Methods for producing alfalfa mosaic virus-like particles using a plant expression system and their applications

Abstract

The present invention relates to a technique for producing AMV (Alfalfa mosaic virus) in the form of VLPs (virus-like particles) isolated from a plant transformed with a recombinant vector for plant expression targeted to chloroplasts, and provides a recombinant vector including a polynucleotide encoding a recombinant protein in which a chloroplast targeting protein and an AMV capsid protein are fused. Also provided are a transformed plant transformed with the recombinant vector, a method for isolating and purifying a target protein from the transformed plant, and a method for producing virus-like particles using the same.

Description

[Technical Field]

[0001] The present invention relates to a method for producing virus-like particles (VLPs) using plants and its applications. More specifically, the present invention relates to a method for producing recombinant Alfalfa mosaic virus (AMV) capsid protein in plants using a highly efficient expression vector, and a method for producing the same in VLPs.

[0002] This application claims priority based on Korean Patent Application No. 10-2021-0025902, filed on February 25, 2021, the entire contents of which are incorporated herein by reference in their entirety in the specification and drawings. [Background technology]

[0003] Virus-like particles (VLPs) are particles that are assembled in a form similar to that of an actual virus through the binding of viral structural proteins. VLPs do not contain viral genes during assembly, making them a safe antigen that cannot replicate when injected into the body. In addition, due to their high immunogenicity, research and development of vaccines that apply VLPs to various pathogens is actively underway.

[0004] Like viruses, VLPs can be divided into enveloped VLPs (hereinafter referred to as "envelop VLPs") and non-enveloped VLPs (hereinafter referred to as "non-envelop VLPs") depending on whether they have an envelope. Enveloped VLPs use the host cell membrane as an envelope, expressing viral proteins as antigens on the outer surface, and are produced by budding onto the outside of the cell through a process of fusion with the host cell membrane. Envelope VLPs are characterized by their relatively larger particle size compared to non-envelope VLPs, and by expressing antigens from various pathogens on the cell membrane, VLPs that simultaneously express various antigens can be produced. Representative envelope VLPs include influenza virus, retrovirus, and hepatitis C virus VLPs.

[0005] Non-envelope VLPs do not require the cell membrane of host cells and are composed of one or more capsid proteins from non-envelope viruses. Like envelope VLPs, non-envelope VLPs are characterized by their ability to simultaneously express various antigens, but they are less flexible in simultaneously expressing antigens from other pathogens. Representative non-envelope VLPs include hepatitis B virus, human papillomavirus, hepatitis E virus, and rotavirus VLPs.

[0006] Currently, expression systems used for VLP production include bacteria, yeast, insect cells, plant cells, and cells from higher animals (mammals, birds, etc.). Bacterial expression systems are the most widely used for producing recombinant proteins, but they have the disadvantage of not being able to perform post-translational modification (PTM) of proteins similar to those in mammals, making them unsuitable for VLP production. However, due to their low production costs, they are primarily used for non-enveloped VLPs, which can be produced simply by combining capsid proteins. Yeast is also widely used for VLP production, and is also being used to develop non-enveloped VLPs. Hepatitis B virus, human papillomavirus, etc. The development of VLPs, such as viruses, has been carried out using yeast.

[0007] Insect cells undergo a PTM process very similar to that of mammalian cells, except for some glycosylation patterns, and can be used to produce both enveloped and non-enveloped VLPs. In general, they have the advantage of producing higher amounts of protein than higher cells. They have been used to develop VLPs against various viruses, including influenza virus and human papillomavirus.

[0008] Plant cells are an expression system that has just begun to be studied, but there are prospects for the development of VLPs. It has many possibilities from this point of view. It can reliably eliminate various sources of contamination such as viruses, oncogenes, and enterotoxins that may occur during the production of VLP proteins. In addition, when demand for useful substances increases sharply, it has absolute advantages over conventional animal cell systems in terms of the equipment technology and costs required for mass production, enabling mass production at low cost in the shortest possible time. In addition, it has PTMs similar to those of mammalian cells, making it possible to produce both enveloped and non-enveloped VLPs.

[0009] However, despite the advantages described above, the biggest obstacle to protein production from plant cells is the relatively unoptimized isolation and purification methods compared to other hosts, including animal cells and microorganisms. Accordingly, much research has been conducted, and various methods have been attempted to increase protein productivity from plant cells. For example, Patent Document 1 discloses that a recombinant vector containing a cellulose-binding domain 3 and a protein synthesized in a plant transformed with the recombinant vector can be bound to cellulose to isolate a fusion protein containing the target protein, and that the target protein can be efficiently separated from the cellulose-binding domain by treating the fusion protein with enterokinase.

[0010] Furthermore, as another example of a protein separation and purification technique capable of producing a target protein, Patent Document 2 has demonstrated that when a physiologically active protein or peptide is conjugated to an immunoglobulin Fc fragment, the solubility of the physiologically active protein or peptide is improved compared to a physiologically active protein or peptide not conjugated to an immunoglobulin Fc fragment.

[0011] Therefore, the production of useful bioactive substances from plants based on the above-mentioned conventional technology can replace conventional production methods using animal cells or microorganisms. First, it shortens the time required to produce useful bioactive substances and dramatically reduces production costs. Second, it reliably eliminates sources of contamination, such as cytotoxicity, that can occur during the isolation and purification process of proteins synthesized in animal cells or microorganisms. Third, once commercialized, it can be stored for long periods in the form of seeds, reducing storage and preservation costs. Fourth, it can be transported in the form of safe seeds, allowing for rapid supply to regions and countries in need when urgent issues arise. Fifth, in the event of a sudden increase in demand for bioactive substances, it does not require extensive technology to build the production facilities and systems required for mass production, making it easy to mass-produce. Compared to production systems using animal cells, it has the advantage of dramatically reducing installation costs and product production costs, enabling mass production in the shortest time possible and ensuring sufficient supply to meet demand.

[0012] As mentioned above, although various techniques are available for effectively synthesizing, isolating, and purifying useful physiologically active substances, including medically applicable VLP proteins and industrially valuable enzymes, from plants, each protein has different inherent properties, making it undesirable to apply a specific method to all of them at once, and individual research is therefore required. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] Korean Patent No. 10-1848082 [Patent Document 2] Korean Patent Publication No. 10-2015-0113934 Summary of the Invention [Problem to be solved by the invention]

[0014] The present invention has been made to solve the above-mentioned problems of the prior art. In producing recombinant Alfalfa Mosaic Virus (AMV) that forms virus-like particles (VLPs) of a target protein in a transformed plant, it has been found that fusing a Rubisco transit peptide to the target protein so that it is targeted to chloroplasts in order to increase the expression level of the protein at the translation stage, and attaching polyhistidine for separation and purification, increases the protein expression level and the efficiency of separation and purification. It has been found that this can be used to dramatically increase the production efficiency of a target protein in a transformed plant.

[0015] Furthermore, we have confirmed through various experimental methods that virus-like particles are formed when the composition, pH, and NaCl concentration of a solution containing the isolated and purified AMV are appropriately adjusted. Through the elucidation of the high-resolution tertiary structure of such virus-like particles, we have established a VLP platform technology that can be utilized in the development of vaccines and therapeutic agents, thereby completing the present invention.

[0016] Therefore, an object of the present invention is to provide a polynucleotide encoding a Rubisco transit peptide comprising the amino acid sequence represented by SEQ ID NO: 2, and an AMV (Alfalfa) comprising the amino acid sequence represented by SEQ ID NO: 4. The present invention provides a recombinant vector for expression in plants, which contains a polynucleotide encoding a capsid protein of a mosaic virus.

[0017] Another object of the present invention is to provide a transformed plant transformed with the recombinant vector of the present invention.

[0018] A further object of the present invention is to provide a method for separating and purifying the recombinant AMV capsid protein and the recombinant AMV capsid protein produced thereby.

[0019] It is yet another object of the present invention to provide a method for producing recombinant AMV virus-like particles (VLPs) containing the recombinant AMV capsid protein, and the recombinant AMV virus-like particles produced thereby.

[0020] It is yet another object of the present invention to provide a method for producing a vaccine composition comprising the recombinant AMV virus-like particle of the present invention.

[0021] It is yet another object of the present invention to provide a drug delivery vehicle comprising the recombinant AMV virus-like particle of the present invention.

[0022] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0023] In order to achieve the above-mentioned object of the present invention, there is provided a recombinant vector for expression in plants, which comprises a polynucleotide encoding a Rubisco transit peptide comprising the amino acid sequence represented by SEQ ID NO: 2, and a polynucleotide encoding an AMV (Alfalfa mosaic virus) capsid protein comprising the amino acid sequence represented by SEQ ID NO: 4.

[0024] In one embodiment of the present invention, the polynucleotide encoding the Rubisco transit peptide comprises the base sequence represented by SEQ ID NO: 1, and the polynucleotide encoding the AMV capsid protein may comprise the base sequence represented by SEQ ID NO: 3 or 10, but is not limited thereto.

[0025] In another embodiment of the present invention, the recombinant vector may further comprise a polynucleotide encoding a polyhistidine tag comprising the amino acid sequence represented by SEQ ID NO: 6, but is not limited thereto.

[0026] In yet another embodiment of the present invention, the polyhistidine tag may be, but is not limited to, linked to the C-terminus or N-terminus of the polynucleotide encoding the AMV capsid protein.

[0027] In yet another embodiment of the present invention, the recombinant vector may have (1) a polynucleotide encoding a Rubisco transit peptide, a polynucleotide encoding an AMV capsid protein, and a polynucleotide encoding a polyhistidine tag linked in sequence between the promoter and the terminator; or (2) a polynucleotide encoding a Rubisco transit peptide, a polynucleotide encoding a polyhistidine tag, and a polynucleotide encoding an AMV capsid protein linked in sequence, but the order of ligation is not limited.

[0028] The present invention also provides a transformed plant transformed with the recombinant vector according to the present invention.

[0029] The present invention also provides a method for isolating and purifying a recombinant AMV capsid protein, comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to the present invention; (Step 2) mixing the transformed plant obtained in the step (Step 1) with a protein extraction buffer solution to prepare a plant mixture; (Step 3) recovering the mixture obtained in (Step 2) from which plant debris has been removed; (Step 4) Injecting the mixture obtained in (Step 3) into an agarose-filled column to adsorb the polyhistidine-tagged recombinant AMV capsid protein onto the agarose; (Step 5) injecting a washing solution into the column to wash it; and (Step 6) Injecting an elution solution into the column to elute the recombinant protein adsorbed to the agarose.

[0030] In one embodiment of the present invention, the protein extraction buffer solution may satisfy one or more of the following characteristics, but is not limited to: (1) 10 to 100 mM Tris, 100 to 1500 mM magnesium chloride (MgCl), 0.01 to 1% Triton (registered trademark) X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenylenediamine), her), and 5 to 300 mM imidazole; or (2) pH is 7 to 9.

[0031] In another embodiment of the present invention, the agarose may be, but is not limited to, Ni-IDA (Iminodiacetic acid) agarose.

[0032] In yet another embodiment of the present invention, the step (Step 1) may be, but is not limited to, transforming a plant using bacteria into which the recombinant vector has been introduced.

[0033] In yet another embodiment of the present invention, the bacterium may be, but is not limited to, Agrobacterium tumefaciens.

[0034] In yet another embodiment of the present invention, the plant may be, but is not limited to, a dicotyledonous plant selected from the group consisting of Arabidopsis thaliana, soybean, tobacco, eggplant, chili pepper, potato, tomato, Chinese cabbage, cabbage, and lettuce; or a monocotyledonous plant selected from the group consisting of rice, barley, wheat, rye, corn, sugarcane, oats, and onion.

[0035] The present invention also provides a recombinant AMV capsid protein eluted by the separation and purification method of the present invention.

[0036] In one embodiment of the present invention, the recombinant AMV capsid protein may satisfy one or more of the following characteristics, but is not limited to: (1) it exists in a trimer structure; or (2) the trimer exhibits a molecular weight of approximately 100 kDa upon size-exclusion chromatography.

[0037] The present invention also provides a method for producing a recombinant AMV virus-like particle (VLP), comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to the present invention; (Step 2) mixing the transformed plant obtained in the step (Step 1) with a protein extraction buffer solution to prepare a plant mixture; (Step 3) recovering the mixture obtained in (Step 2) from which plant debris has been removed; (Step 4) Injecting the mixture obtained in (Step 3) into an agarose-filled column to adsorb the polyhistidine-tagged recombinant AMV capsid protein onto the agarose; (Step 5) Injecting a washing solution into the column to wash it; (Step 6) injecting an elution solution into the column to elute the recombinant protein adsorbed to the agarose; and (Step 7) The protein eluted in (Step 6) is converted into virus-like particles.

[0038] In one embodiment of the present invention, the virus-like particles can be produced in Step 7 by replacing the protein extraction buffer solution containing the recombinant AMV capsid protein with a buffer solution for assembling virus-like particles, but is not limited thereto.

[0039] In another embodiment of the present invention, the buffer solution for virus-like particle assembly may satisfy one or more of the following characteristics, but is not limited to: (1) containing 20 to 100 mM sodium pyrophosphate and 100 to 1000 mM sodium chloride (NaCl); or (2) having a pH of 6.9 to 7.5.

[0040] In yet another embodiment of the present invention, the production method may further comprise, but is not limited to, a step of performing size exclusion chromatography to purify the self-assembled recombinant AMV virus-like particles.

[0041] The present invention also provides recombinant AMV virus-like particles produced by the method for producing virus-like particles according to the present invention.

[0042] In one embodiment of the present invention, the recombinant AMV virus-like particle may satisfy one or more of the following characteristics, but is not limited to these: (1) it appears to have a molecular weight of approximately 2,000 kDa or more by size exclusion chromatography; (2) it has a diameter of 10 nm or more and 40 nm or less when observed under a transmission electron microscope after staining with a negative staining method; or (3) when observed under a cryogenic transmission electron microscope and the 3-dimensional structure is reconstructed, it is a spherical VLP (3x20) in which a total of 20 trimeric complexes are assembled through a regular arrangement.

[0043] The present invention also provides a method for producing a vaccine composition comprising a recombinant AMV virus-like particle, the method comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to the present invention; (Step 2) isolating and purifying the recombinant AMV capsid protein from the transformed plant obtained in (Step 1); (Step 3) Producing virus-like particles from the recombinant AMV capsid protein obtained in (Step 2); (Step 4) clarifying the 3D structure of the virus-like particles obtained in (Step 3) using a cryo-electron microscope; and (Step 5) Producing a vaccine composition containing the virus-like particles.

[0044] In one embodiment of the present invention, the method for preparing the vaccine composition may further comprise, but is not limited to, the step of adding an adjuvant.

[0045] In another embodiment of the present invention, the method for producing the vaccine composition may satisfy one of the following characteristics, but is not limited to: (1) the recombinant vector in (Step 1) further comprises a polynucleotide encoding an antigen protein, and the polynucleotide encoding the antigen protein is linked to the N-terminus of the polynucleotide encoding the AMV capsid protein; or (2) (Step 5) comprises a step of conjugating the antigen protein to the surface of the virus-like particle.

[0046] The present invention also provides a vaccine composition produced by the method for producing a vaccine composition.

[0047] That is, the present invention provides a vaccine composition comprising the AMV virus-like particle and an antigen as active ingredients.

[0048] The present invention also provides use of the AMV virus-like particles for vaccine production.

[0049] The present invention also provides uses of the AMV virus-like particles for antigen presentation and delivery.

[0050] The present invention also provides a preventive or therapeutic use for a disease, which comprises administering a vaccine composition containing the AMV virus-like particle and an antigen as active ingredients to an individual in need thereof.

[0051] The present invention also provides a method for producing a drug delivery vehicle comprising a recombinant AMV virus-like particle, the method comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to the present invention; (Step 2) isolating and purifying the recombinant AMV capsid protein from the transformed plant obtained in (Step 1); (Step 3) replacing the recombinant AMV capsid protein solution obtained in (Step 2) with a first buffer solution to prepare a recombinant AMV capsid protein trimer; (Step 4) adding a drug to the solution of (Step 3); and (Step 5) The first buffer solution of (Step 4) is replaced with a second buffer solution to produce virus-like particles.

[0052] In one embodiment of the present invention, the first buffer solution and the second buffer solution in Step 5 may satisfy the following characteristics, but are not limited to: (1) the first buffer solution contains 10-100 mM Tris and 100-500 mM sodium chloride, and has a pH of 6.9-7.5; and (2) the second buffer solution contains 20-100 mM sodium pyrophosphate and 100-1000 mM sodium chloride, and has a pH of 6.9-7.5.

[0053] The present invention also provides a drug delivery system produced by the above-described method for producing a drug delivery system.

[0054] The present invention also provides uses of the recombinant AMV virus-like particles of the present invention for drug delivery.

[0055] The present invention also provides a use of the recombinant AMV virus-like particle for the production of a drug delivery vehicle.

[0056] The present invention also provides a method for drug delivery, comprising administering the recombinant AMV virus-like particles loaded with a drug to an individual in need thereof. [Effects of the Invention]

[0057] The present invention relates to a method for highly efficient production of recombinant AMV from plants using a plant expression vector targeted to chloroplasts, and a method for producing recombinant AMV VLPs using the same. Due to the high-resolution clarification of its tertiary structure, the recombinant AMV VLPs of the present invention can be used as a platform for the development of vaccines, therapeutic agents, drug delivery vehicles, and the like. Furthermore, the use of a plant expression system in the present invention significantly reduces production costs and reliably blocks various sources of contamination (viruses, oncogenes, enterotoxins, etc.) that may occur in conventionally well-known methods (methods in which proteins are produced from animal cells or microorganisms and then separated and purified). Furthermore, the present invention relates to a method for producing recombinant AMV VLPs from eukaryotic cells, which maintains physiological activity by incorporating the eukaryotic protein synthesis pathway in which post-translational modification processes, which are essential in animal cells, occur. It is advantageous in that it can produce proteins that are already in use, and unlike animal cells or bacteria, it can be managed as a seed stock even during the commercialization stage. Furthermore, when demand for the substance suddenly increases, it is more efficient and economical in terms of the equipment technology and costs required for mass production compared to conventional production systems using animal cells or bacteria, so it has the advantage of being able to mass-produce and supply in a short period of time to meet the demand. [Brief explanation of the drawings]

[0058] [Figure 1]1 is a diagram showing six types of expression cassettes for recombinant AMV capsid proteins for expressing recombinant AMV capsid proteins in plants according to one embodiment of the present invention. The N-terminus (top row) of Fig. 1 is a plant expression vector cassette designed to enable expression of AMV capsid proteins in the cytosol, endoplasmic reticulum, or chloroplast in a form in which a 6xHis (polyhistidine tag) is attached to the N-terminus of the polynucleotide encoding the AMV capsid protein. The C-terminus (bottom) of Figure 1 shows a cassette of a plant expression vector designed to enable expression of the AMV capsid protein in the cytoplasm, endoplasmic reticulum, or chloroplast in a form in which 6xHis is attached to the C-terminus of the polynucleotide encoding the AMV capsid protein (H, polyhistidine tag; HSP-T, Hsp terminator; NR, Rubisco transit peptide; NB, New BiP signal peptide; HDEL, ER retention signal peptide). [Figure 2] FIG. 2 shows the results of Western blotting to confirm the expression of recombinant AMV capsid proteins by six different plant expression vectors in a plant according to one embodiment of the present invention. [Figure 3] FIG. 3 shows the results of separation and purification of recombinant AMV capsid protein using affinity chromatography according to one embodiment of the present invention, as determined by SDS-PAGE and Western blotting. [Figure 4A] Figure 4A shows the results of size exclusion chromatography performed to confirm the conditions for converting the recombinant AMV capsid protein separated and purified in the present invention from the trimeric form to the VLP form, and Figure 4B shows the results of size exclusion chromatography performed in Example 4 using buffer solution 1. [Figure 4B]Figure 4B shows the results of size exclusion chromatography performed to confirm the conditions for converting the recombinant AMV capsid protein separated and purified in the present invention from the trimeric form to the VLP form, and Figure 4C shows the results of size exclusion chromatography equilibrated with buffer solution 2 in Example 4. [Figure 5] FIG. 5 shows images of recombinant AMV VLPs isolated and purified in the present invention, taken with a transmission electron microscope after treatment under various pH conditions. [Figure 6A] 6A shows images of recombinant AMV VLPs isolated and purified in the present invention taken using cryo-electron microscopy, with Figure 6A showing the 2D (two-dimensional) class average of AMV VLPs. [Figure 6B] Figure 6A shows an image of the recombinant AMV VLP isolated and purified in the present invention, taken using cryo-electron microscopy. Figure 6B shows the icosahedral 3D structure of the AMV VLP, constructed based on a 2D class average. [Figure 6C] Figure 6A shows an image of the recombinant AMV VLP isolated and purified in the present invention, taken using a cryo-electron microscope (Cryo-electron microscopy). Figure 6B shows the results of measuring the resolution based on the tertiary structure of the AMV VLP. [Figure 6D] Figure 6A shows images of the recombinant AMV VLP isolated and purified in the present invention taken using cryo-electron microscopy, and Figure 6B shows a flowchart illustrating the image processing and model building procedures for the AMV VLP. [Figure 7A]7A shows the overall structure, monomer structure, and amino acid positions of the recombinant AMV VLP analyzed by cryo-electron microscopy and structural analysis software in the present invention. Figure 7A shows the local resolution of the cryo-EM map. [Figure 7B] In the present invention, the overall structure, monomer structure, and amino acid positions of the recombinant AMV VLP were analyzed using cryo-electron microscopy and structural analysis software. Figure 7B shows the B-factor of the atomic model. [Figure 7C] Figure 7C shows the overall structure, monomer structure, and amino acid positions of the recombinant AMV VLP analyzed using cryo-electron microscopy and structural analysis software. [Figure 7D] Figure 7D shows the overall structure, monomer structure, and amino acid positions of the recombinant AMV VLP analyzed using cryo-electron microscopy and structural analysis software in the present invention. The amino acid sequence in the recombinant AMV capsid monomer and the electron density map of representative amino acid residues analyzed at high resolution are shown. DETAILED DESCRIPTION OF THE INVENTION

[0059] The inventors have confirmed that when producing recombinant AMV capsid protein, which is a target protein that forms virus-like particles, in transformed plants, the expression level of the protein is increased by fusing a Rubisco transit peptide to the target protein so that it is targeted to chloroplasts during the translation stage, and by attaching a polyhistidine tag for separation and purification, the protein expression level and separation and purification efficiency are increased. They confirmed that this can be used to dramatically increase the production efficiency of the target protein in transformed plants, and further confirmed that the AMV capsid protein can successfully form VLPs by adjusting the composition of the buffer solution, thereby completing the present invention.

[0060] Therefore, in one embodiment of the present invention, the pCAMBIA1300 plant expression vector (1) was constructed to enable the cytoplasmic expression of AMV capsid proteins fused to polyhistidine tags at the N-terminus and C-terminus, respectively. The same protein was fused to (1) with Rbc-TP (Rubisco transit peptide) to enable expression in the chloroplast, or to (1) with the NB (New BiP) signal peptide to enable expression in the endoplasmic reticulum. Furthermore, a total of six plant expression vectors were constructed, each designed to express the AMV capsid protein at the N-terminus or C-terminus of the AMV capsid protein-encoding polynucleotide in each organelle (see Example 1).

[0061] In another embodiment of the present invention, the plant expression vector was transformed into Agrobacteria, which was then injected into the abaxial surface of Nicotiana benthamiana leaves to express the recombinant AMV capsid protein in the plant (see Example 2).

[0062] In yet another embodiment of the present invention, the recombinant AMV capsid protein was separated and purified from Nicotiana benthamiana leaves expressing the recombinant AMV capsid protein using a column packed with Ni-IDA (iminodiacetic acid) agarose resin, and the composition of the buffer solution used to produce virus-like particles was adjusted to induce self-assembly of the AMV capsid protein. Specifically, in the first buffer solution, the AMV capsid protein formed a trimer. It was confirmed that in the second buffer solution, the AMV capsid protein assembles into virus-like particles (see Examples 3 and 4).

[0063] In yet another example of the present invention, the composition containing the AMV virus-like particles obtained above was examined using a transmission electron microscope to confirm the stability of VLPs due to changes in pH, and it was confirmed that the VLP form was stably maintained between pH 5 and pH 7 (see Example 5).

[0064] In yet another example of the present invention, the high-resolution tertiary structure of the AMV VLP was confirmed using cryo-electron microscopy (see Examples 6 and 7).

[0065] Thus, the present invention provides a recombinant vector for expression in plants, which comprises a polynucleotide encoding a Rubisco transit peptide having the amino acid sequence represented by SEQ ID NO: 2, a polynucleotide encoding a polyhistidine tag (6xHis-tag) having the amino acid sequence represented by SEQ ID NO: 6, and a polynucleotide encoding an AMV capsid protein having the amino acid sequence represented by SEQ ID NO: 4.

[0066] The term "Rubisco transit peptide" as used herein refers to the N-terminal transit peptide of the small subunit of Rubisco (ribulose-1,5-bisphosphate carboxylase / oxygenase), which is preferably encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, and most preferably encoded by a polynucleotide represented by SEQ ID NO: 1. However, it may also be encoded by a nucleotide sequence having 80% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity to the nucleotide sequence of SEQ ID NO: 1. For example, polynucleotides with 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity are included. The "percent sequence identity" for a polynucleotide is determined by comparing the comparison region with two optimally aligned sequences. A portion of the polynucleotide sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimally aligned sequence of the two sequences. The Rubisco transit peptide is used to transport recombinant proteins expressed in transformed plants into chloroplasts. During expression, a portion of the sequence may be truncated, leaving only a few amino acids. More specifically, when a recombinant AMV capsid protein fused with a Rubisco transit peptide according to the present invention is expressed in a plant, the first 54 amino acids of the Rubisco transit peptide may be truncated and removed, leaving only the amino acid sequence represented by SEQ ID NO: 2. In this case, additional amino acids, preferably glycine or isoleucine, may be inserted between the Rubisco transit peptide sequence and the AMV capsid protein sequence to align the DNA frame.

[0067] The term "NB (New BiP)" as used herein is used to transport an expressed recombinant protein into the endoplasmic reticulum, and preferably refers to a gene comprising the nucleotide sequence of SEQ ID NO: 9, most preferably the gene represented by SEQ ID NO: 9, but may also comprise a nucleotide sequence having 80% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity to the nucleotide sequence of SEQ ID NO: 9. When the NB gene is expressed, the entire sequence is truncated, and no amino acids of BiP remain.

[0068] As used herein, the term "transit peptide" or "signal peptide" refers to an amino acid sequence that can direct the transport or localization of a protein to a specific organelle, cellular compartment, or outside the cell. This term includes all transit peptides and nucleotide sequences that encode transit peptides.

[0069] As used herein, the term "AMV" refers to Alfalfa mosaic virus, an icosahedral, non-enveloped DNA virus. AMV is a virus that causes mosaic-shaped lesions in plants, but is known not to cause any infection or lesions in humans or animals. In the present invention, "AMV" is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 or 10, more preferably by a polynucleotide represented by SEQ ID NO: 3 or 10, and most preferably by a polynucleotide represented by SEQ ID NO: 3. However, it may also be encoded by a nucleotide sequence having 80% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity to the nucleotide sequence of SEQ ID NO: 3 or 10.

[0070] As used herein, the term "polynucleotide" refers to an oligomer or polymer containing two or more linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), generally linked together by phosphodiester bonds. Polynucleotides also include, for example, nucleotide analogs, or DNA and RNA derivatives containing "backbone" bonds other than phosphodiester bonds, such as phosphotriester, phosphoramidate, phosphorothioate, thioester, or peptide bonds (peptide nucleic acids). Polynucleotides include single-stranded and / or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs of either RNA or DNA.

[0071] As used herein, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of effecting the expression of the DNA in a suitable host. A vector may be a plasmid, a phage particle, or simply a potential genome insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases may be integrated into the genome itself. Because the plasmid is currently the most commonly used form of vector, the terms "plasmid" and "vector" may sometimes be used interchangeably. However, the present invention includes other forms of vectors that are known in the art or have equivalent functions to those known.

[0072] The Rubisco transit peptide of the present invention may comprise the amino acid sequence represented by SEQ ID NO: 2, and the AMV capsid protein may comprise the amino acid sequence represented by SEQ ID NO: 4. Furthermore, variants of SEQ ID NO: 2 are included within the scope of the present invention as the amino acid sequence of the Rubisco transit peptide of the present invention. Specifically, the peptide may comprise an amino acid sequence that has 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity with the amino acid sequence of SEQ ID NO: 2. Furthermore, variants of SEQ ID NO: 4 are included within the scope of the present invention as the amino acid sequence of the AMV capsid protein of the present invention. Specifically, the protein may comprise an amino acid sequence that has 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity with the amino acid sequence of SEQ ID NO: 4.

[0073] Furthermore, the recombinant vector of the present invention contains a polynucleotide encoding a polyhistidine tag containing the amino acid sequence represented by SEQ ID NO:6. The polyhistidine tag may further be encoded by a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO:5, and is most preferably encoded by the polynucleotide shown in SEQ ID NO:5.

[0074] The polyhistidine tag is included in addition to the target protein AMV of the present invention for easier isolation, and representative tagging genes that can replace this include Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, Myc tag, S tag, SBP tag, IgG Fc tag, CTB tag, Softag 1 tag, Softag 3 tag, Strep tag, TC tag, V5 tag, VSV tag, and Xpress tag.

[0075] Furthermore, the polyhistidine tag may be linked to the N-terminus or C-terminus of the polynucleotide encoding the AMV capsid protein, and preferably to the C-terminus.

[0076] Furthermore, the recombinant vector of the present invention may be configured such that (1) a polynucleotide encoding a Rubisco transit peptide, a polynucleotide encoding an AMV capsid protein, and a polynucleotide encoding a polyhistidine tag are sequentially linked between a promoter and a terminator; or (2) a polynucleotide encoding a Rubisco transit peptide, a polynucleotide encoding a polyhistidine tag, and a polynucleotide encoding an AMV capsid protein are sequentially linked, but the order of ligation is not limited. In the case of (1), the entire insert sequence inserted into the recombinant vector is encoded by a polynucleotide containing the nucleotide sequence of SEQ ID NO: 11, most preferably by the polynucleotide represented by SEQ ID NO: 11, but may also be encoded by a nucleotide sequence having 80% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity to the nucleotide sequence of SEQ ID NO: 11.

[0077] The promoters include the pEMU promoter, the MAS promoter, the histone promoter, the Clp promoter, the cauliflower mosaic virus (cauliflower Examples of suitable terminators include the 35S promoter derived from the cauliflower mosaic virus, the 19S RNA promoter derived from the cauliflower mosaic virus, a plant actin protein promoter, a ubiquitin protein promoter, a Cytomegalovirus (CMV) promoter, a Simian virus 40 (SV40) promoter, a Respiratory syncytial virus (RSV) promoter, and an Elongation factor-1 alpha (EF-1α) promoter, with the CaMV 35S promoter being the most preferred. Examples of suitable terminators include nopaline synthase (NOS), the rice amylase RAmy1 A terminator, the patholin terminator, the octopine gene terminator of Agrobacterium tumefaciens, and the rrnB1 / B2 terminator of Escherichia coli, with the HSP terminator being the most preferred. However, these examples of promoters and terminators are merely illustrative and not limiting.

[0078] In another aspect of the present invention, there is provided a transformed plant transformed with the recombinant vector according to the present invention.

[0079] As used herein, the term "transformation" refers to the general term for changing the genetic properties of an organism by injecting DNA, and a "transgenic plant" refers to a plant that has been transformed by injecting an exogenous gene into it using molecular genetic methods. The plant body is a plant body produced by the method of the present invention, and preferably a plant body transformed with the recombinant expression vector of the present invention. There are no limitations on the plant body as long as it is capable of achieving the object of the present invention.

[0080] In addition, the transformed plant according to the present invention can be produced by methods such as transformation, transfection, Agrobacterium-mediated transformation, particle gun bombardment, sonication, electroporation, or PEG (Polyethylene glycol)-mediated transformation, but there are no limitations as long as the method allows for injection of the vector of the present invention.

[0081] In another aspect, the present invention provides a method for isolating and purifying a recombinant AMV capsid protein, comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to the present invention; (Step 2) mixing the transformed plant obtained in the step (Step 1) with a protein extraction buffer solution to prepare a plant mixture; (Step 3) recovering the mixture obtained in (Step 2) from which plant debris has been removed; (Step 4) Injecting the mixture obtained in (Step 3) into an agarose-filled column to adsorb the polyhistidine-tagged recombinant AMV capsid protein onto the agarose; (Step 5) injecting a washing solution into the column to wash it; and (Step 6) Injecting an elution solution into the column to elute the recombinant protein adsorbed to the agarose.

[0082] The present invention also provides a recombinant AMV capsid protein eluted by the above separation and purification method.

[0083] The protein extraction buffer solution according to the present invention may contain 10-100 mM Tris, 100-1500 mM magnesium chloride (MgCl), 0.01-1% Triton® X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenylether), and 5-300 mM imidazole. More preferably, the protein extraction buffer solution may contain 10-50 mM Tris, 500-1200 mM MgCl, 0.01-0.7% Triton® X-100, and 5-100 mM imidazole. Most preferably, the protein extraction buffer solution may contain 20 mM Tris, 1 M MgCl, 0.5% Triton® X-100, and 10 mM imidazole. In the present invention, Tris is HCl, and may be titrated to a pH of 8.0 (Tris-HCl).

[0084] The protein extraction buffer solution according to the present invention may have a pH of 7 to 9, preferably 7.5 to 8.5. Most preferably, the protein extraction buffer solution has a pH of 8.

[0085] The agarose according to the present invention may be Ni-IDA (iminodiacetic acid) agarose.

[0086] In addition, in the present invention, the step (Step 1) may involve transforming a plant body using a bacterium into which a recombinant vector has been introduced, and the bacterium may preferably be Agrobacterium tumefaciens.

[0087] In the present invention, the plant may be a dicotyledonous plant selected from the group consisting of Arabidopsis thaliana, soybean, tobacco, eggplant, chili pepper, potato, tomato, Chinese cabbage, cabbage, and lettuce; or a monocotyledonous plant selected from the group consisting of rice, barley, wheat, rye, corn, sugarcane, oats, and onion.

[0088] The term "plant" as used herein refers to any plant capable of mass-producing the recombinant protein of the present invention, without limitation. More specifically, it may be a plant selected from the group of plants listed above, preferably tobacco. The tobacco in the present invention is a plant of the Nicotiana genus, and is not particularly limited to a particular species as long as it is capable of overexpressing a protein. The present invention can be carried out by selecting an appropriate variety suited to the transformation method and the purpose of mass-producing the protein. For example, varieties such as Nicotiana benthamiana L. or Nicotiana tabacum cv. Xanthi can be used.

[0089] The term "capsid" as used herein refers to a protein that constitutes the viral coat. Naturally, capsids self-assemble to form viral coats, and the viral genome is packaged within the coat. Virus-like particles consist of only capsids without the viral genome. The recombinant AMV capsid protein of the present invention may exist individually (monomers), in a multimeric form (e.g., trimers), or in a self-assembled form in virus-like particles. Furthermore, the trimer of the recombinant AMV capsid protein of the present invention may be found to have a molecular weight of approximately 100 kDa by size-exclusion chromatography. Throughout this specification, the term "approximately" refers to ±10%. Therefore, a molecular weight of approximately 100 kDa means 90 kDa to 110 kDa.

[0090] The present invention also provides a method for producing a recombinant AMV virus-like particle (VLP), comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to the present invention; (Step 2) mixing the transformed plant obtained in the step (Step 1) with a protein extraction buffer solution to prepare a plant mixture; (Step 3) recovering the mixture obtained in (Step 2) from which plant debris has been removed; (Step 4) Injecting the mixture obtained in (Step 3) into an agarose-filled column to adsorb the polyhistidine-tagged recombinant AMV capsid protein onto the agarose; (Step 5) Injecting a washing solution into the column to wash it; (Step 6) injecting an elution solution into the column to elute the recombinant protein adsorbed to the agarose; and (Step 7) The protein eluted in (Step 6) is converted into virus-like particles.

[0091] The present invention also provides recombinant AMV virus-like particles produced by the above-mentioned production method. provide.

[0092] The recombinant AMV virus-like particles may also appear by size exclusion chromatography with a molecular weight of greater than about 2,000 kDa.

[0093] Furthermore, the recombinant AMV virus-like particle may be spherical or circular with a diameter of 10 nm to 40 nm, preferably 20 nm to 30 nm, when observed under a transmission electron microscope after staining with a negative staining method.

[0094] Furthermore, when the recombinant AMV virus-like particle is observed under an ultra-low temperature transmission electron microscope and the 3-dimensional structure is reconstructed, it may be a spherical VLP (3x20) in which a total of 20 trimeric complexes are assembled in a regular arrangement.

[0095] In the present invention, in Step 7, virus-like particles can also be produced by replacing the protein extraction buffer solution containing the recombinant AMV capsid protein with a buffer solution for virus-like particle assembly, in which the recombinant AMV capsid protein self-assembles into virus-like particles in the buffer solution for virus-like particle assembly.

[0096] Specifically, the method for producing virus-like particles may include replacing a protein extraction buffer solution with a virus-like particle assembly buffer solution using a filter and concentrating the solution so that the recombinant AMV capsid protein can self-assemble into virus-like particles.

[0097] In the present invention, the buffer solution for virus-like particle assembly may contain 20 to 100 mM sodium pyrophosphate and 100 to 1000 mM sodium chloride (NaCl). Preferably, the buffer solution may contain 30 to 70 mM sodium pyrophosphate and 300 to 700 mM NaCl. Most preferably, the buffer solution may contain 50 mM sodium pyrophosphate and 500 mM NaCl. In this specification, the term "buffer solution for virus-like particle assembly" is used interchangeably with "second buffer solution."

[0098] The pH of the buffer solution may be 6.9 to 7.5, preferably 6.9 to 7.2, and most preferably 7.0.

[0099] The method for producing virus-like particles may further comprise the step of performing size exclusion chromatography to purify the self-assembled recombinant AMV virus-like particles.

[0100] The present invention also provides a method for producing a vaccine composition comprising a recombinant AMV virus-like particle, the method comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to the present invention; (Step 2) isolating and purifying the recombinant AMV capsid protein from the transformed plant obtained in (Step 1); (Step 3) Producing the recombinant AMV capsid protein obtained in the (Step 2) step into virus-like particles; and (Step 4) clarifying the 3D structure of the virus-like particles obtained in (Step 3) using a cryo-electron microscope; and (Step 5) Producing a vaccine composition containing the virus-like particles.

[0101] That is, the present invention provides a vaccine composition comprising the recombinant AMV virus-like particle of the present invention and an antigen protein as active ingredients.

[0102] In the present invention, the term "vaccine" refers to a biological preparation containing an antigen that induces an immune response in the body, and is an immunogen that induces immunity in the body by being injected or orally administered to humans or animals for the prevention of infectious diseases. The animal may be a human or a non-human animal, and the non-human animal may be, but is not limited to, a pig, cow, horse, dog, goat, sheep, etc.

[0103] The "vaccine composition" or "drug delivery vehicle" of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and sterile injectable solutions, respectively, by conventional methods. When formulating, these can be prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc., and can be prepared by mixing the lecithin-like emulsifier with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration can be suspensions, oral solutions, emulsions, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to water and liquid paraffin, which are frequently used simple diluents. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, and lyophilized preparations. Examples of non-aqueous preparations and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0104] Routes of administration of the vaccine composition or drug delivery vehicle of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The vaccine composition of the present invention can also be administered in the form of a suppository for rectal administration.

[0105] The dosage of the vaccine composition, pharmaceutical composition, or drug delivery system according to the present invention is selected taking into consideration the age, weight, sex, physical condition, etc. of the individual. The amount required to induce an immune protective response in an individual without particular side effects can vary depending on the recombinant protein used as the immunogen and the optional presence of excipients.

[0106] In the present invention, the method for preparing the vaccine composition may further include the step of adding an adjuvant.

[0107] In the present invention, "adjuvant" refers to a substance or composition that is added to a vaccine or a pharmaceutically active ingredient to increase or influence the immune response. Typically, it generally refers to a carrier or auxiliary substance for an immunogen and / or other pharmaceutically active substance or composition. Typically, the term "adjuvant" should be interpreted in a broad sense, and refers to a wide range of substances or strategies that can enhance the immunogenicity of an antigen that is incorporated into or administered with the adjuvant. In addition, adjuvants include, but are not limited to, immune enhancers. e potentiator), antigen delivery system, or a combination of these.

[0108] Furthermore, the method for producing the vaccine composition can satisfy one of the following characteristics: (1) the recombinant vector in Step 1 further comprises a polynucleotide encoding an antigen protein, and the polynucleotide encoding the antigen protein is linked to the N-terminus of the polynucleotide encoding the AMV capsid protein; or (2) Step 5 comprises a step of conjugating the antigen protein to the surface of the virus-like particle.

[0109] In the present invention, the term "antigen protein" refers to a protein that induces antibody production when the vaccine composition according to the present invention is administered to the body. The antigen protein may be a pathogenic protein or a recombinant protein, but it may be any protein that can induce antibody production. The antigen protein is displayed on the surface of the vaccine composition according to the present invention. When a polynucleotide encoding the antigen protein is linked to the N-terminus of a polynucleotide encoding an AMV capsid protein, the antigen protein is co-expressed with the recombinant AMV capsid protein from the recombinant vector according to the present invention and assembled together to be displayed on the surface of the capsid protein virus-like particle during assembly of the virus-like particle. Alternatively, the antigen protein can be presented in the vaccine composition by conjugating the antigen protein to the surface of the recombinant AMV virus-like particle after preparing the virus-like particle according to the present invention. Any method known in the art for conjugating a foreign protein to the surface of the virus-like particle can be used without limitation.

[0110] The present invention also provides a vaccine composition produced by the method for producing a vaccine composition.

[0111] The vaccine composition may further include an adjuvant. The adjuvant may be alum, but any aluminum salt suitable for use as an adjuvant can be used in the present invention. Aluminum salts include aluminum hydroxide (Al(OH)3), aluminum phosphate (AlPO4), aluminum hydrochloride, aluminum sulfate, ammonium aluminum, potassium aluminum, and aluminum silicate. Preferably, aluminum hydroxide or aluminum phosphate is used as the aluminum salt adjuvant.

[0112] The present invention also provides a method for producing a drug delivery vehicle comprising a recombinant AMV virus-like particle, the method comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to the present invention; (Step 2) isolating and purifying the recombinant AMV capsid protein from the transformed plant obtained in (Step 1); (Step 3) replacing the recombinant AMV capsid protein solution obtained in (Step 2) with a first buffer solution to prepare a recombinant AMV capsid protein trimer; (Step 4) adding a drug to the solution of (Step 3); and (Step 5) The first buffer solution of (Step 4) is replaced with a second buffer solution to produce virus-like particles.

[0113] The drug delivery vehicle of the present invention comprises a recombinant AMV virus-like particle of the present invention containing a drug, and delivers the drug to a target site via the virus-like particle.

[0114] The drug may include, without limitation, all kinds of drugs, such as anti-cancer drugs, and two or more drugs may be simultaneously carried on the virus-like particles according to the present invention.

[0115] In the method for producing a drug delivery vehicle according to the present invention, the first buffer solution and the second buffer solution in Step 5 can satisfy the following characteristics: (1) the first buffer solution contains 10-100 mM Tris and 100-500 mM sodium chloride, and has a pH of 6.9-7.5; and (2) the second buffer solution contains 20-100 mM sodium pyrophosphate and 100-1000 mM sodium chloride, and has a pH of 6.9-7.5.

[0116] Specifically, a plant is transformed with the recombinant vector of the present invention, and the recombinant AMV capsid protein is isolated and purified in a protein extraction buffer solution. The buffer is then replaced with a first buffer solution, which causes the recombinant AMV capsid protein monomer to form a trimer. The first buffer solution may contain 10-100 mM Tris and 100-500 mM sodium chloride (NaCl). Preferably, the buffer solution may contain 10-50 mM Tris and 200-400 mM NaCl. Most preferably, the buffer solution may contain 25 mM Tris and 300 mM NaCl. In the present invention, Tris may be titrated with HCl (Tris-HCl).

[0117] Next, after adding a drug to the recombinant AMV capsid protein trimer, the first buffer solution is replaced with a second buffer solution to produce drug-loaded recombinant AMV virus-like particles. The drug may be contained inside the virus-like particle or bound to the surface of the virus-like particle, but the location is not limited. The second buffer solution may contain 20-100 mM sodium pyrophosphate and 100-1000 mM sodium chloride (NaCl). Preferably, the buffer solution may contain 30-70 mM sodium pyrophosphate and 300-700 mM NaCl. Most preferably, the buffer solution may contain 50 mM sodium pyrophosphate and 500 mM NaCl.

[0118] The first buffer solution and the second buffer solution may have a pH of 6.9 to 7.5, preferably 6.9 to 7.2, and most preferably pH 7.

[0119] The terms and words used in this specification and claims should not be interpreted as being limited to their ordinary or dictionary meanings, but should be interpreted as having meanings and concepts that correspond to the technical idea of ​​the present invention, based on the principle that the inventor can appropriately define the concepts of terms in order to explain his / her invention in the best possible way.

[0120] Preferred examples are provided below to aid in understanding the present invention. However, the following examples are provided merely to facilitate understanding of the present invention, and the content of the present invention is not limited to the following examples. [Example]

[0121] Example 1: Construction of a plant expression vector for expressing recombinant AMV capsid protein As shown in the cleavage map in Figure 1, a recombinant plant expression vector was constructed to enable expression of the recombinant AMV capsid protein in plants.

[0122] More specifically, the gene information for the AMV capsid protein was obtained, and a gene (SEQ ID NO: 4) was synthesized with a sequence optimized for expression in Nicotiana benthamiana. To confirm the expression pattern of AMV capsid proteins depending on the location and target organelles, six expression vectors were constructed as follows.

[0123] A cytoplasmically targeted recombinant AMV capsid protein plant expression vector was constructed by placing a polynucleotide (SEQ ID NO: 3) encoding the AMV capsid protein between the CaMV35S promoter gene and Hsp terminator of the pCAMBIA1300 vector, and sequentially ligating a polynucleotide (SEQ ID NO: 5) encoding six consecutive histidines before (N-terminus) or after (C-terminus) the vector.

[0124] A chloroplast-targeted recombinant AMV capsid protein plant expression vector was constructed by sequentially ligating a polynucleotide encoding the Rubisco transit peptide (SEQ ID NO: 1) and a polynucleotide encoding the AMV capsid protein (SEQ ID NO: 3) between the CaMV35S promoter gene and Hsp terminator of the pCAMBIA1300 vector. To separate the N-terminal and C-terminal polyhistidine tags, a polynucleotide encoding six consecutive histidines (SEQ ID NO: 5) was inserted before and after the AMV.

[0125] The endoplasmic reticulum-targeted recombinant AMV capsid protein plant expression vector was constructed by ligating, in order, a polynucleotide encoding the NB (New BiP) signal peptide (SEQ ID NO: 9), a polynucleotide encoding the AMV capsid protein (SEQ ID NO: 3), and a polynucleotide encoding the HDEL (His-Asp-Glu-Leu) peptide (SEQ ID NO: 7) between the CaMV35S promoter gene and Hsp terminator of the pCAMBIA1300 vector. To separate the N-terminal and C-terminal polyhistidine tags, a polynucleotide encoding six consecutive histidines (SEQ ID NO: 5) was inserted before and after the AMV.

[0126] Example 2: Confirmation of expression of recombinant AMV capsid protein 2.1. Transient expression of plant expression vectors The plant expression vector prepared in Example 1 was transformed into Agrobacterium strain LBA4404 by electroporation. The transformed Agrobacterium was cultured in 5 mL of YEP liquid medium (10 g yeast extract, 10 g peptone, 5 g NaCl, 50 mg / L kanamycin, and 25 mg / L rifampicin) at 28°C for 16 hours with shaking. Then, 1 mL of the primary culture was inoculated into 50 mL of fresh YEP medium and cultured at 28°C for 6 hours with shaking. The cultured Agrobacterium was harvested by centrifugation (7,000 rpm, 4°C, 5 minutes) and suspended in infiltration buffer (10 mM MES (pH 5.7), 10 mM MgCl2, 200 μM acetosyringone) to an optical density (OD) of 1.0 at 600 nm. Agroinfiltration was carried out by injecting the Agrobacterium suspension into the abaxial surface of Nicotiana benthamiana leaves using a syringe with the needle removed.

[0127] 2.2. Confirmation of expression of recombinant AMV capsid protein in transformed plants Proteins were extracted from the leaves of the plants prepared in Example 2.1 and centrifuged. The proteins in the water-soluble fraction (Supernatant; S), the proteins in the pellet fraction (Pellet; P), and the fraction containing both the water-soluble fraction and the pellet (Total; T) were separated and the expression of the recombinant AMV capsid protein was confirmed by Western blotting. More specifically, 30 μL of each fraction was mixed with SDS sample buffer and then diluted with 1000 μL of PBS. Next, the mixture was electrophoresed on a 10% SDS-PAGE gel to confirm the protein bands separated by size. These were transferred to a PVDF membrane and then washed with 5% skim milk (skim milk). After blocking with PBS (milk), an antibody reactive with polyhistidine was bound to the ECL solution according to the method provided by the manufacturer, and the expression of the recombinant AMV capsid protein was confirmed.

[0128] As a result, as shown in Figure 2, we confirmed that recombinant AMV capsid protein fused with a Rubisco transit peptide for chloroplast targeting was expressed with high efficiency. Over 90-95% of the expressed recombinant AMV capsid protein was observed in the soluble fraction, with approximately 5-10% of the AMV capsid protein observed in the pellet fraction. In comparison, recombinant AMV capsid proteins targeted to the cytoplasm and endoplasmic reticulum were expressed with lower efficiency than constructs targeted to chloroplasts. Therefore, the following examples were conducted using recombinant AMV capsid proteins (His-tagged at the N- or C-terminus) fused with a Rubisco transit peptide for chloroplast targeting.

[0129] Example 3: Separation and purification of recombinant AMV capsid protein expressed in transformed plants 50 g of each Nicotiana benthamiana leaf expressing the recombinant AMV capsid protein fused with a polyhistidine tag (His-tag) at the N-terminus or C-terminus prepared in Example 2.1 was added to a protein extraction solution [20 mM Tris-HCl, pH 8.0, 1 M MgCl, 10 mM imidazole, 0.5% Triton®]. The tissue was disrupted using a blender, and then centrifuged at 13,000 rpm at 4°C for 30 minutes to collect the protein extracts. To separate and purify the recombinant AMV capsid protein from the protein extracts obtained under the two conditions (polyhistidine tag fused to the N-terminus or C-terminus), affinity chromatography was simultaneously performed using a column packed with Ni-IDA (iminodiacetic acid) agarose resin. After loading 10 mL of resin into the column, it was equilibrated with 100 mL of wash solution (20 mM Tris-HCl, pH 8.0, 1 M MgCl2, 10 mM imidazole). Each recovered protein extract was applied to two different columns, equilibrated, and the resin was washed with 100 mL of wash solution. The recombinant AMV capsid protein was then eluted with elution solution (20 mM Tris-HCl, pH 8.0, 1 M MgCl2, 50 mM, 100 mM, or 300 mM imidazole). The elution solution containing each recombinant AMV capsid protein was confirmed by electrophoresis (SDS-PAGE), Coomassie staining, and Western blot analysis.

[0130] As a result, as shown in Figure 3, it was confirmed that the recombinant AMV capsid protein fused with a His-tag at the C-terminus had a higher degree of purification and final recovery rate than the recombinant AMV capsid protein fused with a His-tag at the N-terminus. Therefore, the following examples were carried out using the recombinant AMV capsid protein fused with a His-tag at the C-terminus.

[0131] Example 4: Confirmation of VLP formation conditions for recombinant AMV capsid protein Recombinant AMV capsid protein forms recombinant AMV virus-like particles through self-assembly, so size exclusion chromatography was performed to test and confirm conditions for the formation of recombinant AMV virus-like particles.

[0132] Five mg of the eluate containing the isolated and purified recombinant AMV capsid protein recovered in Example 3 was dialyzed against a first buffer solution (25 mM Tris-HCl, pH 7.0, 300 mM NaCl) and then loaded onto a size exclusion chromatography column equilibrated with the first buffer solution, yielding one peak fraction. The molecular weight was calculated using markers, and a trimer structure of approximately 100 kDa was confirmed (Fig. 4A).

[0133] In addition, 5 mg of the eluate containing the recombinant AMV capsid protein recovered in Example 3 was dialyzed against a second buffer solution (50 mM sodium pyrophosphate, pH 7.0, 500 mM NaCl) and then loaded onto a size-exclusion chromatography column equilibrated with the second buffer solution to confirm a change in the size of the AMV capsid protein (Figure 4B). Compared to the first buffer solution, the second buffer solution showed a decrease in the existing peak (P2), while a new peak (P1) was formed. SDS-PAGE confirmed that both P1 and P2 were recombinant AMV capsid proteins.

[0134] Example 5: Verification of recombinant AMV virus-like particles Recombinant AMV capsid protein from the P1 fraction separated by size exclusion chromatography was diluted to a final concentration of 100 nM in buffer solution 2 (50 mM sodium pyrophosphate, pH 7.0, 500 mM NaCl) for negative staining. 5 μL of the protein solution was incubated with a carbon-coated copper grid for 1 minute, after which the protein solution was removed using filter paper and washed three times with triple-distilled water. The carbon-coated copper grid was then incubated with plasma for 1 minute to convert its hydrophobic properties to hydrophilic ones, allowing the protein to adhere well to the carbon. Finally, the grid was incubated with 1% uranyl acetate for 1 minute for negative staining. The staining solution was then removed using filter paper and the grid was dried at room temperature for 12 hours. The prepared grids were imaged at 20,000x magnification using a transmission electron microscope (Tecnai T10, Philips), and virus-like particles composed of recombinant AMV capsid proteins were observed, as shown in Figure 5 (pH 7.0).

[0135] In addition, to confirm VLP stability under changing pH conditions, we tested the buffer solution at various pH levels (3.0, 5.0, 9.0). We found that VLP stability decreased at pH 3.0 and pH 9.0, whereas VLP stability was maintained at pH 5.0 and pH 7.0. The results are shown in Figure 5.

[0136] Example 6: Structure of recombinant AMV capsid protein 6.1. Sample Vitrification and Data Collection Cryo-electron microscopy was used to analyze the VLP structure of the recombinant AMV capsid protein confirmed in Example 5. 3 μl of recombinant AMV capsid protein VLP was applied to a negatively glow-discharged porous carbon grid (Quantifoil R1.2 / 1.3 Cu 200 mesh, SPI) at 15 mA for 1 minute, blotted for 6.5 seconds using a Vitrobot Mark IV device at 4°C and 100% humidity, and then rapidly fixed by immersion in liquid ethane cooled with liquid nitrogen. Cryo-EM images of AMV VLPs were obtained by 300 kV Titan Krios (Thermo Fisher Scientific, USA) transmission electron microscopy using a Falcon 3EC direct electron detector (Thermo Fisher Scientific, USA) and collected using Automatic Data Acquisition Software (EPU; Thermo Fisher Scientific, USA) (see Figure 6A).

[0137] 6.2. Image Processing and Model Building for AMV VLP Tertiary Structure Analysis CryoSPARC v2 software was used to analyze the recombinant AMV capsid protein VLP images collected in Example 6.1. Beam-induced motion correction and dose weighting were corrected using the motion correction included in the software, and then the contrast transfer function (CTF) parameters were measured using the patch CTF. Images with motion drift or very low or very low defocus were unusable for image processing, so they were visually inspected and removed. 482,224 AMV VLPs were extracted from 1,803 images and analyzed in 2D classes. An average image was created. Using this image as a template, images with low correlation were further removed, and 279,755 AMV VLPs were finally selected and used to create a new 2D class average (Figure 6A, bottom panel). An icosahedral 3D structure was reconstructed based on the new 2D image (see Figure 6B). The resolution was measured based on the tertiary structure of the AMV VLP, and the results are shown in Figure 6C. A high-resolution structure of 2.41 Å was obtained. The image processing and model building procedures are shown in Figure 6D as a flow chart. Furthermore, the overall structure, monomer structure, and amino acid positions of the recombinant AMV VLP were analyzed using cryo-electron microscopy and structural analysis software, and the results are shown in Figures 7A to 7D.

[0138] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not limiting. [Industrial Applicability]

[0139] The present invention relates to a method for efficiently producing recombinant AMV in plants using a chloroplast-targeted plant expression vector and a method for producing recombinant AMV VLPs using the same. Due to the high-resolution clarification of the tertiary structure, the recombinant AMV VLPs of the present invention can be used as a platform for the development of vaccines, therapeutic agents, and drug delivery vehicles. Furthermore, the use of a plant expression system in the present invention significantly reduces production costs and reliably blocks various contaminants (e.g., viruses, oncogenes, and enterotoxins) that may occur in conventional methods (protein production in animal cells or microorganisms followed by isolation and purification). Furthermore, the present invention includes a eukaryotic protein synthesis pathway in which post-translational modification processes, which are essential in animal cells, occur, enabling the production of proteins that maintain physiological activity. Unlike animal cells or bacteria, the present invention is advantageous in that it can be managed as a seed stock during commercialization. Furthermore, when demand for the substance suddenly increases, the present invention has the advantage that it is more efficient and economical in terms of the equipment technology and costs required for mass production compared to conventional production systems using animal cells or bacteria, allowing for mass production and supply in a short period of time to meet demand. Therefore, the present invention is expected to be useful in various industrial fields that utilize virus-like particles, including vaccine and drug delivery technologies.

Claims

1. A recombinant vector for expression in plants, comprising a polynucleotide encoding a Rubisco transit peptide comprising the amino acid sequence represented by SEQ ID NO: 2, and a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 3 encoding an AMV (Alfalfa mosaic virus) capsid protein, A recombinant vector for expression in plants, in which a polynucleotide encoding the Rubisco transit peptide and a polynucleotide encoding the AMV capsid protein are operably linked so that the Rubisco transit peptide and the AMV capsid protein are expressed as a fusion protein.

2. The recombinant vector for expression in plants according to claim 1, wherein the polynucleotide encoding the Rubisco transit peptide comprises the base sequence represented by SEQ ID NO:

1.

3. The recombinant vector for expression in plants according to claim 1, further comprising a polynucleotide encoding a polyhistidine tag comprising the amino acid sequence represented by SEQ ID NO:

6.

4. The recombinant vector for expression in plants according to claim 3 , wherein the polyhistidine tag is linked to the C-terminus or N-terminus of the polynucleotide encoding the AMV capsid protein.

5. The recombinant vector has, between the promoter and the terminator: (1) a polynucleotide encoding a Rubisco transit peptide, a polynucleotide encoding an AMV capsid protein, and a polynucleotide encoding a polyhistidine tag are linked in sequence; or (2) A recombinant vector for plant expression according to claim 3, in which a polynucleotide encoding a Rubisco transit peptide, a polynucleotide encoding a polyhistidine tag, and a polynucleotide encoding an AMV capsid protein are linked in sequence.

6. A transformed plant transformed with the recombinant vector according to any one of claims 1 to 5.

7. A method for isolating and purifying a recombinant AMV capsid protein, comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to any one of claims 1 to 5; (Step 2) mixing the transformed plant obtained in (Step 1) with a protein extraction buffer solution to prepare a plant mixture, the protein extraction buffer solution comprises 10-100 mM Tris, 100-1500 mM magnesium chloride (MgCl 2 ), 0.01-1% Triton® X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenylether), and 5-300 mM imidazole, and has a pH of 7-9; (Step 3) Recovering the mixture obtained in (Step 2) from which plant debris has been removed; (Step 4) Injecting the mixture obtained in (Step 3) into a column filled with agarose, and adsorbing the polyhistidine-tagged recombinant AMV capsid protein to the agarose; (Step 5) Injecting a washing solution into the column to wash it; and (Step 6) Injecting an elution solution into the column to elute the recombinant protein adsorbed to the agarose.

8. 8. The separation and purification method according to claim 7, wherein the agarose is Ni-IDA (iminodiacetic acid) agarose.

9. The method of claim 7, wherein the step (Step 1) comprises transforming a plant using a bacterium into which the recombinant vector has been introduced.

10. 10. The method for separation and purification according to claim 9, wherein the bacterium is Agrobacterium tumefaciens.

11. 8. The method for separation and purification according to claim 7, wherein the plant body is a dicotyledonous plant selected from the group consisting of Arabidopsis thaliana, soybean, tobacco, eggplant, chili pepper, potato, tomato, Chinese cabbage, cabbage, and lettuce; or a monocotyledonous plant selected from the group consisting of rice, barley, wheat, rye, corn, sugarcane, oats, and onion.

12. A method for producing recombinant AMV virus-like particles (VLPs), comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to any one of claims 1 to 5; (Step 2) mixing the transformed plant obtained in the (Step 1) step with a protein extraction buffer solution to prepare a plant mixture; (Step 3) Recovering the mixture obtained in (Step 2) from which plant debris has been removed; (Step 4) Injecting the mixture obtained in (Step 3) into a column filled with agarose, and adsorbing the polyhistidine-tagged recombinant AMV capsid protein to the agarose; (Step 5) injecting a washing solution into the column to wash it; (Step 6) injecting an elution solution into the column to elute the recombinant protein adsorbed to the agarose; and (Step 7) A step of preparing virus-like particles by replacing the protein extraction buffer solution containing the recombinant protein eluted in (Step 6) with a buffer solution for assembling virus-like particles, the virus-like particle assembly buffer solution comprises 20-100 mM sodium pyrophosphate and 100-1000 mM sodium chloride (NaCl), and has a pH of 6.9-7.

5.

13. The method of claim 12, further comprising performing size exclusion chromatography to purify the self-assembled recombinant AMV virus-like particles.

14. A recombinant AMV virus-like particle produced by the method of claim 12, which satisfies one or more of the following characteristics: (1) It appears by size exclusion chromatography with a molecular weight of approximately 2,000 kDa or more; (2) When stained with a negative staining method and observed under a transmission electron microscope, the diameter is 10 nm or more and 40 nm or less; or (3) When observed under cryogenic transmission electron microscopy, the 3-dimensional structure was reconstructed to reveal a spherical VLP (3x20) in which a total of 20 trimeric complexes were assembled in a regular arrangement.

15. 1. A method for producing a vaccine composition comprising recombinant AMV virus-like particles, comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to any one of claims 1 to 5; (Step 2) isolating and purifying the recombinant AMV capsid protein from the transformed plant obtained in (Step 1); (Step 3) preparing virus-like particles from the recombinant AMV capsid protein obtained in (Step 2) using a virus-like particle assembly buffer solution containing 20-100 mM sodium pyrophosphate and 100-1000 mM sodium chloride (NaCl) and having a pH of 6.9-7.5; (Step 4) clarifying the 3D structure of the virus-like particles obtained in the (Step 3) step using a cryo-electron microscope; and (Step 5) Producing a vaccine composition containing the virus-like particle.

16. The method of claim 15, further comprising adding an adjuvant.

17. 16. The method of claim 15, wherein the method satisfies one of the following characteristics: (1) The recombinant vector of the step (Step 1) further comprises a polynucleotide encoding an antigen protein, and the polynucleotide encoding the antigen protein is linked to the N-terminus of the polynucleotide encoding the AMV capsid protein; or (2) (Step 5) includes a step of conjugating an antigen protein to the surface of the virus-like particle.

18. A vaccine composition comprising recombinant AMV virus-like particles produced by the production method of claim 15, A vaccine composition, wherein the recombinant AMV virus-like particle satisfies one or more of the following characteristics: (1) It appears by size exclusion chromatography with a molecular weight of approximately 2,000 kDa or more; (2) When stained with a negative staining method and observed under a transmission electron microscope, the diameter is 10 nm or more and 40 nm or less; or (3) When observed under cryogenic transmission electron microscopy, the 3-dimensional structure was reconstructed, revealing a spherical VLP (3x20) in which a total of 20 trimeric complexes were assembled in a regular arrangement.

19. A method for producing a drug delivery vehicle comprising a recombinant AMV virus-like particle, comprising the steps of: (Step 1) transforming a plant body with the recombinant vector according to any one of claims 1 to 5; (Step 2) isolating and purifying the recombinant AMV capsid protein from the transformed plant obtained in (Step 1); (Step 3) replacing the recombinant AMV capsid protein solution obtained in (Step 2) with a first buffer solution containing 10-100 mM Tris and 100-500 mM sodium chloride and having a pH of 6.9-7.5 to prepare a recombinant AMV capsid protein trimer; (Step 4) adding a drug to the solution of (Step 3); and (Step 5) Replacing the first buffer solution of (Step 4) with a second buffer solution containing 20 to 100 mM sodium pyrophosphate and 100 to 1000 mM sodium chloride and having a pH of 6.9 to 7.5 to produce virus-like particles.

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