Patent Application
20220133879
The present invention relates to a porcine epidemic diarrhea virus vaccine composition which includes a porcine epidemic diarrhea virus S1 protein having an amino acid sequence represented by SEQ ID NO: 5 as an active ingredient, and the like This application claims priority to and the benefit of Korean Patent Application No. 10-2019-0044008, filed on Apr. 16, 2019, the disclosure of which is incorporated herein by reference in its entirety.
Porcine epidemic diarrhea (PED) is a fatal and highly contagious porcine intestinal disease, which is characterized by acute diarrhea, vomiting and dehydration, and causes high mortality in newborn piglets. This disease was first reported in England in 1971, and had spread rapidly throughout Europe, and is assumed to have crossed over to the Asian continent in the 1980s. The coronavirus-analog initially designated as CV777 was identified as the causative agent in Belgium in 1978.
In order to prevent such PED, the need to develop a subunit vaccine as a safer, more economical, and more quickly acting strategy in the onset of a disease is emerging. The subunit vaccine is used as an antigen protein of a vaccine by mass production of a structural or non-structural protein with known antigenicity, which constitutes a pathogenic microorganism, in recombinant microorganisms (E. coli or a different expression system).
In Korea, live attenuated PED vaccines are generally used, but the biggest problem of such vaccines is that, although attenuated, viruses are alive, so they recover their toxicity and become pathogenic, and thus may have problems in safety. In addition, in the development of a vaccine, for attenuation, it takes relatively a long time to subculture 100 times in animal cells, so it is difficult to respond quickly to viruses with a high mutation rate and the cost for vaccine development is also relatively high because of the use of animal cell culture.
Meanwhile, vaccines for preventing such disease are produced generally using animal cells, rather than bacteria due to protein folding or glycosylation. However, since the method of producing a vaccine using animal cells needs a large amount of costs for facility expansion for mass production, vaccine production is not easy, and most vaccines are expensive. In addition, inactivated virus vaccines produced using animal cells are not only difficult to store, but also have the disadvantage of being highly likely to be contaminated with viruses that can infect animals. However, unlike animal cells, since plant cells have a very low possibility of being contaminated with viruses that can infect animals, can be mass-produced at any time as long as a cultivation area is secured, and stored for a long-time in a plant body, it is expected that stable and inexpensive vaccine production is possible.
The present invention is deduced to solve the above-described problems according to the conventional art, and directed to providing a recombinant PED virus protein that not only enables efficient production using a plant body, but also exhibits high immunogenicity and virus neutralization ability, a vaccine composition including the same, and a method of preparing the protein.
However, technical problems to be solved in the present invention are not limited to the above-described problems, and other problems which are not described herein will be fully understood by those of ordinary skill in the art from the following descriptions.
The present invention provides a PED virus vaccine composition, which includes a PED virus protein having an amino acid sequence represented by SEQ ID NO: 5 as an active ingredient. The PED virus protein may also include a functional equivalent of the amino acid sequence represented by SEQ ID NO: 5 in the scope of the present invention, and the functional equivalent refers to a polypeptide which has at least 60% or more, preferably 70% or more, more preferably 80% or more, and most preferably 90% or more sequence homology with the amino acid sequence as a result of the addition, substitution or deletion of an amino acid, and exhibits a substantially the same activity as the amino acid sequence represented by SEQ ID NO: 5, and as long as it is an amino acid sequence of a PED virus protein that can be stably produced using a plant body, the present invention is not limited thereto.
In addition, the present invention provides a feed composition for preventing or treating PED, which includes the PED virus protein as an active ingredient.
In addition, the present invention provides a method of preventing or treating PED by administering the PED virus protein into an individual.
In addition, the present invention provides a use of the PED virus protein for preventing or treating PED.
In addition, the present invention provides a use of the PED virus protein for preparing a PED vaccine or drug.
In addition, the present invention provides a vector for expressing a PED virus protein, which includes a polynucleotide encoding an amino acid sequence represented by SEQ ID NO: 5. The polynucleotide is preferably a polynucleotide sequence represented by SEQ ID NO: 1.
In one embodiment of the present invention, in the vector, a promoter gene and a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 5 may be operably linked sequentially.
In another embodiment of the present invention, the vector may have a structure shown in
In still another embodiment of the present invention, the vector may further include a gene encoding one or more selected from the group consisting of an endoplasmic reticulum (ER) signal peptide, an Fc fragment and a chaperone binding protein (BiP), but the present invention is not limited thereto.
In yet another embodiment of the present invention, the ER signal peptide may be one selected from the group consisting of peptide sequences represented by KDEL, HDEL, SEKDEL, KHEDL, KEEL, and SEHEDL, but the present invention is not limited thereto.
In yet another embodiment of the present invention, the promoter may be a cauliflower mosaic virus-derived 35S promoter, a cauliflower mosaic virus-derived 19S RNA promoter, 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, an elongation factor-1 alpha (EF-1α) promoter, a pEMU promoter, a MAS promoter, a histone promoter, or a Clp promoter, but the present invention is not limited thereto.
In yet another embodiment of the present invention, a recombinant expression vector may further include a polynucleotide encoding a chaperone binding protein (BiP), a gene encoding a His-Asp-Glu-Leu (HDEL) peptide, and a gene encoding a porcine immunoglobulin Fc fragment.
In addition, the present invention provides a transformant which is transformed with the vector.
In one embodiment of the present invention, the transformant is preferably a microorganism such as E. coli, Bacillus, Salmonella or a yeast, insect cells, animal cells including human cells, an animal such as a mouse, a rat, a dog, a monkey, a pig, a horse or a cow, Agrobacterium tumefaciens, or a plant, and more preferably, a food crop such as rice, wheat, barley, corn, bean, potato, red bean, oat or sorghum; a vegetable crop such as Arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, oriental melon, pumpkin, green onion, onion, or carrot; a specialty crop such as ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut or rapeseed; a fruit tree such as an apple tree, a pear tree, a date tree, peach, grape, tangerine, persimmon, plum, apricot or banana; and a flower such as a rose, a carnation, a chrysanthemum, lily, or tulip, but as long as the transformant is an organism that can be transformed with the vector of the present invention, the present invention is not limited thereto.
In addition, the present invention provides a method of producing a PED virus protein, which includes: (a) culturing the transformant; and (b) isolating and purifying a PED virus protein from the transformant or culture solution. The transformant is preferably a cell itself, a plant body or a cultured product including cells, and the culture solution is preferably a culture solution from which cells are removed after cell culture. As long as the culture solution includes a recombinant antigen of the present invention, the present invention is not limited thereto.
In one embodiment of the present invention, the purification of step (b) may be purification using an aqueous fraction, but the present invention is not limited thereto.
A recombinant PED virus S1 protein of the present invention is not only effectively expressed in a plant body, but also has high water solubility, is easily isolated and purified, and acts as an antigen in the body, thereby exhibiting high immunogenicity and virus neutralization ability. Therefore, the recombinant PED virus S1 protein is expected to be widely used in various fields. In addition, due to the significant immunogenicity and virus neutralization ability in the body, the recombinant PED virus S1 protein can be used as a novel PED vaccine composition.
In the present invention, using a gene of a PED virus protein represented by SEQ ID NO: 1, it was confirmed that a PED virus protein having high immunogenicity in a plant body can be effectively produced and isolated. Accordingly, since the PED virus protein of the present invention can be stably and effectively mass-produced, it is expected to provide inexpensive and stable PED vaccines.
The “antigen” used herein is the generic term for all materials causing an immune response in the body, and preferably a virus, a chemical, a bacterium, a pollen, a cancer cell, shrimp, or a partial peptide or protein thereof, and as long as the material is any material that can cause an immune response in the body, the present invention is not limited thereto.
The “porcine epidemic diarrhea (PED) virus” used herein belongs to the family of Coronaviridae, and has single-stranded RNA as a genome, a length of approximately 28 Kb, and encodes three main constituent proteins, such as a spike protein, a membrane or envelope protein, and a nucleocapsid protein. The term “spike protein” used herein is a main constituent protein of the PED virus, and has biologically important functions of recognizing target cells and fusing a cellular membrane with the virus. A partial gene of the spike protein may be used as an attenuated antigenic determinant (neutralization epitope), and in the maturation process, the spike protein is frequently cleaved into a receptor-binding subunit S1 and a membrane-binding subunit S2. Particularly, the S1 domain of the S protein may include a receptor-binding domain (RBD) mediating specific binding for a cell receptor.
The term “vaccine” used herein is a biological agent containing an antigen causing an immune response to a living body, and refers to an immunogen which generates immunity in the living body by injection or oral administration into a human or animal to prevent an infectious disease. The animal is a human or a non-human animal, and the non-human animal refers to a pig, a cow, a horse, a dog, a goat or sheep, but the present invention is not limited thereto.
The term “expression vector” used herein refers to a vector that can express a peptide or protein encoded by a heterologous nucleic acid inserted into the vector, and preferably, a vector manufactured to express a porcine Fc fragment-fused target antigen (in the present invention, a PED virus protein). The “vector” refers to any medium for introducing and/or transferring a base into a host cell in vitro, ex vivo or in vivo, and may be a replicon to which a different DNA fragment is bound to induce the replication of the bound fragment, and the “replicon” is a genetic unit (e.g., a plasmid, a phage, a cosmid, a chromosome, or a virus) which serves as a self-unit of DNA replication in vivo, that is, is replicable by being self-controlled. The recombinant expression vector of the present invention preferably includes a promoter, which is a transcription initiating factor to which an RNA polymerase binds, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome-binding site and a sequence that regulates the termination of transcription and translation, and a terminator, more preferably, further includes a M17 5′ UTR site gene for increasing a protein synthesis level, a BiP gene for transferring a target protein to the ER, an Fc fragment that increases an expression level and solubility of a target protein, an ER signal peptide (the same meaning as ER targeting sequence) gene that minimize degradation of a protein for the protein to be stably maintained in the ER, and a cloning site, and still more preferably, further includes a tag gene for easily isolating a recombinant protein, and a marker gene for selecting an antibiotic-resistant gene to select a transformant.
The “PED virus protein” used herein may include an amino acid sequence of SEQ ID NO: 5, and preferably, is represented by the amino acid sequence of SEQ ID NO: 5. According to one embodiment of the present invention, due to having high solubility, the PED virus protein is easily isolated and purified, inhibits the aggregation of a recombinant protein, and thus is effective for maintaining the physiological or pharmacological activity of a recombinant protein.
In addition, the protein of the present invention may be encoded by a gene base sequence represented by SEQ ID NO: 1. In addition, the gene variant is included in the scope of the present invention. Specifically, the gene may include a base sequence having 70% or more, more preferably, 80% or more, and most preferably, 90% or more sequence homology with the base sequence of SEQ ID NO: 1. For example, the protein of the present invention includes a polynucleotide having 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 homology.
The “BiP gene” is a part of a BiP sequence used to move the expressed recombinant protein to the ER, and is preferably a gene including a base sequence of SEQ ID NO: 2, and most preferably a gene represented by SEQ ID NO: 2, and consists of a base sequence having 80% or more, more preferably, 90% or more, and still more preferably 95% or more sequence homology with the base sequence of SEQ ID NO: 2. The “% in sequence homology” with respect to a polynucleotide is identified by comparing a comparative region with the optimally aligned sequence, and in the comparative region, a part of the polynucleotide sequence may include an addition or deletion (that is, a gap) compared with the reference sequence (without an addition or deletion) for the optimal alignment of the sequence. The “BIP gene” is used to move the expressed recombinant protein to the ER, and may only have some amino acids remaining after a part of the sequence is deleted when expressed.
The “ER signal peptide” refers to an amino acid sequence which allows a protein to be recognized by signal recognition particles on the ER to translocate into the ER lumen. In the present invention, there is no limitation to the type and amino acid sequence of the ER signal peptide, as long as it is a plant ER signal peptide known to those of ordinary skill in the art, and examples of the ER signal peptide in the documents disclosed in US 20130295065 and WO2009158716 may be referenced. In the present invention, the “ER signal peptide” is preferably any one polypeptide selected from the group consisting of KDEL (SEQ ID NO: 6), HDEL (SEQ ID NO: 7), SEKDEL (SEQ ID NO: 8), KHEDL (SEQ ID NO: 9), KEEL (SEQ ID NO: 10), and SEHEDL (SEQ ID NO: 11), and most preferably, a polypeptide represented by HDEL (His-Asp-Glu-Leu, SEQ ID NO: 7), and encoded by SEQ ID NO: 4. In addition, as the ER signal peptide of the present invention, a variant of SEQ ID NO: 4 is included in the scope of the present invention. Specifically, the gene consists of a base sequence having preferably 90% or more, more preferably, 95% or more, and most preferably 98% or more sequence homology with the base sequence of SEQ ID NO: 4. The “% in sequence homology” with respect to a polynucleotide is identified by comparing a comparative region with the optimally aligned sequence, and in the comparative region, a part of the polynucleotide sequence may include an addition or deletion (that is, a gap) compared with the reference sequence (without an addition or deletion) for the optimal alignment of the sequence. A binding site of the ER signal peptide is characterized by being added (or linked) to the C-terminus of a protein to be expressed or synthesized in plant cells.
The “Fc fragment” refers to a part of an immunoglobulin without an antigen-binding site, which remains after only heavy chains (H chains) are linked by S—S bonds when the immunoglobulin is digested by papain, and the Fc fragment of the present invention is preferably a porcine Fc fragment, and more preferably, a porcine Fc fragment represented by SEQ ID NO: 3. However, there is no limitation as long as it is an Fc fragment which increase an expression level and solubility of a target protein when fused with the target protein. In addition, as the Fc fragment of the present invention, a variant of SEQ ID NO: 3 is included in the scope of the present invention. Specifically, the gene consists of a base sequence having preferably 90% or more, more preferably 95% or more, and most preferably 98% or more sequence homology with the base sequence of SEQ ID NO: 3. The “% in sequence homology” with respect to a polynucleotide is identified by comparing a comparative region with the optimally aligned sequence, and in the comparative region, a part of the polynucleotide sequence may include an addition or deletion (that is, a gap) compared with the reference sequence (without an addition or deletion) for the optimal alignment of the sequence.
The “cloning site” encompasses inserts for linking/distinguishing genes in a vector, and is preferably a region represented by “ggatcctg” or “ccccgggca” in SEQ ID NO: 12, “RIL, Arg Ile Leu” located at the 8th to 10th positions of SEQ ID NO: 13, or “PRA, Pro Arg Ala” located at 727th to 729th positions of SEQ ID NO: 13, but the present invention is not limited thereto.
In the present invention, the viral protein is preferably provided in a fused form with an Fc fragment. The term “fusion” means both chemical and genetic fusion, and in the present invention, preferably, refers to genetic fusion. The “genetic fusion” refers to a link that is formed by a linear covalent bond formed through the genetic expression of a DNA sequence encoding a protein.
The gene for tagging may include, for example, an Avi tag, a calmodulin tag, a polyglutamate tag, an E tag, a FLAG tag, a HA tag, a His tag, a Myc tag, a S tag, a SBP tag, an IgG-Fc tag, a CTB tag, a Softag 1 tag, a Softag 3 tag, a Strep tag, a TC tag, a V5 tag, a VSV tag, and an Xpress tag, and the IgG-Fc tag may be derived from a human, a mouse, a rabbit or a pig.
A marker gene for selection may be, for example, a herbicide-resistant gene such as glyphosate or phosphinothricin, an antibiotic-resistant gene such as kanamycin, G418, bleomycin, hygromycin or chloramphenicol, or an aadA gene, and the promoter may be, for example, a pEMU promoter, a MAS promoter, a histone promoter, a Clp promoter, a cauliflower mosaic virus-derived 35S promoter, a cauliflower mosaic virus-derived 19S RNA promoter, a plant actin protein promoter, a ubiqitin protein promoter, a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) promoter, a respiratory syncytial virus (RSV) promoter, or an elongation factor-1 alpha (EF-1α) promoter, and the terminator may be, for example, a nopaline synthase (NOS) terminator, a rice amylase RAmy1 A terminator, a faceolin terminator, a terminator of an octopine gene of Agrobacterium tumafaciens, or an rmB1/B2 terminator of E. coli, but the present invention is not limited thereto.
The “vector” used herein may have a structure shown in
The “vector” used herein is preferably a gene having an amino acid sequence SEQ ID NO: 13, and most preferably an amino acid sequence represented by SEQ ID NO: 13, and consists of an amino acid sequence having 80% or more, more preferably 90% or more, and still more preferably 95% or more sequence homology with the sequence of SEQ ID NO: 13.
In addition, the amino acid sequence may be encoded by a gene sequence represented by SEQ ID NO: 12, but the present invention is not limited thereto. Specifically, the gene may consist of a base sequence having 90% or more, more preferably 95% or more, and most preferably 98% or more sequence homology with the base sequence of SEQ ID NO: 12. The “% in sequence homology” with respect to a polynucleotide is identified by comparing a comparative region with the optimally aligned sequence, and in the comparative region, a part of the polynucleotide sequence may include an addition or deletion (that is, a gap) compared with the reference sequence (without an addition or deletion) for the optimal alignment of the sequence.
The “transformation” used herein encompasses a change in genetic properties of a living organism by injected DNA, the “transgenic organism” is a living organism produced by injecting an external gene through a molecular genetic method, and preferably, a living organism transformed by a recombinant expression vector of the present invention. The living organism is a living thing such as a microorganism, a eukaryotic cell, an insect, an animal or a plant without limitation, and preferably, E. coli, Salmonella, Bacillus, yeast, an animal cell, a mouse, a rat, a dog, a monkey, a pig, a horse, a cow, Agrobacterium tumefaciens or a plant, but the present invention is not limited thereto.
The “plant” used herein is a plant that can produce a protein in a large amount, and more specifically, may be selected from the group consisting of tobacco, Arabidopsis thaliana, corn, rice, soybean, canola, alfalfa, sunflower, sorghum, wheat, cotton, peanut, tomato, potato, lettuce and pepper, and preferably tobacco. The tobacco in the present invention is a Nicotiana genus plant, which can overexpress a protein, but there is no particular limitation on the type of tobacco, and the present invention may be implemented by selecting a suitable species for a transforming method and the purpose of mass-production of a protein. For example, a species such as Nicotiana benthamiana L. or Nicotiana tabacum cv. Xanthi may be used.
The transformant may be prepared by transformation, transfection, Agrobacterium-mediated transformation, particle gun bombardment, sonication, electroporation and polyethylene glycol (PEG)-mediated transformation, but there is no limitation as long as the transformant is prepared by any method of injecting the vector of the present invention.
The “solubility” used herein refers to a degree of solubilizing a target protein or peptide in a solvent suitable for being administered to the human body. Specifically, the solubility indicates a degree of saturation of a solute in a given solvent at a specific temperature. The solubility may be measured by determining a saturation concentration of a solvent, and for example, the solubility may be determined by adding an excessive amount of solute in a solvent, stirring and filtering the solution, and measuring a concentration using an UV spectrophotometer or HPLC, but the present invention is not limited thereto. High solubility is preferable for isolation and purification of a recombinant protein, and has advantages of inhibition of the aggregation of the recombinant protein and maintenance of physiological or pharmacological activity of the recombinant protein.
The “prevention” used herein refers to all actions of inhibiting or delaying the occurrence of PED by administration of a recombinant PED virus protein according to the present invention.
The “treatment” used herein refers to all actions involved in improving or beneficially changing symptoms of PED by administration of a protein according to the present invention.
The “individual” used herein refers to a subject to which a recombinant PED virus protein of the present invention can be administered, but the individual is not limited.
The “vaccine composition” used herein may be formulated in an oral preparation such as such as a powder, granules, a tablet, a capsule, a suspension, an emulsion, a syrup, or an aerosol, or a sterile injectable solution according to a conventional method. The vaccine composition may be formulated with a diluent or an excipient such as a filler, a thickening agent, a binder, a wetting agent, a disintegrant, a surfactant, which are conventionally used. A solid preparation for oral administration may be a tablet, pill, powder, granule or capsule, and such a solid formulation may be obtained by mixing at least one of excipients such as starch, calcium carbonate, sucrose, lactose and gelatin with a lecithin-like emulsifier. In addition, in addition to the simple excipient, lubricants such as magnesium stearate and talc may also be used. As a liquid preparation for oral administration, a suspending agent, a liquid for internal use, an emulsion or a syrup is used, and other than a commonly used simple diluent such as water or a liquid paraffin, various excipients, for example, a wetting agent, a sweetening agent, a fragrance and a preservative may be included. As a preparation for parenteral administration, a sterile aqueous solution, a non-aqueous agent, a suspending agent, an emulsifier, or a freeze-drying agent is included. As a non-aqueous agent or suspending agent, propylene glycol, polyethylene glycol, a vegetable oil such as olive oil, or an injectable ester such as ethyl oleate may be used.
In addition, the “adjuvant” used herein generally refers to any material increasing a humoral and/or cellular immune response to an antigen. Here, the vaccine composition of the present invention may further include an “adjuvant.” The adjuvant may be, for example, a complete Freund's adjuvant (CFA), an incomplete Freund's adjuvant (IFA), alum, oil, Lipid A, monophosphoryl lipid A, a bacterial agent such as Bacillus-Calmette-Guerrin (BCG), a nucleic acid such as CpG-DNA, dsRNA, a bacterial component agent such as tuberculin, a natural high molecular material such as keyhole limpet hemocyanin or yeast mannan, a muramyl tripeptide or a muramyl dipeptide or a derivative thereof, alum, a nonionic block copolymer, or a cytokine such as interleukin 2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), interferon-α (IFN-α), or interferon-0 (IFN-0), and a combination of at least one or two thereof, and in one embodiment of the present invention, an IMS1313 oil adjuvant was used.
The vaccine composition or pharmaceutical composition of the present invention may be formulated in an oral preparation such as a powder, granules, a tablet, a capsule, a suspension, an emulsion, a syrup or an aerosol, a preparation for external use, a suppository or a sterile injectable solution.
Administration routes for the vaccine composition according to the present invention may include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, local, sublingual or rectal administration. Oral or parenteral administration is preferable. The term “parenteral” used herein means subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrabursal, intrastemal, intradural, intralesional and intracranial injection techniques. The vaccine composition of the present invention may be administered in the form of a suppository for rectal administration.
A dosage of the vaccine composition or pharmaceutical composition of the present invention is selected in consideration of an individual's age, weight, sex, or body condition. The amount required for inducing an immunoprotective response in an individual without a separate side reaction may vary according to the presence of any recombinant protein used as an immunogen and any excipient. Generally, each dose contains 0.1 to 1000 μg, and preferably, 0.1 to 100 μg of the recombinant protein of the present invention per mL of a sterile solution. In the case of the vaccine composition, if necessary, optionally repeated antigen stimulation may be performed after an initial dose.
The “feed composition” used herein is a feed including a recombinant PED virus S1 protein of the present invention, and the feed may be a byproduct of pork, beef or chicken as well as corn, rice, common rice straw, wild grass, grass, ensilage, dry grass, or native grass, but the present invention is not limited thereto, and there is no limitation as long as it is a feed used for raising livestock. Methods for adding and mixing a recombinant PED virus S1 protein of the present invention with such a feed may include mechanical mixing, adsorption and occlusion, but the present invention is not limited thereto.
Hereinafter, to help in understanding the present invention, exemplary examples will be suggested. However, the following examples are merely provided to more easily understand the present invention, and not to limit the present invention.
A recombinant plant expression vector was constructed to express a PED virus S1 protein in a plant body as shown in
2.1. Transient Expression of Plant Expression Vector
The plant expression vector prepared in Example 1 was transformed into the Agrobacterium strain LBA4404 by electroporation. The transformed Agrobacterium was cultured with shaking in 5 mL of a yeast extract peptone (YEP) liquid medium (10 g of yeast extract, 10 g of peptone, 5 g of NaCl, 50 mg/L of kanamycin, and 25 mg/L of rifampicin) at 28° C. for 16 hours, 1 mL of a primary culture solution was inoculated into 50 mL of a fresh YEP medium and cultured with shaking at 28° C. for 6 hours. The cultured Agrobacterium was collected through centrifugation (7,000 rpm, 4° C., 5 min), and resuspended in an infiltration buffer (10 mM MES (pH 5.7), 10 mM MgCl2, 200 μM acetosyringone) to an O.D. of 1.0 at a wavelength of 600 nm. The Agrobacterium suspension was injected into the back side of a Nicotiana benthamiana leaf using a needle-free syringe to perform Agro-infiltration.
2.2. Confirmation of Expression of Recombinant PED Virus S1 Protein in Plant Body
A protein was extracted from the plant leaf prepared in Example 2.1 and centrifuged, and then a protein in an aqueous fraction (S) contained in a solution and a protein in a pellet (P) fraction were identified by western blotting. More specifically, 30 μL of each fraction was mixed with an SDS sample buffer, and then heated. In addition, the fraction was loaded on a 10% SDS-PAGE gel for electrophoresis to isolate proteins by size, and the isolated protein was transferred to a PVDF membrane and blocked with 5% skim milk, an anti-pig secondary antibody recognizing pFc2 was bound to the membrane, and treated with an ECL solution according to a method provided by a manufacturer to identify the expression of the recombinant PED virus S1 protein. The result is shown in
As shown in
From the above result, it was confirmed that the vector for expressing a recombinant PED virus S1 protein of the present invention can effectively express the recombinant PED virus S1 protein in a plant body, and since the recombinant PED virus S1 protein prepared using the vector has high solubility, it is easily isolated and purified, and the aggregation of the recombinant protein is inhibited to be effective in maintaining physiological or pharmacological activity of the recombinant protein.
200 mL of a protein extraction solution (50 mM Tris (pH 7.2), 150 mM NaCl, 0.1% Triton X-100, 1× protease inhibitor) was added to the 40 g of the Nicotiana ventamia prepared in Example 2.1, the mixture was subjected to tissue disruption using a blender, and centrifuged at 13,000 rpm for 20 minutes at 4° C. to recover a protein extract. For isolation and purification of the expressed recombinant PED virus protein, affinity chromatography was performed with a column filled with a protein A-sepharose resin. The column was filled with 5 mL of the resin, and equilibrated with 50 mL of a washing solution (50 mM Tris (pH 7.2), 150 mM NaCl). After the recovered protein extract was applied to the column, 100 mL of a washing solution was flowed to wash the resin, the recombinant protein was eluted with an elution solution (0.1 M sodium citrate (pH 3.0), 150 mM NaCl), and the protein eluate was neutralized by adding a neutralization solution (1 M Tris (pH 9.0)). The elution solution containing the recombinant protein was replaced with physiological saline (PBS) using a 30 kD-sized filter and concentrated, thereby obtaining an isolated and purified recombinant PED virus protein. The isolated and purified protein was confirmed by Coomassie staining after SDS-PAGE (
As shown in
The recombinant protein of the present invention was well purified without a large difference from the conventional protein. Such a result shows that, when the protein was expressed in a plant, a problem of being likely to reduce production efficiency due to a modified sugar structure was not found, and the protein according to the present invention is well produced in a plant.
To confirm whether the recombinant PED virus S1 protein antigen induces an antibody in vivo and thus has immunogenicity, experiments were carried out with five 6-week-old female guinea pigs per group. More specifically, for a negative control, physiological saline was administered, for a positive control, a commercially available PED vaccine (Joongang Vaccine) was administered, and for an experimental group, the recombinant PED virus S1 protein was subcutaneously administered at a dose of 150 μg twice every three weeks. Upon injection of the antigen, the same amount of an IMS1313 adjuvant (SEPPIC) was mixed and injected. Blood was collected from the jugular vein or heart before antigen administration and at two weeks after the second administration, followed by isolating a serum, and storing it in a refrigerator at −20° C. The production of a specific antibody against the PEDV S1 protein in each serum was identified using an ELISA plate coated with a His tag-fused PEDV-S1 recombinant protein, and the result is shown in
As shown in
It was confirmed whether blood antibodies induced by the administration of the recombinant antigen protein have PED virus neutralization ability using the serum prepared in Example 4. More specifically, in the neutralizing antibody test, a PEDV QIAP1401 strain virus and Vero cells as an infection host were used. Serum was isolated from the collected blood, immobilized at 57° C. for 30 minutes, and then diluted 2-fold with Minimum Essential Media (MEM) Eagle containing 10 μg/ml of trypsin. Equal amounts of 316 TCID50/0.1 mL of the PEDV virus and the diluted serum were mixed for neutralization at 37° C. for 90 minutes. Immediately before the end of culture, the Vero cells prepared in a monolayer state in a 96-well plate were washed with PBS three times, 100 uL of the serum-virus mixture was added, and then cultured at 37° C. for 2 hours. After culture, 100 uL of a medium containing 2 μg/mL trypsin was added, and the virus was cultured at 37° C. for 3 to 5 days until a cytopathic effect (CPE) was exhibited. A neutralizing antibody titer was measured by observing the serum dilution factor of a well immediately before the CPE was exhibited under a microscope (Table 1).
As a result, a neutralizing antibody titer by the recombinant PEDV-S1 antigen protein is 16.0 (Log 2) on average, and a neutralizing antibody titer by the commercially available vaccine is 7.2 (Log 2), showing that the formation of neutralizing antibody titer by the recombinant PEDV-S1 antigen protein is higher than that by the commercially available vaccine.
From the above results, it was confirmed that the recombinant PED virus S1 protein of the present invention is not only effectively expressed in a plant body, but also has high water solubility, and thus the protein is easily isolated and purified, and serves as an antigen in the body, thereby exhibiting high immunogenicity and virus neutralization ability. Therefore, it was confirmed that the protein can be used as a novel PED vaccine composition.
Hereinafter, preparation examples of the pharmaceutical composition and feed composition of the present invention will be described, but the examples are merely provided to specifically described the present invention, rather than limit the invention.
1.1. Preparation of Powder
A powder was prepared by mixing the above components and filling an airtight pouch with the mixture.
1.2. Preparation of Tablet
A tablet was prepared by mixing the above components and compounding the mixture according to a conventional method of preparing a tablet.
1.3. Preparation of Capsule
A capsule was prepared by mixing the above components and filling a gelatin capsule with the mixture according to a conventional method of preparing a capsule.
1.4. Preparation of Injectable Preparation
An injectable preparation was prepared with the above contents of the components per ampoule (2 mL) according to a conventional method of preparing an injectable preparation.
1.5. Preparation of Liquid
A liquid was prepared by adding and dissolving each component in purified water, adding an appropriate amount of a lemon flavor and mixing them together, adding purified water thereto to a total volume of 100 mL, and then pouring the resulting solution into a brown bottle and sterilizing the solution according to a conventional method of preparing a liquid.
A feed was prepared by mixing the above components according to a conventional feed preparation method.
It should be understood by those of ordinary skill in the art that the above description of the present invention is exemplary, and the exemplary embodiments disclosed herein can be easily modified into other specific forms without departing from the technical spirit or essential features of the present invention. Therefore, the exemplary embodiments described above should be interpreted as illustrative and not limited in any aspect.
As a recombinant PED virus S1 protein of the present invention may be effectively expressed in a plant body, and have high water solubility, it is easily isolated and purified, and also serves as an antigen in the body to exhibit high immunogenicity and virus neutralization ability, and therefore it is expected to be used in various fields. Moreover, due to significant immunogenicity and virus neutralization ability in the body, the protein is expected to be used as a novel PED vaccine composition, and thus is available in the industry.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0044008 | Apr 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/004954 | 4/13/2020 | WO | 00 |
