Porcine epidemic diarrhea virus s protein and related product

Abstract

The present application relates to the technical field of veterinary biological products. Particularly provided are a porcine epidemic diarrhea virus S protein and a related product. The amino acid sequence of a porcine epidemic diarrhea virus S protein fragment is as shown in SEQ ID NO. 4. A trimer tag is fused at the C terminus of a protective antigen in a form of the truncated porcine epidemic diarrhea virus S protein, so as to construct a trimer S protein; and after expression and purification, animal evaluation is performed to determine the immunogenicity. Upon verification, a vaccine prepared from the trimer S protein has the advantages of high safety, good immunogenicity, batch-to-batch consistency, capability of inducing piglets to generate good immune responses, etc., and therefore can effectively treat or prevent porcine epidemic diarrhea.

Description

Porcine epidemic diarrhea virus S protein and related products

[0001] This application claims priority to Chinese patent application number 2024101016994, filed on January 24, 2024, entitled “Porcine epidemic diarrhea virus S protein and related products,” the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present 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

[0003] Porcine epidemic diarrhea (PED) is an infectious enteric disease of pigs characterized by watery diarrhea, vomiting, and dehydration. It primarily causes fatal diarrhea in newborn piglets, with a mortality rate reaching 100%. The primary pathological change is the loss and shortening of villi in the small intestine, ultimately leading to death from dehydration and malnutrition.

[0004] The causative agent of the disease is porcine epidemic diarrhea virus (PEDV), a single-stranded positive-sense enveloped RNA virus belonging to the order Nidovirales, family Coronaviridae, and genus Alphacoronavirus.

[0005] The S protein of PEDV is a type I glycoprotein and a receptor-binding protein that plays an important role in viral entry, virus-host interaction, and immunogenicity assessment. Furthermore, the S protein contains multiple B cell epitopes and is the primary structural protein that induces the production of neutralizing antibodies and provides immune protection. Therefore, the S protein is the primary target for the development of live attenuated coronavirus vaccines and subunit vaccines.

[0006] Vaccination is one of the primary measures for preventing, controlling, and even eradicating porcine epidemic diarrhea virus (PEDV). Subunit vaccines, which do not contain nucleic acid, are safer and do not cause persistent or latent infection. The immune response generated can be distinguished from wild-type infection, facilitating disease control and eradication.

[0007] Therefore, it is of great practical significance to develop a production method for porcine epidemic diarrhea virus subunit vaccine with low production cost, high production efficiency and good vaccine immune effect. Summary of the Invention

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

[0009] In order to achieve the above objectives, this application adopts the following technical solutions.

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

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

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

[0013] Biological materials related to the above-mentioned S protein fragment or recombinant protein S-T4, including any one of the following:

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

[0015] (2) an expression cassette containing the nucleic acid fragment of (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] The use of the above-mentioned 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, comprising an immunizing amount of the above-mentioned 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 an adjuvant, a lyoprotectant, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative;

[0024] Preferably, the adjuvant comprises: one or more of: aluminum gel adjuvant, saponin, avridine, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic acid or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, 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 of the adjuvant ranges from 5% V / V to 60% V / V, preferably 30% V / V to 60% V / V, more preferably 50% V / V;

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

[0027] Preferably, the immunostimulant comprises α-interferon, β-interferon, γ-interferon, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor or interleukin-2.

[0028] Compared with the prior art, the technical effects of this application are:

[0029] This application uses bioinformatics software to analyze the structural and functional domains of the porcine epidemic diarrhea virus (PEDV) S protein. A trimer tag is fused to the C-terminus of the protective antigen of a truncated form of the PEDV S protein to construct a trimeric S protein. After expression and purification, immunogenicity was assessed in animals. The vaccine prepared with this trimeric S protein demonstrated high safety, good immunogenicity, batch-to-batch stability, and the ability to induce a robust immune response in piglets, effectively treating or preventing PED. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following further illustrates the various technical features of the present application and the relationships between them with reference to the accompanying drawings. The accompanying drawings are exemplary, and some technical features are not shown in actual proportion. In addition, some drawings may omit technical features that are commonly used in the technical field to which the present application belongs and are not essential for understanding and implementing the present application, or additional technical features that are not essential for understanding and implementing the present application may be shown. In other words, the combination of the various technical features shown in the accompanying drawings is not intended to limit the present application. In addition, throughout the present application, the same figure numbers refer to the same content. The specific description of the drawings is as follows:

[0031] FIG1 is an electron micrograph of the PEDV S recombinant protein of Example 3;

[0032] Figure 2 is a liquid phase detection result diagram of the third PEDV S recombinant protein in Example 3; wherein 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 DESCRIPTION

[0033] Explanation of relevant terms in this application:

[0034] The term "porcine epidemic diarrhea virus (PEDV)" is a single-stranded, positive-sense enveloped RNA virus belonging to the order Nidovirales, family Coronaviridae, genus Coronavirus. The virus particles are polymorphic, tend to be round, and have 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 the organelles, followed by cell dysfunction and atrophy of the intestinal villi, resulting in a reduction in the absorption surface area. The content of alkaline phosphatase in the small intestinal mucosa is significantly reduced, which leads to nutrient absorption disorders. It is an osmotic diarrhea. Severe diarrhea causes dehydration and the death of sick 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 antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to corresponding products in vivo and in vitro.

[0036] The term "S protein" refers to a type I glycoprotein and receptor-binding protein of PEDV, containing an extracellular domain, a transmembrane region, and an intracellular domain. It plays an important role in virus entry, virus-host interaction, and immunogenicity assessment. 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 provide immune protection.

[0037] The term "T4 trimer tag" is also known as the T4 phage fibritin foldon trimerization sequence. Each subunit consists of 27 amino acids. The domain is composed of three identical subunits, each of which contains a β-hairpin structure.

[0038] The present application provides a porcine epidemic diarrhea virus S protein fragment, the amino acid sequence of which is shown in SEQ ID NO.4.

[0039]

[0040] This application selects the amino acid fragment from position 26 to position 1322 of the extracellular region of the PEDV S protein as the antigen target, which has been verified to be effective in preventing porcine epidemic diarrhea.

[0041] Further research in this application found that by modifying the above-mentioned S protein fragment and adding a trimer tag, the antigen recombinant protein can form a trimer form, which has a good immune effect.

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

[0043]

[0044] The present application also provides a porcine epidemic diarrhea virus antigen, which is a trimer structure of the recombinant protein S-T4. The trimer structure can present the original structure of the protein as much as possible, which is conducive 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 containing 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 protein of the present application, with the advantages of rapid and efficient production. The nucleic acid fragment of the present application can be obtained by primer amplification or artificial synthesis. The nucleotide sequence encoding the recombinant protein S-T4 is shown in SEQ ID NO.2.

[0046]

[0047] The recombinant protein S-T4 or porcine epidemic diarrhea virus antigen provided in this application is used to prepare related products for preventing porcine epidemic diarrhea virus infection, such as vaccines for preventing infection, antibody detection reagents, etc.

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

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

[0050] The term "immunizing amount" should be understood as an "immunologically effective amount", also known as an immunoprotective amount or an effective amount to produce 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 immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of the disease, including adverse health consequences or complications caused by infection caused 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 assessed by, for example, measuring antibody titers, lymphocyte proliferation assays, or directly assessed by monitoring signs or symptoms after challenge with a wild-type strain, and the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs of the subject such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The 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, but 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 porcine epidemic diarrhea virus antigen can be prepared by a eukaryotic expression system, or 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 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 properties of the compound used, preferably an adjuvant.

[0054] The term "adjuvant" may include compounds selected from alum adjuvants; 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; and 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 deuterene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di-(caprylate / deuterate), glycerol tri-(caprylate / deuterate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearates. The oil is used in combination with an emulsifier to form an emulsion. Emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, esters of mannide (e.g. anhydrous mannitol oleate), esters of fatty glycols, esters of polyglycerols, esters of propylene glycol, and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which may be ethoxylated, and polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic products, in particular L 121. See Hunter et al., The theory and practical application of adjuvants (Ed. by DES Stewart-Tull, John Wiley and Sons, New York, 1995: 51-94) and Todd et al., Vaccine (1997, 15: 564-570). For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of "Vaccine design, the Subunit and adiuvant approach" edited by Powell M and Newman M (Plenum Press, 1995) can be used.

[0056] The term "polymer of acrylic acid or methacrylic acid" preferably refers to a cross-linked polymer of acrylic acid or methacrylic acid, in particular cross-linked with a polyalkenyl ether of sugar or a polyol, compounds known as carbomers (trade name Carbopol) (Phameuropa, 1996, 8 (2)). A person skilled in the art may also refer to US Pat. No. 2,909,462, which describes such acrylic acid polymers cross-linked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. Preferred groups are those containing 2 to 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 Carbopol (BF Goodrich, Ohio, USA) and are particularly suitable. They are cross-linked with allyl sucrose or with allyl pentaerythritol. Among these, Carbopol 974P, 934P and 971P may be mentioned, with Carbopol 971P being most preferably used.

[0057] The term "copolymers of maleic anhydride and alkenyl derivatives" also takes into account copolymers of maleic anhydride and ethylene such as EMA (Monsanto), these polymers dissolving in water to produce an acidic solution, which is neutralized, preferably to physiological pH, in order to produce an adjuvant solution into which the immunogenic, immunogenic or vaccinal composition itself can be incorporated.

[0058] The term "adjuvant" also includes, but is not limited to, the 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, and the like.

[0059] In a preferred embodiment, the adjuvant comprises one or more of mineral oil, aluminum gel adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic acid or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, 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, Montanide ISA 201 or Gel adjuvant.

[0060] In some embodiments, the concentration of the adjuvant ranges from 5% V / V to 60% V / V, preferably 30% to 60% V / V, and more preferably 50% V / V. The concentration of the adjuvant may range from, 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 an ingredient, other than an excipient, that protects the efficacy of a pharmaceutical active ingredient during the freeze-drying process and post-lyophilization storage. Lyoprotectants can be selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

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

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

[0064] The present application will be further described below in conjunction with specific embodiments, and the advantages and features of the present application will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present application in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present application may be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements shall fall within the scope of protection of the present application.

[0065] All chemical reagents used in the examples of this application were of analytical grade and purchased from Sinopharm Group. The experimental methods described in this application, unless otherwise specified, are conventional methods; the biological materials described, unless otherwise specified, are commercially available.

[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. SignalP 5.0 and TMHMM online tools were used to analyze the protein's signal peptide and transmembrane regions, respectively. The extracellular domain fragment from amino acids 26 to 1322 was selected as the antigenic target. A Kozak sequence was added before the ATG at the 5' end of the sequence, followed by the HSA protein signal peptide sequence, and a 6×His nucleotide sequence before the stop codon at the 3' end. This nucleotide sequence was synthesized by GenScript and optimized to mouse-preferred codons. The nucleotide sequence, as shown in SEQ ID NO. 1, was cloned into the pUC57 vector using the Xba I / Hind III restriction sites.

[0068]

[0069] The synthesized plasmid was recovered by Xba I / Hind III digestion and ligated to the pcDNA3.1 vector with the same restriction sites using T4 DNA ligase. The ligation product was transformed into DH5α competent E. coli cells, and single clones were picked from the transformation plates and identified by PCR. Clonal cultures that showed positive results from PCR were sent to GeneWeiZ for sequencing analysis, and clones with correct sequencing were selected for the next step. The S-T4 nucleotide sequence is shown in SEQ ID NO. 2, and the amino acid sequence is shown in SEQ ID NO. 3.

[0070] The cloned cells that were sequenced correctly were inoculated into 50 mL of culture at a 1:1000 ratio and cultured overnight at 37°C. The plasmid was extracted using the Omega Plasmid Extraction Kit. The plasmid was verified for accuracy using Xba I / Hind III assays, and its concentration was determined using an ELISA reader. The results showed that the recombinant expression plasmid had the correct band size after enzyme digestion, as expected.

[0071] Example 2 Preparation of eukaryotic expression recombinant protein

[0072] The recombinant plasmid prepared in Example 1 was transfected into Expi-CHO cells at a plasmid concentration of 0.8 μg / mL (25 mL). Fed-batch culture was performed on the second day of transfection. A small sample was collected on the seventh day of transfection for SDS-PAGE and Western blotting, revealing protein expression in the secretory supernatant. The expression product was harvested on the ninth day of transfection.

[0073] The harvested Expi-CHO cell expression product was centrifuged at 6500 rpm for 20 minutes, and the culture supernatant was purified using a His affinity chromatography column (GE, USA). Contaminants were then eluted using 10 mM imidazole and 50 mM imidazole in 20 mM Na2HCO3 (pH 7.2) (containing 0.15 M sodium chloride), respectively. The target protein was eluted using 500 mM imidazole in 20 mM Na2HCO3 (pH 7.2) (containing 0.15 M sodium chloride). Protein peaks were collected and analyzed by SDS-PAGE. The results showed that the Expi-CHO expression system could express soluble and secreted PEDV S protein, and nickel column purification yielded a soluble protein with a molecular weight of approximately 250 kDa, which differed from the molecular weight of the S protein (145.1 kDa) determined by amino acid sequence analysis, indicating that the expressed S protein was heavily glycosylated.

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

[0075] 1. Non-denaturing polyacrylamide gel electrophoresis

[0076] The purified protein obtained in Example 2 was used to prepare a non-denaturing polyacrylamide gel with a separating gel concentration of 6% and a stacking gel concentration of 4%. The sample volume was 20 μL, and electrophoresis was performed at 80 V for 30 minutes, then at 160 V for approximately 2 hours. The electrophoresis was terminated by observing the 250 KDa protein marker indicator band move down to the bottom of the gel. The results were displayed after staining with Coomassie Brilliant Blue. The results showed that the monomeric protein size was observed to be around the 250 KDa protein marker position, and a protein aggregate band was present above the separating gel, indicating that the obtained protein was in an aggregated state.

[0077] 2. Negative Staining Electron Microscopy Analysis

[0078] The S protein purified by molecular sieve was incubated on a copper grid for 1 minute, stained with 1% uranyl acetate for 45 seconds, and then observed using transmission electron microscopy. The results showed that the S protein was present in an aggregated form, indicating that the purified S protein can form aggregates (Figure 1).

[0079] 3. Liquid chromatography detection and analysis

[0080] The purified protein obtained in Example 2 was taken and filtered before detection using a Shimadzu high performance liquid chromatograph. The chromatographic column was a Saifen SRT-C SEC500 chromatographic column, PBS was used as the mobile phase, the column was balanced for 30 minutes, the flow rate was set to 0.5 ml / min, the injection volume was 50 μl, and the detection time for each protein was 40 minutes. The results showed (as shown in Figure 2) that there were three peaks in the protein without the addition of the T4 trimer tag, with peak times of 14 minutes, 16 minutes (750 KDa), and 18 minutes (250 KDa), respectively, which were consistent with the protein sizes of higher aggregates, trimers, and monomers, respectively. The recombinant protein with the addition of the T4 trimer tag had a peak time of about 16 minutes, which was consistent with the peak time of the 750 KDa protein, indicating that the recombinant protein existed in the form of a trimer.

[0081] Example 4 Subunit vaccine preparation

[0082] The porcine epidemic diarrhea virus antigen prepared above (a trimer form of the antigen recombinant protein S-T4) was slowly added to the adjuvant while stirring at 17,500 rpm for 5 minutes. Adjuvants suitable for use in the present invention can be any adjuvant known to those skilled in the art. In the present invention, a biphasic adjuvant (water-in-oil-in-water emulsion) was selected, such as Montanide ISA 201 adjuvant. Specific ratios are shown in Table 1.

[0083] Table 1 Subunit vaccine ratio

[0084] Example 5 Evaluation of the immunogenicity of subunit vaccines in piglets

[0085] Twenty piglets were randomly divided into four groups of five. Groups 1-3 were immunized with vaccine 1, vaccine 2, and vaccine 3, respectively. Group 4 was immunized with PBS solution as a control, with 2 ml injected intramuscularly into the neck. Three weeks after the first immunization, a second immunization was performed. Blood was collected three weeks after the first immunization and two weeks after the second immunization for determination of PEDV IgG and neutralizing antibodies.

[0086] PEDV IgG ELISA antibody test

[0087] 1) Antigen Plate Preparation: Porcine epidemic diarrhea virus S protein was used to coat the ELISA plate at 50 ng / well / 100 μl, and the coating solution was discarded the next day. The plate was washed once with 1× detergent, dried, and stored at 2-8°C until use.

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

[0089] 3) Add 100 μl of the diluted sample to the corresponding wells and gently shake to mix. Simultaneously, set up two positive and negative control wells, 100 μl / well.

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

[0091] 5) Wash the plate. Carefully remove the sealing film and discard the reaction solution. Add 300 μl of 1× wash solution to each well, soak for about 30 seconds, and discard the wash solution. Repeat this wash cycle four times, patting the plate dry as much as possible after the final wash.

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

[0093] 7) Wash the plate. Carefully remove the sealing film and discard the reaction solution. Add 300 μl of 1× Wash Solution to each well, soak for approximately 30 seconds, and discard the solution. Repeat this wash cycle four times, patting the plate dry as much as possible after the final wash.

[0094] 8) Color Development: Add 50 μl of Color Development Solution A and 50 μl of Color Development Solution B to each well, shake gently to mix, and incubate at 37°C for 15 minutes.

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

[0096] 10) Test eligibility criteria: Positive control OD value ≥ 0.5, negative control OD value < 0.1. Result determination: S / P = sample OD value / mean OD value of positive control; S / P ≥ 0.4, considered positive; S / P < 0.4, considered negative.

[0097] The results of PEDV IgG ELISA antibody detection in the serum of piglets after immunization with PEDV subunit vaccine are shown in Table 2.

[0098] Table 2 Results of PEDV IgG ELISA antibody detection in serum

[0099] PEDV neutralizing antibody detection

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

[0101] 2) Monolayer Cell Preparation Vero cells were digested and dispersed according to conventional methods, sampled and counted, and plated on 96-well cell culture plates at 20,000 cells / well, with 100 μL per well. The cells were cultured in a 37°C incubator and allowed to grow into a well-defined monolayer.

[0102] 3) Virus dilution: Take 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: The sample to be tested was serially diluted 2-fold using DMEM maintenance medium containing 2% fetal bovine serum. The diluted sample was mixed with an equal volume of indicator virus diluted to the working concentration.

[0104] 5) Neutralization: Mix the sample prepared in 4) with the indicator virus and mix them evenly. Place them together with the virus control in a 37°C incubator for 1 hour, shaking them 2 to 3 times during the process.

[0105] 6) Sample Addition: Remove the prepared Vero cell monolayer plate from the incubator and discard the liquid inside. Remove the sample, which has been neutralized for 1 hour, from the 37°C incubator and inoculate it onto four wells of each dilution, using 100 μL per well. Set up virus control wells, sample toxicity control wells, and normal cell control wells. Place the plate in a CO2 incubator and incubate daily. Determine the results after 96 hours.

[0106] 7) Result evaluation: All virus control wells should show CPE (cytopathic effect), while normal cell control wells and test serum toxicity control wells should show no CPE. Greater sample dilution will result in more wells showing cytopathic effect. Count the number of wells showing cytopathic effect at each sample dilution and calculate the results using the Reed-Muench method.

[0107] The results of neutralizing antibody detection in the serum of piglets after immunization with PEDV subunit vaccine are shown in Table 3.

[0108] Table 3 Results of neutralizing antibody detection in serum

[0109] Example 6 Evaluation of passive immune protection in sows and piglets immunized with subunit vaccines

[0110] Three sows negative for PEDV antigen and antibody were selected and vaccinated with 22 ml of vaccine each. The vaccination schedule was an initial immunization 35-40 days before farrowing and a secondary immunization 15-20 days before farrowing. Serum and milk were collected on the day of farrowing for neutralizing antibody testing. Serum samples were inactivated in a 56°C waterbath for 30 minutes and then stored at 2-8°C. Milk samples were inactivated in a 56°C waterbath for 60 minutes and then stored at 2-8°C. Neutralizing antibody test results are shown in Table 4.

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

[0112] Three piglets were farrowed by three sows, and three piglets were selected from each litter. Blood was collected 7, 14, 21, and 28 days after farrowing for neutralizing antibody assays. The results of the neutralizing antibody assays are shown in Table 5.

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

[0114] To evaluate piglet protection against challenge, five piglets born from sow 1001 were challenged with a virulent PEDV strain 28 days after farrowing. Five 28-day-old piglets negative for PEDV antigens and antibodies were simultaneously selected as challenge controls. The challenge evaluation results showed that 5 / 5 of the challenge control group exhibited symptoms of anorexia, diarrhea, or vomiting, while 5 / 5 of the piglets born from sow 1001 showed no abnormal clinical symptoms. Detailed results are shown in Table 6.

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

[0116] Unless otherwise defined, all technical and scientific terms used in this application are the same as those commonly understood by those skilled in the art to which this application belongs. In the event of any inconsistency, the meaning described in the full text of this application or the meaning derived from the content recorded in the full text of this application shall prevail. In addition, the terms used in this description are only for the purpose of describing the embodiments of the present 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, characterized in that, 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, wherein, 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, Block co-polymer, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, 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.

Images