Method for preparing foot-and-mouth disease virus-like particles by using yeast and foot-and-mouth disease virus-like particles prepared by method

Abstract

The invention relates to a method for preparing foot-and-mouth disease virus-like particles by using yeast and the foot-and-mouth disease virus-like particles prepared by the method. Relates to a recombinant vector for preparing a foot and mouth disease virus-like particle containing a coding sequence formed by sequentially fusing VP4, VP2, VP3, VP1, 2A and 3C of a foot and mouth disease virus, and a method for preparing the foot and mouth disease virus-like particle by using yeast transformed by the recombinant vector.

Description

Technical Field

[0001] The present invention relates to a method for preparing foot-and-mouth disease virus-like particles (FMDV-like particles) by using yeast and the FMDV-like particles prepared by the method.

[0002] This product is the result of research supported by the Korea Research Foundation's Science and Engineering Research Infrastructure Project (Project No.: 2017R1A6A1A03015876). Background Art

[0003] Foot-and-mouth disease (FMD) is a highly contagious disease caused by the foot-and-mouth disease virus (FMDV) that infects more than 70 species of even-toed ungulates, including pigs, cattle, sheep, goats, and African buffalo. FMDV is a positive-sense single-stranded RNA virus belonging to the genus Aphthovirus of the family Picornaviridae with an 8.5 kb genome encoding four structural proteins (VP1-VP4), ten nonstructural proteins (L pro , 2A, 2B, 2C, 3A, 3B 1-3 、3C pro , 3D pol) and a number of cleavage intermediates. After initial translation, the precursor P1 polypeptide is cleaved and processed into capsid protein subunits VP0, VP1, and VP3 by the activity of 3C protease, which is generated from the nonstructural precursor P3 polypeptide. During maturation, capsid protein VP0 self-cleaves into capsid proteins VP2 and VP4. VP1, VP2, and VP3 are exposed on the surface of the virus, while VP4 is located internally. FMDV particles are smooth, spherical, lack an envelope, and have a diameter of approximately 30 nm. The FMDV capsid has a pseudo-T=3 (T=pseudo3) icosahedral structure composed of 60 tightly packed asymmetric protomers. FMDV structural proteins are responsible for capsid assembly, viral stability, cell binding, and antigenic specificity, playing a central role in viral infection and recognition. FMDV is divided into seven major serotypes (A, O, C, Asia 1, South African Territories (SAT) 1, SAT2, and SAT3), which are distributed irregularly throughout the world. Furthermore, numerous subtypes and variants have evolved from each major serotype. Serotypes O, A, and C are the most prevalent in Europe, the United States, Asia, and Africa. While there is no cross-immunity between the seven serotypes, some cross-immunity exists within the various subtypes of the same serotype. Given the antigenic diversity between serotypes, further research is needed to control and prevent FMD through vaccine development.

[0004] Commercially available FMD vaccines consist of purified, inactivated whole virus preparations, but these have drawbacks such as quality control and residual infectivity, prompting the development of recombinant vaccine alternatives. Empty capsid vaccines, such as virus-like particles (VLPs), avoid the use of virus in vaccine preparation while preserving epitope structure. While more complex than simple subunit vaccines, they remain a highly attractive option. VLPs self-assemble from multiple copies of viral structural proteins similar to those found in natural virus particles, arranged in a specific order. VLPs contain the full repertoire of immunogenic sites found in intact viruses but lack the infectious viral genome. VLPs can be produced naturally in the laboratory in cell culture or through heterologous expression and self-assembly of capsid proteins. Various heterologous expression systems, including bacteria, baculovirus, insect larvae, mammalian cells, and plants, have been investigated for producing recombinant FMDV VLPs. The formation of recombinant FMDV VLPs requires the expression of VP0, VP3, and VP1, which are viral structural proteins. This can be achieved by expressing a polyprotein comprising a single structural protein or a P1-2A precursor and a 3C protease. The latter is generally considered to be more efficient than the former.

[0005] Yeast Saccharomyces cerevisiae ( Saccharomyces cerevisiae The availability of a wealth of genetic, molecular, and cellular information, along with established protocols for gene modification, makes it a highly attractive system for heterologous expression research. Furthermore, as a single-cell microorganism, it offers advantages such as high heterologous protein expression levels, ease of gene manipulation and scale-up, the establishment of large-scale culture systems, and the potential for post-translational modification and secretion. In the field of biotechnology, Saccharomyces cerevisiae, the most popular eukaryotic host for exogenous gene expression, is classified as a Generally Recognized as Safe (GRAS) organism, offering the advantage of worry-free application of products expressed in it. Furthermore, the potent adjuvant properties of Saccharomyces cerevisiae derivatives make yeast an attractive heterologous expression system for vaccine production and development.

[0006] On the other hand, Korean Patent Publication No. 2019-0032299 discloses a "Foot-and-mouth disease virus-like particle vaccine and its preparation method" using an expression plasmid and insect cell system prepared by cloning a single ORF of the P12A3C gene of FMDV under the codon optimization of the SV40 promoter in a vector, and Korean Patent Publication No. 2019-0072138 discloses a "Fusion protein containing an antigenic determinant derived from foot-and-mouth disease virus and a virus-like particle containing the same", which relates to a fusion protein and a virus-like particle containing the fusion protein. , particles composed of influenza virus M1 protein and enveloped virus-like particles, the fusion protein comprising: at least one of the antigenic determinants at amino acid positions 132-162 of VP1 derived from foot-and-mouth disease virus and at least one of the antigenic determinants at amino acid positions 192-211 of VP1 derived from foot-and-mouth disease virus; and a peptide composed of the transmembrane region and the cytoplasmic intraloop region of the hemagglutinin protein, but there is no record of the present invention's "method for preparing foot-and-mouth disease virus-like particles using yeast and foot-and-mouth disease virus-like particles prepared by the method". Summary of the Invention

[0007] (1) Technical issues to be resolved

[0008] The present invention was derived from the above requirements. The inventors constructed a construct (P1-2A-3C) composed of VP4, VP2, VP3, VP1, 2A and 3C of foot-and-mouth disease virus fused in sequence, and transformed Saccharomyces cerevisiae using a recombinant vector containing the coding sequence of the fusion construct optimized in yeast codons.

[0009] Yeast transformants were used to test whether the structural protein P1 polyprotein precursor of FMDV was processed into capsid subunits by 3C protease and whether the capsid proteins were assembled into VLPs. The results confirmed that the P1 polyprotein expressed in yeast was also processed into capsid subunit proteins by the translated 3C protease and then assembled into VLPs. It was also confirmed that the prepared VLPs maintained antigenicity capable of inducing an immune response, thereby completing the present invention.

[0010] (2) Technical solution

[0011] In order to solve the technical problem, the present invention provides a recombinant vector for preparing foot-and-mouth disease virus-like particles, comprising a coding sequence formed by sequentially fusion of VP4, VP2, VP3, VP1, 2A and 3C of foot-and-mouth disease virus.

[0012] Furthermore, the present invention provides a host cell transformed by the recombinant vector.

[0013] Furthermore, the present invention provides a method for preparing foot-and-mouth disease virus-like particles and foot-and-mouth disease virus-like particles prepared by the method, wherein the method comprises the step of culturing host cells transformed by the recombinant vector.

[0014] Furthermore, the present invention provides a vaccine composition for preventing foot-and-mouth disease, comprising the foot-and-mouth disease virus-like particles as an active ingredient.

[0015] Furthermore, the present invention provides a method for diagnosing foot-and-mouth disease infection, comprising the step of reacting a culture of host cells transformed by the recombinant vector or the foot-and-mouth disease virus-like particles with serum isolated from an individual suspected of being infected with foot-and-mouth disease.

[0016] Furthermore, the present invention provides a method for inducing immunity of cloven-hoofed animals against foot-and-mouth disease, comprising the step of administering host cells transformed by the recombinant vector or a culture thereof or the foot-and-mouth disease virus-like particles to the cloven-hoofed animals.

[0017] Furthermore, the present invention provides a feed additive composition for enhancing immunity of even-toed ungulates, comprising a host cell transformed by the recombinant vector or a culture thereof as an effective ingredient.

[0018] (3) Beneficial effects

[0019] The foot-and-mouth disease virus-like particles prepared by the method of the present invention self-assemble in yeast cells, showing a morphological structure similar to the original foot-and-mouth disease virus and maintaining antigenic properties, but do not contain genetic material that causes infection. Therefore, they can be used as an active ingredient in a vaccine composition for preventing foot-and-mouth disease, and it is expected that their application can be expanded in the development of therapeutic agents or methods for diagnosing foot-and-mouth disease infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Part (A) is a schematic diagram of the yeast episomal expression vector pYEGPD-TER, showing the 2μ yeast replication origin, URA3 (encodes orotidine 5-phosphate decarboxylase), and Amp R markers, Figure 1 Part (B) shows the sequence around the junction including the promoter / start codon of P1, P1 / 2A, and 2A / 3C in the pYEGPD-P1-2A-3C construct. Figure 1 Part (C) shows the sequence of P1 in the pYEGPD-P1 construct.

[0021] Figure 2 The following table shows the results of polymerase chain reaction (PCR) of yeast transformants and the results of agarose gel electrophoresis of PCR products using primers for 3C sequence amplification. PC is the PCR product using pYEGPD-P1-2A-3C as a template, and NC is the PCR product using a mock (vector-only) transformant.

[0022] Figure 3 Northern blot analysis of the yeast transformant TpYEGPD-P1-2A-3C-3 confirms the temporal transcriptional pattern of P1-2A-3C. Blots 1 through 4 represent total RNA from transformants on days 1, 2, 3, and 5 of culture. rRNA represents an equal amount of RNA loaded in each blot. GPD (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control.

[0023] Figure 4 Results of Western blotting analysis of P1-2A-3C polyprotein expression in yeast transformants without antibodies against VP3 (Part (A)) and VP2 (Part (B)). Blots 1 and 2 show protein samples from two selected yeast transformants cultured at 30°C for 3 days. PC represents capsid proteins (VP1 and VP2) expressed in E. coli, and NC represents protein from a mock (vector-only) transformant. (C) Quantification of VP2 protein expression levels in yeast transformants with differential P1-2A-3C polyprotein expression using a densitometer. Figure 4 Part (B) and Figure 5 Results of Part (C)).

[0024] Figure 5 Western blotting analysis of P1-2A-3C polyprotein expression in yeast transformants using antibodies against VP3 (part (A)), VP1 (part (B)), VP2 (part (C)), and 3C (part (D)) is shown. Blots 1 to 7 show protein samples from seven selected yeast transformants cultured at 30°C for 3 days. PC represents the capsid proteins (VP1, VP2, VP3) and 3C protease (expressed as ) expressed in E. coli. ), NC is the protein of mock (only vector) transformant. Figure 5 Part (E) shows a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel stained with Coomassie Brilliant Blue. When expressing proteins in E. coli, a vector containing a histidine tag is used for expression and purification. Therefore, the size of the protein expressed in yeast transformants may differ slightly from that on the blot.

[0025] Figure 6 Analysis of P1 polyprotein expression in yeast transformants. (A) shows the results of Western blotting using an anti-VP3 antibody. PC represents VP0-VP3 proteins expressed in yeast, PC1 represents VP3 protein expressed in E. coli, and NC represents protein samples from mock (vector-only) transformants. Blots 1 to 5 represent protein samples from five yeast transformants cultured at 30°C for three days. (B) shows a photograph of an SDS-PAGE gel stained with Coomassie Brilliant Blue.

[0026] Figure 7 Western blot analysis of yeast expressing the P1-2A-3C polyprotein for VLP measurement using an anti-VP3 antibody against serially diluted samples of known concentrations of E. coli-expressed VP3 was compared with Western blot analysis of yeast-expressed samples using an anti-VP3 antibody. Blots 1 through 4 contain 0.5 μg, 1.0 μg, 2.0 μg, and 3.0 μg of E. coli-expressed VP3 protein, respectively. Blot 5 contains a 100 μg protein sample from yeast transformants expressing the P1-2A-3C polyprotein. The arrow indicates the VP3 protein. To measure the amount of yeast-expressed VP3 protein, densitometry of the cross-reactive blot intensity was performed.

[0027] Figure 8Western blotting results of partially purified VLPs using anti-VP3 antibodies. (A) shows the results of a Western blotting of a sample that was not heated before loading on the gel, and (B) shows the results of a Western blotting of a sample that was heated before loading on the gel. Blots 1 to 4 represent selected samples. PC and NC represent protein samples from yeast transformants expressing the P1-2A-3C polyprotein and mock (vector-only) transformants, respectively.

[0028] Figure 9 This is an electron micrograph of purified VLPs, showing icosahedral viral particles with a diameter of approximately 30 nm. The arrow indicates the VLP.

[0029] Figure 10 The results of recombinant foot-and-mouth disease virus (FMDV) VLPs assay using a commercially available FMDV diagnostic kit are shown in Figure 1. (A) shows the results of assaying total soluble protein (1) isolated from yeast transformants and VLPs (2) partially purified after ultracentrifugation using a commercially available FMDV diagnostic kit. (B) shows the results of assaying the lowest concentration using serial dilutions of total soluble protein from yeast transformants. NC represents a total soluble protein sample prepared from mock (vector-only) transformants. DETAILED DESCRIPTION

[0030] To achieve the purpose of the present invention, the present invention provides a recombinant vector for preparing foot-and-mouth disease virus-like particles, comprising a coding sequence formed by sequentially fusion of VP4, VP2, VP3, VP1, 2A and 3C of foot-and-mouth disease virus.

[0031] The term "virus-like particle (VLP)" as used herein refers to particles that resemble actual viruses by combining viral structural proteins. VLPs do not contain the viral genome during assembly and are unable to multiply upon injection into the body, offering the advantage of being a safe antigen.

[0032] In the recombinant vector for preparing foot-and-mouth disease VLP of the present invention, the coding sequence formed by sequentially fusion of VP4, VP2, VP3, VP1, 2A and 3C of foot-and-mouth disease virus can consist of the base sequence of SEQ ID NO: 2, but is not limited thereto.

[0033] The base sequence of SEQ ID NO: 1 was synthesized by optimizing yeast codons for the sequential fusion of coding sequences composed of VP4, VP2, VP3, VP1, 2A, and 3C of foot-and-mouth disease virus. During the search for an optimal sequence to improve expression of the base sequence of SEQ ID NO: 1, the base sequence of SEQ ID NO: 2 of the present invention was discovered. Compared to the base sequence of SEQ ID NO: 1, the base sequence comprises four synonymous substitutions and one non-synonymous substitution. Specifically, the four synonymous substitutions are mutations of G, C, C, and G at bases 12, 18, 636, and 639 of SEQ ID NO: 1 to T, T, T, and A, respectively. The non-synonymous substitution is a mutation of CTG to CCT at bases 379 to 381 of SEQ ID NO: 1, encoding a leucine (Leu) to proline (Pro) amino acid.

[0034] The term "codon optimization" as used herein refers to altering the codons of a protein-encoding polynucleotide to optimize their preferred usage in a specific organism in order to more efficiently express the encoded protein. While the genetic code is degenerate, with most amino acids represented by a number of so-called "synonymous" codons, codon usage in a given organism is not random but rather favors specific codon triplets. This codon usage preference is further enhanced by association with defined genes, genes of common function or ancestral origin, proteins with low copy numbers relative to highly expressed proteins, and the overall protein-coding region of an organism's genome.

[0035] The term "recombinant" as used herein refers to the ability of a cell to replicate a foreign nucleic acid, express such nucleic acid or a foreign peptide, or express a protein encoded by a foreign nucleic acid. Recombinant cells can express genes or gene fragments not found in the cell's native form in either sense or antisense form. Furthermore, recombinant cells can express genes found in the cell's native state, but in modified forms that have been artificially introduced into the cell.

[0036] Furthermore, the term "vector" is used to refer to DNA segments or nucleic acid molecules that are transported into cells. Vectors can replicate DNA and reproduce independently in host cells. The term "transporter" is often used interchangeably with "vector." The term "expression vector" refers to a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a specific host cell. Key characteristics of such expression vectors include an origin of replication, a promoter, a terminator, a marker gene, and a translation control element.

[0037] In one embodiment of the present invention, the recombinant vector for preparing foot-and-mouth disease VLPs may be a yeast episome expression vector pYEGPD-TER, but is not limited thereto.

[0038] The recombinant vectors of the present invention can be constructed using methods well known to those skilled in the art. These methods include laboratory recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. To induce mRNA synthesis, the DNA sequence can be operably linked to an appropriate promoter within the vector. The term "operably linked" refers to a nucleic acid segment being linked to another nucleic acid segment so that its function or expression is affected by the other nucleic acid segment.

[0039] Furthermore, the present invention provides a host cell transformed by the recombinant vector.

[0040] In the present invention, preferably, the host cell can be a yeast ( Saccharomyces ) genus, Candida genus, Cryptococcus ( Cryptococcus ) genus, Hansenula ( Hansenula ) genus, Kluyveromyces ( Kluyveromyces ) or Pichia pastoris ( Pichia ) genus, more preferably, it can be a yeast of the genus Saccharomyces, and even more preferably, it can be a yeast of the genus Saccharomyces ( Saccharomyces cerevisiae ), but not limited to this.

[0041] The recombinant vector of the present invention can be delivered into the host cell, i.e., the transformation method can be carried out by the calcium chloride (CaCl2) method, the Hanahan method (Hanahan, D., 1983 J. Mol. Biol. 166:557-580), electroporation or the lithium acetate method (Gietz D et al., 1992 Nucleic Acids Res. 20:1425), but is not limited thereto.

[0042] Furthermore, the present invention provides a method for preparing foot-and-mouth disease virus-like particles and foot-and-mouth disease virus-like particles prepared by the method, wherein the method comprises the step of culturing host cells transformed by the recombinant vector.

[0043] In the method for preparing foot-and-mouth disease VLP of the present invention, the recombinant vector, transformation method and host cell are as described above.

[0044] In the method for preparing foot-and-mouth disease VLPs of the present invention, the transformed host cells can be cultured in a culture medium suitable for expressing the target protein using known techniques. Suitable culture media can be purchased commercially or prepared based on the components and composition ratios described in publications such as the catalog of the American Type Culture Collection, but are not limited thereto.

[0045] Furthermore, after the host cell culture step, the method for preparing foot-and-mouth disease VLPs of the present invention may further include, but is not limited to, the step of isolating and purifying the foot-and-mouth disease VLPs from the host cell culture. For example, the VLPs may be isolated from the culture by conventional separation methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Furthermore, the isolated protein may be purified by various well-known methods including chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion), dialysis, electrophoresis, fractionated solubility (e.g., ammonium sulfate precipitation), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or extraction.

[0046] The foot-and-mouth disease (FMD) VLPs of the present invention are non-envelope VLPs, lacking an envelope and not requiring a host cell membrane. They are composed of one or more capsid proteins from non-enveloped viruses. The capsid is the protein that makes up the viral coat, and the capsid naturally self-assembles to form the viral outer shell.

[0047] Furthermore, the present invention provides a vaccine composition for preventing foot-and-mouth disease, comprising the foot-and-mouth disease virus-like particle as an active ingredient.

[0048] In the present invention, the term "prevention" refers to all actions to inhibit or delay the occurrence, spread and recurrence of foot-and-mouth disease by administering the vaccine composition of the present invention.

[0049] Furthermore, the vaccine composition for preventing foot-and-mouth disease of the present invention may further comprise a pharmaceutically acceptable carrier and / or adjuvant, and in addition to the carrier, may further comprise an excipient and / or a diluent.

[0050] The term "pharmaceutically acceptable" as used herein refers to substances that are physiologically acceptable and generally do not cause gastrointestinal disturbances, vertigo, allergic reactions, or similar reactions when administered to a subject. Examples of the carrier, excipient, and diluent include lactose, glucose, sucrose, sorbitol, mannitol, erythritol, xylitol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Furthermore, fillers, anticoagulants, lubricants, wetting agents, flavorings, emulsifiers, and preservatives may also be included.

[0051] The term "adjuvant" is a substance that is administered for the purpose of enhancing the immune response of a vaccine and non-specifically promotes the immune response to an antigen during the initial activation of immune cells. It refers to a preparation that, although not an immunogen, increases the activity of cells in the host immune system (Warren et al., 1986, Annu. Rev. Immunol. 4:369). The adjuvant includes any of a variety of adjuvants, and can include Freund's adjuvant, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, MF59, AS04 (a mixture of MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum salt), AS03 (a mixture of DL-α-tocopherol, squalene, and polysorbate 80), AS01 (a mixture of MPL and saponin QS-21), AS02, muramyl dipeptide and its derivatives, flagellin, Poly I:C (polyinosinic: polycytidylic acid), and the like. acid), ISCOMs or ISCOM-matrix (Matrix), but is not limited thereto.

[0052] To achieve rapid, sustained, or delayed release of the active ingredient upon administration to a subject, the vaccine composition of the present invention can be formulated using methods known in the art. Dosage forms include powders, granules, tablets, emulsions, syrups, sprays, soft or hard gelatin capsules, sterile injectable solutions, and sterile powders.

[0053] The vaccine composition of the present invention can be administered by various routes, for example, orally, parenterally, by suppository, transdermally, intravenously, intraperitoneally, intramuscularly, intralesionally, nasally, or intrathecally. Furthermore, it can be administered using a sustained-release or continuous or repeated-release implantable device. The administration frequency can be once daily or divided into several doses, as desired, and the duration of administration is not particularly limited.

[0054] Furthermore, the present invention provides a method for diagnosing foot-and-mouth disease infection, comprising the step of reacting a culture of host cells transformed by the recombinant vector of the present invention or the foot-and-mouth disease virus-like particle with serum isolated from an individual suspected of being infected with foot-and-mouth disease.

[0055] In the method for diagnosing foot-and-mouth disease infection of the present invention, the recombinant vector, transformation method, host cells and foot-and-mouth disease VLP are as described above. In the present invention, the culture of the host cells transformed by the recombinant vector contains foot-and-mouth disease VLP.

[0056] The method for diagnosing foot-and-mouth disease infection of the present invention can diagnose whether an individual suspected of being infected with foot-and-mouth disease is infected or not based on the antigen-antibody reaction, and can be achieved by the following steps: serum isolated from a suspected individual of foot-and-mouth disease is cultured with a transformed host cell containing foot-and-mouth disease VLP or isolated under conditions that can form an antigen-antibody complex; After the purified foot-and-mouth disease VLPs react, the formation of antigen-antibody complexes is detected.

[0057] The term "antigen-antibody complex" in the present invention refers to a complex of antibodies against foot-and-mouth disease virus (FMDV) present in serum and the foot-and-mouth disease VLP of the present invention bound thereto. The formation of such an antigen-antibody complex can be detected by conventional immunoassay methods such as immunoprecipitation or enzyme-linked immunosorbent assay (ELISA, enzyme-linked immunosorbent assay).

[0058] Furthermore, the present invention provides a method for inducing immunity of cloven-hoofed animals against foot-and-mouth disease, comprising the step of administering to the cloven-hoofed animals the host cells transformed with the recombinant vector of the present invention or the culture thereof or the foot-and-mouth disease virus-like particles.

[0059] In the immune induction method of the present invention, the recombinant vector, transformation method, host cell, and foot-and-mouth disease VLP are as described above. In addition, the culture may include a culture fluid of the transformed host cells, a concentrated solution of the culture fluid, or a dried product thereof. In the present invention, the culture of the host cell transformed by the recombinant vector contains foot-and-mouth disease VLP.

[0060] The term "administration" in the present invention refers to providing the specified transformed host cells of the present invention or their culture or isolated and purified foot-and-mouth disease VLP to an even-toed ungulate individual by any appropriate method. In addition, the transformed host cells or their culture or isolated and purified foot-and-mouth disease VLP can be administered in an immunologically effective amount. The "immunologically effective amount" refers to a sufficient amount that can show a preventive effect on foot-and-mouth disease and an amount that has no side effects or does not cause severe or excessive immune responses. The exact administration concentration can be easily determined by relevant practitioners based on factors well-known in the medical field such as the age, weight, health status, gender, individual sensitivity to drugs, administration route, administration method, etc. of the subject individual, and the administration can be performed once or several times.

[0061] In the immunity induction method of the present invention, the even-toed ungulate animal may be cattle, pigs, sheep, goats, sika deer, camels, bison or reindeer, but is not limited thereto.

[0062] Furthermore, the present invention provides a feed additive composition for enhancing immunity of even-toed ungulates, comprising a host cell transformed by the recombinant vector of the present invention or a culture thereof as an effective ingredient.

[0063] In the feed additive composition of the present invention, the recombinant vector, transformation method, and host cell are as described above. Furthermore, the culture may include a culture fluid of the transformed host cells, a concentrated solution of the culture fluid, or a dried product thereof. In the present invention, the culture of the host cells transformed by the recombinant vector contains foot-and-mouth disease VLPs.

[0064] The feed additive composition of the present invention can be added to feed. "Feed" can refer to any natural or artificial feed, ready-to-eat feed, or the like, or a component of such ready-to-eat feed, intended for consumption, ingestion, digestion, or equivalent thereof by an animal. The type of feed is not particularly limited, and feed commonly used in the art to which the present invention pertains can be used. Non-limiting examples of the feed include: plant-based feeds such as cereals, root vegetables, food processing byproducts, algae, fiber, pharmaceutical byproducts, oils, starches, cucurbits, or cereal byproducts; and animal-based feeds such as proteins, inorganic substances, oils, minerals, single-cell proteins, zooplankton, or food.

[0065] Furthermore, in the present invention, in addition to the microorganisms expressing the target protein on their cell surfaces, the feed additive composition may also contain various nutrients such as vitamins, amino acids, minerals, antioxidants, antibiotics, antibacterial agents and other additives as needed, and its form may be in an appropriate state such as powder, granules, particles, suspension, etc.

[0066] In this specification, when a part is referred to as “comprising” a certain structural element, unless otherwise stated, it means that other structural elements may also be included, rather than excluding other structural elements.

[0067] The present invention will be described in detail below by way of examples. However, the following examples are only for the purpose of illustrating the present invention, and the present invention is not limited to the following examples.

[0068] Materials and methods

[0069] 1. Microbial strains and culture conditions

[0070] Escherichia coli TOP10 strain was used for gene cloning. Purified antigens for each FMDV capsid protein were prepared using E. coli BL21 and M15 strains. Saccharomyces cerevisiae 2805 strain (MATa pep4::HIS3 prb1-Δcan1 GAL2 his3 ura3-52) was used for heterologous expression. Yeast and E. coli culture were performed according to conventional methods (So KK et al., 2021 Biotechnol Lett. 43:1895-1904).

[0071] 2. Construction and transformation of yeast expression vector

[0072] A fusion gene construct (P1-2A-3C) consisting of the polyprotein precursors P1 and 2A and proteinase 3C of FMDV serotype O (GenBank accession no. AY593823.1) was chemically synthesized at Bioneer (Daejeon, South Korea) using codons optimized for yeast expression (SEQ ID NO: 1). For subsequent replication, the construct was cloned by PCR using the primers in Table 1. Bam The HI and SalI restriction enzyme sites were introduced into the 5' and 3' ends of the synthetic gene (SEQ ID NO: 1), respectively. The fusion construct was cloned into the yeast episome expression vector pYEGPD-TER containing the GPD promoter and the GAL7 (galactose-1-phosphate uridyl transferase) terminator to construct pYEGPD-P1-2A-3C ( Figure 1 Similarly, the yeast expression recombinant vector pYEGPD-P1 was designed using the P1 gene alone as a control group. The recombinant vectors carrying the P1-2A-3C gene or the P1 gene were introduced into the yeast using the lithium acetate transformation method (Gietz D et al., 1992). S. Cerevisiae 2805 strain. Yeast transformed with the empty vector pYEGPD-TER served as a negative control. To analyze the presence of the recombinant vector, colony PCR and E. coli back-transformants were performed according to previous methods (So KK et al., 2021).

[0073] Table 1

[0074] Primer information used in the present invention

[0075]

[0076]

[0077] 3. Northern blot analysis

[0078] Total RNA was extracted as previously described (Lim YY et al., 2003 J Microbiol Biotechnol. 13:537-543) and quantified using a Multiskan Go ultraviolet (UV) spectrophotometer (Thermo Scientific, Finland). 30 μg of total RNA was loaded onto a 1.2% formaldehyde-agarose gel, subjected to electrophoresis, and then transferred to an Amersham Hybond™ membrane. Northern blots were performed using modified Church's buffer (250 mM sodium hydrogen phosphate (Na2HPO4), 1 mM ethylenediaminetetraacetic acid (EDTA), 7% sodium dodecyl sulfate (SDS), 0.17% phosphoric acid (H3PO4), and 1% hydrolyzed casein) followed by hybridization with an α-[³²P]-labeled probe prepared using a random labeling kit.

[0079] 4. Western blot analysis

[0080] FMDV antigen expression and purification were performed by expressing viral capsid proteins (VP1, VP2, and VP3) and 3C protease in E. coli. The gene encoding VP1 was cloned into the pCold II vector (TaKaRa, Japan), and the genes encoding VP2, VP3, and 3C were cloned into the pQE-30 vector (Qiagen, USA). The recombinant expression vectors were transformed into the recommended host (in the case of pCold II, BL21-CodonPlus) according to the manufacturer's instructions. ® -RIL (M15 in the case of pQE-30) was used to express the recombinant protein. Ni-NTA agarose columns (Invitrogen, USA) were used to purify the E. coli-expressed proteins in 8 M urea buffer. The purity of each antigen was confirmed by SDS-PAGE and anti-His tag antibodies. Anti-FMDV antibodies were raised by injecting purified VP1, VP, VP3, and 3C proteins with Freund's complete adjuvant and Freund's incomplete adjuvant into 8-week-old BALB / c mice (Charles River Laboratory, USA). Antisera were collected four days after the third booster injection.

[0081] Protein samples from yeast transformants were prepared in the same manner as previously reported (Mo AY et al., 2005 Biotechnol Bioprocess Eng. 10:576-581). 150 μg of total protein was run on a 12% SDS-PAGE gel and then transferred to a nitrocellulose membrane. The target proteins (VP1, VP2, VP3, and 3C) were detected using the corresponding antibodies (anti-VP1, anti-VP2, anti-VP3, and anti-3C). Alkaline phosphatase-deficient goat anti-mouse IgG (Sigma-Aldrich, USA) was used as a secondary antibody. Chromogenic detection was performed using BCIP / NBT in TMN buffer (100 mM Tris, pH 9.5, 5 mM magnesium chloride (MgCl2), 100 mM sodium chloride (NaCl)).

[0082] 5. Observation of VLPs using an electron microscope

[0083] To remove cellular debris, the yeast lysate treated with magnetic beads was centrifuged. The supernatant was ultracentrifuged at 119,000 × g for 2 hours at 4°C to pellet the viral particles. The recovered pellets were loaded onto a sucrose density gradient (100–500 mg / ml at 100 mg / ml intervals) and subjected to additional ultracentrifugation at 70,000 × g for 4 hours at 4°C. The collected fractions were dialyzed against PBS and analyzed by SDS-PAGE and Western blotting. The viral particle structure was observed using an H-7650 transmission electron microscope (Hitachi, Japan) after negative staining with 2% uranyl acetate.

[0084] 6. Immune response to expressed VLPs

[0085] The commercially available serotype-specific FMDV antigen detection kit VDRG was used according to the manufacturer's instructions. ® The antigenicity of yeast-derived VLPs was examined using the FMDV 3Diff / PAN Ag Rapid Kit (MEDIAN Diagnostics Inc., South Korea).

[0086] Example 1. Construction of yeast ( S.cerevisiae ) transformed VLP expression vector

[0087] To express the target gene, the yeast episomal expression vector pYEPGPD-TER (Bal J, et al., 2018 Microb Cell Fact. 17:146), which is used to express various heterologous proteins, was used. GPD constitutive promoters and GAL7 The expression cassette begins with the terminator of the gene and a 2μ yeast replication origin sequence that can replicate independently from chromosomal DNA for high copy number vectors. For yeast expression, the DNA sequence of the fusion construct (P1-2A-3C) of P1-2A from the Turkish variant of FMD serotype O virus (O1 / Manisa / TUR / 69, GenBank accession no. AY593823.1) and the precursor protein for 3C were codon-optimized and chemically synthesized into a single construct, which was then cloned using the pGEM T-easy vector, which was designated pP1-2A-3C. To clone the expression cassette using the vector pYEPGPD-TER, the P1-2A-3C gene was added during PCR amplification. Bam HI and Sal I restriction enzyme position. The 2.9 kb PCR amplicon resulting from the above results was cloned into the pGEM T-easy vector by sequencing analysis. Bam HI and Sal I treated pYEPGPD-TER vector with Bam HI and Sal The P1-2A-3C ligation treated with I was verified by sequencing to produce a recombinant vector for yeast expression (pYEPGPD-P1-2A-3C) and then used in yeast transformation.

[0088] Example 2. Yeast transformation analysis

[0089] Twenty putative yeast transformants were screened in uracil-deficient selective medium. The presence of pYEGPD-P1-2A-3C was confirmed by PCR using the colonies as a source of DNA template in the reaction mixture. All PCR-tested colonies showed the expected 640 bp PCR amplicon (using primers for 3C sequence amplification). Figure 2 Furthermore, the presence of the relevant recombinant plasmid pYEGPD-P1-2A-3C in yeast cells was verified by E. coli transformation of the plasmid DNA isolated from the yeast transformants and restriction enzyme analysis of the plasmid DNA isolated from the E. coli transformants.

[0090] Furthermore, for the screening of transformants, Western blotting was performed using anti-VP3 or anti-VP2 antibodies using cell-free extracts of yeast transformants as samples. As a result, no expression of VP3 and VP2 was confirmed, and some yeast transformants ( Figure 4 ), some yeast transformants showing high expression levels of VP3 and VP2 were confirmed ( Figure 5 ). Sequencing analysis of plasmids isolated from yeast transformants expressing different VP3 and VP2 levels revealed that the P1-2A-3C coding sequence from transformants expressing low VP3 and VP2 levels did not differ from the cloned base sequence of SEQ ID NO: 1. However, in the P1-2A-3C coding sequence from transformants expressing high VP3 and VP2 levels, two silent mutations were confirmed in the VP4 coding sequence, as well as two silent mutations and one missense mutation in the 3C coding sequence. Considering the reports that single synonymous codon substitution can affect gene expression levels or protein folding (Chamary, JV, et al., Nat. Rev. Genet. (2006) 7, 98-108; Marin, M., Biotechnol. J. (2008) 3, 1047-1057; Saunders, R., Deane, CM, Nucleic Acids Res. (2010), 38, 6719-6728; Plotkin, JB, Kudla, G., Nat. Rev. Genet. (2011) 12, 32-42), it is judged that the silent mutation of the four bases affects the expression of FMDV capsid protein (VP3). The P1-2A-3C coding sequence with the base mutation is designated as the base sequence of SEQ ID NO: 2, and yeast transformants with the base sequence of SEQ ID NO: 2 are screened and confirmed for subsequent experiments.

[0091] The accumulation of the target gene transcriptome in the selected transformants was measured using cells cultured for 3 days. Northern blotting of the 13 selected transformants showed accumulation of the P1-2A-3C transcriptome in all transformants (not shown). Variations in the transcription levels of the P1-2A-3C gene were observed between the transformants, and it was determined that this was caused by differences in the copy number of the plasmid. Therefore, for subsequent analysis, 7 transformants (TpYEGPD-P1-2A-3C-2, -3, -4, -5, -6, -7 and -11) showing high transcription levels were selected. The growth curves of each selected transformant were measured over 5 days. No significant differences were confirmed between the transformants. More specifically, similar numbers of cells (i.e., 3.0-4.0×10 per liter) were observed when the stationary phase was reached after 3 days of culture. 11 These results indicate that the selected transformants did not have growth abnormalities caused by the expression of the recombinant protein P1-2A-3C.

[0092] The transcription pattern of P1-2A-3C over time was investigated by Northern blot analysis of TpYEGPD-P1-2A-3C-3, which was selected as a representative of the 7 transformants. Figure 3 As shown in Figure 2, the transcriptome accumulation of P1-2A-3C reached a peak after 48 hours of culture and then gradually decreased, while the internal control group ( GPD The transcriptome of these genes remains at high levels until the late stationary phase (up to 5 days after culture).

[0093] Example 3. Expression and processing analysis of target gene protein products

[0094] To confirm the expression of the protein product encoded by the P1-2A-3C gene, cell-free extracts of yeast transformants of the seven selected strains were used as samples and Western blotting was performed using an anti-VP3 antibody.

[0095] Using anti-VP3 antibody, a specific antibody-reactive band was observed at 24 kDa, which corresponds to the estimated size of VP3 capsid protein ( Figure 5 As a result of using anti-VP1 antibody, a cross-reactive protein band was detected at 24 kDa, which is equivalent to the estimated size of VP1 capsid protein ( Figure 5 (B) of the sample), but the band was not as strong as the reaction with anti-VP3 antibody. Anti-VP2 antibody cross-binded to two distinct protein bands at 37kDa and 24kDa. The intermediate viral capsid precursor VP0 contains VP4 and VP2. Therefore, the cross-reactive 37kDa and 24kDa bands were judged to be VP0 (VP4-VP2) and VP2 capsid proteins ( Figure 5(C)). VP0 processing is known to occur during the maturation cleavage of capsids packaged by genomic RNA, so the observation of a protein band equivalent in size to VP2 in addition to the VP0 band is particularly interesting. The additional VP2 band indicates that some VP0 cleavage and capsid assembly occur in the absence of genomic RNA. Furthermore, although the anti-3C antibody detected a 23 kDa cross-reactive protein band, it showed very low intensity and was considered to be minimal expression of the 3C protein ( Figure 5 (D)). These results suggest that the 2A peptide modifies the protein's translational machinery by cooperating with the N-terminal serine residue of the 3C protein, thereby allowing the release of the early P1-2A polyprotein from the ribosome while allowing the translation of the downstream 3C protein to be restarted through a new translation effect. Therefore, despite co-translation from a monocistronic transcriptome, the production of additional 3C protein is possible. Furthermore, the 2A / 3C linker (where proline is replaced with serine, which has been reported to reduce cleavage efficiency to a lower level than the authentic 2A / 2B linker) Figure 1 ) results, it is predicted that only a small amount of additional 3C protein will be produced (ribosome jumping efficiency is reduced). Taking into account the adverse effects of 3C protease on host cells, the reduction in 3C protein expression can be seen as beneficial to the growth of yeast cells, which is consistent with the normal cell growth results of the transformants. As a control group, an expression vector pYEGPD-P1 similarly designed to pYEGPD-P1-2A-3C and based on the same expression vector (pYEGPD-TER) was used to construct a recombinant yeast designed to express only the P1 gene product. Regardless of whether anti-VP1, -VP2 or -VP3 antibodies were used, protein blot analysis of cell-free extracts (CFE) of the three transformants induced by the results showed the presence of an uncleaved polyprotein around 83 kDa ( Figure 6 ), whereby the protein product of the P1 gene is expressed as a single polyprotein without additional treatment. In contrast, it was found that after the P1-2A-3C gene is translated to produce the polyprotein P1, it is decomposed into the capsid proteins of VP0, VP3, and VP1 by the 3C protease that is simultaneously translated in yeast.

[0096] The expression level of VLPs was measured indirectly by comparing the band intensity with serial dilutions of known concentrations of E. coli expressing VP3. Figure 7As shown, the band intensity of processed VP3 in yeast-expressed VLPs ranged from approximately 0.5 μg to 1.0 μg, equivalent to 0.5% of the protein preparation. The inventors therefore hypothesized that the scaled-up culture of the recombinant yeast produced VLPs equivalent to 15 mg of VP3. Furthermore, the amount of VLPs was measured using a commercially available detection kit and compared with known amounts of FMDV. The results, consistent with previous results using VP3 subunits, showed that approximately 50 mg of VLPs were produced per liter of culture.

[0097] Example 4. Electron Microscopic Analysis of VLPs

[0098] Partial purification of VLPs from yeast cells was performed by sucrose density gradient ultracentrifugation. Two distinct bands were observed in the middle of the sucrose gradient after ultracentrifugation, and 200 μl fractions were collected. Ultracentrifugation fractions were used for Western blot analysis prior to electron microscopy.

[0099] The results are as follows Figure 8 As shown, when the samples of each component were not heated before loading, a large single band of over 100 kDa, corresponding to the VLP, was observed. However, when the samples of each component were heated before loading, bands corresponding to the expected size of the capsid subunits were observed. These results indicate that the FMDV capsid is highly sensitive to elevated temperatures, consistent with previous findings that it is degraded under these conditions. These results can be interpreted as indicating that the polyprotein expressed in yeast is properly processed and assembled into VLPs, which are then degraded upon heating.

[0100] Electron microscopy (EM) analysis of partially purified virus preparations revealed the presence of hollow isometric particles approximately 30 nm in diameter, with characteristic black spots associated with heavy metal staining ( Figure 9 ), and the particles were similar in size to FMDV virions (Dong H, et al., 2021 J Virol. 95:e0017721). These results suggest that the P1 precursor polyprotein expressed in yeast is processed into capsid subunit proteins by the co-translational 3C protease and then assembled into non-ionic particles.

[0101] Example 5. Analysis of the immune response to expressed VLPs

[0102] Use the commercially available FMDV antigen detection kit (VDRG ®The antigenicity of VLPs expressed in yeast was examined using the FMDV 3Diff / PAN Ag Rapid Kit (MEDIA Diagnostics Inc., Korea). Antigen-antibody reactions were detected not only with partially purified VLPs but also with proteins prepared from recombinant yeast ( Figure 10 In addition, the protein sample prepared from the recombinant yeast was detected by the kit up to a concentration of 2 μg ( Figure 10 (B)). In particular, the kit's serotype determination results confirmed that the yeast-expressed VLPs produced in the present invention were serotype O. This result suggests that the antigenicity of the VLPs is maintained by preserving the integrity of native FMDV particles. Therefore, it is believed that the VLPs produced by the present method induce an immune response and possess potent antigenicity capable of protecting against additional FMDV infection.

Claims

1. A recombinant vector for preparing foot-and-mouth disease virus-like particles, characterized in that: It contains a coding sequence formed by the fusion of VP4, VP2, VP3, VP1, 2A and 3C of foot-and-mouth disease virus in sequence.

2. The recombinant vector for preparing foot-and-mouth disease virus-like particles according to claim 1, characterized in that The coding sequence formed by sequentially fusing VP4, VP2, VP3, VP1, 2A and 3C of foot-and-mouth disease virus consists of the base sequence of SEQ ID NO:

2.

3. A host cell, characterized in that It is transformed from the recombinant vector for preparing foot-and-mouth disease virus-like particles according to claim 1 or 2.

4. The host cell according to claim 3, characterized in that The host cell is Saccharomyces cerevisiae.

5. A method for preparing foot-and-mouth disease virus-like particles, characterized in that: The method comprises the step of culturing a host cell transformed by the recombinant vector for preparing foot-and-mouth disease virus-like particles according to claim 1 or 2.

6. The method for preparing foot-and-mouth disease virus-like particles according to claim 5, characterized in that: After the host cell culture step, the method further comprises the step of isolating and purifying foot-and-mouth disease virus-like particles from the host cell culture.

7. A foot-and-mouth disease virus-like particle, characterized in that: The virus-like particles are prepared by the method for preparing the foot-and-mouth disease virus-like particles according to claim 5.

8. A vaccine composition for preventing foot-and-mouth disease, characterized in that: The invention comprises the foot-and-mouth disease virus-like particles according to claim 7 as an active ingredient.

9. The vaccine composition for preventing foot-and-mouth disease according to claim 8, characterized in that The vaccine composition further comprises a pharmaceutically acceptable carrier or adjuvant.

10. A method for diagnosing foot-and-mouth disease infection, characterized in that: The method comprises reacting a culture of host cells transformed by the recombinant vector for preparing foot-and-mouth disease virus-like particles according to claim 1 or the foot-and-mouth disease virus-like particles according to claim 7 with serum isolated from an individual suspected of being infected with foot-and-mouth disease.

11. A method for inducing immunity of even-toed ungulates against foot-and-mouth disease, characterized in that: The method comprises the step of administering the host cell transformed by the recombinant vector for preparing foot-and-mouth disease virus-like particles according to claim 1 or the culture thereof or the foot-and-mouth disease virus-like particles according to claim 7 to an even-toed ungulate.

12. A feed additive composition for enhancing immunity of even-toed ungulates, characterized in that: The invention comprises a host cell transformed by the recombinant vector for preparing foot-and-mouth disease virus-like particles according to claim 1 or a culture thereof as an effective ingredient.

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