A recombinant protein subunit vaccine constructed by combining salmonella flagellin and porcine epidemic diarrhea coe region

Abstract

The application belongs to the technical field of biological medicine, and discloses a recombinant protein subunit vaccine constructed by combining Salmonella flagellin and a COE region of porcine epidemic diarrhea virus (PEDV). The application specifically discloses a fusion protein, wherein the fusion protein comprises Salmonella flagellin FliC L3A a mutant and a PEDV structural protein connected to the C terminal of the Salmonella flagellin FliC L3A The subunit vaccine obtained by emulsifying the fusion protein and the adjuvant can effectively prevent the G2b subfamily of PEDV, initiate a downstream immune response, induce a series of immune responses of the body, make the mucosal immune system of the body produce a large number of PEDV-specific antibodies to resist the invasion of external pathogens, and has the effect of specific immune protection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a recombinant protein subunit vaccine constructed by combining Salmonella flagellin and the COE region of porcine epidemic diarrhea. Background Technology

[0002] Since its discovery, porcine epidemic diarrhea virus (PEDV) has caused significant economic losses worldwide and has had a major impact on the pig farming industry. It has a mortality rate of up to 100% in piglets under 10 days old, putting considerable pressure on disease prevention.

[0003] Currently, the most common method for preventing PEDV is vaccination, as there is no specific antidote. Common vaccination methods are mainly traditional vaccines: live attenuated vaccines and inactivated vaccines. However, current traditional vaccines have problems such as poor protective efficacy, insufficient mucosal immune induction, and low IgA production, resulting in low protection rates in piglets. Summary of the Invention

[0004] This invention aims to at least solve one of the technical problems existing in the prior art. Addressing the issues of insufficient protective efficacy and poor mucosal immunity in currently available traditional vaccines, this invention proposes a new method to create a subunit vaccine that provides strong protection for piglets by constructing a fusion protein.

[0005] The first aspect of the present invention is to provide a fusion protein.

[0006] A second aspect of the present invention aims to provide biomaterials related to the fusion protein described in the first aspect of the present invention.

[0007] The third aspect of this invention aims to provide the application of the fusion protein described in the first aspect or the biomaterial described in the second aspect.

[0008] The fourth aspect of this invention is to provide a vaccine.

[0009] The fifth aspect of this invention is to provide a method for preparing the vaccine described in the fourth aspect of this invention.

[0010] The sixth aspect of this invention is to provide an antibody.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a fusion protein comprising Salmonella flagellin FliC. L3A The mutant and the PEDV structural protein, which is linked to the Salmonella flagellate protein FliC. L3AThe C-terminus of the mutant.

[0012] In some embodiments of the present invention, the Salmonella flagellin FliC L3A The amino acid sequence of the mutant is shown in SEQ ID NO:4.

[0013] In some embodiments of the present invention, the Salmonella flagellin FliC L3A The coding gene sequence of the mutant is shown in SEQ ID NO:3.

[0014] In some embodiments of the present invention, the PEDV structural protein includes the PEDV S protein.

[0015] In some embodiments of the present invention, the PEDV structural protein includes the COE region of the PEDV S protein.

[0016] In some embodiments of the present invention, the amino acid sequence of the core neutralizing epitope of the PEDV S protein is shown in SEQ ID NO:2.

[0017] In some embodiments of the present invention, the gene encoding the core neutralizing epitope COE region of the PEDV S protein is shown in SEQ ID NO:1.

[0018] In some embodiments of the present invention, the PEDV structural protein is linked to the Salmonella flagellate protein FliC via a linker peptide. L3A Mutant.

[0019] In some embodiments of the present invention, the linker peptide includes at least one of GSS, GSGGGS, GGSGGS, GGGGS, GGGSGGSG, GGGGGGG, GGSGGSGGSGGSGGS, GGGGSG ...

[0020] In some embodiments of the present invention, the fusion protein further includes a tag protein sequence that assists in expression and / or purification and / or detection.

[0021] In some embodiments of the present invention, the tag protein sequence includes at least one of His6, GST, MBP, FLAG, HA, Strep, and fluorescent tag protein sequences.

[0022] In some embodiments of the present invention, the fluorescent tag protein sequence includes at least one of GFP, RFP, BFP, CFP, YFP, and OFP.

[0023] In some embodiments of the present invention, the GFP also includes EGFP.

[0024] In some embodiments of the present invention, the fusion protein contains Salmonella flagellate protein FliC. L3A The mutant, PEDV structural protein, and / or tag protein sequences are linked by linker peptides.

[0025] In some embodiments of the present invention, the linker peptide includes at least one of GSS, GSGGGS, GGSGGS, GGGGS, GGGSGGSG, GGGGGGG, GGSGGSGGSGGSGGS, GGGGSG ...

[0026] In some preferred embodiments of the present invention, the linker peptide is GGSGGS.

[0027] In some embodiments of the present invention, the amino acid sequence of the fusion protein is as follows: a1)SEQ ID NO:6; or a2) An amino acid sequence of SEQ ID NO:6 with one or more amino acid substitutions and / or deletions and / or additions that have the same function as the protein shown in SEQ ID NO:6.

[0028] In some embodiments of the present invention, the amino acid sequence of the fusion protein further includes an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with SEQ ID NO:6 and having the same function as the protein shown in SEQ ID NO:6.

[0029] A second aspect of the present invention aims to provide biomaterials related to the fusion protein described in the first aspect of the present invention, said biomaterials comprising at least one of a1) to a16): a1) A nucleic acid molecule encoding the fusion protein described in the first aspect of the present invention; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A carrier containing the nucleic acid molecules described in a1); a4) A carrier containing the expression box described in a2); a5) Transgenic cell lines containing the nucleic acid molecules described in a1); a6) Transgenic cell lines containing the expression cassette described in a2); a7) A transgenic cell line containing the vector described in a3); a8) A transgenic cell line containing the vector described in a4); a9) Microorganisms containing the nucleic acid molecules described in a1); a10) contains microorganisms containing the expression cassette described in a2); a11) contains microorganisms that contain the carrier described in a3); a12) contains microorganisms that contain the carrier described in a4); a13) Viruses containing the nucleic acid molecules described in a1); a14) A virus containing the expression cassette described in a2); a15) contains a virus containing the vector described in a3); a16) contains a virus containing the vector described in a4); The transgenic cell line does not contain propagation material.

[0030] In some embodiments of the present invention, the sequence of the nucleic acid molecule includes SEQ ID NO:5.

[0031] In some embodiments of the present invention, the sequence of the nucleic acid molecule further comprises a nucleic acid molecule that has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with SEQ ID NO:5 and encodes the fusion protein.

[0032] A third aspect of the present invention aims to provide the use of the fusion protein described in the first aspect of the present invention or the biomaterial described in the second aspect of the present invention in any of the following: a1) Prepare drugs for the prevention and / or treatment of porcine epidemic diarrhea; a2) Drugs used to induce an immune response to porcine epidemic diarrhea virus antigens.

[0033] In some embodiments of the present invention, the drug includes a vaccine.

[0034] A fourth aspect of the present invention is to provide a vaccine comprising the fusion protein described in the first aspect of the present invention.

[0035] In some embodiments of the invention, the vaccine further includes an immunologically and pharmaceutically acceptable carrier or adjuvant.

[0036] In some embodiments of the present invention, the adjuvant includes at least one of aluminum adjuvant, emulsion adjuvant, liposome adjuvant, virus-like particle adjuvant, immunostimulatory complex, and immunostimulator.

[0037] In some embodiments of the present invention, the aluminum adjuvant includes aluminum hydroxide and aluminum phosphate.

[0038] In some embodiments of the present invention, the emulsion adjuvant includes at least one of oil-in-water adjuvant, water-in-oil adjuvant, and water-in-oil-in-water adjuvant.

[0039] In some embodiments of the present invention, the immunologically and pharmaceutically acceptable vectors include viral and non-viral vectors.

[0040] In some embodiments of the present invention, the immunologically and pharmaceutically acceptable viral vector includes at least one of adenovirus vectors, retroviruses, lentiviruses, herpesviruses, and virus-like particles.

[0041] In some embodiments of the present invention, the non-viral vector includes at least one of naked DNA, liposomes, nanocarriers, and exosomes.

[0042] In some preferred embodiments of the present invention, the adjuvant is a water-in-oil-in-water adjuvant.

[0043] A fifth aspect of the present invention is to provide a method for preparing the vaccine described in the fourth aspect of the present invention, comprising the following steps: mixing the fusion protein described in the first aspect of the present invention with an adjuvant to obtain a vaccine.

[0044] In some embodiments of the present invention, the adjuvant includes at least one of aluminum adjuvant, emulsion adjuvant, liposome adjuvant, virus-like particle adjuvant, immunostimulatory complex, and immunostimulator.

[0045] In some embodiments of the present invention, the aluminum adjuvant includes aluminum hydroxide and aluminum phosphate.

[0046] In some embodiments of the present invention, the emulsion adjuvant includes at least one of oil-in-water adjuvant, water-in-oil adjuvant, and water-in-oil-in-water adjuvant.

[0047] In some embodiments of the present invention, the adjuvant is a water-in-oil-in-water adjuvant.

[0048] In some embodiments of the present invention, the mass ratio of the fusion protein to the adjuvant is 1:1 to 3.

[0049] In some preferred embodiments of the present invention, the mass ratio of the fusion protein to the adjuvant is 1:1.

[0050] A sixth aspect of the present invention is to provide an antibody comprising an antigen induced by a fusion protein described in the first aspect of the present invention or a biological material described in the second aspect of the present invention.

[0051] The beneficial effects of this invention are: This invention utilizes protein design to synthesize the porcine intestinal Salmonella flagellate protein mutant FliC. L3AThe fusion protein FliC was obtained by fusing the PEDV COE region. L3A -COE-C, utilizing the spontaneous formation of homopolymers by flagellin proteins, exposes the COE domain of the PEDV neutralizing epitope, causing it to aggregate into a bouquet-like structure. This activates the pathogen-associated molecular pattern response in the organism, further enhancing the immune response and improving immunity. The subunit vaccine obtained by emulsifying the fusion protein and adjuvant provided by this invention can effectively prevent PEDV of the G2b subfamily, initiate downstream immune responses, and induce a series of immune responses in the body. This results in the production of a large number of PEDV-specific antibodies by the mucosal immune system to resist the invasion of foreign pathogens, thus achieving a specific immune protection effect. Attached Figure Description

[0052] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The diagram shows the predicted structure of the complex protein, where A is a side view of the complex protein monomer and B is a side and top view of the flagellin polymer.

[0053] Figure 2 Fluorescence results for screening cell lines with stable transfection of the complex protein (Figure) Figure 2 SDS-PAGE Coomassie Brilliant Blue gel image of complex protein (A) Figure 2 (B) and Western blot results ( Figure 2 (C), the fluorescence microscope used has a magnification of 20x objective lens.

[0054] Figure 3 PEDV-specific IgG in the blood of mice immunized with recombinant vaccine ( Figure 3 Medium A), IgA ( Figure 3 B), IgM ( Figure 3 C), IFN-γ ( Figure 3 (middle D) and IL-4 ( Figure 3 The results at the E level are plotted. The letters in the plot represent significance; different letters indicate significant differences, while the same letter indicates no significant differences. Detailed Implementation

[0055] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0056] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0057] This invention selects the COE region of PEDV G2b subfamily strain CH / JSLYG / 02 / 2021 as the research object. The nucleic acid sequence of its S1 protein COE region is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2. Mutations were performed on the FliC protein at sites L503A, L505A, and L506A to obtain FliC. L3A Mutant. This mutant can better stimulate the mucosal immune response and improve the protection rate of piglets. Its nucleic acid sequence is shown in SEQ ID NO:3, and its amino acid sequence is shown in SEQ ID NO:4. The COE region is linked to FliC via a GS-linker (GGSGGS). L3A The C-terminus of the protein forms a complex protein, the nucleic acid sequence of which is shown in SEQ ID NO:5, and the amino acid sequence in SEQ ID NO:6. The structural prediction results of the complex protein are as follows: Figure 1 As shown, this complex protein design utilizes the property of flagellin to spontaneously form homopolymers, exposing the COE domain to form a bouquet-like structure, which further enhances the body's immune response.

[0058] The nucleic acid sequence of the COE region is as follows: ATATTCTTTCGTGACCCTGCCCTCTTTCAACGACCACTCCTTCGTAAACATTACAGTTAGTGCCTCTTTCGGCGGTCACTCCGGGGCCAATCTGATAGCTTCCGACACCACTATCAACGGACTCTCCAGCTTCTGCGTTGATACCCGGCAATTCACAATCTCCCTGTTTTATAACGTTACCAACAGCTACGGATATGTCTCCAAGTCCCAGGA TTCCAACTGTCCCTTTACCTTGCAGAGTGTTAACGACTACTTGAGCTTCTCCAAATTCTGCGTGTCTACCTCACTCCTGGCATCAGCTTGTACCATAGACTTGTTCGGGTACCCTGAGTTTGGGTCTGGCGTGAAATTCACTAGCCTGTACTTTCAGTTCACAAAGGGCGAGCTGATAACTGGCACCCCTAAACCACTGGAGGGGGTG (SEQ ID NO:1).

[0059] The amino acid sequence of the COE region is as follows: ISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGLSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGV (SEQ ID NO: 2).

[0060] FliC L3A The nucleic acid sequence of the mutant is:

[0061] FliC L3A The amino acid sequence of the mutant is as follows: GSAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYDVKDTAVTTKAYANNGTTLDVSGLDDAAIKAATGGTNGTASVTGGAVKFDADNNKYFVTIGGFTGADAAKNGDYEVNVATDGTVTLAAGATKTTMPAGATTKTEVQELKDTPAVVSADAKNALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTSYTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO:4).

[0062] Composite protein FliC L3A The nucleic acid sequence of -COE-C is as follows: GGTGGTTCCGGGGGCTCC ATATCTTTCGTGACCCTGCCCTCTTTCAACGACCACTCCTTCGTAAACATTACAGTTAGTGCCTCTTTCGGCGGTCACTCCGGGGCCAATCTGATAGCTTCCGACACCACTATCAACGGACTCTCCAGCTTCTGCGTTGATACCCGGCAATTCACAATCTCCCTGTTTTATAACGTTACCAACAGCTACGGATATGTCTCCAAGTCCCAGGATTCCAACTGTCCCTTTACCTTGCAGAGTGTTAACGACTACTTGAGCTTCTCCAAATTCTGCGTGTCTACCTCACTCCTGGCATCAGCTTGTACCATAGACTTGTTCGGGTACCCTGAGTTTGGGTCTGGCGTGAAATTCACTAGCCTGTACTTTCAGTTCACAAAGGGCGAGCTGATAACTGGCACCCCTAAACCACTGGAGGGGGTG (SEQ ID NO:5, the underlined part is the nucleic acid sequence of the linker peptide).

[0063] Composite protein FliC L3A The amino acid sequence of -COE-C is: (SEQ ID NO:6).

[0064] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0065] Example 1 (1) Protein structure design Using the G2b subfamily PEDV strain CH / JSLYG / 02 / 2021 as the research object, the COE region of its S protein sequence (GeneBank: MZ161072.1) was selected and linked to the porcine enteric Salmonella choleraesuis flagellate protein mutant FliC using a GS-linker. L3A The target protein FliC was designed by attaching EGFP to the C-terminus of the complex protein via a TEV restriction site (ENLYFQG) and a 6×His tag (HHHHHH) to the N-terminus of the fluorescent molecule for protein purification.L3A -COE-C.

[0066] (2) Cloning of the target gene The target gene was constructed using the PiggyBac-dual-EGFP vector, which is a PiggyBac transposase system. Stable cell lines were constructed by co-transfecting CHO-K1 adherent cells (Chinese hamster ovary cell line, donated by Dr. Wang Aibo of the Guangdong Haida Group Animal Husbandry and Fisheries Research Center) with the transposase expression vector PB200PA-1. Primers required for inserting the target gene into the vector are shown in Table 1. The cloning template used in this invention includes FliC... L3A Both the PiggyBac-dual-EGFP and COE gene fragments were synthesized by Shanghai Sangon Biotech Co., Ltd. The PiggyBac-dual-EGFP and PB200PA-1 vectors were purchased from Hunan Fenghui Biotechnology Co., Ltd. PBDE-F / R primers were used to amplify the PiggyBac-dual-EGFP vector, and linearized vectors were obtained by PCR for subsequent ligation. EGFP(PBDE)-F and TEV(His)-R primers were used for amplifying the EGFP fragment, and His(TEV)-F and COE(PBDE)-R primers were used for amplifying the FliC gene fragment. L3A -COE fusion gene sequence. PCR amplification was performed using Merck's KOD ONE PCR MasterMix (catalog number: KMM-101NV), and the reaction system is shown in Table 2, while the amplification program is shown in Table 3.

[0067] Table 1 Primers required for PEDV S complex protein gene amplification

[0068] Table 2 Reaction System

[0069] Table 3 Amplification System

[0070] Since the amplified gene fragment contains homologous sequences at both its 3' and 5' ends, this invention uses Seamless Clone recombinase from Beyotime Biotechnology Co., Ltd. (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: B522232) for seamless cloning. The insert fragment and linearized vector were mixed at a mass ratio of 3:1, and an equal volume of recombinase was added. The mixture was then incubated in a PCR instrument at 55°C for 15 min for ligation. The ligation product was transformed into DH5α Escherichia coli competent cells for plasmid amplification. The specific operational steps are as follows: 1) Place DH5α competent Escherichia coli cells at -80℃ on ice until they thaw; 2) Take 2 μL of the correctly sequenced plasmid and add it to DH5α competent cells and place them on ice for 30 min; 3) After an ice bath of 30 min, the competent cells were placed in a 42℃ water bath for 90 s for heat shock; 4) After heat shock, place the competent cells on ice for 2-3 minutes; 5) In a clean bench, spread competent cells onto an antibiotic plate and incubate overnight at 37°C.

[0071] After colony PCR, plasmids were extracted from positive monoclonal colonies and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing verification. Recombinant plasmids with correct sequencing results were used for the construction of stable cell lines mentioned in subsequent plasmids.

[0072] Example 2 (1) Construction of stable cell lines After obtaining the vector plasmid, plasmid extraction was performed to obtain the target gene plasmid suitable for transfection. During transfection, adherent CHO-K1 cells showed a survival rate of over 90% and a density of approximately 80%. DMEM / F12 medium (Thermo Fisher Scientific, catalog number: 10565042) was slowly aspirated, and 10 mL of fresh, PS-free (penicillin-streptomycin) resistant medium was added, and the cells were cultured overnight. Transfection was performed the following day, using a liposome (Hieff Trans® Liposomal Transfection Reagent, Yeasen) to plasmid ratio of 20 μL : 10 μg, targeting cells 10 cm². 2 Prepare mixtures A and B in round dishes: A: 20 μL liposomes + 1000 μL Opti-MEM medium (Thermo Fisher Scientific, catalog number: 11058021); B: 10 μg plasmid + 1000 μL Opti-MEM medium.

[0073] After preparation, let solution A stand at room temperature for 5 minutes. Then, slowly add solution A to solution B and mix. Vortex to mix thoroughly and let stand at room temperature for 20 minutes. Finally, pipette the mixture of A and B and add it dropwise evenly to the cells to be transfected. Change the medium overnight and add about 15 mL of complete culture medium (DMEM / F12 medium + 10% FBS). After culturing for 24-48 hours, the cell fluorescence can be observed.

[0074] (2) Screening of monoclonal cell lines Observe fluorescence 48 hours after transfection, then aspirate the medium and replace it with complete medium containing 10 μg / mL puromycin, and continue culturing in a 37°C, 5% CO2 incubator. Observe cell floating and adherent cell density daily, changing the selection medium every 2 days on average, for approximately 15-30 days. Once cell density remains constant, growth rate is normal, and the proportion of fluorescent cells stabilizes, resistance selection is complete.

[0075] After screening, fluorescence flow cytometry (FACS) cell sorting is performed. The specific steps are as follows: 1) Digest adherent CHO-K1 cells with trypsin, centrifuge (300g, 5min) after stopping digestion with complete medium (DMEM / F12 medium + 10% FBS), resuspend in PBS containing 1-2% FBS, filter through a 40μm sieve into FACS tubes, and adjust the density to 1-5×10⁶ cells / mL. 6 cells / mL. Pre-add 100 μL of complete culture medium to each well of a 96-well plate.

[0076] 2) Use a 100μm nozzle for the flow cytometer and select "Single Cell" mode. Establish the gating logic: FSC / SSC main group → SSC-W / SSC-H de-adhesion → GFP positive gate (use untransfected cells as a negative control). Set up a 96-well plate and sort one GFP positive cell in each well.

[0077] 3) After sorting, place the 96-well plates in an incubator and let them stand for 3-7 days without moving them. If the culture medium turns yellow on the 5th-7th day, carefully aspirate 50μL of the old solution and add an equal amount of fresh, preheated culture medium.

[0078] 4) Once the single clonal clusters have grown to 30-50% of the bottom area of ​​the wells (approximately 10-14 days), they are digested with trypsin and then transferred to 48-well plates for stepwise amplification. Simultaneously, microscopy is used to confirm that the cells from day 0-1 are of single-cell origin.

[0079] Screening fluorescence results are shown in Figure 2 In Figure A, all images were taken at approximately 14 days of age for monoclonal positive cell lines. The left image is a GFP fluorescence image, and the right image is a corresponding bright field image.

[0080] (3) Protein purification Protein purification from CHO-K1 monoclonal cells was performed using affinity chromatography. First, the cell pellet was resuspended in lysis buffer (150 mM NaCl + 25 mM Tris-HCl, pH 8.0), sonicated, and then centrifuged at 12000 g for 30 min at 4 °C. The supernatant was collected. The supernatant was incubated with a Ni-NTA affinity chromatography column at 4 °C, and the flow-through was collected. The column was then washed three times with 30 mL of washing buffer (150 mM NaCl + 25 mM Tris-HCl, pH 8.0 + 30 mM imidazole), 10 mL each time, and the flow-through was collected after each wash. Elution was then performed three times with 15 mL of elution buffer (150 mM NaCl + 25 mM Tris-HCl, pH 8.0 + 300 mM imidazole), 5 mL each time, and the eluted fraction was collected to obtain the target protein solution. To remove the fusion tag, TEV protease (Thermo Fisher Scientific, catalog number: 12575023) was added and the reaction was carried out at 4°C for 16-18 hours. Finally, the digestion product was passed through a Ni-NTA chromatography column to separate and remove the TEV protease and uncleaved fusion protein, ultimately obtaining high-purity, tag-free target protein.

[0081] Protein purification results (SDS-PAGE) are shown below. Figure 2 The results of Western blot analysis for B1 are shown below. Figure 2 In the case of C, SDS-PAGE electrophoresis, Coomassie brilliant blue staining, and Western blot analysis showed that the location and size of the purified protein were consistent with the expected size. The concentration was detected by the A280 absorption peak, and the final expression level was approximately 1.38 g / L.

[0082] Example 3 (1) Vaccine preparation After obtaining the target protein at a concentration of approximately 200 μg / mL, the preparation of the vaccine formulation was immediately commenced. Adjuvant 201 (ISA201, SEPPIC (Shanghai) Specialty Chemicals Co., Ltd.) was selected as the immunostimulant and emulsified with the target protein at an equal volume ratio. The specific procedure was as follows: In a clean bench, the target protein solution, restored to room temperature, was transferred to a sterile beaker, followed by the addition of an equal volume of adjuvant 201. After initial mixing, a sterilized magnetic stir bar was placed on the beaker. The beaker was then placed on the magnetic stir bar and stirred continuously at 300 rpm for 10 minutes at 31°C to ensure thorough emulsification of the aqueous and oil phases, forming a homogeneous formulation. After stirring, the formulation was dispensed under aseptic conditions and stored at 4°C for later use. Strict aseptic principles were maintained throughout the entire process and subsequent use to ensure the stability and reliability of the formulation.

[0083] Since the 201 adjuvant and the target protein are diluted in equal amounts, the final vaccine concentration is approximately 100 μg / mL.

[0084] (2) Mouse immunization experiment Twelve male Balb / c mice aged 6-8 weeks were randomly divided into three groups: COE injection group, negative control group, and FliC group. L3A -COE-N control group (FliC) L3A -COE-N is a COE connection in FliC. L3A (protein N-terminus) and FliC L3A -COE-C experimental group. Mice were subcutaneously immunized at three points (subcutaneous injection in the neck and hind legs). 100 μL of the test substance (the vaccine prepared in step (1) of Example 3) was injected into the neck, and 50 μL of the test substance was injected into each of the inner sides of the hind legs. The immunization was carried out three times, with an interval of 14 days between each immunization (N=3). Blood was collected from the tail vein one week after the first and second immunizations, and from the orbital vein two weeks after the third immunization. After centrifugation to separate the serum from the collected blood samples, the serum levels of PEDV-specific IgG, IgA and IgM antibodies were detected using commercial ELISA kits (antibody detection kits were purchased from Quanzhou Ruixin Biotechnology Co., Ltd., catalog numbers: RXW201452M, RXW203434M, RX2D2012106). Meanwhile, the concentrations of Th1 / Th2 cytokines such as interferon-γ (IFN-γ) and interleukin-4 (IL-4) in mouse serum were measured using cytokine detection kits (purchased from Wuhan Yilairuit Biotechnology Co., Ltd., catalog numbers E-EL-M0048 and E-EL-M0043, respectively) to systematically evaluate the levels of vaccine-induced humoral and cellular immune responses.

[0085] The results are as follows Figure 3 As shown, FliC L3A -COE-C experimental group and FliC L3A The levels of IgG, IgA, IgM, IFN-γ, and IL-4 in the COE-N control group were significantly higher than those in the COE-only injection group. L3A The levels of IgG, IFN-γ, and IL-4 in the COE-C experimental group were significantly higher than those in the FliC group. L3A -COE-N control group ( Figure 3 China A Figure 3 D, Figure 3 (E). During three immunizations, FliC L3A The IgA antibody titer in the COE-C experimental group was significantly higher than that in the FliC group. L3A -COE-N control group ( Figure 3 (B) FliC L3A -IgM antibody titer and FliC in the COE-C experimental groupL3A There was no significant difference between the -COE-N control group and the control group. Figure 3 (C)

[0086] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A fusion protein comprising Salmonella flagellin FliC L3A The mutant and the PEDV structural protein, which is linked to the Salmonella flagellate protein FliC. L3A The C-terminus of the mutant.

2. The fusion protein according to claim 1, characterized in that, The Salmonella flagellin FliC L3A The amino acid sequence of the mutant is shown in SEQ ID NO:

4.

3. The fusion protein according to claim 1, characterized in that, The PEDV structural protein includes the PEDV S protein; preferably, the PEDV structural protein includes the core neutralizing epitope (COE) region of the PEDV S protein; preferably, the amino acid sequence of the core neutralizing epitope (COE) region of the PEDV S protein is shown in SEQ ID NO:

2.

4. The fusion protein according to claim 1 or 3, characterized in that, The PEDV structural protein is linked to Salmonella flagellin FliC via a linker peptide. L3A The mutant; and / or, the fusion protein further includes a tag protein sequence that assists in expression and / or purification and / or detection; preferably, the tag protein sequence includes at least one of His6, GST, MBP, FLAG, HA, Strep and fluorescent tag protein sequences.

5. The fusion protein according to claim 4, characterized in that, The amino acid sequence of the fusion protein is as follows: a1)SEQ ID NO:6; or a2) An amino acid sequence of SEQ ID NO:6 that has undergone substitution and / or deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO:

6.

6. A biomaterial relating to the fusion protein of any one of claims 1 to 5, said biomaterial comprising at least one of a1) to a16): a1) A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 5; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A carrier containing the nucleic acid molecules described in a1); a4) A carrier containing the expression box described in a2); a5) Transgenic cell lines containing the nucleic acid molecules described in a1); a6) Transgenic cell lines containing the expression cassette described in a2); a7) A transgenic cell line containing the vector described in a3); a8) A transgenic cell line containing the vector described in a4); a9) Microorganisms containing the nucleic acid molecules described in a1); a10) contains microorganisms containing the expression cassette described in a2); a11) contains microorganisms that contain the carrier described in a3); a12) contains microorganisms that contain the carrier described in a4); a13) Viruses containing the nucleic acid molecules described in a1); a14) A virus containing the expression cassette described in a2); a15) contains a virus containing the vector described in a3); a16) contains a virus containing the vector described in a4); The transgenic cell line does not contain propagation material; Preferably, the sequence of the nucleic acid molecule includes SEQ ID NO:

5.

7. The use of the fusion protein according to any one of claims 1 to 5 or the biomaterial according to claim 6 in any of the following: a1) Prepare drugs for the prevention and / or treatment of porcine epidemic diarrhea; a2) Drugs used to induce an immune response to porcine epidemic diarrhea virus antigens; Preferably, the drug includes a vaccine.

8. A vaccine comprising the fusion protein according to any one of claims 1 to 5; preferably, the vaccine further comprising an immunologically and pharmaceutically acceptable carrier or adjuvant.

9. A method for preparing the vaccine according to claim 8, comprising the following steps: mixing the fusion protein and adjuvant according to any one of claims 1 to 5 to obtain the vaccine.

10. An antibody, characterized in that, The antibody comprises an antigen induced by the fusion protein of any one of claims 1 to 5 or the biological material of claim 6.

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