Recombinant protein, nucleic acid molecule, recombinant bacteriophage and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a recombinant protein, a nucleic acid molecule, a recombinant bacteriophage and application thereof. The invention 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 of the recombinant protein in preparation of vaccines. A vaccine prepared by injecting the recombinant protein can effectively prevent coronavirus infection. According to the invention, the PEDV S protein is selected as a candidate antigen for epitope design, two targeting peptides connected with the specific S protein epitope are selected, the M13KE phage is selected as a delivery carrier of the PEDV epitope, and animal experiments are utilized to evaluate the effect of the PEDV vaccine. The result shows that the lymphocyte of the vaccinated mouse shows an obvious proliferation reaction to the PEDV antigen; and the IFN-gamma level in the serum of the vaccinated mouse is obviously increased.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a recombinant protein, a nucleic acid molecule and a recombinant bacteriophage and applications thereof. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the Coronaviridae family and the genus Alphacoronavirus. It is an enveloped single-stranded positive-strand RNA virus with polymorphic virus particles and a diameter of about 95-190nm. The viral genome is about 28kb long and encodes four structural proteins, including spike glycoprotein (S protein), envelope protein (E protein), membrane protein (M protein) and nucleocapsid protein (N protein); two non-structural proteins of the polyprotein complex pp1a and pp1ab; and an ORF3 auxiliary protein.

[0003] PEDV is the main pathogen of porcine epidemic diarrhea (PED), which mainly causes intestinal contact infectious diseases in pigs, characterized by vomiting, diarrhea and dehydration. It was first discovered in a fattening pig farm in the UK in 1971, and had a greater impact on 4-5 week old pre-weaned piglets. Subsequently, it also became popular in many countries in Europe and Asia. In 2013, the outbreak began in North America, and PEDV became a global epidemic virus and seriously damaged the global breeding industry.

[0004] For the prevention and control of PEDV, in addition to biological control, vaccination is currently the main means of preventing and controlling PEDV infection. Commercial vaccines are mainly whole virus inactivated vaccines and attenuated vaccines, but the protection effect on the current epidemic strains is not very ideal. The reasons may be as follows: inactivated vaccines cannot produce effective mucosal immunity after intramuscular injection, and attenuated vaccines have reduced intestinal stability and proliferation ability after attenuation, resulting in limited protection. In addition, some farms also use reverse feeding to prevent and control. Although it has a protective effect to a certain extent, it also poses the risk of infection by other viruses. Therefore, it is extremely important to develop a vaccine that can effectively prevent and control PEDV. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant protein, nucleic acid molecule and recombinant phage and their application to solve the problems existing in the above-mentioned commercial vaccine or vaccine development process. The vaccine prepared by using the recombinant protein, nucleic acid molecule and recombinant phage provided by the present invention is injected to induce 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 object, the present invention provides the following solutions:

[0007] The present invention provides a recombinant protein, which comprises 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, which encodes the above-mentioned recombinant protein.

[0009] The present invention provides a biological material containing the above nucleic acid molecules.

[0010] Preferably, the biological material comprises a recombinant bacteriophage.

[0011] Further preferably, the recombinant phage vector comprises a basic phage which is M13KE phage.

[0012] The present invention provides the use of the above-mentioned recombinant protein, the above-mentioned nucleic acid molecule or the above-mentioned biological material in the preparation of vaccines.

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

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

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

[0016] Preferably, the vaccine further comprises pharmaceutically acceptable excipients.

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

[0018] Further preferably, the porcine coronavirus comprises porcine epidemic diarrhea virus.

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

[0020] The present invention provides a recombinant protein, which includes BXT1-PEYC1 with an amino acid sequence as shown in SEQ ID NO.6 and BXT2-PEYC2 with an amino acid sequence as shown in SEQ ID NO.10. The vaccine prepared by using the recombinant protein provided by the present invention is injected to induce the production of antigen-specific PEDV IgG antibodies, PEDV-specific T cells and neutralizing antibodies; the neutralizing antibodies can bind to the antigens on the surface of PEDV, thereby preventing PEDV from adhering to target cell receptors, preventing PEDV from invading cells, and effectively preventing coronavirus infection. In order to study the vaccine development and antiviral treatment strategies of PEDV, in a specific embodiment of the present invention, the inventors selected the dominant epitope of the S protein of PEDV as a candidate antigen for epitope design, and selected two targeting peptides BXT1 and BXT2 connected to the dominant epitope of the S protein to enhance the binding ability of the dominant epitope to target host cells. At the same time, in order to further improve the delivery ability and immune effect of the epitope antigen, the M13KE phage is selected as the delivery carrier of the dominant epitope. The animal immunization experiment was used to monitor the activation effect of phage vaccine on mouse lymphocyte proliferation and IFN-γ level after immunization to evaluate the immune effect of the vaccine. The results showed that the lymphocytes of mice after vaccination showed a significant proliferation response to PEDV antigens, indicating that the vaccine can effectively activate T cell-mediated immunity; at the same time, the IFN-γ level in the serum of mice after vaccination increased significantly, indicating that the vaccine can effectively induce the antiviral effect of cells.

[0021] Moreover, the present invention is based on a specific targeting peptide (BXT1 and BXT2), and an in vivo immunization study was conducted by nasal drops and oral gavage. This method can help epitope vaccines target the host mucosa and provide immune protection against PEDV. And vaccines based on phage display technology can promote dose-saving immunization practices, which will improve its practicality as a vaccine platform from the perspective of production and sales. The present invention provides new insights into vaccine development to improve the effectiveness of vaccines through dose dilution immunization practices. This method can effectively reduce the cost of vaccine production and make vaccines more economical. In the future, as research deepens, this immunization strategy will be applied to more vaccines, providing strong support for the development and application of vaccines.

[0022] At the same time, the recombinant protein designed by the present invention has a low molecular weight, is easy to express and operate, and is convenient for laboratory research and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0024] Figure 1 Prediction of transmembrane region and epitope of PEDV S protein; (a) is the prediction of transmembrane region of PEDV S protein; (b) is the three-dimensional structure of PEDV S protein (PDB ID: 6U7K);

[0025] Figure 2 A flowchart for the construction of recombinant phage;

[0026] Figure 3 The 3D structure of the ligand peptide GS epitope of the pIII structural protein on the M13KE recombinant phage; red represents the recombinant peptide connected to the targeting peptide BXT1; blue represents the recombinant peptide connected to the targeting peptide BXT2;

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

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

[0029] Figure 6 is the PEDV neutralization titer of serially diluted sera of immunized mice; (a) is the PEDV neutralization titer of serially diluted sera of BALB / c mice immunized by intranasal drops; (b) is the PEDV neutralization titer of serially diluted sera of BALB / c mice immunized by oral gavage; **p<0.01; PBS is the group treated with PBS, referred to as the PBS group; M13KE is the group treated with M13KE phage, referred to as the M13KE group; M13KE-BXT1 is the group treated with recombinant phage M13KE-BXT1, referred to as M13KE-BXT1 group; M13KE-BXT1-PEYC1 is a group treated with recombinant phage M13KE-BXT1-PEYC1, referred to as M13KE-BXT1-PEYC1 group; M13KE-BXT2 is a group treated with recombinant phage M13KE-BXT2, referred to as M13KE-BXT2 group; M13KE-BXT2-PEYC2 is a group treated with recombinant phage M13KE-BXT2-PEYC2, referred to as M13KE-BXT2-PEYC2 group;

[0030] Figure 7is the proliferation of lymphocytes in the spleen of immunized mice; (a) is the proliferation level of T cells in the spleen of BALB / c mice immunized by intranasal drops; (b) is the proliferation level of T cells in the spleen of BALB / c mice immunized by oral gavage; ****p<0.0001, ns=not significant; PBS is the group treated with PBS, referred to as the PBS group; M13KE is the group treated with M13KE phage, referred to as the M13KE group; M13KE-BXT1 is the group treated with the recombinant phage M13KE-BXT1, referred to as M1 3KE-BXT1 group; M13KE-BXT1-PEYC1 is a group treated with recombinant phage M13KE-BXT1-PEYC1, referred to as M13KE-BXT1-PEYC1 group; M13KE-BXT2 is a group treated with recombinant phage M13KE-BXT2, referred to as M13KE-BXT2 group; M13KE-BXT2-PEYC2 is a group treated with recombinant phage M13KE-BXT2-PEYC2, referred to as M13KE-BXT2-PEYC2 group;

[0031] Figure 8 is the content of IFN-γ in the spleen of immunized mice; (a) is the content of IFN-γ in the spleen of BALB / c mice immunized by intranasal drops; (b) is the content of IFN-γ in the spleen of BALB / c mice immunized by oral gavage; *p<0.05, ****p<0.0001, ns=not significant; PBS is the group treated with PBS, referred to as the PBS group; M13KE is the group treated with M13KE phage, referred to as the M13KE group; M13KE-BXT1 is the group treated with the recombinant phage M13KE-BXT1 , referred to as M13KE-BXT1 group; M13KE-BXT1-PEYC1 is the group treated with recombinant phage M13KE-BXT1-PEYC1, referred to as M13KE-BXT1-PEYC1 group; M13KE-BXT2 is the group treated with recombinant phage M13KE-BXT2, referred to as M13KE-BXT2 group; M13KE-BXT2-PEYC2 is the group treated with recombinant phage M13KE-BXT2-PEYC2, referred to as M13KE-BXT2-PEYC2 group. DETAILED DESCRIPTION

[0032] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

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

[0037] Example 1

[0038] 1. Materials and methods

[0039] 1.1. The systems and websites 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 prediction of the surface region of PEDV S protein 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 follows:

[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. Strains, phages, cells, strains, and serum

[0053] M13KE phage and Escherichia coli ER2738 were purchased from New England Biolabs; Escherichia coli ER2738 chemically competent cells and Escherichia coli ER2738 electroporation competent cells were prepared and stored in this 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 stored in this laboratory.

[0054] 1.5. Construction of M13KE phage display single epitope

[0055] In this example, the targeting peptide BXT1 (CAKSMGDIVC, SEQ ID NO.1) targeting lung epithelial cells and the targeting peptide BXT2 (CDCRGDCFC, SEQ ID NO.2) targeting intestinal dendritic cells are connected 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 sequence encoding each recombinant peptide, i.e., the DNA fragment of each recombinant peptide, was amplified using polymerase chain reaction (PCR) primers (primer sequences are shown in Table 1). The M13KE phage genome was double-digested with Kpn I and Eag I restriction endonucleases to remove the partial gene sequence encoding the PIII protein. The DNA fragment of each recombinant peptide was cloned into the M13KE phage and expressed on the capsid protein (pIII) of the M13KE phage to obtain the M13KE recombinant phage vector.

[0057] Take out the ER2738 chemical competent cells from the -80℃ refrigerator, place them on ice for 5 minutes, and then gently add 20-30ng of the M13KE recombinant phage vector. After transformation, resuspend the bacterial solution and spread it on an LB / IPTG / X-gal / tetracycline culture dish (IPTG was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A100487-0005; X-gal was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A100428-0100); tetracycline was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: A430165-0001), and then invert the culture dish in a bacterial constant temperature incubator at 37℃ for overnight culture.

[0058] On the second day, blue phage spots were selected and inoculated into 50 mL of LB liquid medium, cultured in a bacterial culture shaker at 37°C for 8 h, then centrifuged at 4,000 rpm for 15 min, the supernatant was collected, filtered with a 0.22 μm filter, and stored at 4°C. Subsequently, the recombinant phage was amplified and the genome was extracted, and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0059] Table 1 Primer sequences

[0060]

[0061]

[0062] 1.6. Amplification and purification of recombinant phage

[0063] Select blue recombinant phage spots and inoculate them in 20 mL LB medium, and culture them overnight at 37°C and 220 rpm. The overnight culture was centrifuged at 10,000 rpm for 20 min, and the supernatant was collected. The supernatant was added with 1 / 6 volume of a mixture containing PEG8000 and NaCl (the volume percentage of PEG8000 in the mixture was 20%, and the concentration of NaCl was 2.5 M; PEG8000 was purchased from Sangon Biotech (Shanghai) Co., Ltd., item number: A600433-0500; NaCl was purchased from Sangon Biotech (Shanghai) Co., Ltd., item number: A610476-0001), and precipitated overnight at 4°C. The phage precipitated overnight was centrifuged at 4°C and 10,000 rpm for 20 min, the supernatant was discarded, and the precipitate was resuspended in 1 mL of TBS buffer, which was the recombinant phage solution after amplification. The recombinant phage titer was determined by the double-layer plate method and stored briefly at 4°C.

[0064] 1.7. ELISA analysis of recombinant phage

[0065] PEDV polyclonal antibody (100 μL, 3 ng / μL, company: Guangzhou Qianxun Biotechnology Co., Ltd., catalog number: Pb-002) was added to the ELISA plate and incubated overnight at 4°C. The liquid was then discarded and the plate was washed with Tween-containing TM The ELISA plate was washed 6 times with 20% PBST, 100 μL of 5% skim milk was added, and incubated at 37°C for 1 h to block nonspecific binding. The liquid was discarded, the ELISA plate was washed 6 times with PBST, and then 100 μL of M13KE recombinant phage (10 8 pfu / mL) and wild-type M13KE phage (10 8 pfu / mL) and added PBS, which were recorded as M13KE-BXT1 group, 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, and incubated at 37°C for 1 h; a PBS group (PBS only) was also set up. The solution was discarded, and the ELISA plate was rinsed with PBST for 6 times, and 100 μL of 1:5000 diluted Anti-M13 Antibody (HRP) (Sino Biological) antibody was added, and incubated at 37°C for 1 h. Discard the solution, wash the ELISA plate 6 times with PBST, add 100 μL of 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 phages in mice

[0067] The titers of M13KE recombinant phage and wild-type M13KE phage were adjusted to 1.0 × 10 13 pfu / mL, stored at 4°C.

[0068] Specific pathogen-free (SPF) BALB / c mice (3-4 weeks old) were purchased from the Experimental Animal Center of Shanghai Jiao Tong University (Shanghai, China). The mice (48 in total) were randomly divided into 8 groups, 6 in each group, and the specific groups are shown in Table 2. The mice received intranasal immunization and oral gavage immunization with recombinant phages, respectively.

[0069] Booster immunizations were performed at weeks 2, 4, and 6. Blood samples were collected from the eye sockets of immunized mice the evening before weeks 2, 4, 6, and 8, stored overnight at 4°C, and serum was collected by brief centrifugation and stored at -20°C.

[0070] Table 2 Immunization groups of mice

[0071]

[0072] 1.9 Analysis of IgG Levels

[0073] The amount of IgG antibodies produced by mice infected with PEDV was evaluated by indirect ELISA. 20 μg / mL PEDV strain (PEDV / CHSH / 2019, published in "Isolation, Identification and Genetic Analysis of Porcine Epidemic Diarrhea Virus Shanghai Strain PEDV / CHSH / 2019" (Wang Na, Ji Likai, Wang Jian, et al. Isolation, Identification and Genetic Analysis of Porcine Epidemic Diarrhea Virus Shanghai Strain PEDV / CHSH / 2019 [J]. Chinese Journal of Zooinfectious Diseases, 2023, 31(03): 46-53. DOI: 10.19958 / j.cnki.cn31-2031 / s.2023.03.026.)) was used as an antigen coated on a 96-well polystyrene microplate, and then stained with 0.1% Tween TM 20 (PBST) PBS three times, blocked with 5% skim milk, and incubated at 37 ° C for 2 hours. Incubate at 37 ° C for 1 hour. Discard the solution, wash the ELISA plate with PBST for 6 times, add 100 μL of 1:5000 diluted Peroxidase AffiniPure GoatAnti-Mouse IgG (H+L), and incubate at 37 ° C for 1 hour. Discard the solution, wash the ELISA plate with PBST for 6 times, add 100 μL of 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 neutralization ability of antibodies induced by recombinant phages against viruses, the neutralization level of antibodies in mouse serum was determined while keeping the virus concentration constant. Mouse serum was inactivated at 56°C for 30 min, diluted twice, and 50 μL of serum was mixed with 50 μL of PEDV / CHSH / 2019 (100 TCID 50 ) and incubated at 37°C for 1 h, of which 100 μL was transferred to a 96-well cell culture plate (for overnight culture of Vero monolayer cells) and adsorbed at 37°C for 1 h. The supernatant was discarded, the cells were rinsed twice with PBS, 100 μL of maintenance solution was added to each well, and the culture plate was placed in a 5% CO2 37°C incubator for 72 h to observe the cytopathic effect (CPE).

[0076] 1.11. Lymphocyte proliferation test

[0077] At week 8, the spleen was removed from the immunized mice and placed in PBS. A large number of cavities were poked in the spleen with a sterile needle tip, and the PBS containing lymphocytes was carefully aspirated and slowly mixed with 5 mL of ficoll separation solution (1.2 g / mL). The mixture was centrifuged at 600 rpm for 25 min to obtain a liquid divided into three layers. The middle layer liquid was carefully aspirated, washed with PBS, and centrifuged at 600 rpm for 5 min. The red blood cell suspension was lysed with 0.75% Tris-NH4Cl (pH 7.4), washed three times with PBS, and then the spleen cells were 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 cell / mL spleen cell suspension was added to a 96-well cell culture plate (100 μL), and 100 μL of culture medium containing or not containing PEDV protein (20 μg / mL) was added to each group to stimulate 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] After stimulating the cells with 20 μg / mL PEDV protein for 72 h, the cell culture supernatant (from step "1.11, lymphocyte proliferation assay") was collected. The IFN-γ content in the supernatant was assessed by a commercial mouse IFN-γ enzyme-linked immunosorbent assay kit (Sangon Biotech (Shanghai) Co., Ltd.) according to the manufacturer's instructions. The cytokine concentration was calculated based on the standard curve of each ELISA plate.

[0080] 2. Results

[0081] 2.1 Bioinformatics analysis of PEDV S protein

[0082] We analyzed the S protein sequences of PEDV in the NCBI database before November 20, 2022. The purpose was to find out the sequence variations between different strains of PEDV. After removing duplicate and incomplete sequences, the analysis resulted in 1,882 unique PEDV S protein sequences. This dataset was then 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. The similarity of PEDV S protein sequences was further analyzed using MEGA11, and the results showed that the similarity between S protein sequences of different strains exceeded 87.33%. This indicates that there are many conserved similar sequences between PEDV S protein sequences of different strains, and that some potential antigenic epitopes are shared between different strains.

[0083] The PEDV S protein interacts with cell receptors during virus entry and stimulates the host to produce natural neutralizing antibodies. This example uses the DeepTMHMM server to predict the α-helix and β-fold transmembrane protein topology of the PEDV S protein. The results are shown in Figure 1 As shown in A. The results showed that the 20-1325S protein sequence was located in the extracellular region, and therefore, it was a good candidate for a potential neutralizing epitope.

[0084] One of the main challenges in developing new vaccines is the prediction of effective protective antigens. Theoretically, attention to structural antigen localization and the discovery of immunodominant B cell epitopes are extremely important for eliciting potent neutralizing activity and high levels of immune responses. In this example, we used the IEDB tool to predict B cell linear epitopes of the PEDV S protein and screened them to obtain three B cell linear PEDV epitopes (PEYC1, PEYC2, and PEYC3), as shown in Table 3. Compared with the predicted transmembrane regions of these proteins, it was found that the epitopes were all located in the PEDV S external region (20-1,325 amino acids). Among the 1,882 reported PEDV S proteins, the coverage rate of these three predicted epitopes exceeded 85%, making them good candidates for potential neutralizing epitopes (Table 3). We further analyzed whether the epitopes were located on the surface of the PEDV S protein structure, and the results were as shown in Table 3. Figure 1 As shown in B. PEYC1, PEYC2 and PEYC3 epitopes are exposed on the surface of the PEDV S three-dimensional structure, that is, the location of the dominant epitopes in the three-dimensional structure of the PEDV S protein, and they are mainly foreign antigens recognized by immune cells in space.

[0085] Table 3 Prediction of linear epitopes of PEDV S protein in B cells

[0086]

[0087] By specifically binding to receptors on the surface of immune cells, targeting peptides can direct vaccine components to key areas of immune response, thereby improving the immune effect and safety of the vaccine. In this example, in order to achieve targeted delivery of phage recombinant vaccines, targeting peptides BXT1 and BXT2 that target lung epithelial cells and induce targeted phage particles to enter the systemic circulation are reported to be concatenated with B cell linear epitopes of PEDV S protein, respectively. The names and sequences after concatenation are shown in Table 4.

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

[0089]

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

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

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

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

[0094] In order to verify whether the recombinant phage can specifically bind to PEDV positive serum, we used PEDV positive serum as antibody to measure the binding ability of exogenous peptides (i.e., recombinant peptides shown in Table 4) displayed by 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) with anti-PEDV antibodies. The results showed that the recombinant phages M13KE-BXT1-PEYC1 and M13KE-BXT2-PEYC2 had the highest affinity with PEDV positive serum ( Figure 4 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 body to produce neutralizing antibodies against PEDV, we inoculated mice with M13KE-BXT1-PEYC1 recombinant phage by intranasal drops and M13KE-BXT2-PEYC2 recombinant phage by oral gavage.

[0097] 14 days after the last booster immunization, the titer of PEDV antigen-specific IgG antibodies against the immune recombinant phage was evaluated by ELISA, that is, the mouse blood was collected at week 8, and the serum of all mice (BOOST3 mouse serum) was collected for ELISA analysis. The results showed that wild-type M13KE phage and M13KE phage with targeting peptide did not cause mice to produce antigen-specific PEDV IgG antibodies. On the contrary, the antigenic epitope delivered by M13KE phage can induce mice to produce specific IgG antibodies against PEDV, and the recombinant phage carrying PEDV epitopes can effectively induce the host to produce specific antibodies against PEDV ( Figure 5 (a) and (b) in FIG. 5A ). It can be seen that the M13KE recombinant phage with the targeting peptide and epitope induces antigen-specific PEDV IgG antibodies in mice.

[0098] The sera of immune mice were further serially diluted (serum was diluted 2-fold during the experiment) to neutralize live PEDV (PEDV / CHSH / 2019 strain), and the neutralization titer was calculated by reverse dilution. Figure 6As shown, the M13KE-BXT1-PEYC1 group and the M13KE-BXT2-PEYC2 group can stimulate the immunized mice to produce neutralizing antibodies. In the presence of low-fold dilution serum, mouse serum can neutralize live PEDV, but the PBS group, M13KE group, M13KE-BXT1 group and M13KE-BXT2 group cannot stimulate the immunized mice to produce neutralizing antibodies. The immunization strategy of the M13KE-BXT1-PEYC1 group and the M13KE-BXT2-PEYC2 group successfully and effectively induced mice to produce neutralizing antibodies against PEDV. It can be seen that the recombinant phage M13KE-BXT1-PEYC1 and the recombinant phage M13KE-BXT2-PEYC2 stimulated the immunized mice to produce neutralizing antibodies.

[0099] Two weeks after the third booster immunization, we isolated mouse spleen lymphocytes and stimulated lymphocyte proliferation in vitro with PEDV. Figure 7 As shown. After in vitro PEDV stimulation, the spleen T cells of mice in the M13KE-BXT1-PEYC1 group and the M13KE-BXT2-PEYC2 group had obvious proliferation, but there was no obvious proliferation after stimulation in the M13KE group, the M13KE-BXT1 group and the M13KE-BXT2 group, which was significantly different from the PBS group (p<0.0001). These results indicate that the immunization strategy of the M13KE-BXT1-PEYC1 group and the M13KE-BXT2-PEYC2 group can effectively induce mice to produce specific T cells against PEDV. It can be seen that the immunization strategy of the M13KE-BXT1-PEYC1 group and the M13KE-BXT2-PEYC2 group can effectively induce mice to produce T cells specific to PEDV.

[0100] After 72 h of PEDV stimulation of spleen cells, the cells were centrifuged and the culture supernatant was collected for quantitative detection of IFN-γ. Figure 8 As shown. The results showed that after PEDV stimulation in vitro, IFN-γ in the M13KE-BXT1-PEYC1 group and the M13KE-BXT2-PEYC2 group increased significantly, while IFN-γ in the M13KE group, the M13KE-BXT1 group and the 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. It can be seen that after PEDV stimulation in vitro, IFN-γ in the M13KE-BXT1-PEYC1 and M13KE-BEXT2-PEYC2 groups increased significantly. It can be seen 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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A recombinant protein, characterized in that The recombinant protein includes 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.

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

3. A biological material containing the nucleic acid molecule according to claim 2.

4. The biomaterial according to claim 3, characterized in that The biological material includes a recombinant bacteriophage.

5. Use of the recombinant protein according to claim 1, the nucleic acid molecule according to claim 2 or the biological material according to claim 3 or 4 in the preparation of a vaccine.

6. The use according to claim 5, characterized in that: The vaccine comprises a porcine coronavirus recombinant phage vaccine.

7. The use according to claim 6, characterized in that: The porcine coronavirus includes porcine epidemic diarrhea virus.

8. 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 .

9. The vaccine according to claim 8, characterized in that The vaccine also includes pharmaceutically acceptable excipients.

10. The vaccine according to claim 8, characterized in that The vaccine comprises a porcine coronavirus recombinant phage vaccine.

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