.3.1 Evaluation of the immunogenicity of candidate subunit vaccines in weaned piglets

[0209] 2.3.1.1 Candidate subunit vaccine immunization strategies

[0210] In order to compare the immune effect of spike protein chimeras, TGEV S (replacing D0-NTD) protein was selected as the experimental group, TGEV S-2P and PEDV S-2P as the positive control group, and PBS as the negative control group. The antigen dose of 100 μg / head was fully emulsified with adjuvant to prepare 2 mL / head subunit vaccine for weaned piglets to be immunized. There were 5 pigs in each group. The first and second immunizations were performed on the 0th and 14th days. Blood was collected every 7 days to obtain serum for determination of neutralization titer ( Figure 19 ).

[0211] 2.3.1.2 Neutralizing Antibody Detection for Candidate Subunit Vaccines

[0212] The serum samples collected weekly were used to determine the neutralization titers against TGEV and PEDV, respectively, to analyze the antibody titers and cross-protection. 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 group, the TGEV S (D0-NTD replacement) group produced neutralizing antibody levels close to those of the TGEV S group, with an average neutralizing antibody titer of 1:12129 at the highest on day 21. At the same time, neutralizing antibodies against PEDV were produced, with an average neutralizing antibody titer of 1:57 at the highest on day 28.

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

Claims

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

15.

2. A gene encoding the spike protein chimera as described in claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

16.

4. A recombinant plasmid, characterized in that 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 according to claim 5, characterized in that The recombinant host cell is a recombinant HEK293F cell.

7. Use of the encoding 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 preparing the spike protein chimera according to claim 1.

8. Use of the spike protein chimera as claimed in claim 1 in the preparation of a bivalent subunit vaccine.

9. A method for preparing a bivalent subunit vaccine, characterized in that: The method comprises the steps of uniformly mixing the spike protein chimera described in claim 1 with a vaccine adjuvant to prepare the bivalent subunit vaccine.

10. A bivalent subunit vaccine, characterized in that: The active ingredient includes the spike protein chimera described in claim 1.

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