KR20200145321A - Viral hemorrhagic septicemia virus glycoprotein antigen obtained from transgenic plants and vaccine comprising the same - Google Patents

Abstract

The present invention relates to a viral hemorrhagic septicemia virus glycoprotein antigen obtained from transgenic plants, and a vaccine comprising the same, and more specifically, to: a viral hemorrhagic septicemia virus glycoprotein antigen, in which a glycoprotein of viral hemorrhagic septicemia virus obtained from transgenic plants is fused with an immunoglobulin Fc region with immune cell cognitive ability; and a vaccine comprising the antigen. The viral hemorrhagic septicemia virus glycoprotein antigen provided by the present invention is capable of inducing a very good immunization response in halibut, thereby allowing a desired antibody to be produced in the individual. According to a method provided by the present invention, it is possible to economically and safely produce a viral hemorrhagic septicemia virus glycoprotein antigen from plants.

Description

Viral hemorrhagic septicemia virus glycoprotein antigen obtained from transgenic plants and vaccine comprising the same}

The present invention relates to a viral hemorrhagic sepsis virus glycoprotein antigen obtained from a transgenic plant and a vaccine comprising the same, and more particularly, to a glycoprotein and immune cell recognition ability of a viral hemorrhagic sepsis virus obtained from a transgenic plant. It relates to a viral hemorrhagic sepsis virus glycoprotein antigen and a vaccine comprising the same in which an immunoglobulin Fc region (Fc region) is fused.

Viral Hemorrhagic Septicemia (VHS) is called Egtved disease and is a disease that occurs in freshwater and seawater fish worldwide and causes mass mortality. In Korea, since it was first reported in halibut in 2001, it has been steadily confirmed in Gyeongbuk and Jeju regions, and from late autumn when the water temperature decreases to spring, it causes mortality to not only small fry but also large fish, causing great losses to the aquaculture industry. have. Viral hemorrhagic sepsis is caused by infection with viral hemorrhagic sepsis virus (VHSV), and infected fish such as flounder darken their body color and show symptoms such as abdominal distension and hernia, and die as soon as a week or a month later. As there is a tendency to do so, careful attention is required in the management of aquaculture farms.

The causative virus of viral hemorrhagic sepsis (VHS) is a single-stranded RNA virus, which has an outer membrane of the genus Rhabdoviridae and Novirhabdovirus. It is 50-70×180-240 nm in size, and the model of the virus particle is a bullet-shaped, typical ravdovirus type, round on one side and flat on the other. In academia, genotypes are classified based on the glycoprotein gene sequence of VHSV isolated from freshwater, and these genotypes are American type (Genogroup Ⅰ) and British Isles type (Genogroup Ⅱ) according to geographic separation origin. , European type (Genogroup III), three genotypic distinctions have been generalized. According to the neutralizing antibody test, it is divided into three serotypes, and cross-reaction occurs between these serotypes. The genotype isolated in Korea is found to be similar to the virus isolated in North America and Japan belonging to Genogroup I. In addition, VHSV isolated from flounder has a genetic distinction from the virus isolated from freshwater fish, but not serologically.

Viral hemorrhagic sepsis is a aquatic animal infectious disease under Article 2 of the Fishery Animal Disease Control Act.It is a viral disease that causes halibut mortality in low water temperatures below 15℃. Since the mortality rate is high and contagious is strong, discharge is prohibited if it is detected in seedlings, and other areas It is a disease that is managed by restricting its movement to.

Since viral hemorrhagic sepsis is transmitted at a rapid rate once onset, prevention of the disease is very important, but there is a limit to suppressing the occurrence of disease only by improving the breeding environment and breeding management. Therefore, it is urgent to develop a vaccine as a more fundamental preventive measure.

On the other hand, vaccines are drugs used to generate an immune response against antigens for the purpose of defense against pathogen infection. Recently, vaccines developed mainly use recombinant proteins as antigens. Recombinant proteins have fewer side effects and safer than live attenuated vaccines or dead cell vaccines, but have a problem of low immunogenicity.

Accordingly, there is an increasing need for antigenic proteins against viral hemorrhagic sepsis viruses capable of exhibiting a sufficient immune response while using a recombinant protein as an antigen.

Accordingly, the present inventors have conducted intensive research to develop a recombinant protein antigen capable of inducing an excellent immunization response against the viral hemorrhagic sepsis virus of flounder. As a result, the Fc-fusion body combined with the glycoprotein of the viral hemorrhagic sepsis virus was incorporated into the endoplasmic reticulum. It was found that when the storage was induced and expressed in plants, a remarkably improved immunization reaction was induced, and the present invention was completed.

Accordingly, an object of the present invention is (a) preparing a transgenic plant expressing a recombinant protein comprising a glycoprotein of a viral hemorrhagic sepsis virus and an Fc fragment of an antibody; And (b) to provide a viral hemorrhagic sepsis virus glycoprotein antigen prepared by a method comprising the step of obtaining a protein using the transgenic plant.

Another object of the present invention is to provide a viral hemorrhagic sepsis vaccine composition of flounder comprising a viral hemorrhagic sepsis virus glycoprotein antigen.

Another object of the present invention is a virus comprising the step of transforming a plant cell with a recombinant vector designed to express an antigen including a glycoprotein of a viral hemorrhagic sepsis virus and an Fc fragment of an antibody It is to provide a method for producing a transgenic plant that produces a sexual hemorrhagic sepsis virus glycoprotein antigen.

Another object of the present invention is to provide a plant that produces a viral hemorrhagic sepsis virus glycoprotein antigen prepared by the method.

In order to achieve the object of the present invention, the present invention (a) prepares a transgenic plant expressing a recombinant protein including a glycoprotein of a viral hemorrhagic sepsis virus and an Fc fragment of an antibody. Step to do; And (b) it provides a viral hemorrhagic sepsis virus glycoprotein antigen prepared by a method comprising the step of obtaining a protein using the transgenic plant.

In order to achieve another object of the present invention, the present invention provides a viral hemorrhagic sepsis vaccine composition of flounder comprising a viral hemorrhagic sepsis virus glycoprotein antigen.

In order to achieve another object of the present invention, the present invention transforms plant cells with a recombinant vector designed to express an antigen including a glycoprotein of a viral hemorrhagic sepsis virus and an Fc fragment of an antibody. It provides a method for producing a transgenic plant that produces a viral hemorrhagic sepsis virus glycoprotein antigen comprising the step of:

In order to achieve another object of the present invention, the present invention provides a plant that produces a viral hemorrhagic sepsis virus glycoprotein antigen prepared by the above method.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of: (a) preparing a transformed plant expressing a recombinant protein including a glycoprotein of viral hemorrhagic sepsis virus (VHSV) and an Fc fragment of an antibody; And (b) it provides a viral hemorrhagic sepsis virus glycoprotein antigen prepared by a method comprising the step of obtaining a protein using the transgenic plant.

The causative virus of viral hemorrhagic sepsis (VHS) is a single-stranded RNA virus, which has an outer membrane of the genus Rhabdoviridae and Novirhabdovirus. It is 50-70×180-240 nm in size, and the model of the virus particle is a bullet-shaped, typical ravdovirus type, round on one side and flat on the other. In academia, genotypes are classified based on the glycoprotein gene sequence of VHSV isolated from freshwater, and these genotypes are American type (Genogroup I) and British Isles type (Genogroup II) according to geographic separation origin. , European type (Genogroup III), three genotypic distinctions have been generalized. According to the neutralizing antibody test, it is divided into three serotypes, and cross-reaction occurs between these serotypes. The genotype isolated in Korea is found to be similar to the virus isolated in North America and Japan belonging to Genogroup I. In addition, VHSV isolated from flounder has a genetic distinction from the virus isolated from freshwater fish, but not serologically.

In the present invention, the type of the VHSV glycoprotein is not particularly limited, and may include all of the VHSV glycoproteins known to date or the VHSV glycoproteins to be newly identified in the future. The specific sequence of the VHSV glycoprotein can be easily searched by a person skilled in the art from the NCBI genebank, and not only the entire sequence of VHSV but also the antigenic VHSV fragment can be included in the VHSV glycoprotein of the present invention. It can be understood clearly.

Preferably, in the present invention, the VHSV glycoprotein may be a VSHV glycoprotein consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof.

In the VHSV glycoprotein antigen provided by the present invention, the VHSV glycoprotein acts as an antigen that induces a substantial immune response, that is, humoral and/or cellular immunity (T cell response).

In the present invention, the term'antigen' induces a state of sensitization and/or immunoreactivity upon introduction by contact with appropriate cells, and in a verifiable manner with the immune cells and/or antibodies of the subject thus sensitized in vivo or in vitro. Refers to all reacting substances. In the present invention, the term'antigen' may be used collectively with the same meaning as the term'immunogen', and preferably promotes the host immune system to generate a secretory, humoral and/or cellular immune response specific to the antigen. It refers to a molecule containing one or more epitopes capable of. In addition, in the present invention, the term'antigenicity' or'immunogenicity' refers to the property of the antigen or immunogen and means a property that causes a secretory, humoral and/or cellular immune response.

The term'immune reaction' is a self-defense system existing in an animal's body, and is a biological phenomenon in which various substances or living organisms invading from the outside are distinguished from oneself and the invader is removed. The surveillance system for self-defense is largely formed by two mechanisms, one is humoral immunity and the other is cellular immunity. Humoral immunity is achieved by antibodies present in the serum, and the antibodies play an important role in removing invading foreign antigens by binding them. On the other hand, cellular immunity is made by several types of cells belonging to the lymphatic system, which are responsible for the direct destruction of invading cells or tissues. Thus, humoral immunity is mainly effective against foreign substances such as bacteria, viruses, proteins, and complex carbohydrates existing outside cells, and cellular immunity exerts its function in various parasites, tissues, intracellular infections, and cancer cells. This double defense system is mainly carried out by two types of lymphocytes, such as B cells and T cells. B cells produce antibodies, and T cells participate in cellular immunity. This immune response by B cells or T cells is an immune system that responds to antigens once invading the body, but works when the same type of antigen continues to exist or repeatedly invades. Thus, this immune response is a specific response to a specific antigen. In addition to these antigen-specific immune responses, there is also a kind of natural immune response that directly reacts to destroy attacking cells even if the body has not been exposed to any antigen. These immune responses include neutrophil, macrophage, and natural killer (NK) cells. It is characterized by being involved in this and exerting various functions without being limited by the type of target cell.

The'epitope' refers to the simplest form of an antigenic determinant on a complex antigenic molecule, which is a specific portion of an antigen recognized by an antibody or T cell receptor.

In the present invention, the Fc fragment (Fc antibody fragment) includes a CH2 domain and/or a CH3 domain derived from an antibody Fc fragment, and refers to a portion capable of binding to an antigen-presenting cell (APC).

In the present invention, the term'antibody' is used interchangeably with'immunoglobulin (hereinafter, referred to as "Ig"), and is a generic term for a protein that selectively acts on an antigen and is involved in biological immunity. Antibodies consist of two pairs of light and heavy chains. The light and heavy chains of these antibodies are polypeptides consisting of several domains. In a whole antibody, each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant regions include heavy chain constant domains CH1, CH2 and CH3 (antibody classes IgA, IgD and IgG) and optionally heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain (VL) and a light chain constant domain (CL). The variable domains VH and VL can be further subdivided into more conserved, hypervariable regions called complementarity determining regions (CDRs) interspersed within regions called framework regions (FR). Each VH and VL consists of 3 CDRs and 4 FRs, in the following sequence arranged from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Janeway, CA, Jr. et al. (2001). Immunobiology., 5th ed., Garland Publishing; and Woof, J., Burton, D., Nat Rev Immunol 4 (2004) 89-99). The two pairs of heavy and light chains (HC/LC) can specifically bind to the same antigen. Therefore, the whole antibody is a bivalent, monospecific antibody.

There are five types of mammalian antibody heavy chains denoted by the Greek letters α, δ, ε, γ, and μ (Janeway, C.A., JR., et al., (2001). Immunobiology., 5th ed., Garland Publishing). The type of heavy chain present defines the class of antibodies; These chains are found in IgA, IgD, IgE, IgG and IgM antibodies, respectively (Rhoades R.A., Pflanzer RG (2002). Human Physiology, 4th ed., Thomson Learning). The distinct heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids. Each heavy chain has two regions, a constant region and a variable region. The constant regions are identical in all antibodies of the same isotype, but are different in antibodies of different isotypes. The heavy chains γ, α and δ have a constant domain composed of three constant domains CH1, CH2 and CH3 (in a row), and a hinge site to add flexibility (Woof, J., Burton, D., Nat Rev Immunol 4 ( 2004) 89-99); The heavy chains μ and ε have a constant region consisting of four constant domains CH1, CH2, CH3 and CH4 (Janeway, C.A., Jr., et al., (2001). Immunobiology., 5th ed., Garland Publishing). The variable regions of the heavy chain are different for antibodies produced by different B cells, but are the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and consists of a single antibody domain. There are only two types of light chains in mammals, called lambda (λ) and kappa (κ). The light chain has two contiguous domains of one constant domain CL and one variable domain VL. The approximate length of the light chain is 211 to 217 amino acids.

It is understood that, unless otherwise specified in the specification of the present invention, IgG is described as a representative basic structure of an antibody.

The Fc fragment of the present invention may be derived from any one selected from the group consisting of IgG, IgA, IgD, IgE and IgM, and preferably may be an IgG-derived Fc fragment. The IgG can be further divided into IgG1, IgG2, IgG3 and IgG4, and the Fc fragment of the present invention may most preferably be an Fc fragment derived from IgG1.

In the present invention, the term'Fc fragment' is a fragment obtained when an immunoglobulin (Ig) molecule is decomposed into papain, and the variable region (VL) and constant region (CL) of the light chain, the variable region (VH) and heavy chain constant region of the heavy chain This is the region from which 1(CH1) has been removed. That is, the Fc fragment refers to a dimer of two CH2-CH3 chains, and the two chains form a dimer structure by disulfide bonds. In addition, the Fc fragment may include all or part of a hinge region peptide in the heavy chain constant region. In addition, as long as it has substantially the same or improved effect with the native type, it may be an extended Fc fragment including part or all of the heavy chain constant region 1 (CH1) and/or the light chain constant region 1 (CL1). Further, it may be a fragment from which some considerably long amino acid sequences corresponding to CH2 and/or CH3 have been removed.

The'antibody Fc fragment' of the present invention is not limited in its kind and amino acid sequence as long as it is an antibody Fc fragment peptide known to those skilled in the art, and may be, for example, a polypeptide represented by SEQ ID NO: 2 (Fc fragment sequence of human IgG1) In addition, it may be a polypeptide represented by SEQ ID NO: 3 to which a hinge region is added to the sequence.

The term'antigen presenting cell' of the present invention is an antigen-induced event that functions primarily by internalizing antigens, processing antigens, and presenting antigenic epitopes to lymphocytes in the context of major histocompatibility complex (MHC) class I or II molecules. Refers to the helper cells of. The interaction between APC and antigen is an essential step in the induction of immunity because it allows lymphocytes to contact and recognize antigenic molecules and activate them. Exemplary APCs include macrophages, monocytes, Langerhans cells, interlocked dendritic cells, vesicular dendritic cells, and B cells.

The'antibody Fc fragment' of the present invention includes at least one or more antibody Fc fragment-derived CH2 and CH3 domains, and thus can bind to the Fc receptor on APC. The antibody Fc fragment has an Fc receptor binding site, and binds to an Fc receptor (FcγR, etc.) on APC at the Fc receptor binding site.

The VHSV glycoprotein and the Fc fragment of the antibody may be directly or indirectly linked by genetic fusion means. Accordingly, the VHSV glycoprotein antigen of the present invention may include the use of a linking molecule (linker) that connects the VHSV glycoprotein and the Fc fragment of the antibody. Illustrative linker molecules include leucine zippers, and biotin/avidin. For example, other linkers that can be used for the VHSV glycoprotein antigen are peptide sequences. Such peptide linkers are generally about 2 to about 40 amino acids (eg, about 4 to 10 amino acids) in length. Illustrative peptide linkers may include the amino acid sequence'SRPQGGGS'. Other linkers are known in the art and are generally rich in glycine and/or alanine, taking into account the flexibility between the regions to which they connect.

The VHSV glycoprotein antigen of the present invention may be monomeric or dimeric. US 8,465,745 for the dimeric chimeric antigen; US 8,029,803 and Korean Patent Registration No. 10-1054851 may be referred to.

The VHSV glycoprotein antigen of the present invention is preferably an antibody comprising (i) a VHSV glycoprotein (ii) an antibody hinge region and (iii) a CH2 domain and a CH3 domain from the N-terminal direction to the C-terminal direction. It may be a dimeric protein in which the Fc region is sequentially linked by peptide linkage.

In the step (a) of the present invention, in order to express the antigenic protein in a manner unique to the present invention that induces plant-specific multimerization, the above-described VHSV glycoprotein antigen is an endoplasmic reticulum storage induction sequence (or endoplasmic reticulum residual signal peptide, ER retention signal peptide) may additionally be included. The type of the endoplasmic reticulum storage induction sequence is not limited as long as it is a plant endoplasmic reticulum storage induction sequence known to those skilled in the art, and may be referred to WO 2009158716 and the following documents; Pagny et al., Signals and mechanisms for protein retention in the endoplasmic reticulum, Journal of Experimental Botany, Vol. 50, No. 331, pp. 157-64, February 1999.

The endoplasmic reticulum storage induction sequence of the present invention may be preferably KDEL (SEQ ID NO: 4), HDEL (SEQ ID NO: 5), and 1 to 5 amino acids added to the sequence (for example, SEKDEL of SEQ ID NO: 6, sequence KHDEL of Number 7, KEEL of SEQ ID NO: 8, SEHDEL of SEQ ID NO: 9, etc.), most preferably KDEL represented by SEQ ID NO: 4.

By inserting the nucleotide encoding the endoplasmic reticulum storage induction sequence into a specific gene (a chimeric antigen expression gene in the present invention), the endoplasmic reticulum storage induction sequence such as KDEL can be exposed at the end of the amino acid sequence of the final product. This induces the produced protein to be present in the endoplasmic reticulum within the transformed cell without being secreted outside the plant cell. The protein produced in the host cell into which the specific gene is introduced is stored in the endoplasmic reticulum by a sequence such as KDEL, and undergoes a post-translational modification that can be made in the plant. This plays an important role in solving the problem of increasing the expression level of antibody proteins in cells and the difference in sugar structures between heterogeneous species, which plays an important role in the immune response between heterogeneous species.

The site of insertion of the endoplasmic reticulum storage induction sequence (KDEL) is not limited as long as it does not affect the immunogenicity or antibody binding ability of the VHSV glycoprotein antigen according to the present invention. The insertion site of the endoplasmic reticulum storage induction sequence is not limited thereto, but may preferably be a C-terminal site of the target binding domain including the antibody Fc fragment.

The term'transformation' refers to a modification of the genotype of a host cell by introduction of an exogenous polynucleotide, and refers to the introduction of an exogenous polynucleotide into a host cell regardless of the method used for the transformation. The foreign polynucleotide introduced into the host cell may be integrated and maintained or not integrated into the genome of the host cell, and the present invention includes both.

The term'introduction' refers to an manipulation of artificially inserting a gene or group of genes into a cell that targets and expressing the group of genes or adding another gene (group) to the genome of the cell. The introduction of these genes is by means of transduction (bacteria) by bacteriophage, an indirect method via a soil bacterium Agrobacterium spp., genegun, electroporation, microinjection, etc. In addition to this, a person skilled in the art can selectively use a known gene introduction technique according to the characteristics of the target cell and the inserted gene.

Transformation in the step (a) of the present invention means introducing the polynucleotide encoding the VSVH glycoprotein antigen into plant cells, and in step (a) of the present invention, a transformed plant expressing a recombinant protein 'Preparation' may be performed by a known plant cell transformation method, but is not limited thereto, for example, a recombinant vector by inserting a desired gene (polynucleotide encoding a glycoprotein antigen) into a vector (vertor) It may be a method of constructing (or constructing), transforming the recombinant vector into a strain of genus Agrobacterium, and then infecting the strain into plant cells.

The vector generally comprises one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence, and is preferably an expression vector. The expression vector is a form of a vector capable of expressing a selected polynucleotide. A polynucleotide sequence is “operably linked” to the regulatory sequence when the regulatory sequence affects the expression (eg, level, timing or location of expression) of the polynucleotide sequence. . The regulatory sequence is a sequence that affects the expression (eg, level, timing or location of expression) of the nucleic acid to which it is operably linked. The regulatory sequence exerts its effect, for example, directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., the regulatory sequence and/or polypeptides that bind to the nucleic acid). Can be done. The regulatory sequences include promoters, enhancers and other expression control elements.

The standard recombinant DNA and molecular cloning techniques, which are molecular biology techniques known in the art, are described in the following documents (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2 ^nd ed ed ed. ., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989; by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1984; and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience, 1987).

After stable transformation, the transformed plants are then propagated. This reproduction means increasing the number of plants. The propagation of the plant is not limited as long as the characteristics of the regenerated plant and the characteristics expressed by the parent transgenic plant are the same, but may preferably be micro-proliferation. The microproliferation is a method of growing second generation plants from a single tissue sample cut from a selected parent plant or cultivar. This method enables mass reproduction of plants that have a desired tissue and express the desired protein. The newly created plant is genetically identical to the original plant and has all of the characteristics of the original plant. Microproliferation enables mass production of superior plant material in a short period of time and enables rapid proliferation of selected crops while preserving the characteristics of the original transgenic or transgenic plant. Advantages of plant cloning methods include the rapidity of plant growth and the superiority and uniformity of the resulting plants.

The plant is not limited as long as the'plant' is a plant into which foreign genes can be introduced, but for example, monocotyledons such as rice, wheat, barley, bamboo shoots, corn, taro, asparagus, onion, garlic, green onion, leek, soothing, There are hemp and ginger. Examples of dicotyledonous plants include, but are not limited to, Arabidopsis, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, parsley, parsley, Chinese cabbage, cabbage, radish, It can be watermelon, melon, cucumber pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, buzzfoot trefoil, potato, duckweed, perilla, pigeon bean and pea. Preferably garlic, broccoli, tobacco, Arabidopsis, Chinese cabbage, cabbage, onion, green onion, zucchini, radish, carrot. It may be a cucumber, and most preferably it may be tobacco ( Nicotiana tabacum ). Tobacco has a high protein content, an easy cultivation method and an advantage of obtaining a large amount of biomass.

In the step (b),'obtaining a protein from a plant' may be performed by a known plant cell-derived protein obtaining method, but is not limited thereto, but, for example, by crushing and crushing the mated plant, an extraction buffer (buffer) ) May be a method of homogenizing. The extraction buffer may be a known plant protein extraction buffer, but is not limited thereto, but may be, for example, phosphate buffered saline (PBS), or Tris-HCl pH 8, dithiothreitol (DTT ), protease inhibitors (e.g. aprotinin, pepstatin, leupeptine, phenyl methyl sulphonyl fluoride) and [(N-(N-(L -3-trans-carboxyoxylane (carboxyoxirane)-2-carbonyl)-L lucil)-agmantine] and the like.

In the step (b), the timing of obtaining the protein component from the transgenic plant produced in the step (a) may be preferably the anterior leaf phase to the maturation phase of the plant. Through such collection at a specific time, it is possible to mass-produce the immunogenic multimeric antigen protein of the present invention with higher efficiency, and to obtain a target protein (immunogenic multimeric antigen protein) having excellent effects.

The obtained period is the adult period of flowering of the tobacco plant, and may preferably be about 14 weeks.

In the present invention, a protein purification process (step c) may be further included after step (b). The'protein purification' may be purified in a conventional manner, for example, salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.), dialysis, gel Techniques such as filtration, ion exchange, column chromatography such as reverse phase column chromatography and ultrafiltration may be applied alone or in combination (Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc. , San Diego, CA (1990)). By the protein purification process, only the immunogenic complex protein of the present invention (ie, the large quaternary structure antigen-antibody complex) can be obtained at a high concentration.

The VHSV glycoprotein antigen of the present invention produced according to the above method exhibits remarkably improved immunogenicity when compared to the VHSV glycoprotein antigen produced in animal cells, etc., which is affected by the glycosylation property of the glycoprotein antigen derived from the plant. Originated.

The present invention also provides a vaccine composition for viral hemorrhagic sepsis of flounder comprising the VHSV glycoprotein antigen.

In the present invention, the term'vaccine' or'vaccine composition' refers to a composition that stimulates an immune response, and has the same meaning as an immunogenic composition and is used interchangeably herein. The vaccine includes both prophylactic and therapeutic vaccines. Prophylactic vaccines induce an immune response prior to exposure to a substance containing the antigen, thus increasing the ability to resist an antigen-carrying substance or cell, in order to induce a greater immune response when an individual is exposed to the antigen. Means that. Therapeutic vaccine is used by administration to an individual who already has a disease associated with the antigen of the vaccine. The therapeutic vaccine provides an increased ability to fight antigen-carrying diseases or cells, thereby increasing the individual's immune response to the antigen. Can increase. Preferably, the vaccine composition in the present invention may be a preventive vaccine.

The vaccine composition of the present invention may be administered alone or may be administered in combination with a known compound having an effect of preventing and treating a target disease. In addition, the vaccine composition of the present invention is characterized by comprising the immunogenic complex protein, and may further contain a pharmaceutically acceptable carrier, excipient, or diluent.

The term "carrier" refers to a substance that facilitates addition of a compound into cells or tissues. The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery substances used for oral administration of the peptide preparation. In addition, the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and formulations may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

In addition, the vaccine composition comprising the immunogenic complex protein of the present invention can be administered to an individual in need thereof in an effective amount and used for immunization.

The individual may preferably be a flounder.

The'immunization' is that when the VHSV glycoprotein antigen according to the present invention is administered to an individual, secretory, humoral and/or cellular immune responses to the VHSV glycoprotein antigen are induced in the individual, Through this immunization, a preventive or therapeutic effect on VHS is shown.

The'effective amount' is an amount showing a preventive or therapeutic effect on the target disease of the vaccine composition of the present invention, and secretory, humoral and/or cellular immune responses to the immunogenic complex protein of the present invention in the administered individual It means an amount sufficient to induce.

The total effective amount of the protein of the present invention may be administered to an individual as a single dose, and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time. In addition, the content of the active ingredient may be varied depending on the purpose of administration. The effective dose is for each individual in consideration of various factors such as the type and severity of the target disease, the route of administration and the number of administrations, as well as the age, weight, health status, sex, severity of the disease, diet and excretion rate of the subject in need of administration. Since the effective dosage is determined, those skilled in the art will be able to determine the appropriate effective dosage according to the purpose of administration. It can also be determined by monitoring the efficacy of the therapy using an assay that determines the activity of immune cells after administration of the protein according to the present invention or by using a well-known in vivo assay. The pharmaceutical composition of the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

On the other hand, the vaccine of the present invention is preferably inoculated by injection when the size of the flounder is 13 to 17 cm. The immersion method is known as a vaccination method, but the immersion method shows lower efficiency than the injection method in terms of increased expression of immune-related genes and mortality. In addition, it is advantageous to inoculate the vaccine of the present invention at a high temperature (eg 18 to 22°C) compared to a low temperature (eg 10 to 14°C) during treatment. Treatment at low temperatures shows lower efficiency compared to treatment at high temperatures in terms of increased expression of immune-related genes and mortality. Most preferably, the vaccine of the present invention can be inoculated by high temperature/injection.

The present invention also includes the step of transforming a plant cell with a recombinant vector designed to express an antigen containing a glycoprotein of a viral hemorrhagic sepsis virus and an Fc antibody fragment of an antibody. It provides a method for producing a transgenic plant that produces a sepsis virus glycoprotein antigen.

A detailed description of the method for producing a transgenic plant that produces the viral hemorrhagic sepsis virus glycoprotein antigen may be referred to the foregoing.

In the preparation method of the present invention, the antigen may further include an endoplasmic reticulum storage inducing sequence, which may be referred to as described above.

The present invention also provides a plant that produces a viral hemorrhagic sepsis virus glycoprotein antigen prepared by the above method.

In the present invention, it should be understood that the plant includes a passaged plant derived from the plant, cells and seeds obtained from the plant.

For the plant, reference may be made to the foregoing.

The viral hemorrhagic sepsis virus glycoprotein antigen provided by the present invention induces a very excellent immunization reaction in flounder, so that the desired antibody can be produced in an individual. According to the method provided by the present invention, viral hemorrhagic activity in plants Sepsis virus glycoprotein antigen can be produced economically and safely.

Figure 1 is a plant expression vector pBI121 vector endoplasmic reticulum retention signal peptide endoplasmic reticulum retention signal peptide in an Fc-fusion protein in which a glycoprotein (indicated as VHSVG) and an IgG Fc fragment are fused to a viral hemorrhagic sepsis virus antigen of flounder. , KDEL) was added and shown in a schematic diagram (collectively expressed as'pBI VHSVG-FcK') (A), and the protein structure and sugar structure (B) produced therefrom are shown.
2 is a series of procedures for obtaining a transgenic plant expressing the viral hemorrhagic sepsis virus vaccine protein (VHSVG-FcK) of flounder.
3A is a viral hemorrhagic sepsis virus vaccine of flounder through polymerase chain reaction (PCR) obtained by obtaining genomic DNA from a plant obtained by transformation using a plant expression vector pBI VHSVG-FcK It was confirmed whether the gene was inserted or not.
3B is a Western blot confirming the expression of the viral hemorrhagic sepsis vaccine protein (VHSVG-FcK) of flounder, the target protein, using a plant obtained by transformation using the plant expression vector pBI VHSVG-FcK. will be.
4A is a SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) for a protein purified from biomass obtained by mass-producing tobacco plants expressing the viral hemorrhagic sepsis virus vaccine protein (VHSVG-FcK) of flounder. It was confirmed through.
4B is a result of confirming the purified flounder viral hemorrhagic sepsis virus vaccine protein (VHSVG-FcK) by Western blot using an anti-VHSV antibody.
4C is a result of confirming the purified flounder viral hemorrhagic sepsis virus vaccine protein (VHSVG-FcK) by Western blot using an anti-IgG Fc antibody.

5A is a schematic diagram of a series of experiments for verifying the immunogenic effect of the protein by immunizing mice with a viral hemorrhagic sepsis virus vaccine protein of flounder.
5B is a result of confirming the binding affinity of antibodies in serum obtained from mice immunized with a viral hemorrhagic sepsis virus vaccine protein (VHSVG-FcK) of flounder through an enzyme-linked immunosorbent assay. to be.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are for illustrative purposes only, and the present invention is not limited thereto.

<Example 1>

Preparation of vector for expression of VHSV vaccine in tobacco plant expression system

VHSVG by adding an endoplasmic reticulum retention signal peptide (KDEL) to an Fc-fusion protein in which VHSVG, a glycoprotein of the viral hemorrhagic sepsis virus of flounder and human IgG1 Fc fragment, are fused to a plant expression vector (pBI121 vector). It was constructed to express -FcK gene. The gene was ligated so that it could be placed and operated together with the cauliflower mosaic virus 35S promoter (labeled E/35S-P) and an AMV translation enhancer. The schematic diagram of pBI VHSVG-FcK is as shown in Fig. 1A. Fig. 1B shows an expected schematic diagram of a protein to be expressed through the vector and a schematic diagram of an expected sugar structure.

In addition, in the protein to be expressed through the vector, VHSV consists of the amino acid sequence represented by SEQ ID NO: 1, the human IgG1 Fc fragment fused with VHSV is represented by SEQ ID NO: 3, and the endoplasmic reticulum storage induction sequence is represented by SEQ ID NO: 4. It could be expected to consist of an amino acid sequence.

<Example 2>

Tobacco Plant Transformation for VHSV Vaccine Production

The pBI VHSVG-FcK vector expressing the VHSV vaccine constructed in <Example 1> was introduced using the Agrobacterium transformation method. Specifically, Agrobacterium tumefaciens LBA4404 was used as the strain, and the vector was introduced into the strain through electroporation using MicroPulser Electroporator (Bio-Rad, Hercules, CA). Agrobacterium- mediated plant transformation was performed using Agrobacterium into which the vector was introduced, and at this time, the plant used was a tobacco plant ( Nicotiana tabacum ). The transformed tobacco plant leaves induce callus in a regeneration media containing 100 mg/L kanamycin antibiotic and 250 mg/L cefotaxime antibiotic. The shoots derived from callus were transferred to the magenta vessel GA-7 (Sigma, St. Louis, MO), and then the roots and stems were induced, and the processes are as shown in FIG. 2.

<Example 3>

Confirmation of VHSV vaccine gene insertion in transgenic tobacco plants

After extracting genomic DNA from the leaves of the transgenic plant obtained in Example 2, insertion of the VHSV vaccine gene was confirmed through polymerase chain reaction (PCR).

Specifically, the leaves of the transgenic plant expressing the VHSV vaccine protein were transferred to about 100 mg or so, frozen with liquid nitrogen, and pulverized. The plant was separated genomic DNA using a DNA extraction kit (iNtRON Biotechnology, Seoul, Korea). Using the isolated genomic DNA as a template, PCR was performed using a pair of primers of SEQ ID NO: 10 and SEQ ID NO: 11.

-F: 5'-GTCGACATGGAATGGAATACTTTTTC-3' (SEQ ID NO: 10)

-R: 5'-GCCAAATGTTTGAACGATCGG-3' (SEQ ID NO: 11)

PCR was carried out under conditions of reacting at 94°C for 2 minutes, followed by reaction at 94°C for 20 seconds, 59°C for 30 seconds, and 72°C for 2 minutes 30 times, and then at 72°C for 5 minutes. The amplified PCR product was confirmed by electrophoresis on a 1% agarose gel.

As a result, as shown in Fig. 3A, it is seen that the gene band appears on 2,286 bp in the transgenic plants (marked as T807, T808, T809, T810, T814, T815, T816, T817), the VHSVG-FcK gene is inserted I confirmed that it was done.

<Example 4>

Confirmation of VHSV Vaccine Protein Expression in Transgenic Tobacco Plants

The expression of the VHSV vaccine protein was confirmed by Western blot analysis using the leaves of the transgenic plant obtained in <Example 2>.

Specifically, after collecting about 100 mg of the leaves of the transgenic plant, 300 μl of 1X PBS (137 mM NaCl, 10 mM Na ₂ HPO ₄ , 2.7 mM KCl, and 2 mM KH ₂₎ corresponding to a volume of 3 times the weight. PO ₄ ) was added and then crushed. Mix 16 μl of the pulverized sample and 4 μl of 5X sample buffer (1 M Tris-HCl, 50% glycerol, 10% SDS, 5% 2-mercaptoethanol, and 0.1% bromophenol blue), boil at 100°C for 10 minutes, and then 10% Electrophoresis was performed by loading 20 μl on the SDS-PAGE gel. Transfer the SDS-PAGE gel to a nitrocellulose membrane (NC membrane) at 100 V for 1 hour, and use 5% skim milk (Sigma, St. Louis, MO) for 1 hour and 30 minutes. Blocking was carried out. As the antibody, goat anti-human IgG, Fcγ fragment antibody (Jackson ImmunoResearch, West Grove, PA) to which horseradish peroxidase (HRP) is bound was used, and this was diluted in a ratio of 1:5,000. Treated for 2 hours. After that, after washing 4 times for 10 minutes each using a solution containing 0.5% Tween 20 in 1X TBS (Tris-buffered saline), the film was reacted with chemiluminescence substrate (Bio-Rad, Hercules, CA). It was observed by sensitization.

As a result, as shown in FIG. 3B, it is seen that a band appears at a position of 70 kDa or more in the leaves of the transgenic plants (marked as T807, T808, T809, T810, T814, T815, T816, T817), VHSV vaccine It was confirmed that the protein was expressed.

<Example 5>

Purification of VHSV vaccine protein from tobacco plants obtained from mass production

A plant expressing the VHSV vaccine protein selected in Example 4 was mass-produced and protein purification was performed from the obtained tobacco plants.

Specifically, plants expressing the VHSV vaccine protein were mass-produced in a green house as shown in FIG. 2 to harvest and secure large amounts of leaves among biomass.

250 g of plant leaves expressing the VHSV vaccine protein were pulverized in 750 ml of the extraction solution (37.5 mM Tris-HCl pH 7.5, 50 mM NaCl, 15 mM EDTA, 75 mM sodium citrate, and 0.2% sodium thiosulfate). After centrifugation at 4°C for 30 minutes at 8,800xg to recover the supernatant, the pH of the solution was adjusted to 5.1 using acetic acid. Thereafter, the solution was centrifuged at 4° C. and 10,200xg for 30 minutes to obtain a supernatant, and the pH was adjusted to 7.0 using 3M Tris. After that, the solution was precipitated to become an 8% saturated solution using ammonium sulfate, and the solution was stirred at 4° C. for 2 hours. The supernatant was recovered by centrifugation at 4°C for 30 minutes at 8,800xg, precipitated to a 22.6% saturated solution with ammonium sulfate, and stirred at 4°C for 14 hours or longer. Then, centrifugation was performed at 4°C for 30 minutes at 8,800xg, and the precipitate was resuspended in an extraction solution of 1/10 the volume of the supernatant, and the supernatant was recovered by centrifugation at 4°C for 30 minutes at 10,200xg. . The obtained solution was purified using protein A Sepharose 4 Fast Flow (GE Healthcare, Sweden, NJ), and the purified sample was confirmed through SDS-PAGE.

As a result, as shown in Fig. 4A, when purified using the leaves of a plant expressing the VHSV vaccine protein, samples 1 and 2 of the purified samples (indicated as 1, 2, 3, 4, 5) At the position of 70 kDa or more, the target protein band could be observed. On the other hand, the target protein band could not be identified in the sample (column through, indicated as CT) that passed through the column.

<Example 6>

Confirmation of VHSV vaccine protein purified from tobacco plants

Western blot was performed to secondly verify that the sample purified in Example 5 was a VHSV vaccine protein.

Specifically, the purified sample was subjected to SDS-PAGE, blotting, and blocking processes in the same manner as in the method presented in <Example 4>. As the primary antibody of FIG. 4B, rabbit anti-VHSV antibody (Amsbio, Abingdon, UK) was diluted at a ratio of 1:1,000 and treated for 2 hours, and the secondary antibody was combined with HRP. Anti-rabbit IgG (H+L) antibody (Bethyl, Montgomery, TX) was diluted in a ratio of 1:5,000 and treated for 1 hour and 30 minutes. As the antibody of FIG. 4C, goat anti-human IgG and Fcγ fragment antibody (Jackson ImmunoResearch, West Grove, PA) bound to HRP were diluted at a ratio of 1:5,000 and treated for 2 hours.

As a result, as shown in FIGS. 4B and 4C, two antibodies respectively confirming the VHSVG portion and the Fc portion of a sample purified from a plant expressing the VHSV vaccine protein (labeled as 1, 2, 3, 4, 5) When treated with, all of the target protein bands could be observed at positions of 70 kDa or more.

<Example 7>

VHSV vaccine protein antigenicity verification

In order to verify the antigenic effect of the VHSV vaccine protein (VHSVG-FcK) purified from <Example 5>, an experiment was performed to immunize mice.

Specifically, for the experimental design, a plant-derived VHSV vaccine protein (VHSVG-FcK) was prepared as an experimental group, and 1X PBS was prepared as a control group. This was injected into 8-week-old BALB/c female rats by intraperitoneal injection, and when administered once, the amount of the sample was mixed with aluminum hydroxide, an immunostimulant, in a certain ratio, so that the protein concentration was contained within a total volume of 300 μl. I did. A total of 3 injections were performed at 14-day intervals, the first blood collection was performed 10 days after the second injection, and the second blood collection was performed 14 days after the first blood collection. After separating the serum from each blood, it was stored at -70°C. The experimental design is as shown in Fig. 5A.

<Example 8>

Confirmation of binding affinity of antibodies in serum (ELISA)

Enzyme-linked assay (Enzyme-linked) to confirm the binding affinity of antibodies in serum obtained from mice immunized with the viral hemorrhagic sepsis virus vaccine protein (VHSVG-FcK) of flounder in Example 7 above. immunosorbent assay, ELISA).

Specifically, plant-derived VHSVG vaccine protein (VHSVG-FcK) was mixed with 0.2 M sodium carbonate/sodium bicarbonate solution, and then coated on a 96 well plate (Nunc, Rochester, NY) to a concentration of 100 ng, Placed at 4° C. for 16 hours. After washing 4 times using a solution containing 0.5% Tween 20 in 1X PBS (Phosphate-buffered saline), blocking was performed at room temperature for 2 hours with 5% skim milk solution. . As the primary antibody, the serum obtained in <Example 7> was diluted 2 times from 1 μl and treated at room temperature for 2 hours, and the secondary antibody was an anti-mouse IgG Fc antibody (Jackson ImmunoResearch, West Grove) conjugated with HRP. , PA) was diluted in a ratio of 1:5,000 and treated for 2 hours. After washing 4 times using a solution containing 0.5% Tween 20 in 1X PBS, a TMB substrate solution (Seracare, Milford, MA) was treated for 5 minutes, and a TMB stop solution (Seracare, Milford, MA) was added to 450 nm. Absorbance was measured at.

As a result, as shown in Figure 5B, the first serum obtained by injection of the plant-derived VHSV vaccine protein (VHSVG-FcK) and the group treated with the second serum showed high absorbance in a concentration-dependent manner, but by injecting 1X PBS The obtained first serum and the second serum treated group showed remarkably low absorbance. Through this, it was confirmed that the anti-VHSVG-FcK antibody against the plant-derived VHSV vaccine protein (VHSVG-FcK) was generated in the serum in the experimental group, and at the same time, it was confirmed that it had binding capacity.

The viral hemorrhagic sepsis virus glycoprotein antigen provided by the present invention induces a very excellent immunization reaction in flounder, so that the desired antibody can be produced in an individual. According to the method provided by the present invention, viral hemorrhagic activity in plants The sepsis virus glycoprotein antigen can be produced economically and safely, so it has high industrial applicability.

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> Viral hemorrhagic septicemia virus glycoprotein antigen obtained from transgenic plants and vaccine comprising the same <130> NP19-0064 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 507 <212> PRT <213> Artificial Sequence <220> <223> Viral hemorrhagic septicemia virus glycoprotein (VHSVG) <400> 1 Met Glu Trp Asn Thr Phe Ser Leu Val Ile Leu Val Ile Ile Ile Lys 1 5 10 15 Ser Thr Thr Ser Gln Ile Thr Gln Arg Pro Pro Val Glu Asn Ile Ser 20 25 30 Thr Tyr His Val Asp Trp Asp Thr Pro Leu Tyr Thr His Pro Ser Asn 35 40 45 Cys Arg Lys Asn Ser Phe Val Pro Ile Arg Pro Asp Gln Leu Arg Cys 50 55 60 Pro His Glu Phe Glu Asp Thr Asn Lys Gly Leu Val Ser Val Pro Ala 65 70 75 80 Gln Ile Ile His Leu Pro Leu Ser Val Thr Ser Val Ser Ala Val Ala 85 90 95 Asn Gly His Tyr Leu His Arg Val Thr Tyr Arg Val Thr Cys Ser Thr 100 105 110 Asn Phe Phe Gly Gly Gln Thr Ile Glu Lys Thr Ile Leu Glu Ala Lys 115 120 125 Leu Ser Arg Gln Glu Ala Ile Asn Glu Ala Gly Lys Asp His Glu Tyr 130 135 140 Pro Phe Phe Pro Glu Pro Ser Cys Ile Trp Ile Lys Asp Asn Val His 145 150 155 160 Lys Asp Ile Thr His Tyr Tyr Lys Thr Pro Lys Thr Val Ser Ile Asp 165 170 175 Leu Tyr Ser Arg Lys Phe Leu Asn Pro Asp Phe Ile Glu Gly Val Cys 180 185 190 Thr Thr Ser Pro Cys Pro Thr His Trp Gln Gly Val Tyr Trp Ile Gly 195 200 205 Ala Thr Pro Gln Ala His Cys Pro Thr Ser Glu Thr Leu Gln Gly His 210 215 220 Leu Phe Thr Arg Thr His Asp His Arg Val Val Lys Ala Ile Val Ala 225 230 235 240 Gly His His Pro Trp Gly Leu Thr Met Ala Cys Lys Val Thr Phe Cys 245 250 255 Gly Thr Glu Trp Ile Lys Thr Asp Leu Gly Asp Leu Ile Gln Val Thr 260 265 270 Gly Gln Gly Gly Ala Asn Lys Leu Ser Pro Lys Lys Cys Val Asn Thr 275 280 285 Asp Val Gln Met Arg Gly Ala Thr Asp Asp Phe Ser Tyr Leu Asn His 290 295 300 Leu Ile Thr Asn Met Ala Gln Arg Thr Glu Cys Leu Asp Ala His Ser 305 310 315 320 Asp Ile Thr Ala Ser Gly Lys Ile Ser Pro Phe Leu Leu Ser Lys Phe 325 330 335 Arg Pro Ser His Pro Gly Pro Gly Lys Ala His Tyr Leu Leu Asp Gly 340 345 350 Gln Ile Met Arg Gly Glu Cys Asp Tyr Glu Ala Val Val Ser Ile Asn 355 360 365 Tyr Asn Ser Ala Gln Tyr Lys Thr Val Asn Asn Ile Trp Lys Ser Trp 370 375 380 Asn Arg Ile Asp Asn Asn Thr Asp Gly Tyr Asp Gly Met Ile Phe Gly 385 390 395 400 Asp Lys Leu Ile Ile Pro Asp Ile Glu Lys Tyr Gln Ser Ile Tyr Asp 405 410 415 Ser Gly Met Leu Val Gln Arg Asn Leu Val Gly Ile Pro His Pro Ser 420 425 430 Ile Val Phe Val Ser Asn Thr Ser Asp Leu Ser Thr Asn Tyr Ile His 435 440 445 Thr Asn Leu Ile Pro Ser Asp Trp Ser Phe Asn Trp Ser Leu Trp Pro 450 455 460 Ser Leu Ser Gly Met Gly Val Val Gly Gly Ala Phe Leu Leu Leu Val 465 470 475 480 Leu Cys Cys Cys Cys Arg Ala Ser Pro Pro Pro Pro Ser Tyr Gly Ile 485 490 495 Pro Met Gln Gln Phe Ser Arg Ser Gln Thr Val 500 505 <210> 2 <211> 192 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 Fc region <400> 2 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 1 5 10 15 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 20 25 30 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 35 40 45 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 50 55 60 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 65 70 75 80 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 85 90 95 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 100 105 110 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 115 120 125 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 130 135 140 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 145 150 155 160 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 165 170 175 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 180 185 190 <210> 3 <211> 237 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 Fc region with hinge <400> 3 Ala Ala Ala Asp Leu Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 1 5 10 15 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 20 25 30 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 35 40 45 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 50 55 60 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 65 70 75 80 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 85 90 95 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 100 105 110 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 115 120 125 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 130 135 140 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 145 150 155 160 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 165 170 175 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 180 185 190 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 195 200 205 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 210 215 220 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 225 230 235 <210> 4 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide for inducing endoplasmic reticulum storage <400> 4 Lys Asp Glu Leu One <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide for inducing endoplasmic reticulum storage <400> 5 His Asp Glu Leu One <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide for inducing endoplasmic reticulum storage <400> 6 Ser Glu Lys Asp Glu Leu 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Peptide for inducing endoplasmic reticulum storage <400> 7 Lys His Asp Glu Leu 1 5 <210> 8 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide for inducing endoplasmic reticulum storage <400> 8 Lys Glu Glu Leu One <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide for inducing endoplasmic reticulum storage <400> 9 Ser Glu His Asp Glu Leu 1 5 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for VHSVG <400> 10 gtcgacatgg aatggaatac tttttc 26 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for VHSVG <400> 11 gccaaatgtt tgaacgatcg g 21

Claims (11)

(a) preparing a transgenic plant expressing a recombinant protein including a glycoprotein of a viral hemorrhagic sepsis virus and an Fc antibody fragment; And
(b) A viral hemorrhagic sepsis virus glycoprotein antigen prepared by a method comprising the step of obtaining a protein using the transgenic plant.
The viral hemorrhagic sepsis virus glycoprotein antigen according to claim 1, wherein the immunogenic antigen protein further comprises an endoplasmic reticulum storage inducing sequence.
The viral hemorrhagic sepsis virus glycoprotein antigen according to claim 1, wherein the viral hemorrhagic sepsis virus glycoprotein further comprises an immunoglobulin hinge region, a heavy chain CH1 domain, or a linker.
The viral hemorrhagic sepsis virus glycoprotein antigen according to claim 1, wherein the antibody Fc fragment comprises an IgG hinge region, a CH2 domain, and a CH3 domain specific for a glycoprotein of a viral hemorrhagic sepsis virus.
The viral hemorrhagic sepsis virus glycoprotein antigen according to claim 1, wherein the glycoprotein of the viral hemorrhagic sepsis virus comprises the amino acid sequence of SEQ ID NO: 1.
The viral hemorrhagic sepsis virus glycoprotein antigen according to claim 1, wherein the antibody Fc fragment consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
The viral hemorrhagic sepsis virus glycoprotein antigen according to claim 1, wherein the plant is tobacco ( Nicotiana tabacum ).
The viral hemorrhagic sepsis vaccine composition of flounder comprising the viral hemorrhagic sepsis virus glycoprotein antigen of any one of claims 1 to 7.
Viral hemorrhagic sepsis virus glycoprotein comprising transforming plant cells with a recombinant vector designed to express an antigen containing a glycoprotein of a viral hemorrhagic sepsis virus and an Fc antibody fragment of an antibody Method for producing an antigen-producing transgenic plant.
The method of claim 9, wherein the antigen further comprises an antifoaming agent storage inducing sequence.
A plant producing a viral hemorrhagic sepsis virus glycoprotein antigen prepared by the method of claim 9 or 10.

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