Recombinant proteins, nucleic acid molecules and recombinant bacteriophages and uses thereof

Abstract

The present application relates to the field of biological medicine, and particularly relates to a recombinant protein, a nucleic acid molecule and a recombinant bacteriophage and application thereof. The present application provides a recombinant protein with an amino acid sequence as shown in SEQ ID NO. 6, a recombinant protein with an amino acid sequence as shown in SEQ ID NO. 10 and application thereof in preparing a vaccine. Injection of the vaccine prepared by using the recombinant protein can effectively prevent coronavirus infection. The present application selects PEDV S protein as a candidate antigen for epitope design, selects two target peptides connected with specific S protein epitopes, selects M13KE bacteriophage as a delivery carrier of PEDV epitopes, and uses animal experiments to evaluate the effect of the PEDV vaccine. The results show that the lymphocytes of the vaccinated mice exhibit a significant proliferation response to PEDV antigens; the IFN-gamma level in the serum of the vaccinated mice is significantly increased.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a recombinant protein, nucleic acid molecule, and recombinant bacteriophage and their applications. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the family Coronaviridae, genus Alphacoronavirus. It is an enveloped, single-stranded, positive-sense RNA virus with pleomorphic viral particles, approximately 95-190 nm in diameter. The virus genome is approximately 28 kb in length, encoding four structural proteins: spike glycoprotein (S protein), envelope protein (E protein), membrane protein (M protein), and nucleocapsid protein (N protein); two non-structural proteins, the polyprotein complexes pp1a and pp1ab; and one ORF3 accessory protein.

[0003] Porcine epidemic diarrhea (PED) is the main pathogen of this disease, primarily causing transmissible intestinal infections in pigs, characterized by vomiting, diarrhea, and dehydration. It was first discovered in UK fattening pig farms in 1971, with a significant impact on pre-weaning piglets aged 4-5 weeks. Subsequently, it spread to many countries in Europe and Asia. An outbreak began in North America in 2013, and PEDV became a globally prevalent virus, severely damaging the global livestock industry.

[0004] Besides biosecurity measures, vaccination is currently the primary means of controlling PEDV infection. Commercially available vaccines are mainly inactivated whole-virus vaccines and attenuated live vaccines, but their protective efficacy against currently circulating strains is not ideal. The reasons for this may be as follows: inactivated vaccines do not produce effective mucosal immunity after intramuscular injection, and attenuated live vaccines, after being weakened, have reduced intestinal stability and proliferative capacity, resulting in limited protective efficacy. Furthermore, some farms use reverse feeding for control, which, while offering some protection, also carries the risk of other viral infections. Therefore, developing a vaccine that can effectively control PEDV is extremely important. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant protein, nucleic acid molecule, and recombinant phage, and their applications, to address the problems existing in the development of commercial vaccines or vaccines. Injection of a vaccine prepared using the recombinant protein, nucleic acid molecule, and recombinant phage provided by this invention induces the production of a large number of antigen-specific PEDV IgG antibodies and neutralizing antibodies, thereby achieving the effect of preventing and controlling PEDV.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention provides a recombinant protein comprising BXT1-PEYC1 with an amino acid sequence as shown in SEQ ID NO.6 and / or BXT2-PEYC2 with an amino acid sequence as shown in SEQ ID NO.10.

[0008] The present invention provides a nucleic acid molecule that encodes the above-mentioned recombinant protein.

[0009] This invention provides a biomaterial containing the above-mentioned nucleic acid molecules.

[0010] Preferably, the biomaterial includes recombinant bacteriophage.

[0011] More preferably, the recombinant phage vector includes the M13KE phage as the basic phage.

[0012] This invention provides the application of the above-described recombinant protein, the above-described nucleic acid molecule, or the above-described biological material in the preparation of vaccines.

[0013] Preferably, the vaccine comprises a porcine coronavirus recombinant phage vaccine.

[0014] Preferably, the porcine coronavirus includes porcine epidemic diarrhea virus.

[0015] The present invention provides a vaccine comprising the above-described recombinant protein, the above-described nucleic acid molecule, or the above-described biological material.

[0016] Preferably, the vaccine also includes pharmaceutically acceptable excipients.

[0017] Preferably, the vaccine comprises a porcine coronavirus recombinant phage vaccine.

[0018] More preferably, the porcine coronavirus includes porcine epidemic diarrhea virus.

[0019] The present invention discloses the following technical effects:

[0020] This invention provides a recombinant protein comprising BXT1-PEYC1 (amino acid sequence as shown in SEQ ID NO. 6) and BXT2-PEYC2 (amino acid sequence as shown in SEQ ID NO. 10). Injection of a vaccine prepared using the recombinant protein provided by this invention induces the production of antigen-specific PEDV IgG antibodies, PEDV-specific T cells, and neutralizing antibodies. The neutralizing antibodies can bind to antigens on the surface of PEDV, thereby preventing PEDV from adhering to target cell receptors and preventing PEDV from invading cells, effectively preventing coronavirus infection. To study PEDV vaccine development and antiviral treatment strategies, in a specific embodiment of this invention, the inventors selected the dominant epitope of the PEDV S protein as a candidate antigen for epitope design, and selected two targeting peptides, BXT1 and BXT2, linked to the dominant epitope of the S protein to enhance the binding ability of the dominant epitope to host cells. Simultaneously, to further improve the delivery capability and immune effect of the epitope antigen, M13KE bacteriophage was selected as the delivery vector for the dominant epitope. Animal immunization experiments were conducted to monitor the activation effects of phage vaccine on mouse lymphocyte proliferation and IFN-γ levels, and to evaluate the vaccine's immunization efficacy. Results showed that mouse lymphocytes exhibited a significant proliferative response to PEDV antigen after vaccination, indicating that the vaccine effectively activated T cell-mediated immunity. Simultaneously, serum IFN-γ levels in vaccinated mice significantly increased, indicating that the vaccine effectively induced antiviral effects in cells.

[0021] Furthermore, this invention utilizes specific targeting peptides (BXT1 and BXT2) to conduct in vivo immunization studies via nasal drops and gavage. This method helps epitope vaccines target the host mucosa, providing immune protection against PEDV. Moreover, vaccines based on phage display technology can facilitate dose-saving immunization practices, which will improve their practicality as a vaccine platform from a production and marketing perspective. This invention provides new insights into vaccine development to improve vaccine efficacy through dose-dilution immunization practices. This method can effectively reduce vaccine production costs, making vaccines more economical. In the future, as research progresses, this immunization strategy will be applied to more vaccines, providing strong support for vaccine development and application.

[0022] Meanwhile, the recombinant protein designed in this invention has a low molecular weight, is easy to express and manipulate, and is convenient for laboratory research and clinical application. Attached Figure Description

[0023] 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.

[0024] Figure 1 Predictions of transmembrane regions and epitopes for PEDV S protein; where (a) is the predicted transmembrane region of PEDV S protein; and (b) is the three-dimensional structure of PEDV S protein (PDB ID: 6U7K).

[0025] Figure 2 This is a flowchart illustrating the construction process of recombinant bacteriophages.

[0026] Figure 3 The image shows the 3D structure of the pIII structural protein ligand peptide GS epitope on the M13KE recombinant phage; red represents the recombinant peptide linked to the target peptide BXT1; and blue represents the recombinant peptide linked to the target peptide BXT2.

[0027] Figure 4 ELISA analysis of M13KE recombinant phage binding to PEDV-positive serum; (a) ELISA analysis of the BXT1 epitope group; (b) ELISA analysis of the BXT2 epitope group; **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant; PBS is the group treated with PBS, abbreviated as PBS group; M13KE Phage refers to the group treated with M13KE phage, abbreviated as M13KE group; M13KE-BXT1 refers to the group treated with recombinant phage M13KE-BXT1, abbreviated as M13KE-BXT1 group; M13KE-BXT1-PEYC1 refers to the group treated with recombinant phage M13KE-BXT1-PEYC1, abbreviated as M13KE-BXT1-PEYC1 group; M13KE-BXT1-PEYC2 refers to the group treated with recombinant phage M13KE-BXT1-PEYC2, abbreviated as M13KE-BXT1-PEYC2 group; M13KE-BXT1-PEYC3 refers to the group treated with recombinant phage M13KE-BXT1-PEYC3, abbreviated as M1 3KE-BXT1-PEYC3; M13KE-BXT2 is the group treated with recombinant phage M13KE-BXT2, abbreviated as M13KE-BXT2 group; M13KE-BXT2-PEYC1 is the group treated with recombinant phage M13KE-BXT2-PEYC1, abbreviated as M13KE-BXT2-PEYC1 group; M13KE-BXT2-PEYC2 is the group treated with recombinant phage M13KE-BXT2-PEYC2, abbreviated as M13KE-BXT2-PEYC2 group; M13KE-BXT2-PEYC3 is the group treated with recombinant phage M13KE-BXT2-PEYC3, abbreviated as M13KE-BXT2-PEYC3;

[0028] Figure 5For antibody titer analysis; where (a) is the titer of antigen-specific IgG antibodies against PEDV in the serum of BALB / c mice immunized by intranasal administration; (b) is the titer of antibody against PEDV in the serum of BALB / c mice immunized by gavage; ****p<0.0001; PBS is the group treated with PBS, abbreviated as PBS group; M13KE is the group treated with M13KE phage, abbreviated as M13KE group; M13KE-BXT1 is the group treated with recombinant phage M13KE-BXT1, abbreviated as M1 Group 3KE-BXT1; Group M13KE-BXT1-PEYC1 is a subgroup treated with recombinant phage M13KE-BXT1-PEYC1, abbreviated as Group M13KE-BXT1-PEYC1; Group M13KE-BXT2 is a subgroup treated with recombinant phage M13KE-BXT2, abbreviated as Group M13KE-BXT2; Group M13KE-BXT2-PEYC2 is a subgroup treated with recombinant phage M13KE-BXT2-PEYC2, abbreviated as Group M13KE-BXT2-PEYC2;

[0029] Figure 6 The PEDV neutralizing titer in serially diluted immunized mouse serum is given; (a) represents the PEDV neutralizing titer in serially diluted serum of BALB / c mice immunized by intranasal administration; (b) represents the PEDV neutralizing titer in serially diluted serum of BALB / c mice immunized by gavage; **p<0.01; PBS refers to the group treated with PBS, abbreviated as PBS group; M13KE refers to the group treated with M13KE phage, abbreviated as M13KE group; M13KE-BXT1 refers to the group treated with recombinant phage M13KE-BXT1, abbreviated as M13KE group. Group M13KE-BXT1; Group M13KE-BXT1-PEYC1 is a subgroup treated with recombinant phage M13KE-BXT1-PEYC1, abbreviated as Group M13KE-BXT1-PEYC1; Group M13KE-BXT2 is a subgroup treated with recombinant phage M13KE-BXT2, abbreviated as Group M13KE-BXT2; Group M13KE-BXT2-PEYC2 is a subgroup treated with recombinant phage M13KE-BXT2-PEYC2, abbreviated as Group M13KE-BXT2-PEYC2;

[0030] Figure 7The proliferation of lymphocytes in the spleen of immunized mice was measured. (a) represents the T cell proliferation level in the spleen of BALB / c mice immunized by intranasal administration; (b) represents the T cell proliferation level in the spleen of BALB / c mice immunized by gavage. ****p<0.0001, ns=not significant; PBS refers to the group treated with PBS, abbreviated as PBS group; M13KE refers to the group treated with M13KE phage, abbreviated as M13KE group; M13KE-BXT1 refers to the group treated with recombinant phage M13KE-BXT1, abbreviated as M1 Group 3KE-BXT1; Group M13KE-BXT1-PEYC1 is a subgroup treated with recombinant phage M13KE-BXT1-PEYC1, abbreviated as Group M13KE-BXT1-PEYC1; Group M13KE-BXT2 is a subgroup treated with recombinant phage M13KE-BXT2, abbreviated as Group M13KE-BXT2; Group M13KE-BXT2-PEYC2 is a subgroup treated with recombinant phage M13KE-BXT2-PEYC2, abbreviated as Group M13KE-BXT2-PEYC2;

[0031] Figure 8 The values ​​represent the IFN-γ content in the spleen of immunized mice; (a) represents the IFN-γ content in the spleen of BALB / c mice immunized by intranasal administration; (b) represents the IFN-γ content in the spleen of BALB / c mice immunized by gavage; *p<0.05, ****p<0.0001, ns = not significant; PBS refers to the group treated with PBS, abbreviated as PBS group; M13KE refers to the group treated with M13KE phage, abbreviated as M13KE group; M13KE-BXT1 refers to the group treated with recombinant phage M13KE-BXT1. The M13KE-BXT1 group is abbreviated as M13KE-BXT1; the M13KE-BXT1-PEYC1 group is abbreviated as M13KE-BXT1-PEYC1; the M13KE-BXT2 group is abbreviated as M13KE-BXT2; the M13KE-BXT2-PEYC2 group is abbreviated as M13KE-BXT2-PEYC2. Detailed Implementation

[0032] 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.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] 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.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Example 1

[0038] 1. Materials and Methods

[0039] 1.1 The system and website used for sequence similarity analysis of PEDV S protein (S protein of porcine epidemic diarrhea virus) are as follows:

[0040] NCBI: National Center for Biotechnology Information (nih.gov); MEGA11.

[0041] 1.2 The system and website used for PEDV S protein surface region prediction are as follows:

[0042] DeepTMHMM: DTU / DeepTMHMM-BioLib.

[0043] 1.3 The systems and websites used for protein structure prediction are as follows:

[0044] AlphaFold2: AlphaFold2.ipynb-Colaboratory(google.com).

[0045] 1.4 PEDV linear epitope prediction includes linear epitope prediction and antigenicity prediction, as detailed below:

[0046] The systems and websites used for linear epitope prediction are as follows:

[0047] Epitope Length: 10aa; Score: 0.85, http: / / sysbio.unl.edu / SVMTriP;

[0048] The systems and websites used for antigenicity prediction are as follows:

[0049] TARGET ORGANISM: Virus;

[0050] THRESHOLD: 0.1, http: / / www.ddg-pharmfac.net / vaxijen / VaxiJen / VaxiJen.htm;

[0051] IEDB.org: Free epitope database and prediction resource.

[0052] 1.4. Bacteriological strains, bacteriophages, cells, viral strains, serum

[0053] M13KE bacteriophages and Escherichia coli ER2738 were purchased from New England Biolabs; Escherichia coli ER2738 chemocompetent cells and Escherichia coli ER2738 electrotransformation competent cells were prepared and preserved in our laboratory; rabbit anti-PEDV positive serum was purchased from Guangzhou Qianxun Biotechnology Co., Ltd.; goat anti-rabbit His-tag monoclonal antibody was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; Vero cells were preserved in our laboratory.

[0054] 1.5 Construction of M13KE phage display monoepithelialis

[0055] In this embodiment, the targeting peptides BXT1 (CAKSMGDIVC, SEQ ID NO.1) targeting lung epithelial cells and BXT2 (CDCRGDCFC, SEQ ID NO.2) targeting intestinal dendritic cells are linked to epitopes (PEYC1 (SEQ ID NO.3), PEYC2 (SEQ ID NO.4), and PEYC3 (SEQ ID NO.5)) to form recombinant peptides.

[0056] The gene sequences encoding each recombinant peptide, i.e., the DNA fragments of each recombinant peptide, were amplified using polymerase chain reaction (PCR) primers (primer sequences are detailed in Table 1). The M13KE phage genome was double-digested with Kpn I and Eag I restriction endonucleases to remove portions of the gene sequence encoding the PIII protein. The DNA fragments of each recombinant peptide were cloned into M13KE phages and expressed on the M13KE phage capsid protein (pIII) to obtain the M13KE recombinant phage vector.

[0057] ER2738 chemocompetent cells were removed from a -80°C freezer and placed on ice for 5 minutes. Then, 20-30 ng of the M13KE recombinant phage vector was gently added. After transformation, the bacterial culture was resuspended and plated on LB / IPTG / X-gal / tetracycline culture dishes (IPTG purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A100487-0005; X-gal purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A100428-0100; tetracycline purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A430165-0001). The culture dishes were then inverted and incubated overnight at 37°C in a bacterial incubator.

[0058] The next day, blue phage spots were selected and inoculated into 50 mL of LB liquid medium. The culture was carried out in a shaker at 37°C for 8 h, followed by centrifugation at 4,000 rpm for 15 min. The supernatant was collected, filtered through a 0.22 μm filter, and stored at 4°C. Recombinant phage amplification and genome extraction were then performed, and the samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0059] Table 1 Primer sequences

[0060]

[0061]

[0062] 1.6 Amplification and purification of recombinant phages

[0063] Blue recombinant phage spots were selected and inoculated into 20 mL LB medium, and cultured overnight at 37°C and 220 rpm. The overnight culture was centrifuged at 10,000 rpm for 20 min, and the supernatant was collected. 1 / 6 volume of a mixture containing PEG8000 and NaCl was added to the supernatant (PEG8000 was 20% by volume, and NaCl was 2.5 M; PEG8000 was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A600433-0500; NaCl was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A610476-0001). The mixture was incubated overnight at 4°C. The phage precipitate was centrifuged at 10,000 rpm for 20 min at 4°C, the supernatant was discarded, and the precipitate was resuspended in 1 mL LTB buffer. This was the amplified recombinant phage solution. The titer of the recombinant phage was determined using the bilayer plate method, and the solution was briefly stored at 4°C.

[0064] 1.7 ELISA Analysis of Recombinant Bacteriophages

[0065] Add PEDV polyclonal antibody (100 μL, 3 ng / μL, Guangzhou Qianxun Biotechnology Co., Ltd., catalog number: Pb-002) to the ELISA plate and incubate overnight at 4°C. Then discard the liquid and use a Tween-containing solution. TM Wash the ELISA plate 6 times with PBST, add 100 μL of 5% skim milk, and incubate at 37°C for 1 h to block nonspecific binding. Discard the liquid, wash the ELISA plate 6 times with PBST, and then add 100 μL of M13KE recombinant phage (10 μL / well) to each well of the 96-well ELISA plate. 8 pfu / mL) and wild-type M13KE phage (10 pfu / mL) 8 Add PBS to the M13KE-BXT1 group (pfu / mL), and label them M13KE-BXT1-PEYC1 group, M13KE-BXT1-PEYC2 group, M13KE-BXT1-PEYC3 group, M13KE-BXT2 group, M13KE-BXT2-PEYC1 group, M13KE-BXT2-PEYC2 group, and M13KE-BXT2-PEYC3 group, respectively. Incubate at 37°C for 1 hour. A PBS group (using only PBS) is also included. Discard the solution, wash the ELISA plate 6 times with PBST, add 100 μL of 1:5000 diluted Anti-M13 Antibody (HRP) (Sino Biological) antibody, and incubate at 37°C for 1 hour. Discard the solution, wash the ELISA plate 6 times with PBST, add 100 μL of the colorimetric substrate, incubate at 37 °C for 15 min, then add 50 μL of 5% sulfuric acid to stop the reaction, and measure the OD. 450 .

[0066] 1.8 Immunological studies of recombinant bacteriophages in mice

[0067] The titers of recombinant M13KE phage and wild-type M13KE phage were adjusted to 1.0 × 10⁻⁶. 13 pfu / mL, store at 4℃.

[0068] Specific pathogen-free (SPF) BALB / c mice (3-4 weeks old) were purchased from the Laboratory Animal Center of Shanghai Jiao Tong University (Shanghai, China). Forty-eight mice were randomly divided into eight groups of six each, as shown in Table 2. Mice received intranasal immunization and gavage immunization with recombinant phage, respectively.

[0069] Boosted immunizations were administered at weeks 2, 4, and 6. Blood samples were collected from the orbital pits of immunized mice the night before weeks 2, 4, 6, and 8, stored overnight at 4°C, followed by brief centrifugation to collect serum, which was then stored at -20°C.

[0070] Table 2. Mouse Immunization Groups

[0071]

[0072] 1.9 IgG Level Analysis

[0073] The amount of IgG antibodies produced in mice infected with PEDV was assessed using an indirect ELISA method. 20 μg / mL of the PEDV strain (PEDV / CHSH / 2019, published in "Isolation, Identification and Genetic Analysis of Shanghai Strain of Porcine Epidemic Diarrhea Virus PEDV / CHSH / 2019" (Wang Na, Ji Likai, Wang Jian, et al. Isolation, Identification and Genetic Analysis of Shanghai Strain of Porcine Epidemic Diarrhea Virus PEDV / CHSH / 2019 [J]. Chinese Journal of Animal Infectious Diseases, 2023, 31(03):46-53. DOI:10.19958 / j.cnki.cn31-2031 / s.2023.03.026.)) was used as antigen and plated onto 96-well polystyrene microplates. Then, a solution containing 0.1% Tween was applied. TM Wash the plate three times with PBST, block with 5% skim milk, and incubate at 37°C for 2 hours. Incubate at 37°C for 1 hour. Discard the solution, wash the ELISA plate six times with PBST, add 100 μL of 1:5000 diluted Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L), and incubate at 37°C for 1 hour. Discard the solution, wash the ELISA plate six times with PBST, add 100 μL of the colorimetric substrate, incubate at 37°C for 15 minutes, then add 50 μL of 5% sulfuric acid to stop the reaction, and measure the OD. 450 .

[0074] 1.10 Virus neutralization test

[0075] To evaluate the neutralizing ability of recombinant phage-induced antibodies against the virus, antibody neutralization levels in mouse serum were measured while maintaining a constant viral concentration. Mouse serum was inactivated at 56°C for 30 min, diluted two-fold, and 50 μL of serum was mixed with 50 μL of PEDV / CHSH / 2019 (100 TCID50) 50 Mix the ingredients and incubate at 37°C for 1 hour. Transfer 100 μL of the mixture into a 96-well cell culture plate (for overnight Vero monolayer cells) and incubate at 37°C for 1 hour. Discard the supernatant, wash the cells twice with PBS, add 100 μL of maintenance medium to each well, and incubate the plate in a 5% CO2 incubator at 37°C for 72 hours. Observe the cytopathic effect (CPE).

[0076] 1.11 Lymphocyte proliferation test

[0077] In week 8, spleens were removed from immunized mice and placed in PBS. Numerous cavities were created in the spleen using a sterile needle. The PBS containing lymphocytes was carefully aspirated and slowly mixed with 5 mL of Ficoll separation buffer (1.2 g / mL). The mixture was centrifuged at 600 rpm for 25 min, resulting in a three-layered liquid. The middle layer was carefully aspirated, washed with PBS, and centrifuged at 600 rpm for 5 min. The erythrocyte suspension was lysed with 0.75% Tris-NH4Cl (pH 7.4), washed three times with PBS, and then resuspended in supplemented DMEM (containing 10% FBS, 14 mM HEPES, 50 mM 2-mercaptoethanol, 100 μg / mL streptomycin, and 100 IU / mL penicillin) to obtain a concentration of 1 × 10⁻⁶ cells. 6 Spleen cells were suspended in 100 μL of 1 / mL medium and added to 96-well cell culture plates. 100 μL of medium containing or without PEDV protein (20 μg / mL) was added to each group to stimulate the spleen cells. After incubation for 24 h, lymphocyte proliferation was detected according to the instructions of the MTT cell proliferation assay kit.

[0078] 1.12. Interferon-γ (IFN-γ) Detection

[0079] Cells were stimulated with 20 μg / mL PEDV protein for 72 h, and the cell culture supernatant was collected (from step “1.11, Lymphocyte Proliferation Assay”). The IFN-γ content in the supernatant was assessed using a commercial mouse IFN-γ enzyme-linked immunosorbent assay kit (Sangon Biotech (Shanghai) Co., Ltd.) according to the manufacturer’s instructions. Cytokine concentrations were calculated based on the standard curve for each ELISA plate.

[0080] 2. Results

[0081] 2.1 Bioinformatics Analysis of PEDV S Protein

[0082] We analyzed the PEDV S protein sequences prior to November 20, 2022, in the NCBI database. The aim was to identify sequence variations among different PEDV strains. After removing duplicates and incomplete sequences, 1,882 unique PEDV S protein sequences were identified. This dataset was subsequently used to study the genetic diversity and potential evolutionary trends of the PEDV population, which is of great significance for the development of PEDV vaccines and therapeutic interventions. Further analysis of the similarity of the PEDV S protein sequences using MEGA11 revealed that the similarity between different strains exceeded 87.33%. This indicates that there are many conserved similar sequences among the PEDV S protein sequences of different strains, and that some potential antigenic epitopes are shared among different strains.

[0083] The PEDV S protein interacts with cellular receptors during viral entry, stimulating the host to produce natural neutralizing antibodies. This example utilizes the DeepTMHMM server to predict the α-helix and β-sheet transmembrane topology of the PEDV S protein. Results are as follows... Figure 1 As shown in A in the figure. The results show that the 20-1325S protein sequence is located in the extracellular region, therefore, it is a good candidate for a potential neutralizing epitope.

[0084] One of the main challenges in developing novel vaccines is predicting effective protective antigens. Theoretically, attention to structural antigen localization and the discovery of immunodominant B-cell epitopes are crucial for eliciting potent neutralizing activity and high-level immune responses. In this embodiment, we used the IEDB tool to predict and screen B-cell linear epitopes of the PEDV S protein, obtaining three linear B-cell PEDV epitopes (PEYC1, PEYC2, and PEYC3), as shown in Table 3. Comparison with the predicted transmembrane regions of these proteins revealed that all epitopes are located in the PEDV S extracellular region (20–1,325 amino acids). These three predicted epitopes represent over 85% coverage of the 1882 reported PEDV S proteins, making them promising candidates for potential neutralizing epitopes (Table 3). We further analyzed whether the epitopes were located on the surface of the PEDV S protein structure, with results as follows... Figure 1 As shown in B, the PEYC1, PEYC2, and PEYC3 epitopes are exposed on the surface of the PEDVS three-dimensional structure, representing the dominant epitopes within the PEDVS protein's three-dimensional structure. Spatially, they are primarily foreign antigens recognized by immune cells.

[0085] Table 3B shows the prediction of linear epitopes of the PEDV S protein in cells.

[0086]

[0087] By specifically binding to receptors on the surface of immune cells, targeting peptides can direct vaccine components to key regions of the immune response, thereby improving the immunogenicity and safety of the vaccine. In this embodiment, to achieve targeted delivery of a phage recombinant vaccine, targeting peptides BXT1 and BXT2, which target lung epithelial cells and induce targeted phage particles to enter systemic circulation, are reported to be tandemly linked to B-cell linear epitopes of the PEDV S protein. The names and sequences of the tandemly linked peptides are shown in Table 4.

[0088] Table 4 Targeting peptides - PEDV linear epitope recombinant peptides

[0089]

[0090] Note: The bolded and underlined parts are the sequences of the target peptides BXT1 and BXT2.

[0091] 2.2 Construction of PEDV S protein epitopes displayed by phages and in vitro antigenicity analysis

[0092] In this embodiment, M13KE phage was used as a vector to display linear epitopes. After cleaving the circular genome of M13KE phage, the target genes encoding the recombinant peptides listed in Table 4 were fused into the M13KE phage genome via homologous recombination. The recombinant phage genome was electroporated into E. coli ER2738 competent cells to obtain recombinant phages (…). Figure 2 ).

[0093] Next, we used AlphaFold 2 to determine the three-dimensional structure of the recombinant peptide located in the pIII structural protein, and the results are as follows. Figure 3 As shown. In Figure 3 In the diagram, red indicates the target peptide epitope linked to the target peptide BXT1, and blue indicates the target peptide epitope linked to the target peptide BXT2. The recombinant peptide is visible outside the M13KE structural protein pIII. This means that immune cells can easily recognize the target peptide-linked epitopes delivered by the M13KE phage vector.

[0094] To verify whether recombinant phages could specifically bind to PEDV-positive serum, we used PEDV-positive serum as an antibody to determine the binding affinity of exogenous peptides (i.e., the recombinant peptides shown in Table 4) of M13KE recombinant phages (M13KE-BXT1, M13KE-BXT1-PEYC1, M13KE-BXT1-PEYC2, M13KE-BXT1-PEYC3, M13KE-BXT2, M13KE-BXT2-PEYC1, M13KE-BXT2-PEYC2, and M13KE-BXT2-PEYC3) to anti-PEDV antibodies. The results showed that recombinant phages M13KE-BXT1-PEYC1 and M13KE-BXT2-PEYC2 exhibited the highest affinity for PEDV-positive serum, respectively (…). Figure 4 (a) and (b) in the text. Therefore, we used recombinant phage M13KE-BXT1-PEYC1 and recombinant phage M13KE-BXT2-PEYC2 as candidate antigens to immunize mice.

[0095] 2.3 Evaluation of efficacy after immunization

[0096] To evaluate whether recombinant phage vaccines can induce the production of neutralizing antibodies against PEDV, we administered M13KE-BXT1-PEYC1 recombinant phage to mice via nasal drops and M13KE-BXT2-PEYC2 recombinant phage via gavage.

[0097] Fourteen days after the final booster immunization, the titer of PEDV antigen-specific IgG antibodies against the recombinant phage was assessed using ELISA. Blood samples were collected from mice at week 8, and serum from all mice (BOOST3 mice) was analyzed by ELISA. Results showed that neither wild-type M13KE phage nor M13KE phage carrying the targeting peptide induced antigen-specific PEDV IgG antibodies in mice. Conversely, the antigenic epitope delivered by M13KE phage induced PEDV-specific IgG antibodies in mice, and the recombinant phage carrying the PEDV epitope effectively induced the host to produce PEDV-specific antibodies. Figure 5 (a) and (b) in the figure. It can be seen that the M13KE recombinant phage with targeting peptides and epitopes induces antigen-specific PEDV IgG antibodies in mice.

[0098] Further serial dilutions were performed on the immunized mouse serum (serum was diluted 2-fold during the experiment) to neutralize live PEDV (PEDV / CHSH / 2019 strain), and the neutralization titer was calculated using the de-dilution. Results are as follows: Figure 6As shown, the M13KE-BXT1-PEYC1 and M13KE-BXT2-PEYC2 groups stimulated the production of neutralizing antibodies in immunized mice. In the presence of low-dilution serum, mouse serum could neutralize live PEDV, but the PBS, M13KE, M13KE-BXT1, and M13KE-BXT2 groups failed to stimulate the production of neutralizing antibodies in immunized mice. The immunization strategy of the M13KE-BXT1-PEYC1 and M13KE-BXT2-PEYC2 groups successfully and effectively induced the production of neutralizing antibodies against PEDV in mice. Therefore, recombinant phages M13KE-BXT1-PEYC1 and M13KE-BXT2-PEYC2 stimulated the production of neutralizing antibodies in immunized mice.

[0099] Two weeks after the third booster immunization, we isolated splenic lymphocytes from mice and stimulated their proliferation in vitro using PEDV. The results were as follows: Figure 7 As shown in the figure, after in vitro PEDV stimulation, splenic T cells in mice in the M13KE-BXT1-PEYC1 and M13KE-BXT2-PEYC2 groups showed significant proliferation, but no significant proliferation was observed in the M13KE, M13KE-BXT1, and M13KE-BXT2 groups, which were significantly different from the PBS group (p<0.0001). These results indicate that the immunization strategy of the M13KE-BXT1-PEYC1 and M13KE-BXT2-PEYC2 groups can effectively induce mice to produce PEDV-specific T cells. Therefore, the immunization strategy of the M13KE-BXT1-PEYC1 and M13KE-BXT2-PEYC2 groups can effectively induce mice to produce PEDV-specific T cells.

[0100] After 72 hours of PEDV stimulation of spleen cells, the cells were centrifuged, and the culture supernatant was collected for quantitative detection of IFN-γ. The results are as follows: Figure 8 As shown in the figure. The results showed that after PEDV in vitro stimulation, IFN-γ in the M13KE-BXT1-PEYC1 group and the M13KE-BXT2-PEYC2 group increased significantly, while IFN-γ in the M13KE group, M13KE-BXT1 group, and M13KE-BXT2 group did not increase, and the difference was significant compared with the control group (p<0.0001). This indicates that the M13KE-BXT1-PEYC1 group and the M13KE-BXT2-PEYC2 group more effectively activated the spleen cell immune response under PEDV stimulation, thereby producing more IFN-γ. Therefore, after PEDV in vitro stimulation, IFN-γ in the M13KE-BXT1-PEYC1 and M13KE-BXT2-PEYC2 groups increased significantly. This demonstrates that the immunization strategies of the M13KE-BXT1-PEYC1 group and the M13KE-BXT2-PEYC2 group can effectively induce cellular immune responses.

[0101] 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 recombinant protein, characterized in that, The recombinant protein is BXT1-PEYC1 with an amino acid sequence as shown in SEQ ID NO.6 or BXT2-PEYC2 with an amino acid sequence as shown in SEQ ID NO.

10.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant protein of claim 1.

3. A biomaterial containing the nucleic acid molecule as described in claim 2.

4. The biomaterial according to claim 3, characterized in that, The biomaterials include recombinant bacteriophages.

5. The use of the recombinant protein of claim 1, the nucleic acid molecule of claim 2, or the biological material of claim 3 or 4 in the preparation of a porcine epidemic diarrhea virus vaccine.

6. A vaccine, characterized in that, The vaccine comprises the recombinant protein of claim 1, the nucleic acid molecule of claim 2, or the biological material of claim 3 or 4.

7. The vaccine according to claim 6, characterized in that, The vaccine also includes pharmaceutically acceptable excipients.

8. The vaccine according to claim 6, characterized in that, The vaccine includes a swine epidemic diarrhea virus vaccine.

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