Recombinant protein of porcine epidemic diarrhea virus and application thereof

Abstract

The present disclosure provides a recombinant protein comprising a signal peptide, an S1 domain of a porcine epidemic diarrhea virus S protein, and a multimerization sequence. The present disclosure also provides a recombinant vaccine prepared from the recombinant protein, which has the advantages of higher safety, no reverse toxicity, stimulation of the body to produce an effective neutralizing antibody concentration, and batch production consistency.

Description

Technical Field

[0001] This disclosure belongs to the field of biotechnology, specifically relating to a recombinant protein of porcine epidemic diarrhea virus, a recombinant vaccine containing the protein, and its applications. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease in pigs caused by the porcine epidemic diarrhea virus (PEDV). This virus belongs to the Coronaviridae family, has an envelope, exhibits pleomorphism, tends to be spherical, with a diameter of 95-190 nanometers, and its nucleic acid type is RNA.

[0003] The main symptoms of porcine epidemic diarrhea (PED) include vomiting, diarrhea, and dehydration. Affected pigs exhibit watery diarrhea, yellow or grayish-yellow in color, with a foul odor, and their body temperature remains generally normal. Pigs of all ages can be infected, with morbidity rates sometimes reaching 100% in suckling piglets, growing pigs, or finishing pigs, especially suckling piglets. Pathological changes are primarily observed in the small intestine, manifesting as intestinal dilation, thin contents, thinning of the intestinal wall, and shortening of intestinal villi.

[0004] Porcine epidemic diarrhea virus (PEDV) is primarily transmitted through the digestive tract, with the fecal-oral route being the main transmission route, but it can also be transmitted through the respiratory tract. The most effective way to prevent this infectious disease is to implement comprehensive prevention and control measures, including isolation, sanitation and disinfection, temperature and humidity control, cardiac support, fluid and electrolyte replacement, and vaccination. Once the disease occurs, symptomatic treatment should be administered to prevent secondary infections.

[0005] Currently, the main targets for vaccination against porcine epidemic diarrhea (PEDV) include pregnant sows and piglets. Prenatal immunization of pregnant sows, allowing the transfer of maternal anti-PEDV antibodies to piglets via the mother-to-child route, is an economical and effective preventative method.

[0006] Therefore, developing new vaccines against PEDV is of great significance. Summary of the Invention

[0007] This disclosure provides a recombinant subunit vaccine based on the S protein of porcine epidemic diarrhea virus (PEDV). Specifically, the S1 domain of the S protein from the PEDV G1a subtype strain CV777 and the G2a subtype strain CH / HNAY / 2015 is used as the main immunogen to construct the recombinant protein and prepare the recombinant subunit vaccine. A His tag protein is added to the C-terminus of the sequence to facilitate purification. Compared with live attenuated and inactivated vaccines against PEDV, the recombinant subunit vaccine has advantages such as higher safety, no retroviral response, high stimulation of the body to produce effective neutralizing antibody concentrations, and high batch-to-batch consistency.

[0008] According to one aspect of this disclosure, a recombinant protein is provided, the recombinant protein comprising the S1 domain and polymerized sequence of the porcine epidemic diarrhea virus S protein.

[0009] In some embodiments, the porcine epidemic diarrhea virus S protein includes the S protein of PEDV G1a subtype strain CV777 and / or the S protein of PEDV G2a subtype strain CH / HNAY / 2015.

[0010] In some embodiments, the S1 domain of the porcine epidemic diarrhea virus S protein includes the amino acid sequence shown in SEQ ID NO: 1 or 2, or an amino acid sequence that is at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to it, or an amino acid sequence that has one or more amino acid substitutions, deletions, or additions compared to it.

[0011] In some embodiments, the polymerization sequence includes a trimer Folden sequence.

[0012] In some embodiments, the polymerized sequence comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it.

[0013] In some embodiments, the recombinant protein further includes a signal peptide.

[0014] In some embodiments, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it.

[0015] In some embodiments, the recombinant protein further includes a purification tag.

[0016] In some implementations, the purification tag includes a His tag.

[0017] In some embodiments, the recombinant protein includes a signal peptide, porcine epidemic diarrhea virus S protein, and a polymerized sequence from the N-terminus to the C-terminus, as well as an optional purification tag.

[0018] In some embodiments, the signal peptide is directly linked to the porcine epidemic diarrhea virus S protein, the porcine epidemic diarrhea virus S protein to the polymerized sequence, and / or the polymerized sequence to the purification tag, or linked via a linker.

[0019] In some embodiments, the linker is a peptide with a length of 2-30 amino acids.

[0020] In some implementations, the connector is selected from (G n S) m (G) n (EAAAK) n Or (XP) n , where n and m are each independently selected from integers from 1 to 10.

[0021] In some embodiments, the recombinant protein has one or more of the amino acid sequences shown in SEQ ID NO: 6-9, or an amino acid sequence that is at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to that sequence, or an amino acid sequence that has one or more amino acid substitutions, deletions, or additions compared to that sequence.

[0022] According to another aspect of this disclosure, a multimeric recombinant protein is provided, the multimeric recombinant protein comprising at least three recombinant protein units, the recombinant protein units comprising the recombinant protein described in this disclosure.

[0023] According to another aspect of this disclosure, an isolated nucleic acid is provided, said isolated nucleic acid encoding the recombinant protein or the multimeric recombinant protein described in this disclosure.

[0024] According to another aspect of this disclosure, an expression vector is provided, said expression vector comprising the isolated nucleic acid described in this disclosure.

[0025] In some embodiments, the expression vector may be a eukaryotic cell expression vector and / or a prokaryotic cell expression vector, such as a retroviral vector, lentiviral vector, bacteriophage vector, adenovirus vector, adeno-associated vector, or herpes simplex vector.

[0026] In some embodiments, the expression vector is present in nanoparticles, liposomes, exogenous bodies, microvesicles, or gene guns.

[0027] According to another aspect of this disclosure, a host cell is provided, the host cell comprising the isolated nucleic acid described in this disclosure, or the expression vector described in this disclosure, or expressing the recombinant protein described in this disclosure or the multimeric recombinant protein described in this disclosure.

[0028] In some embodiments, the host cell is a conventional host cell in the art, as long as the expression vector stably expresses the carried nucleic acid molecule as the recombinant protein or the multimeric recombinant protein described in this disclosure. Preferably, the host cell is a prokaryotic cell and / or a eukaryotic cell, with E. coli cells such as TG1 and BL21 being preferred prokaryotic cells, and HEK293 cells or CHO cells being preferred eukaryotic cells. Transforming the expression vector into the host cell yields the host cell of this disclosure. The transformation method is a conventional transformation method in the art, preferably a chemical transformation, heat shock, or electroporation.

[0029] According to another aspect of this disclosure, a pharmaceutical composition is provided comprising: the recombinant protein or the multimeric recombinant protein described in this disclosure, and a pharmaceutically acceptable carrier.

[0030] In some embodiments, the pharmaceutical composition is in the form of a recombinant vaccine.

[0031] In some implementations, the recombinant vaccine is a recombinant subunit vaccine.

[0032] In some embodiments, the vaccine also includes a buffer solution.

[0033] In some embodiments, the buffer includes physiological saline, 0.01M PBS buffer (e.g., 0.01-0.1M), or HEPES buffer (e.g., 0.01-1M).

[0034] In some embodiments, the vaccine also includes an adjuvant.

[0035] In some embodiments, the adjuvant includes one or more of aluminum adjuvant, Cpg adjuvant, and MF59 adjuvant.

[0036] In some embodiments, the pharmaceutically acceptable carrier may be a carrier conventional in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents, etc.

[0037] In some embodiments, the drug composition may be administered via parenteral, injection, or oral administration. The drug composition may be formulated into a form suitable for administration, such as a solid, semi-solid, or liquid form, and may be an aqueous solution, non-aqueous solution, or suspension, or in the form of powder, tablet, capsule, granule, injection, or infusion. It may be administered intravascularly, subcutaneously, intraperitoneally, intramuscularly, by inhalation, intranasal, airway instillation, or intrapleural instillation. The drug composition may also be administered as an aerosol or spray, for example, via nasal administration; or via intrathecal, intramedullary, or intraventricular administration, or via transdermal, percutaneous, local, enteric, intravaginal, sublingual, or rectal administration. The drug composition may be formulated into various dosage forms as needed, and the physician may determine the beneficial dosage for the patient based on factors such as patient type, age, weight, general disease condition, and route of administration.

[0038] In some embodiments, the recombinant protein or multimeric recombinant protein in the pharmaceutical composition may be administered simultaneously or sequentially with other active ingredients.

[0039] According to another aspect of this disclosure, a kit is provided comprising the recombinant protein described in this disclosure, or the multimeric recombinant protein described herein.

[0040] In some embodiments, the kit further includes a detectable marker that can be linked to the recombinant protein or the multimeric recombinant protein, the detectable marker being linked to the recombinant protein or the multimeric recombinant protein, or being present separately in the kit.

[0041] In some embodiments, the kit also includes a substrate corresponding to a detectable marker and / or instructions for use.

[0042] According to another aspect of this disclosure, the recombinant protein, the multimeric recombinant protein, the isolated nucleic acid, the expression vector, the host cell, the antibody-drug conjugate, the pharmaceutical composition, or the kit described herein are provided for use in screening or preparing medicaments for the prevention, treatment, and / or diagnosis of diseases related to porcine epidemic diarrhea virus infection.

[0043] In some embodiments, the porcine epidemic diarrhea virus infection-related disease includes porcine epidemic diarrhea. Attached Figure Description

[0044] Figure 1 The electrophoresis results according to Example 1 are shown, where lane 1 is the PCDNA3.1 plasmid, lane 2 is the plasmid digested with HindIII-EcoRI, and lane M is the marker.

[0045] Figure 2The results of SDS-PAGE electrophoresis and Western bolt analysis of the trimeric recombinant protein are shown, where lane M is the marker and lane 1 is the trimeric S protein.

[0046] Figure 3 The results of ELISA assays in mice from different drug administration groups are shown.

[0047] Figure 4 The body weight results of mice in different drug administration groups are shown. Detailed Implementation

[0048] The following describes preferred embodiments of this disclosure, but the scope of protection of this disclosure is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of this disclosure. Reagents used, unless otherwise specified, are all commercially available conventional products.

[0049] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0050] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0051] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0052] As used herein, the term "recombinant protein" refers to two or more proteins or fragments thereof that are collinearly linked by their respective peptide backbones using genetic expression or protein synthesis methods that encode protein polynucleotides. Preferably, the proteins or fragments thereof are from different sources.

[0053] The recombinant proteins or fragments thereof disclosed herein may include conserved amino acid substitutions at one or more amino acid residues, such as essential or non-essential amino acid residues. A “conserved amino acid substitution” is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, in this document, essential or non-essential amino acid residues in recombinant proteins or linkers are preferably replaced with another amino acid residue from the same side chain family. In some embodiments, amino acid segments can be replaced with structurally similar segments that differ in the order and / or composition of side chain family members. Alternatively, in some embodiments, mutations can be randomly introduced along all or part of the coding sequence, such as through saturation mutagenesis, and the resulting mutants can be incorporated into the recombinant proteins of the present invention, and the ability of these peptides to bind to desired targets can be screened.

[0054] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0055] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0056] As used herein, the term "linker" refers to a (peptide) linker of natural and / or synthetic origin, composed of linear amino acids. The domains of the recombinant proteins described herein can be linked by linkers, wherein each linker is fused to and / or otherwise linked (e.g., via peptide bonds) with at least two polypeptides or domains. Linkers should have a length suitable for linking two or more monomeric domains in this manner, ensuring that the different domains they link fold correctly and are properly presented to perform their biological functions. In various embodiments, linkers have a flexible conformation. Suitable flexible linkers include, for example, those having glycine, glutamine, and / or serine residues. In some embodiments, the linker may be selected from (Glycine, glutamine, and / or serine residues). n S) m (G) n (EAAAK) n Or (XP) n , where n and m are each independently selected from integers from 0 to 10. For example, n is selected from 0, 1, 2, 3, 4 or 5, m is selected from 1, 2, 3, 4 or 5, and X is any natural amino acid.

[0057] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleotide sequence” refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may include conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0058] As used herein, the term "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, that has been separated from its native environment. In this document, recombinant polynucleotides encoding polypeptides included in vectors are also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules typically found in cells containing the polynucleotide molecule, but which are located extrachromosomally or at chromosomal locations different from their native chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts as described herein, in positive and negative strand form, and in double strand form. Isolated polynucleotides or nucleic acids as described herein further include such molecules synthesized. Additionally, polynucleotides or nucleic acids may be or may include regulatory elements such as promoters, ribosome binding sites, or transcription terminators.

[0059] As used herein, the terms "vector" or "expression vector" and "expression construct" are used interchangeably. A vector is a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. Vectors include vectors that function as self-replicating nucleic acid structures and vectors that are incorporated into the genome of the host cell into which they have been introduced. Expression vectors as used herein include expression cassettes. Expression vectors can perform transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, cellular transcription and / or translation mechanisms generate a ribonucleic acid molecule or protein encoded by the gene. As used herein, the term "expression cassette" refers to a recombinant or synthetically produced polynucleotide having a set of nucleic acid elements that allow a specific nucleic acid to be transcribed in the target cell. Recombinant expression cassettes can be introduced from plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of an expression vector includes the nucleic acid sequence to be transcribed and a promoter.

[0060] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including primary transformed cells and their derived progeny. The nucleic acids of the progeny may not be completely identical to those of the parent cells and may contain mutations. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0061] An "effective amount" of a drug is the amount necessary to produce physiological changes in the cells or tissues to which it is administered. An "effective amount" includes an amount sufficient to improve or prevent symptoms or conditions of a medically diagnosed disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0062] The terms “individual” or “subject” refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, an individual or subject is a human.

[0063] The term "treatment" refers to the administration of an oral or topical therapeutic agent, such as any recombinant protein or multimeric recombinant protein of this disclosure, to a patient who has one or more diseases or symptoms, and the therapeutic agent has a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce the regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent.

[0064] The term "detectable marker" encompasses markers that can be detected directly or indirectly, whether linked to the recombinant protein or the multimeric recombinant protein, or present independently in the kit. Suitable markers include, but are not limited to, molecules detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Suitable markers include, but are not limited to, fluorescent dyes (e.g., GFT and its variants, FITC, TRITC, fluorescein, and rhodamine), electron-dense reagents (e.g., gold), enzymes (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase), molecules containing radionuclides (i.e., radioisotopes), chemiluminescent molecules, electrochemiluminescent molecules, biotin, digoxin / digoxigenin, or haptens, and other entities that are or can be detectable.

[0065] Example Example 1 (1) Carrier construction: Using the S1 domain of the S protein of PEDV G1a subtype strain CV777 and G2a subtype strain CH / HNAY / 2015 as the main immunogen, recombinant trimeric S1 proteins of PEDV G1a subtype strain CV777 and PEDV G2a subtype strain CH / HNAY / 2015 were constructed. Their structures are signal peptide-S1 domain of S protein-trimeric structure sequence-purification tag. The sequences of each domain are shown in Table 1 below.

[0066] The specific steps are as follows: The DNA sequences encoding the recombinant proteins (SEQ ID NO: 7 and 9) were synthesized and spliced ​​onto the PCDNA3.1 plasmid to obtain the recombinant vector. The recombinant vector was digested with HindIII-EcoRI to obtain the digestion products.

[0067] Agarose gel electrophoresis was performed on the PCDNA3.1 plasmid before and after enzyme digestion, and the results are as follows: Figure 1 As shown, the results indicate that the target band is around 2300bp, consistent with the theoretical result, and the DNA sequencing results are consistent with the designed sequence.

[0068] Table 1

[0069] (2) Protein expression and purification The recombinant plasmid constructed in step (1) was transfected into the engineered strain of *E. coli* DH5α, amplified, and then the endotoxin-free plasmid was extracted and transfected into CHO-K1 cells to transiently express the target protein. The plasmid was purified using high-affinity nickel-containing resin.

[0070] CHO transient conversion step: wait for the CHO cell density to reach 3-5 × 10⁻⁵. 6 When the cell density was 3.0 × 10⁶ cells / mL, the cells were resuspended in fresh CHO medium to a density of 3.0 × 10⁶ cells / mL. 6 Cells / mL; Mix 5 mL PEI and 100 μg sterile plasmid DNA with 5 mL KPM and 500 μL transfection reagent (purchased from Merck) to prepare a plasmid-vector complex; Mix the prepared plasmid-vector complex with cultured CHO cells, and collect the supernatant by centrifugation after 10 days of culture.

[0071] Protein purification: The culture supernatant was purified using a 10 mL Ni-NTA pre-packed column (purchased from Tiandi Renhe). After passing the culture supernatant through the pre-packed column at a flow rate of 1 mL / min, impurities were eluted with 30 mL PBS solution at the same flow rate. The target protein was then eluted with 500 mmol imidazole. The elution peak fraction was collected, and the pH was adjusted to between 6.5 and 7.3 with 2 M Tris solution to obtain the purified target protein.

[0072] The purified target protein was subjected to SDS-PAGE electrophoresis and Western blotting. The anti-PEDV antibody in the Western blotting was a commercial monoclonal antibody against PEDV-S protein (purchased from Yuduo Biotechnology, YDD-009-1).

[0073] Test results as follows Figure 2 As shown, the SDS-PAGE results indicate that the trimeric protein band is basically consistent with the theory, and the Western-bolt results show that the band is a PEDV-specific band.

[0074] Example 2 Immunoassay Twenty-five female 8-week-old BALB / C mice were acclimatized for at least 5 days before being immunized.

[0075] Administration route: Immunize the lateral thigh muscle group of the hind limbs on day 0 (D0) and day 14 (D14), 50 μl for each hind limb, 100 μl / animal.

[0076] Dosing grouping: S1: Water-in-oil-in-water adjuvant + 100 μg antigen + 10 μg CpG adjuvant 1 S2: Water-in-oil-in-water adjuvant + 100 μg antigen + 10 μg CpG adjuvant 2 S3: Water-in-oil-in-water adjuvant + 100μg antigen + 10μg CpG adjuvant 3; Among them, the water-in-oil-in-water adjuvant was purchased from Wuhan Renfu Pharmaceutical; CpG adjuvant 1 was CpG2006, CpG adjuvant 2 was CpG1018, and CpG adjuvant 3 was CpG1826.

[0077] Attenuated vaccine: Kefujing, an attenuated vaccine, purchased from Wuhan Keqian Biotechnology Co., Ltd. Inactivated: Femmetra, an inactivated vaccine, purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.

[0078] Clinical observation: Observation once a week. Observation content includes, but is not limited to, death, onset of disease, respiration, secretions, feces, and diet and water intake.

[0079] Blood collection time: Day 21 (D21); All animals were collected at the final blood volume: after anesthesia, the eyeballs were enucleated to obtain as much whole blood as possible (without anticoagulation).

[0080] ELISA detection of mouse serum: Trimeric recombinant protein: 2 μg / mL plate Experimental Methods: The ELISA kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd. Mouse serum was serially diluted and incubated with pre-coated ELISA plates at 37°C for 1 hour, followed by 5 washes. 100 μL of working concentration anti-mouse IgG-HRP antibody (Shanghai Yuduo Biotechnology Co., Ltd.) was added, and the plate was incubated at 37°C for 1 hour, followed by 5 washes. Hydrogen peroxide (Solution A), TMB (TMB), and stop solution were added, and color development was initiated. Data were read within 10 minutes (OD630-OD450, blank well reading <0.006, cutoff value ≥0.05). The results are as follows Figure 3 As shown, the results of the S protein-coated ELISA plate showed that the titer of the trimeric S protein antibody was much higher than that of the existing commercial products Kefujing and Fumeiri.

[0081] The weight results of each treatment group are as follows: Figure 4 As shown in the figure, the results indicated no significant difference in body weight before and after immunization among the groups.

[0082] The technical solutions disclosed herein are not limited to the specific embodiments described above. Any technical modifications made based on the technical solutions disclosed herein shall fall within the protection scope of this disclosure.

Claims

1. A recombinant protein comprising the S1 domain and polymerized sequence of the porcine epidemic diarrhea virus S protein.

2. The recombinant protein according to claim 1, characterized in that, The porcine epidemic diarrhea virus S protein includes the S protein of PEDV G1a subtype strain CV777 and / or the S protein of PEDV G2a subtype strain CH / HNAY / 2015. Preferably, the S1 domain of the porcine epidemic diarrhea virus S protein includes the amino acid sequence shown in SEQ ID NO: 1 or 2, or an amino acid sequence that has at least 80% sequence identity with it, or an amino acid sequence that has one or more amino acid substitutions, deletions or additions compared to it.

3. The recombinant protein according to claim 1 or 2, characterized in that, The polymerizing sequence includes the trimer Folden sequence; Preferably, the polymerized sequence comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it.

4. The recombinant protein according to any one of claims 1-3, characterized in that, The recombinant protein also includes a signal peptide; Preferably, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it; and / or The recombinant protein also includes a purification tag; Preferably, the purification tag includes a His tag.

5. The recombinant protein according to any one of claims 1-4, characterized in that, The recombinant protein comprises, from N-terminus to C-terminus, a signal peptide, porcine epidemic diarrhea virus S protein, a polymerized sequence, and an optional purification tag. Preferably, the signal peptide is directly linked to the porcine epidemic diarrhea virus S protein, the porcine epidemic diarrhea virus S protein is linked to the polymerized sequence, and / or the polymerized sequence is linked to the purification tag, or linked through a linker. Preferably, the linker is a peptide with a length of 2-30 amino acids; More preferably, the connector is selected from (G n S) m (G) n (EAAAK) n Or (XP) n , where n and m are each independently selected from integers from 1 to 10; More preferably, the recombinant protein has one or more of the amino acid sequences shown in SEQ ID NO: 6-9, or an amino acid sequence that is at least 80% identical to it, or an amino acid sequence that has one or more amino acid substitutions, deletions or additions compared to it.

6. A multimeric recombinant protein, characterized in that, The multimeric recombinant protein comprises at least three recombinant protein units, wherein the recombinant protein units comprise any one of claims 1-5.

7. An isolated nucleic acid encoding a recombinant protein according to any one of claims 1-5 or a multimeric recombinant protein according to claim 6.

8. An expression vector comprising the isolated nucleic acid according to claim 7.

9. A host cell comprising the isolated nucleic acid according to claim 7, or the expression vector according to claim 8, or expressing the recombinant protein according to any one of claims 1-5 or the multimeric recombinant protein according to claim 6.

10. A pharmaceutical composition comprising: The recombinant protein of any one of claims 1-5 or the multimeric recombinant protein of claim 6, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, characterized in that, The pharmaceutical composition is in the form of a recombinant vaccine; Preferably, the vaccine further includes a buffer solution and / or an adjuvant; More preferably, the buffer solution includes physiological saline, PBS buffer, or HEPES buffer; More preferably, the adjuvant includes one or more of aluminum adjuvant, Cpg adjuvant, and MF59 adjuvant.

12. A kit comprising the recombinant protein of any one of claims 1-5, or the multimeric recombinant protein of claim 6; Preferably, the kit further includes a detectable marker that can be linked to the recombinant protein or the multimeric recombinant protein, and optionally a substrate corresponding to the detectable marker and / or instructions for use.

13. The recombinant protein of any one of claims 1-5, the multimeric recombinant protein of claim 6, the isolated nucleic acid of claim 7, the expression vector of claim 8, the host cell of claim 9, the pharmaceutical composition of claim 10 or 11, or the kit of claim 12, in the use of screening or preparing medicaments for the prevention, treatment and / or diagnosis of diseases related to porcine epidemic diarrhea virus infection; Preferably, the porcine epidemic diarrhea virus infection-related diseases include porcine epidemic diarrhea.

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