Porcine epidemic diarrhea virus S protein and related products

Abstract

The invention relates to the technical field of veterinary biological products, and particularly provides a porcine epidemic diarrhea virus S protein and related products. The amino acid sequence of the porcine epidemic diarrhea virus S protein fragment is as shown in SEQ ID NO.4. The trimer S protein is constructed by fusing a trimer tag at the C terminal of a protective antigen in a truncated form of the porcine epidemic diarrhea virus S protein. After expression and purification, immunogenicity is judged through animal evaluation, and it is verified that the vaccine prepared from the trimer S protein has the advantages of being high in safety, good in immunogenicity, stable in batches, capable of inducing piglets to generate good immunoreaction and the like, and porcine epidemic diarrhea can be effectively treated or prevented.

Description

Technical Field

[0001] This application relates to the technical field of veterinary biological products, and specifically provides a porcine epidemic diarrhea virus S protein and related products. Background Art

[0002] Porcine epidemic diarrhea (PED) is an infectious disease of the porcine intestine, and its main clinical features are watery diarrhea, vomiting, and dehydration. This disease mainly causes fatal diarrhea in neonatal piglets, and the mortality rate can even reach 100%. The main pathological change is the shedding and shortening of the small intestinal villi in piglets, ultimately resulting in the death of piglets due to dehydration and nutritional deficiency.

[0003] The pathogen of this disease is porcine epidemic diarrhea virus (PEDV), which is a single-stranded positive-strand enveloped RNA virus and belongs to the order Nidovirales, family Coronaviridae, genus Alphacoronavirus.

[0004] The S protein of PEDV is a type I glycoprotein and a receptor-binding protein, which plays an important role in virus entry, virus-host interaction, and immunogenicity evaluation. In addition, the S protein contains multiple B-cell epitopes and is the main structural protein that induces the body to produce neutralizing antibodies and provides immune protection. Therefore, the S protein is the main target for developing live attenuated vaccines and subunit vaccines against coronaviruses.

[0005] Vaccination is one of the main measures for preventing, controlling, and even eradicating porcine epidemic diarrhea virus. Subunit vaccines do not contain nucleic acid substances, have good safety, do not produce persistent infection or latent infection after vaccination, and the immune response generated can be distinguished from wild virus infection, which is conducive to the control and eradication of the disease.

[0006] Therefore, it has important practical significance to develop a production method for porcine epidemic diarrhea virus subunit vaccines with low production cost, high production efficiency, and good vaccine immune effect.

[0007] In view of this, the present application is specifically proposed. Summary of the Invention

[0008] The purpose of this application is to provide a porcine epidemic diarrhea virus S protein and related products to provide a product with good immune effect for preventing porcine epidemic diarrhea virus infection.

[0009] In order to achieve the above purpose, the following technical solutions are adopted in this application.

[0010] A porcine epidemic diarrhea virus S protein fragment, and the amino acid sequence of the S protein fragment is as shown in SEQ ID NO.4.

[0011] A recombinant protein S-T4 of porcine epidemic diarrhea virus antigen, wherein the amino acid sequence of the recombinant protein S-T4 is shown in SEQ ID NO.3.

[0012] A porcine epidemic diarrhea virus antigen, wherein the porcine epidemic diarrhea virus antigen is a trimeric structure of the above recombinant protein S-T4.

[0013] Biomaterials related to the above S protein fragment or recombinant protein S-T4, wherein the biomaterials include any one of the following:

[0014] (1) A nucleic acid fragment encoding the above S protein fragment or recombinant protein S-T4;

[0015] (2) An expression cassette containing the nucleic acid fragment in (1);

[0016] (3) A vector containing the nucleic acid fragment in (1) or the expression cassette in (2);

[0017] (4) A recombinant cell containing the nucleic acid fragment in (1), the expression cassette in (2) or the vector in (3).

[0018] Furthermore, the nucleotide sequence of the nucleic acid fragment encoding the recombinant protein S-T4 in (1) is shown in SEQ ID NO.2.

[0019] Use of the above recombinant protein S-T4 or porcine epidemic diarrhea virus antigen in the preparation of a drug for preventing porcine epidemic diarrhea virus infection.

[0020] Furthermore, the drug is a porcine epidemic diarrhea virus subunit vaccine.

[0021] A porcine epidemic diarrhea virus subunit vaccine, wherein the subunit vaccine includes an immunologically effective amount of the above recombinant protein S-T4 or porcine epidemic diarrhea virus antigen, and a pharmaceutically acceptable carrier.

[0022] Furthermore, the content of the porcine epidemic diarrhea virus antigen is ≥25 μg / ml, preferably 25-100 μg / ml.

[0023] Furthermore, the pharmaceutically acceptable carrier includes at least one of adjuvants, lyoprotectants, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants and preservatives;

[0024] Preferably, the adjuvant includes one or more of: aluminum hydroxide adjuvant, saponin, afoxolaner, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, Gel adjuvant;

[0025] Preferably, the concentration range of the adjuvant is from 5% V / V to 60% V / V, preferably 30% V / V to 60% V / V, and more preferably 50% V / V;

[0026] Preferably, the cryoprotectant is selected from sugars, polyols, polymers, surfactants, salts, amines or amino acids;

[0027] Preferably, the immunostimulant includes alpha-interferon, beta-interferon, gamma-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor or interleukin-2.

[0028] Compared with the prior art, the technical effects of the present application are as follows:

[0029] In the present application, bioinformatics software is used to analyze the domains and functional domains of the S protein of porcine epidemic diarrhea virus. A trimeric tag is fused to the C-terminus of the protective antigen in the truncated form of the S protein of porcine epidemic diarrhea virus to construct a trimeric S protein. After expression and purification, the immunogenicity is judged through animal evaluation, and it is verified that the vaccine prepared from the trimeric S protein has the advantages of high safety, good immunogenicity, stability between batches, and can induce good immune responses in piglets, and can effectively treat or prevent porcine epidemic diarrhea. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following further describes the technical features of the present application and the relationships between them with reference to the drawings. The drawings are exemplary. Some technical features are not shown in actual proportions, and some drawings may omit technical features that are customary in the technical field to which the present application belongs and are not essential for understanding and implementing the present application, or may additionally show technical features that are not essential for understanding and implementing the present application. That is, the combination of the technical features shown in the drawings is not used to limit the present application. In addition, throughout the present application, the content referred to by the same reference numerals is also the same. The specific description of the drawings is as follows:

[0031] Figure 1 It is the electron micrograph of the PEDV S recombinant protein in Example 3 (ii);

[0032] Figure 2It is the graph showing the liquid-phase detection results of the PEDV S recombinant protein in Example 3; among them, S is the purified protein without the addition of the T4 tag (T4 trimer tag); S-T4 is the recombinant protein after the addition of the T4 tag. Detailed implementation manners

[0033] The relevant terms in this application are explained as follows:

[0034] The term "Porcine epidemic diarrhea virus (PEDV)" is a single-stranded positive-sense enveloped RNA virus, belonging to the order Nidovirales, the family Coronaviridae, and the genus Coronavirus. Viral particles are pleomorphic, tending to be round, with an outer envelope. After oral and nasal infection, the virus directly enters the small intestine and replicates in the cytoplasm of the epithelial cells of the small intestine and colon villi. It causes damage to cell organelles, followed by cell dysfunction, intestinal villus atrophy, resulting in a reduction in the absorption surface area, a significant reduction in the content of intestinal mucosal alkaline phosphatase, and thus causing malabsorption of nutrients, belonging to osmotic diarrhea. Severe diarrhea causes dehydration, leading to the death of diseased pigs.

[0035] The term "Antigen" refers to a substance that can induce an immune response in the body, that is, a substance that can be specifically recognized and bound by the antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activate T / B cells, cause them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to the corresponding products in vivo and in vitro.

[0036] The term "S protein" is a type I glycoprotein and a receptor-binding protein of PEDV, containing an extracellular domain, a transmembrane region, and an intracellular domain, and plays an important role in virus entry, virus-host interaction, and immunogenicity evaluation. In addition, the S protein contains multiple B cell epitopes and is the main structural protein that induces the body to produce neutralizing antibodies and provides immune protection.

[0037] The term "T4 trimer tag" is also known as the C-terminal of the T4 phage fibritin or the T4 phage fibritin C-terminal trimerization sequence (T4 phage fibritin foldon trimerization sequence). Each subunit consists of 27 amino acids, and this domain is composed of 3 identical subunits, and each subunit contains 1 β-hairpin structure.

[0038] This application provides a porcine epidemic diarrhea virus S protein fragment, and its amino acid sequence is as shown in SEQ ID NO.4.

[0039]

[0040] In this application, the amino acid fragment at positions 26 to 1322 in the extracellular region of the PEDV S protein is selected as an antigen target, and it has been verified that it can effectively prevent porcine epidemic diarrhea.

[0041] Further research in this application found that by modifying the above S protein fragment and adding a trimer tag, the antigen recombinant protein can form a trimeric form and achieve a good immunization effect.

[0042] In some embodiments, the trimer tag can be a T4 trimer tag with the amino acid sequence: GYIPEAPRDGQAYVRKDGEWVLLSTFL, resulting in the porcine epidemic diarrhea virus antigen recombinant protein S-T4 with the amino acid sequence shown in SEQ ID NO.3.

[0043]

[0044] The present application also provides a porcine epidemic diarrhea virus antigen, which is a trimeric structure of recombinant protein S-T4. The trimeric structure can present the original protein structure as much as possible, which is beneficial to stimulating the body to produce a stronger immune response.

[0045] The present application provides biological materials related to the above-mentioned S protein fragment or recombinant protein S-T4, such as nucleic acid fragments encoding the above-mentioned proteins, expression cassettes containing the nucleic acid fragments, vectors of the expression cassettes (such as cloning plasmids and expression plasmids, etc.), and recombinant cells containing the nucleic acid fragments, expression cassettes or vectors. These biological materials can be directly used as biological modules for the production of the proteins of the present application, and have the advantages of rapidity and high efficiency. The nucleic acid fragments of the present application can be obtained by primer amplification or by artificial synthesis. The nucleotide sequence encoding recombinant protein S-T4 is shown in SEQ ID NO.2.

[0046]

[0047] The recombinant protein S-T4 or the porcine epidemic diarrhea virus antigen provided by the present application is used for preparing related products for preventing porcine epidemic diarrhea virus infection, such as vaccines for preventing infection, antibody detection reagents, and the like.

[0048] The present application provides a subunit vaccine against porcine epidemic diarrhea virus. In addition to the active ingredient mainly being the recombinant protein S-T4 or the porcine epidemic diarrhea virus antigen provided by the present application, it may also contain a pharmaceutically acceptable carrier, aiming to meet different requirements in actual applications such as production, transportation, dosage form, administration method, etc.

[0049] The term "vaccine" refers to a pharmaceutical composition containing a porcine epidemic diarrhea virus protein antigen, and this pharmaceutical composition can induce, stimulate or enhance the immune response of pigs to porcine epidemic diarrhea virus.

[0050] The term "immunizing amount" should be understood as "immunologically effective amount", also known as immunoprotective amount or effective amount for generating an immune response, which is the amount of antigen that can effectively induce an immune response in the recipient, and this amount is sufficient to prevent or improve the signs or symptoms of the disease, including adverse health effects or its complications. The said immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing the signs or symptoms of the disease, including the adverse health outcomes or its complications caused by infections by pathogens. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be indirectly evaluated by, for example, measuring antibody titers, lymphocyte proliferation assays, or directly evaluated by monitoring signs or symptoms after challenge with a wild-type strain, and the protective immunity provided by the vaccine can be evaluated by measuring, for example, the clinical signs of the subject such as reduction in mortality, morbidity, temperature values, the overall physiological condition of the subject, and overall health and performance. The said immune response may include, but is not limited to, inducing cellular and / or humoral immunity.

[0051] In some embodiments, the subunit vaccine contains an immunizing amount of porcine epidemic diarrhea virus antigen, and the content of porcine epidemic diarrhea virus antigen is ≥25 μg / ml, preferably 25 - 100 μg / ml. For example, it can be, but is not limited to, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, 50 μg / ml, 55 μg / ml, 60 μg / ml, 65 μg / ml, 70 μg / ml, 75 μg / ml, 80 μg / ml, 85 μg / ml, 90 μg / ml, 95 μg / ml or 100 μg / ml.

[0052] In the present application, the porcine epidemic diarrhea virus antigen recombinant protein S-T4 or the porcine epidemic diarrhea virus antigen can be prepared by a eukaryotic expression system, or can be prepared by a prokaryotic expression system, a cell expression system or a chemical synthesis method.

[0053] The term "pharmaceutically acceptable carrier" refers to all other components in the subunit vaccine of the present application except for the porcine epidemic diarrhea virus subunit protein antigen, a carrier or diluent that does not stimulate the body and does not hinder the biological activity and characteristics of the compound used, and is preferably an adjuvant.

[0054] The term "adjuvant" may include aluminum hydroxide adjuvant; saponins, such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); water-in-oil emulsions; oil-in-water emulsions; water-in-oil-in-water emulsions; polymers of acrylic acid or methacrylic acid; compounds selected from copolymers of maleic anhydride and alkenyl derivatives.

[0055] The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils resulting from olefin oligomerization, such as squalane or squalene oil, in particular isobutene or decene; linear alkyl esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di-(caprylate / caprate), glyceryl tri-(caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearates. The oil is used in combination with an emulsifier in order to form an emulsion. The emulsifier is preferably a nonionic surfactant, in particular esters of sorbitan, esters of mannitol (such as anhydrous mannitol oleate), esters of aliphatic glycols, esters of polyglycerol, esters of propylene glycol and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic products, especially L121. See "The theory and practical application of adjuvants" by Hunter et al. (Ed. by DES Stewart-Tull, John Wiley and Sons, New York, 1995: 51-94) and "Vaccine" by Todd et al. (1997, 15: 564-570). For example, the SPT emulsion described on page 147 of "Vaccine design, the Subunit and adiuvant approach" by Powell M and Newman M (Plenum Press, 1995) and the MF59 emulsion described on page 183 may be used.

[0056] The term "polymers of acrylic or methacrylic acid" is preferably a cross-linked polymer of acrylic or methacrylic acid, especially cross-linked with a polyalkenyl ether or polyol of sugar, and these compounds are known as Carbomer (trade name Carbopol) (Phameuropa, 1996, 8(2)). Those skilled in the art can also refer to US Patent US2909462, which describes such acrylic polymers cross-linked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, and in which at least 3 hydrogen atoms of the hydroxyl groups are replaced by an unsaturated aliphatic radical having at least 2 carbon atoms. Preferred groups are those containing 2-4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated groups themselves may contain other substituents, such as methyl. These products are sold under the name of Carbopol (BF Goodrich, Ohio, USA) and are particularly suitable. They are cross-linked with allyl sucrose or allyl pentaerythritol. Among these, Carbopol 974P, 934P and 971P may be mentioned, and Carbopol 971P is most preferably used.

[0057] The term "copolymer of maleic anhydride and alkenyl derivatives" also includes the copolymer of maleic anhydride and ethylene, EMA (Monsanto). These polymers dissolve in water to produce an acidic solution, which is preferably neutralized to physiological pH in order to produce an adjuvant solution into which an immunogenic, immunizing or vaccine composition itself can be incorporated.

[0058] The term "adjuvant" also includes, but is not limited to, RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant, etc.

[0059] In a preferred embodiment, the adjuvant includes one or more of mineral oil, aluminum hydroxide adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 201 or Gel adjuvant.

[0060] In some embodiments, the concentration range of the adjuvant is from 5% V / V to 60% V / V, preferably from 30% to 60% V / V, more preferably 50% V / V. The concentration range of the adjuvant can be, but is not limited to, 5% V / V, 10% V / V, 15% V / V, 20% V / V, 25% V / V, 30% V / V, 35% V / V, 40% V / V, 45% V / V, 50% V / V, 55% V / V or 60% V / V.

[0061] The term "lyoprotectant" refers to a component that, in addition to excipients, protects the efficacy of the active pharmaceutical ingredient during the freeze-drying process and storage after freeze-drying. The lyoprotectant can be selected from sugars, polyols, polymers, surfactants, salts, amines or amino acids.

[0062] In some embodiments, the immunostimulant includes alpha-interferon, beta-interferon, gamma-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor or interleukin-2.

[0063] The term "prevention" when referring to porcine epidemic diarrhea virus infection means inhibiting the replication of porcine epidemic diarrhea virus, inhibiting the transmission of porcine epidemic diarrhea virus or preventing porcine epidemic diarrhea virus from colonizing in its host, and alleviating the symptoms of the disease or disorder caused by porcine epidemic diarrhea virus infection.

[0064] The present application will be further described below with reference to specific embodiments, and the advantages and features of the present application will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, but such modifications and replacements all fall within the protection scope of the present application.

[0065] The chemical reagents used in the embodiments of the present application are all of analytical grade and are purchased from the National Pharmaceutical Group. The experimental methods described in the present application are all conventional methods unless otherwise specified; the biological materials described, unless otherwise specified, can be obtained from commercial sources.

[0066] Example 1 Construction of a recombinant plasmid for eukaryotic expression of recombinant protein

[0067] The PEDV S protein is a type I transmembrane protein containing an extracellular domain, a transmembrane region, and an intracellular domain. The signal peptide region and transmembrane region of the protein were analyzed using the online tools SignalP 5.0 and TMHMM, respectively. The amino acid fragment from the 26th to the 1322nd position in the extracellular region of the protein was selected as the antigen target. A Kozak sequence was added before the ATG at the 5' end of the sequence, an HSA protein signal peptide sequence was added after the ATG, and a 6×His nucleotide sequence was added before the stop codon at the 3' end of the sequence. This nucleotide sequence was synthesized by GenScript and optimized to murine-preferred codons. The nucleotide sequence is as shown in SEQ ID NO.1 and was cloned into the pUC57 vector through the Xba I / Hind III restriction enzyme sites.

[0068]

[0069] After the synthetic plasmid was recovered by digestion with Xba I / Hind III, it was ligated into the pcDNA3.1 vector with the same restriction sites using T4 DNA ligase. The ligation product was transformed into competent DH5α Escherichia coli cells, and monoclonal colonies were picked from the transformation plate for identification by colony PCR. The positive clone bacterial solution identified by PCR was sent to Genewiz for sequencing analysis, and finally the correctly sequenced clone was selected for the next step. The S-T4 nucleotide sequence is as shown in SEQ ID NO.2, and the amino acid sequence is as shown in SEQ ID NO.3.

[0070] The correctly sequenced clone bacterial solution was taken out, inoculated at a ratio of 1:1000 into 50 mL, and cultured overnight at 37°C. The cultured bacterial solution was used to extract the plasmid using the Omega Plasmid Midiprep Kit. The correctness of the plasmid was identified using Xba I / Hind III, and the plasmid concentration was measured using a microplate reader. The results showed that the size of the bands after digestion of the constructed recombinant expression plasmid was correct, meeting the expectations.

[0071] Example 2 Preparation of Eukaryotic Expressed Recombinant Protein

[0072] The recombinant plasmid prepared in Example 1 was taken and transfected into Expi-CHO cells at a transfection amount of 0.8 μg / mL and a transfection volume of 25 mL. Fed-batch culture was carried out on the second day after transfection. A small sample was collected on the 7th day after transfection for SDS-PAGE and WB identification, and it was found that the protein was expressed in the secreted supernatant. The expression product was harvested on the 9th day after transfection.

[0073] The harvested Expi-CHO cell expression product was centrifuged at 6500 rpm for 20 min, and then the culture supernatant was taken to a His affinity chromatography column (GE, USA) for purification. Then, the impurity proteins were eluted with 20 mM Na2HCO3 (pH 7.2) (containing 0.15 M sodium chloride) solutions containing 10 mM imidazole and 50 mM imidazole, respectively. The target protein was eluted with a 20 mM Na2HCO3 (pH 7.2) (containing 0.15 M sodium chloride) solution containing 500 mM imidazole. The protein peaks were collected separately and detected by SDS-PAGE. The results showed that the soluble secreted PEDV S protein could be expressed using the Expi-CHO expression system, and the soluble protein could be obtained by purification with a nickel column. The molecular weight was approximately 250 KDa, which was different from the molecular weight analyzed by the amino acid sequence of the S protein (145.1 KDa), indicating that the expressed S protein had a large amount of glycosylation modification.

[0074] Example 3 Identification of Purified PEDV-S Protein

[0075] I. Native Polyacrylamide Gel Electrophoresis

[0076] Take the purified protein obtained in Example 2, prepare a non-denaturing polyacrylamide gel with a separating gel concentration of 6% and a stacking gel concentration of 4%. The sample loading volume is 20 μL. During electrophoresis, the voltage is 80 V for 30 min, and then 160 V for about 2 h. Observe until the 250 KDa protein Marker indicates that the band has moved down to the bottom of the gel to end electrophoresis, and display the results after Coomassie Brilliant Blue staining. The results show that the size of the monomer protein can be observed around the position of the 250 KDa protein Marker, and there is a protein aggregate band above the separating gel, indicating that the obtained protein is in an aggregated state.

[0077] II. Negative staining electron microscopy analysis

[0078] Incubate the S protein purified by molecular sieve on a copper grid for 1 min, stain with 1% uranyl acetate for 45 s, and then observe using a transmission electron microscope. The results show that the S protein exists in an aggregated form, indicating that the purified S protein can form aggregates ( Figure 1 ).

[0079] III. Detection and analysis by liquid chromatograph

[0080] Take the purified protein obtained in Example 2, filter it and then detect it using a Shimadzu high-performance liquid chromatograph. The chromatographic column is a Sepax SRT-C SEC500 chromatographic column, with PBS as the mobile phase. Equilibrate the column for 30 min, set the flow rate to 0.5 ml / min, the sample injection volume to 50 μl, and the detection time for each protein to 40 min. The results show (as Figure 2 ) that the protein without the T4 trimer tag has 3 peaks, and the elution times are 14 min, 16 min (750 KDa), and 18 min (250 KDa) respectively, which correspond to the sizes of higher aggregates, trimers, and monomeric proteins respectively. The elution time of the recombinant protein with the T4 trimer tag is about 16 min, which corresponds to the elution time of the 750 KDa protein, indicating that the recombinant protein exists in the form of a trimer.

[0081] Example 4 Preparation of subunit vaccine

[0082] Slowly add the above-prepared porcine epidemic diarrhea virus antigen (the trimer form of the antigen recombinant protein S-T4) to the adjuvant, and at the same time start the motor and stir at 17500 r / min for 5 min. The adjuvant applicable to the present invention can be an adjuvant well-known to those skilled in the art. In the present invention, the selected adjuvant is a biphasic adjuvant (water-in-oil-in-water emulsion), for example, it can be Montanide ISA 201 adjuvant. The specific ratio is shown in Table 1.

[0083] Table 1 Subunit vaccine ratio

[0084] Evaluation of Immunogenicity of Subunit Vaccine in Piglets

[0085] Twenty piglets were randomly divided into 4 groups of 5 each. Groups 1 - 3 were immunized with Vaccine 1, Vaccine 2, and Vaccine 3 respectively, and Group 4 was immunized with PBS solution as a control. Each was intramuscularly injected with 2 ml in the neck. Three weeks after the first immunization, a second immunization was carried out. Blood samples were collected 3 weeks after the first immunization and 2 weeks after the second immunization to measure PEDV IgG antibody and neutralizing antibody.

[0086] Detection of PEDV IgG ELISA Antibody

[0087] 1) Preparation of antigen plate: Use the S protein of porcine epidemic diarrhea virus, coat the ELISA plate at 50 ng / well / 100 μl, coat overnight at 2 - 8°C. Discard the coating solution the next day, wash once with 1× washing solution, dry in air, and store at 2 - 8°C for later use.

[0088] 2) Sample dilution: Dilute the serum to be tested 100 - fold with serum diluent (take 3 μl of the serum to be tested and add 297 μl of serum diluent), and mix well.

[0089] 3) Sample addition: Add 100 μl of the diluted sample to be tested to the corresponding wells respectively, and gently mix. At the same time, set up 2 wells each for negative and positive controls, 100 μl / well.

[0090] 4) Incubation: After sealing the plate with a sealing film, incubate at 37°C for 30 minutes.

[0091] 5) Plate washing: Carefully remove the sealing film, discard the reaction solution, add 300 μl of 1× washing solution to each well, soak for about 30 s, and discard the washing solution. Repeat this 4 times, and pat dry as much as possible for the last time.

[0092] 6) Addition of enzyme - labeled antibody: Add 100 μl of the enzyme - labeled antibody working solution to each well, and incubate at 37°C for 30 minutes.

[0093] 7) Plate washing: Carefully remove the sealing film, discard the reaction solution, add 300 μl of 1× washing solution to each well, soak for about 30 s, and discard the washing solution. Repeat this 4 times, and pat dry as much as possible for the last time.

[0094] 8) Color development: Add 50 μl each of color - developing solution A and color - developing solution B to each well in sequence, gently mix, and incubate at 37°C for 15 minutes.

[0095] 9) Termination: Add 50 μl of the termination solution to each well, gently mix, and read the OD450nm - 630nm within 10 minutes.

[0096] 10) Conditions for the test to be valid: OD value of positive control ≥ 0.5, OD value of negative control < 0.1. Result determination: S / P = sample OD value / mean OD value of positive control; if S / P ≥ 0.4, it is judged as positive; if S / P < 0.4, it is judged as negative.

[0097] After immunizing piglets with the PEDV subunit vaccine, the detection results of PEDV IgG ELISA antibodies in serum are shown in Table 2.

[0098] Table 2 Detection results of PEDV IgG ELISA antibodies in serum

[0099] Detection of PEDV neutralizing antibody

[0100] 1) Sample treatment: Inactivate the serum to be tested in a water bath at 56 °C for 30 min and store it at 2 - 8 °C for later use.

[0101] 2) Preparation of monolayer cells: Digest and disperse Vero cells by the conventional method, take samples for counting, seed a 96-well cell culture plate, with 20,000 cells per well, 100 μL per well, and culture it in an incubator at 37 °C to form a good monolayer.

[0102] 3) Virus dilution: Take the porcine epidemic diarrhea virus solution with known virus content and dilute it to the working concentration (100 TCID per well 50 )

[0103] 4) Sample dilution: Serially dilute the sample to be tested 2-fold with DMEM maintenance solution containing 2% fetal bovine serum. Mix the diluted sample with the indicator virus diluted to the working concentration in equal volume.

[0104] 5) Neutralization: Mix the sample mixed with the indicator virus in step 4) evenly, and place it in an incubator at 37 °C for 1 h for neutralization together with the virus control, shaking 2 - 3 times during this period.

[0105] 6) Sample addition: Take out the prepared Vero monolayer cell plate from the incubator and discard the liquid in the plate. Take out the sample neutralized for 1 h from the incubator at 37 °C and inoculate it onto the Vero cell culture plate, adding 4 wells for each dilution, 100 μL per well. Set virus control wells, sample toxicity control wells and normal cell control wells, and culture the cell plate in a carbon dioxide incubator, observe it every day, and judge the results at 96 h.

[0106] 7) Result determination: CPE (cytopathic effect) should appear in all virus control wells, and there should be no CPE in normal cell control wells and sample toxicity control wells to make a determination. The more diluted the sample, the more wells with lesions will be. Count the number of wells with lesions for each dilution of the sample and calculate the results by the Reed-Muench method.

[0107] After immunizing piglets with the PEDV subunit vaccine, the detection results of neutralizing antibodies in serum are shown in Table 3.

[0108] Table 3 Neutralizing antibody test results in serum

[0109] Example 6 Evaluation of passive immunoprotection of subunit vaccine-immunized sows and piglets

[0110] Three sows negative for both PEDV antigen and antibody were selected and immunized with 2 ml / head of the vaccine. The immunization schedule was the first immunization 35 - 40 days before parturition and the second immunization 15 - 20 days before parturition. Serum and milk samples of the sows were collected on the day of parturition after parturition to measure neutralizing antibodies. Serum samples were inactivated in a water bath at 56 °C for 30 min and stored at 2 - 8 °C; milk samples were inactivated in a water bath at 56 °C for 60 min and stored at 2 - 8 °C. The neutralizing antibody test results are shown in Table 4.

[0111] Table 4 Neutralizing antibody results in serum and colostrum of immunized sows at parturition

[0112] The piglets born to 3 sows were selected, with 3 piglets per litter. Blood samples were collected on the 7th, 14th, 21st, and 28th days after parturition to measure neutralizing antibodies. The neutralizing antibody test results are shown in Table 5.

[0113] Table 5 Neutralizing antibody results in serum and colostrum of immunized sows at parturition

[0114] For the evaluation of the protection of piglets born to sows against challenge, 5 piglets born to sow 1001 were challenged with a virulent PEDV strain on the 28th day after parturition. Meanwhile, 5 28-day-old piglets negative for both PEDV antigen and antibody were selected as the challenge control. The results of the challenge evaluation were as follows: 5 / 5 in the challenge control group showed symptoms of anorexia, diarrhea, or vomiting, while 5 / 5 of the piglets born to sow 1001 had no abnormal clinical symptoms. The detailed results are shown in Table 6.

[0115] Table 6 Neutralizing antibody results in serum and colostrum of immunized sows at parturition

[0116] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. In case of inconsistency, the meaning stated in the full text of this application or the meaning derived from the content recorded in the full text of this application shall prevail. Additionally, the terms used in this specification are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

Claims

1. A porcine epidemic diarrhea virus S protein fragment, characterized in that, The amino acid sequence of the S protein fragment is as shown in SEQ ID NO.

4.

2. A recombinant porcine epidemic diarrhea virus antigen protein S-T4, characterized in that, The amino acid sequence of the recombinant protein S-T4 is as shown in SEQ ID NO.

3.

3. A porcine epidemic diarrhea virus antigen, characterized in that, The porcine epidemic diarrhea virus antigen is the trimeric structure of the recombinant protein S-T4 described in claim 3.

4. A biological material related to the S protein fragment according to claim 1 or the recombinant protein S-T4 according to claim 2, characterized in that, The biological material includes any one of the following: (1) A nucleic acid fragment encoding the S protein fragment described in claim 1 or the recombinant protein S-T4 described in claim 2; (2) An expression cassette containing the nucleic acid fragment in (1); (3) A vector containing the nucleic acid fragment in (1) or the expression cassette in (2); (4) A recombinant cell containing the nucleic acid fragment in (1), the expression cassette in (2), or the vector in (3).

5. The biomaterial according to claim 4, characterized in that, The nucleotide sequence of the nucleic acid fragment encoding the recombinant protein S-T4 in (1) is as shown in SEQ ID NO.

2.

6. Use of the recombinant protein S-T4 described in claim 2 or the porcine epidemic diarrhea virus antigen described in claim 3 in the preparation of a medicament for preventing porcine epidemic diarrhea virus infection.

7. The application according to claim 6, wherein The medicament is a porcine epidemic diarrhea virus subunit vaccine.

8. A porcine epidemic diarrhea virus subunit vaccine, characterized in that, The subunit vaccine includes an immunologically effective amount of the recombinant protein S-T4 described in claim 2 or the porcine epidemic diarrhea virus antigen described in claim 3, and a pharmaceutically acceptable carrier.

9. The subunit vaccine according to claim 8, characterized in that, The content of the porcine epidemic diarrhea virus antigen is ≥25 μg / ml, preferably 25 - 100 μg / ml.

10. The subunit vaccine according to claim 8 or 9, characterized in that, The pharmaceutically acceptable carrier includes at least one of an adjuvant, a lyoprotectant, an immunostimulant, an antioxidant, a surfactant, a coloring agent, a volatile oil, a buffer, a dispersant, a propellant, and a preservative; Preferably, the adjuvant includes: aluminum hydroxide adjuvant, saponin, avridine, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, Gel adjuvant, etc.; Preferably, the concentration range of the adjuvant is from 5% V / V to 60% V / V, preferably 30% V / V to 60% V / V, more preferably 50% V / V; Preferably, the lyoprotectant is selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids; Preferably, the immunostimulant includes α-interferon, β-interferon, γ-interferon, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin 2.

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