KR101732624B1 - Vaccine composition for classical swine fever from plant and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a recombinant vector for producing plant-derived porcine fever antigen pmE2 protein, a plant transformed with said recombinant vector, a plant-derived porcine fever antigen pmE2 protein expressed in said plant, and uses thereof. A polynucleotide encoding the GP55 protein of the swine fever virus according to the present invention; A recombinant vector comprising a polynucleotide encoding a cellulosic binding domain protein; And plant-derived porcine fever antigen pmE2 protein can be produced with high efficiency when the plant transformed with the recombinant vector is used, and safety and stability are more advantageous than other production methods. In addition, since the plant-derived porcine heat antigen protein pmE2 contains a cellulose-binding domain (CBD) protein, it can be usefully used as an effective marker for discriminating the exposure pathway and antibody production pathway of swine fever virus .

Description

FIELD OF THE INVENTION [0001] The present invention relates to a plant-derived pig fever vaccine composition and a method for producing the same,

The present invention relates to a recombinant vector for producing plant-derived porcine fever antigen pmE2 protein, a plant transformed with said recombinant vector, a plant-derived porcine fever antigen pmE2 protein expressed in said plant, and uses thereof.

Swine fever is a disease caused by classical swine fever virus (CSFV), a disease that does not infect humans or other animals, but is a type of epidemic that can not be cured if swine occurs and most of the infected people die (Penrith, M (2011), Classical Swine Fever (Hog Cholera): Review of Aspects Relevant to Control, Transboundary and Emerging Diseases, 58: 187-196). Swine fever is classified as a Class A disease in the World Animal Health Organization (OIE) and is classified as a Type 1 livestock infectious disease in the domestic livestock epidemic prevention law. It is highly infectious to pigs and has a high mortality rate. And infected with acute, subacute or chronic. Thus, it is an epidemic that prevention is considered to be an important issue so that pigs fever is high in mortality and morbidity and can not guarantee the future of swine industry without eradication of swine fever. Swine fever is the only natural host in which swine fever is transmitted, and swine fever can be transmitted to all age-old pigs susceptible to the virus. In Korea, vaccination is being carried out with a live virus vaccine manufactured by the LOM strain, which is a purified virus infection virus, as a disease control measure. Since live pigs of swine fever can not be distinguished serologically from outdoor viruses, genetically modified vaccines such as marker vaccines are under development worldwide so as to distinguish antibodies against outdoor virus infection and vaccination. Korea is also focusing on the development of practical vaccines that can easily and accurately distinguish antibodies from outdoor infectious agents and vaccines in vaccine use.

On the other hand, the remarkable development of molecular biology and genetic engineering techniques has been applied to the field of plants, and efforts to produce useful physiologically active substances from plants are steadily continuing. The production of useful materials in plants has several advantages: 1) it can dramatically reduce production costs, and 2) it can be used in a variety of ways that can occur in conventional methods (such as the separation and purification of proteins from animal cells or microorganisms) It is also advantageous in that it can control seed stocks as seeds, unlike animal cells or microorganisms, at the commercialization stage. In addition, 4) If the demand of the material rapidly increases, it is absolutely advantageous compared with the existing animal cell system in terms of equipment technology and cost required for mass production, so that it is possible to supply according to the increased demand in the shortest period.

In this background, a method of transforming a plant and producing a useful substance therefrom has a protein synthesis pathway in a plant. Protein synthesis in mammals requires a post-translational modification process. Since plants have a synthesis pathway for eukaryotic proteins, they can produce proteins that are similar to those expressed in mammals. However, in spite of the above-mentioned various advantages, the technology to obtain useful physiologically active substances (medically useful proteins, vaccines and industrially valuable enzymes, etc.) from plants with high efficiency has not been so successful.

Accordingly, the present inventors have conducted studies to improve low efficiency in the production of proteins using plants, and have found that polynucleotides encoding the GP55 protein of swine fever virus; And a polynucleotide encoding a cellulose binding domain (CBD) protein, and using the transgenic plant transformed with the recombinant vector, a plant-derived porcine fever antigen pmE2 protein can be produced with high efficiency , It is confirmed that safety and stability are superior to other production methods in this case. In addition, the present invention has been accomplished by confirming that the above-mentioned pmE2 protein has an effect that can be used as a marker for discriminating the exposure pathway and antibody production pathway of swine fever virus.

An object of the present invention is to provide a recombinant vector for producing plant-derived porcine fever antigen pmE2 protein.

It is also an object of the present invention to provide a transgenic plant for producing a plant-derived porcine fever antigen pmE2 protein transformed with said recombinant vector.

It is also an object of the present invention to provide a plant-derived porcine fever antigen pmE2 protein expressed in said transgenic plant, and a production method thereof.

Also, an object of the present invention is to provide a vaccine for preventing swine fever comprising the pmE2 protein as an active ingredient, a pharmaceutical composition or a feed composition.

It is also an object of the present invention to provide a composition for diagnosing swine fever virus containing the pmE2 protein and a diagnostic kit.

It is also an object of the present invention to provide a method for preventing swine fever by administering the vaccine composition to an animal.

It is another object of the present invention to provide a method for detecting swine fever virus using the recombinant protein and a method for determining the pathogenesis of swine fever virus antibody.

In order to solve the above problems, the present invention provides a polynucleotide encoding a GP55 protein of swine fever virus; And a polynucleotide encoding a cellulosic binding domain protein. The recombinant vector for producing plant-derived porcine heat antigen pME2 protein is provided.

The present invention also provides a transgenic plant for producing a plant-derived porcine fever antigen pmE2 protein transformed with the recombinant vector.

It is also an object of the present invention to provide a plant-derived porcine fever antigen pmE2 protein expressed in said transgenic plant.

(A) transforming Agrobacterium with the recombinant vector; (b) introducing the transformed Agrobacterium into a plant; And (c) isolating and purifying the expressed plant-derived porcine fever antigen pmE2 protein from the plant, wherein the plant-derived porcine fever antigen pmE2 protein is produced.

Also, the present invention provides a vaccine for preventing swine fever comprising the pmE2 protein as an active ingredient, a pharmaceutical or feed composition.

The present invention also provides a composition for diagnosing swine fever virus containing the pmE2 protein and a diagnostic kit.

The present invention also provides a method for preventing swine fever by administering the vaccine composition to an animal.

Also, the present invention provides a method for detecting swine fever virus, characterized in that swine fever virus is detected in a sample through an antigen-antibody reaction using the pmE2 protein.

The present invention also relates to a method for producing a vaccine composition, comprising the steps of: 1) administering the vaccine composition to a specimen, and then collecting blood from the specimen; 2) separating the serum from the blood collected in the step 1); And 3) treating the plant-derived porcine fever antigen pmE2 protein with the antigen to the serum separated in the step 2), thereby reacting the porcine fever virus antibody production pathway.

A polynucleotide encoding the GP55 protein of the swine fever virus according to the present invention; A recombinant vector comprising a polynucleotide encoding a cellulosic binding domain protein; And plant-derived porcine fever antigen pmE2 protein can be produced with high efficiency when the plant transformed with the recombinant vector is used, and safety and stability are more advantageous than other production methods. In addition, since the plant-derived porcine fever antigen pmE2 protein comprises a cellulose-binding domain (CBD) protein, it can be usefully used as an effective marker for discriminating a pathway for virus exposure and an antibody-producing pathway .

Fig. 1 is a diagram showing a cleavage map of a recombinant vector for producing plant-derived porcine fever antigen pmE2 protein.
FIG. 2 shows Western blot analysis of expression of CBD protein in a plant transformed with a recombinant vector.
FIG. 3 is a diagram showing ELISA of the antigenicity of the protein expressed in the transformed plant.
FIG. 4 shows Western blot analysis of the antigenicity of the protein expressed in the transformed plant.
FIG. 5 shows the identification of the plant-derived porcine fever antigen protein pmE2 isolated and purified from the transgenic plant.
FIG. 6 is a graph showing the results of quantitative analysis of a plant-derived porcine fever antigen protein pmE2 isolated and purified from a transgenic plant.
FIG. 7 shows the results of the results obtained after administration of the plant-derived porcine fever antigen pmE2 protein to determine whether a specific antibody against the pig fever virus and a specific antibody against the CBD protein contained in the plant-derived porcine fever antigen protein pmE2 were formed in the obtained serum Fig.
Fig. 8 is a diagram showing antibody titers in a mouse to which a plant-derived swine fever antigen protein pmE2 was administered.
FIG. 9 is a diagram showing the result of confirming the infection pathway of swine fever virus using the plant-derived swine fever antigen protein pmE2.
FIG. 10 is a graph showing the results of confirming availability of the plant-derived porcine heat antigen protein pmE2 to determine the pathway of virus infection through the reaction between different antigens and immunized mouse sera.
Fig. 11 is a diagram showing the schematic contents of the experiment for protecting the plant-expressing proteins.
Fig. 12 is a diagram showing the results of body temperature change of an animal model (pig) after an attack inoculation.
FIG. 13 shows the results of leukocyte count changes in an animal model (pig) after an inoculation.
FIG. 14 is a diagram showing the result of detection of an antigen after blood sampling and sampling in an animal model (pig) after an attack inoculation. FIG.
FIG. 15 is a diagram showing the result of detection of an antigen after autopsy of an animal model (pig) after an attack inoculation. FIG.
FIG. 16 is a diagram showing the result of the test of the protective antibody of an animal model (pig) after an attack inoculation.

The present invention relates to a polynucleotide encoding a GP55 protein of a classical swine fever virus; And a polynucleotide encoding a cellulosic binding domain (CBD) protein. The recombinant vector for producing plant-derived porcine heat antigen pME2 protein is provided.

In the present invention, "expression vector" means a plasmid, virus or other medium known in the art to which a gene or polynucleotide can be inserted or introduced. The polynucleotide according to the present invention may be operably linked to an expression control sequence and the operably linked polynucleotide may be contained in an expression vector containing a selection marker and a replication origin . Said "operably linked" may be a gene and expression control sequence linked in such a way as to enable gene expression when a suitable molecule is coupled to an expression control sequence. "Expression control sequence" means a DNA sequence that regulates the expression of a polynucleotide sequence operably linked to a particular host cell. Such regulatory sequences include promoters for conducting transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation.

In the present invention, "classical swine fever virus" has an antigenic determinant in the E2 glycoprotein, which is a kind of envelope-associated glycoprotein (E protein) of about 12.3 to 12.5 kb in size belonging to pestivirus. Proteins are recognized as relatively important structural proteins. E2 protein of swine fever virus is known to cause virus neutralizing antibody reaction and play an important role in immunologically defense mechanism of swine fever.

The polynucleotide encoding the GP55 protein of the pig fever virus can be represented by SEQ ID NO: 1, and the polynucleotide encoding the cellulose binding domain protein can be represented by SEQ ID NO: 2, but is not limited thereto.

The recombinant vector may be a CaMV 35S promoter gene; 5 'UTR site gene of M17; A polynucleotide encoding a BiP (chaperone binding protein) protein; And a polynucleotide encoding an HDEL (His-Asp-Glu-Leu) protein, but the present invention is not limited thereto.

The 5 'UTR site gene of M17 may be composed of the nucleotide sequence shown in SEQ ID NO: 3, and the nucleotide encoding the BiP protein may be represented by SEQ ID NO: 4, but the present invention is not limited thereto.

The vector may be represented by the following vector map, and may include, but is not limited to, the nucleotide sequence shown in SEQ ID NO: 5.

In the present invention, the polynucleotide or gene consisting of the nucleotide sequence shown in SEQ ID NOS: 1 to 5 may be synthesized artificially by using a nucleic acid synthesizer or the like with reference to the nucleotide sequence of the gene, As a template and performing PCR using an oligonucleotide having a sequence complementary to both ends of the gene or a polynucleotide of interest as a primer. On the other hand, due to codon degeneracy, the polynucleotide or gene of the present invention may exist in various base sequences, all of which are included in the scope of the present invention. Also, variants of the nucleotide sequences shown in SEQ ID NOS: 1 to 5 are included in the scope of the present invention. Specifically, the polynucleotide or gene of the present invention preferably has at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% sequence identity with the nucleotide sequence of SEQ ID NO: And may include nucleotide sequences having homology with each other. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In the present invention, the GP55 protein is one of the E2 antigenic domains of CSFV (Classical Swine Fever Virus). In order to increase the protein synthesis amount of the 5 'UTR region gene of M17, the BiP protein uses a genomic DNA sequence Can be used to transfer proteins to the N-terminal endoplasmic reticulum. In addition, the CBD protein can be used as a hybrid protein tag, and the HDEL protein allows the protein to stay in the endoplasmic reticulum, thereby increasing the folding and assembly by the molecular chaperone, So that the final target protein can be accumulated in the endoplasmic reticulum.

[Degree]

As the vector, it is preferable to use a binary vector such as pCHF3, pPZP, pGA, and pCAMBIA. In one embodiment of the present invention, the pBI121 vector is used, but the present invention is not limited thereto.

The present invention also provides a plant transformed with the recombinant vector.

The method of introducing the recombinant vector of the present invention into a plant is not limited thereto, but a method of introducing Agrobacterium sp . ) - mediated method, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, or PEG (Polyethylenglycol) Can be used. In one embodiment of the invention, Agrobacterium sp . ) -Mediated method was introduced into the plant Arabidopsis thaliana with the recombinant vector of the present invention.

In the present invention, the plant transformed with the recombinant vector can be obtained by a conventional method in the art, such as an ovine reproduction method or an asexual reproduction method. More specifically, the plant of the present invention can be obtained through reproductive propagation, which is a process of producing seeds through the moisture process of flowers and propagating from the seeds. In addition, the plant can be transformed with the recombinant vector according to the present invention, and then can be obtained through the aseptic propagation method, which is a process of inducing callus, roots and soil purification according to a conventional method. That is, the transformed plant transformed with the recombinant vector according to the present invention is pelleted in a suitable medium known in the art, and cultured under appropriate conditions to induce callus formation. When shoots are formed, they are transferred to a hormone-free medium do. After about two weeks, the shoots are transferred to rooting medium to induce roots. The plants according to the invention can be obtained by roots being induced and then transplanted into the soil for purification. The transformed plant in the present invention may include tissues, cells or seeds obtainable therefrom.

In the present invention, the plant may be a dicotyledonous plant or a monocotyledonous plant. The dicotyledonous plants include, but are not limited to, Arabidopsis thaliana, soybean, tobacco, eggplant, pepper, potato, taro, cabbage, radish, cabbage, lettuce, Peanut, pear, strawberry, watermelon, melon, cucumber, carrot and celery, and the monocotyledonous plants include paddy rice, barley, wheat, rye, corn, sugarcane, oats and sheep paddies.

The present invention also provides a plant-derived porcine fever antigen pmE2 protein expressed in said transgenic plant.

The plant-derived porcine fever antigen pmE2 protein according to the present invention is not limited to the fusion of the GP55 protein and the cellulose-binding domain protein of swine fever virus.

In the present invention, the plant-derived porcine fever antigen pmE2 protein includes not only a protein having a native amino acid sequence thereof but also an amino acid sequence variant thereof, within the scope of the present invention. A variant of the plant-derived porcine fever antigen pmE2 protein refers to a protein having a sequence that differs by recombinant native amino acid sequence and deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art. The plant-derived porcine fever antigen pmE2 protein or a variant thereof may be isolated from natural sources or prepared by a recombinant method based on a DNA sequence (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) (Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd ed., 1989).

Further, according to the present invention,

(a) transforming Agrobacterium with the recombinant vector; (b) introducing the transformed Agrobacterium into a plant; And (c) isolating and purifying the expressed plant-derived porcine fever antigen pmE2 protein from the plant, wherein the plant-derived porcine fever antigen pmE2 protein is produced.

The plant may be selected from the group consisting of Arabidopsis, wheat, barley, corn, beans, potatoes, red beans, oats, sorghum, rice, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, Ginseng, tobacco, cotton, sesame seeds, sugar beet, perilla, peanut, rapeseed, apple tree, pear, jujube tree, peach, sheep, grape, citrus, persimmon, apricot, banana, rose, gladiolus, gerbera But may be at least one plant selected from the group consisting of carnation, chrysanthemum, lily, tulip, raglass, red clover, orchardgrass, alfapa, tall fescue and perennialla grass, Do not.

In the present invention, the method of separating and purifying at step (c) may be performed by a known method depending on the physical and chemical properties of the substance. For example, amorphous cellulose (AMC), distillation, Dialysis, pervaporation, chromatography, solvent extraction, reaction extraction, HPLC, and the like, preferably amorphous cellulose, but is not limited thereto.

In one embodiment of the invention, polynucleotides encoding the GP55 protein of swine fever virus; A polynucleotide encoding a cellulosic binding domain protein; CaMV 35S promoter gene; 5 'UTR site gene of M17; A polynucleotide encoding a BiP (chaperone binding protein) protein; And a polynucleotide encoding HDEL (His-Asp-Glu-Leu) protein. The recombinant vector thus prepared was transformed into Arabidopsis thaliana using Agrobacterium, and then the transformed plant was cultured to obtain a plant-derived porcine fever antigen pmE2 protein in which the GP55 protein and the cellulase-binding domain protein of porcine fever virus were fused Was isolated and purified. The plant-derived porcine fever antigen pmE2 protein was expressed at a high level in the transgenic plants and was mass-producible, had immunogenicity against swine fever virus, exhibited a very high potency, Respectively. In addition, it was confirmed that the PME2 protein can be used as a marker for determining the route of virus exposure according to the detection of the CBD protein contained therein. In addition, when the above-mentioned pmE2 protein was inoculated into an animal model, it was confirmed that it was effective in defense against swine fever virus, suppressed virus amplification and propagation, and exhibited excellent defensive ability.

In addition, the present invention provides a vaccine composition or a pharmaceutical composition for preventing swine fever comprising the pmE2 protein as an active ingredient.

The term "vaccine " in the present invention refers to a biological agent containing an antigenic substance that immunizes a living body, and refers to an immunogen that causes immunity to a living body by injection or injection into an organism for prevention of swine fever.

The vaccine composition of the present invention may further comprise an adjuvant in addition to the pmE2 protein. Such adjuvants may be used without limitation as long as they are known in the art, but may include, for example, Freund's complete or incomplete adjuvant to increase its immunity.

The vaccine composition or the pharmaceutical composition according to the present invention can be prepared by incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers for use in the vaccine compositions and pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The vaccine composition or the pharmaceutical composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions, Can be used.

In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin And the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweetening agents, fragrances and preservatives . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Propellants, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of suppositories, witepsol, tween 61, cacao paper, laurin, and glycerogelatin can be used.

The vaccine composition or pharmaceutical composition according to the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dose of the vaccine composition or the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the individual. The amount necessary to induce an immunoprotective response in an individual without adverse side effects may vary depending upon the optional presence of the recombinant protein and excipient used as immunogen. Generally, each dose contains from 0.1 to 1000 [mu] g of protein, preferably from 0.1 to 100 [mu] g per mL of sterilized solution of the recombinant protein of the present invention. In the case of a vaccine composition, antigen stimulation may be optionally repeated in addition to the initial dose as necessary.

The present invention also provides a feed composition for preventing swine fever comprising the pmE2 protein as an active ingredient.

The feeds include, but are not limited to, by-products such as pork, beef, and chicken, as well as corn, rice, common straw, grass, herb, anchor, hay, It is acceptable. Methods for adding an antibody to the pME2 protein of the present invention to such feeds and mixing them include mechanical mixing, adsorption, occlusion, and the like, but the present invention is not limited thereto.

The present invention also provides a composition for diagnosing swine fever virus comprising the pmE2 protein.

The present invention also provides a swine fever virus diagnostic kit comprising the pmE2 protein.

The diagnostic kit may be manufactured to include a reaction solution according to need, which can be easily performed by applying known techniques. The kit of the present invention may further include a reagent necessary for diagnosis of swine fever virus, and the reagent may be, for example, a buffer solution or the like without limitation. In addition, the kit may include a brochure. The manual is a printed document that explains how to use the kit, for example, how to make the buffer, the reaction conditions presented, and so on. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.

The present invention also provides a method for preventing swine fever by administering the vaccine composition to an animal.

The animal is preferably a mammal, more preferably a pig.

Also, the present invention provides a method for detecting swine fever virus, characterized in that swine fever virus is detected in a sample through an antigen-antibody reaction using the pmE2 protein.

The antigen-antibody reaction can be assayed by immunohistochemical staining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assays, immunodiffusion assays, (Complement Fixation Assay), Fluorescence-activated cell sorter (FACS) and protein chip analysis. However, the present invention is not limited thereto.

The sample may be at least one selected from the group consisting of cells, blood, urine, saliva, and tissues, but is not limited thereto.

In addition,

1) administering the vaccine composition to a specimen, and then collecting blood from the specimen; 2) separating the serum from the blood collected in the step 1); And 3) treating the plant-derived porcine fever antigen pmE2 protein with the antigen to the serum separated in the step 2), and reacting the same.

When the GP55 protein of swine fever virus and the antibody for the cellulose binding domain protein are detected together in the reaction of step 3), the antibody formed in the specimen can be determined to be generated by administration of the vaccine composition. In addition, when the antibody against the GP55 protein of the swine fever virus is detected in the reaction in the step 3), it can be determined that it is generated by the infection of the swine fever virus.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Example 1 Production of a recombinant vector containing a porcine fever virus antigen GP55 gene

In order to produce a recombinant vector containing the porcine fever antigen GP55 gene so as to produce a swine fever antigen protein with high efficiency in plants, the following experiment was conducted.

More specifically, as shown in Fig. 1, the CaMV 35S promoter gene in the pBI121 vector; 5 'UTR region gene of M17 (SEQ ID NO: 3); A polynucleotide encoding a BiP (chaperone binding protein) protein (SEQ ID NO: 4); A polynucleotide encoding the GP55 protein (SEQ ID NO: 1); A polynucleotide encoding a Cellulose Binding Domain (CBD) protein (SEQ ID NO: 2); And a polynucleotide encoding HDEL (His-Asp-Glu-Leu) protein.

The GP55 protein is one of the E2 antigenic domains of CSFV (Classical Swine Fever Virus). In order to increase the protein synthesis amount of the 5 'UTR region gene of M17, the BiP protein uses the genomic DNA sequence to amplify the N- Lt; / RTI > In addition, the CBD protein was used as a hybrid protein tag, and the HDEL protein allowed the protein to stay in the endoplasmic reticulum, thereby increasing the folding and assembly by the molecular chaperone to minimize the degradation of the protein , So that the final target protein can be accumulated in the endoplasmic reticulum. The nucleotide sequence of the prepared recombinant vector is shown in SEQ ID NO: 5.

Example  2. Antigenicity of swine fever virus GP55  Protein and CBD  Preparation of Transgenic Plants Expressing Fusion Proteins Containing Proteins

In order to prepare a transgenic plant expressing a fused recombinant protein comprising the antigenic GP55 protein and the CBD protein of swine fever virus, the following experiment was conducted.

More specifically, Arabidopsis thaliana expressing the fused recombinant protein containing the porcine fowl virus GP55 protein and the CBD protein was prepared by the Agrobacterium-mediated transformation method of the recombinant vector prepared in Example 1 above. Since the recombinant vector of Example 1 has resistance to kanamycin in plants, the selection of transformed Arabidopsis thaliana is performed by Western blotting using kanamycin resistance test and antibody against CBD, and then expression of the fusion recombinant protein Respectively. In addition, expression and expression levels of the target protein in the Arabidopsis thaliana are shown in Fig.

As shown in FIG. 2, it was confirmed that the CB55 protein was expressed in transgenic Arabidopsis thaliana through Western blot, and that the GP55 protein of the fused porcine virus was also expressed.

Example  3. Swine fever viruses produced in transgenic plants GP55  Identification of antigenicity of protein

In order to confirm the antigenicity of the swine fever virus GP55 protein in the fused recombinant protein containing the antigenic GP55 protein and the CBD protein of the swine fever virus derived from the transgenic plant prepared in Example 2, Respectively.

More specifically, the homozygous seeds with stable expression of the target protein were finally obtained through generation progression of the transgenic plants prepared in Example 2, and the expression level of the target protein was determined for each seed line to determine the line to be used for the experiment . The seeds of the line thus determined were cultured to obtain a plant, and the protein was extracted using an extraction buffer commonly used for protein extraction for the purpose of ELISA. Antigenic GP55 of porcine fowl virus derived from the transgenic plant prepared in Example 2 above was used for a clinical diagnosis kit (GenoBiotech (currently, median dinostick), CSFV-Ag ELISA kit) used for detection of swine fever antigen In the protein fused with the protein and the CBD protein, the antigenicity of the porcine fowl virus GP55 protein was confirmed, and the ELISA results and the Western blot results are shown in Figs. 3 and 4.

As shown in FIG. 3 and FIG. 4, it was confirmed that the plant-derived proteins prepared in the present invention exhibited remarkable reactivity with clinical diagnostic antibodies for sale in both ELISA and Western blotting experiments. Therefore, in the fusion recombinant protein comprising the antigenic GP55 protein and the CBD protein of the swine fever virus derived from the transgenic plant prepared in Example 2, the swine fever virus GP55 protein was confirmed to have antigenicity. In addition, it was confirmed that the porcine fowl virus GP55 protein was expressed at a high level in transformed plants, and it was confirmed that mass production was possible.

Example  4. From the transgenic plants, the plant-derived porcine fever antigen protein pmE2 (plant-made E2 ) And purification

4-1. Separation purification of fused recombinant protein

Since the transgenic plants prepared according to the present invention were prepared so that the cellulase binding domain (CBD) protein was expressed together, the proteins were separated and purified with high efficiency using cellulose. At this time, amorphous cellulose (AMC) specially prepared from microcrystalline cellulose (MCC) was used as a resin for separation and purification of a target protein.

4-2. Of purified recombinant protein By fraction  Confirm

The recombinant proteins isolated and purified according to Example 4-1 were confirmed by two methods. As a first method, the presence of the target protein, swine fever virus GP55 protein, was confirmed by Western blot. Since CBD is fused to the porcine fowl virus GP55 protein, the reaction to CBD used as the primary antibody indirectly indicates the presence of the GP55 protein in porcine fowl virus. At this time, in each of the fractions of the plant solution containing the protein, the degree of distribution of the GP55 protein of the porcine fowl virus was relatively compared, so that each of the fractions of the gel had the same ratio As shown in FIG. As a second method, each fraction was subjected to Coomassie staining on SDS-PAGE gel to identify protein bands, and the overall pattern was compared with a coma-stained Western blot result. The results are shown in Fig.

As shown in Fig. 5, Western blot analysis confirmed that the GP55 protein binds to the resin significantly. Also, it was confirmed that a small amount remained in the Ub lane. In the E1 and E2 lanes, it was confirmed that most of the proteins were recovered, though the trace amount of protein was washed away by three washing steps. When the recovered protein was confirmed by SDS-PAGE, the band of the GP55 protein was visually confirmed visually, and the nonspecific band was confirmed to be of an undetermined degree of purity.

4-3. Quantification of isolated and purified protein

To further purify the protein solution isolated in 4-1, the protein solution was dialyzed against PBS. The concentrate was then concentrated using a Centrifugal filter tube and the result of performing coma staining on SDS-PAGE gel is shown in FIG. Bovine serum albumin (BSA) solution was used as a standard, and the concentration of protein bands was compared with each other to quantify the target protein (including CBD protein). At this time, the accuracy of the purification and quantification process is reviewed by loading and analyzing the separated protein solution (before dialysis), dialysis protein solution, protein solution after concentration, and solution Respectively. The results are shown in Fig.

As shown in FIG. 6, it was confirmed that the buffer can be exchanged without protein disappearance even after dialysis, and the final vaccine starting protein antigen can be prepared at a desired concentration through concentration, and quantified in comparison with the standard protein.

Thus, the fused recombinant protein comprising the antigenic GP55 protein and the CBD protein of swine fever virus expressed in the transformed plant was named "pmE2 (plant-made E2) ".

Example  5. In a model animal, a plant-derived porcine fever antigen protein pmE2 Immunogenicity and viruses Neutralization ability  Confirm

In order to confirm that the plant-derived swine fever antigen protein pmE2 in animal models induces antibodies in vivo to have immunogenicity and virus neutralizing ability, the following experiment was conducted.

More specifically, a mouse as a model animal was administered the plant-derived porcine heat antigen protein pmE2 protein. PBS was administered to the negative control mice, and the same dose of Freund's adjuvant was administered to the antigens. The serum was taken three times at intervals of 2 weeks. The generation of specific antibodies against the administered antigen was confirmed by using an antibody kit for clinical diagnosis of swine fever virus (Genoviotech (current, median dinostick), CSFV-Ab ELISA kit). The results of judging grades according to the rules of using the kit are shown in Fig. In addition, the virus neutralizing ability in the same mouse serum was requested to the agriculture, livestock and livestock quarantine headquarters, and the result is shown in Fig.

As shown in FIG. 7, it was confirmed that antibody titers recorded positive values in all the condition groups. Thus, it was confirmed that the plant-derived porcine heat antigen protein pmE2 produced by the method of the present invention had immunogenicity against swine fever virus in mice Respectively.

In addition, as shown in Fig. 8, it was confirmed that the activity was very high in all the condition groups.

Therefore, the plant-derived porcine heat antigen protein pmE2 produced according to the present invention produced a specific antibody in mouse, and confirmed that this antibody had high potency of virus neutralizing ability.

Example  6. Plant derived pig fever antigen protein pmE2 Determine Pathway to Pig Fever Virus As a marker  Confirm availability

Since the plant-derived porcine fever antigen protein pmE2 (fused recombinant protein containing the antigenic GP55 protein and the CBD protein of swine fever virus) is fused with the CBD protein, the CBD protein can be used as a marker for determining the pathway for the virus exposure To confirm, the following experiment was carried out.

More specifically, the CBD protein contained in the plant-derived porcine heat antigen protein pmE2 was used as an antigen, and specific antibodies to the above antigens were measured in each of the following sera by ELISA method. Seven sows and 28 piglets in breeding stocks of swine serum and livestock farms included in the kit for clinical diagnosis (GenoBiotech (current, median dinostick), CSFV-Ab ELISA kit) Specific antibody against the CBD protein contained in the plant-derived swine fever antigen protein pmE2 was measured. The age at the time of collection of the sows and the most recent vaccination period were not confirmed. The age at the time of collection of the piglets was about 3 weeks. The results of confirming the possibility of utilizing the plant-derived swine fever antigen protein pmE2 as a marker for determining the exposure pathway of swine fever virus are shown in FIG.

As shown in Fig. 9, in the serum of the mice exposed to the plant-derived protein, a specific antibody against the CBD protein contained in the plant-derived porcine heat antigen protein pmE2 was detected together. On the other hand, the sera of the control sera and the raised sows and piglets contained in the kit were reversely detected as positive values of the specific antibodies against the swine fever virus according to the kit use rules in all the specimens, No antibody was detected. At this time, the specific antibody against swine fever virus detected in sows and piglets could not be identified, but a specific antibody against CBD protein was not detected, and thus it was judged to be independent of the protein used in the present invention.

Therefore, it has been confirmed that the plant-derived porcine fever antigen protein pmE2 of the present invention can be sufficiently used as a marker for determining the route of virus exposure.

Further, it was tried to confirm whether the CBD protein contained in the plant-derived porcine heat antigen protein pmE2 of the present invention generally induces an immune response to form a specific antibody. If the CBD protein is a protein that normally makes antibodies, the formation of CBD antibodies by pmE2 may increase confidence in positively testing whether CSFV antibodies and pmE2 antibodies are produced by outdoor virus infection. To confirm this, a fusion protein of GFP (green fluorescent protein) and CBD was obtained from transformed Escherichia coli. Antibodies to GFP and CBD were detected by ELISA from the sera obtained from the mouse. At this time, several proteins with different antigens were tested.

As shown in Fig. 10, the mouse serum injected with GFP-CBD derived from Escherichia coli showed an increase in the absorbance of various GFP proteins, so that it was confirmed that α-GFP IgG was well produced, and α-CBD IgG against various CBD proteins was also high And it was confirmed that it was generated in the reverse direction.

Therefore, it was confirmed that the CBD protein fused together with the plant-derived porcine heat antigen protein pmE2 of the present invention produces each specific antibody regardless of the kind of the target protein. In addition, the plant-derived swine fever antigen protein pmE2 (a fusion recombinant protein containing the antigenic GP55 protein and the CBD protein of swine fever virus) contains antigenic GP55 protein of swine fever virus and can be used as an antigen. In addition, it was confirmed that CBD protein was included and could be used for detection of each specific antibody. Thus, it was confirmed that the virus can be used for distinguishing whether the virus exposure pathway is a vaccination or a natural infection.

Example  7. Plant-derived pig fever antigen protein in the target animal (pig) pmE2 Defense  Confirm

7-1. Plant-derived swine fever antigen protein in the target animal (pig) pmE2  inoculation

In order to confirm the protective ability of the plant-derived swine fever antigen protein pmE2 in a pig animal model, the following experiment was conducted.

More specifically, as shown in Fig. 11, 8 pigs with pig fever antibody negative were selected and used in the experiment. Four of them were used as a control group in which the plant-derived swine fever antigen protein pmE2 was not inoculated, and two as an experimental group were inoculated once with 100 쨉 g of the plant-derived swine fever antigen protein pmE2. The other two were inoculated twice with 100 ㎍ at 2 - week intervals. To investigate the defense from outdoor viruses, swine fever outbreak virus (Yeoncheonju: separated from domestic wild boar in 2012) was inoculated at a concentration of ^ 6.0 TCID 50 / ml. The attack was carried out on the 14th day after inoculation with the plant - derived porcine fever antigen protein pmE2, and the experiment was completed and autopsied 2 weeks after the inoculation.

7-2. Identification of clinical symptoms of pig after attack

We investigated whether fever, a typical febrile condition, occurs after swine challenge in pigs inoculated with the plant-derived pig fever antigen protein pmE2. The results are shown in Fig.

 As shown in Fig. 12, in the control group of 4 mice inoculated with the pmE2 protein of the present invention, heat started to rise from 3 days after the inoculation, and after 7 days, body temperature exceeded 40 degrees and remained high until 11th day Respectively. On the other hand, in both of the pigs immunized once or twice with the pmE2 protein of the present invention, the animals were observed for 10 days after the inoculation, but no increase in body temperature was observed, and all 4 animals were maintained at 40 degrees or less.

In addition, since leukocyte levels generally fall below 9000 when infected with swine fever virus, the inhibition of leukocyte level reduction through the inoculation of the pmE2 protein of the present invention was examined. The results are shown in Fig.

As shown in Fig. 13, four control pigs showed a decrease in leukocyte counts from day 3 after the attack and seven days after leukocyte attack. On the other hand, it was confirmed that the leukocyte counts of all the pigs injected with the PME2 of the present invention were more than 9000 after 10 days of the inoculation with both the 1-shot and the 2-shot pigs.

Therefore, the clinical symptoms related to swine fever were not observed in the vaccinated individuals, and it was confirmed that the pmE2 protein of the present invention is effective in defense against swine fever virus upon inoculation.

7-3. Blood samples of pigs after attack and confirmation of antigen detection in samples

To confirm whether the pmE2 protein of the present invention inhibits virus propagation after the attack, and to determine whether the pigs were collected and the antigen was detected in the samples, the following experiment was conducted.

More specifically, whole blood, juice, saliva and feces of the control and experimental pigs were collected at 3 days, 5 days, 7 days and 10 days after the inoculation, Respectively. The 5'NCR region was amplified 421 bp at 30 min / 42 ° C, 15 min / 94 ° C, 40 cycles / 30 sec / 94 ° C, 30 sec / 55 ° C, 45 sec / 72 ° C and 5 min / 72 ° C. The results are shown in Fig. 14

As shown in Fig. 14, on the third day after the inoculation, the whole blood was detected in the control pigs and the antigen was detected in the juice, saliva and feces on the fifth day. On the other hand, the pigs immunized once with the pmE2 vaccine of the present invention did not detect any antigen in the juice and saliva, and all the antigens were detected in the whole blood, and there were many positive samples. In addition, one of the pigs inoculated twice with the present invention of pmE2 vaccine had no antigen detected from 3 days to 10 days after the inoculation of the pigs. In one of the pigs, 3 days after the attack inoculation, in the feces, , And showed weak antigen responses in juice after 7 days.

Therefore, it was confirmed that the treatment of the pmE2 protein of the present invention inhibited viral amplification and inhibited propagation in the body.

7-4. Detection of antigens in pigs after attack inoculation

On the 14th day after the inoculation, the control and test pigs were autopsied and the detection of outdoor virus viral antigens attacked in various organs was performed by PCR to investigate the effect of the pmE2 protein of the present invention on the viral amplification of organs in pigs Respectively. The results are shown in Fig.

As shown in Fig. 15, virus antigens attacked in all the pigs of the control group were attacked in one side, heart, kidney, lung, liver, spleen, small intestine, colon, subcutaneous lymph node, inguinal area and mesentery, In pigs injected once, the antigen was detected in the tonsils, sublingual lymph nodes and mesentery, and no viral antigen was detected in the heart, kidney, lung, liver, small intestine and large intestine. In one of the pigs injected with the pmE2 protein of the present invention, the antigen was detected in one of the pigs, and a weak positive response was observed in the sublingual lymph node, inguinal region, and mesentery, whereas no antigen was detected in the other one organs .

Thus, it was confirmed that the pmE2 vaccine of the present invention can protect pigs from virus attack in pigs and is effective in a dose-dependent manner.

7-5. Plant-derived swine fever antigen protein pmE2 Antibody Identification

The defensive antibody titer, which is a key index expressing the protective ability of the swine fever vaccine, was examined, and the results are shown in Fig.

As shown in FIG. 16, in the control group, the antibody titer did not exceed 10 before and after the aggressive inoculation. However, in pigs injected with the plant-derived swine fever antigen protein pmE2 of the present invention, . It is judged that the antibody capable of neutralizing the fever virus was already memorized by the pmE2 of the present invention and boosted by the virus attacking inoculation. In the pigs inoculated with the pmE2 protein of the present invention, 16 and 8 defensive antibodies were detected before the attack, respectively, and 32 values were shown after 5 days from the attack. And then 512 on the 14th day after the attack. In two pigs vaccinated twice with pmE2 vaccine, the value was 16 on the 14th day of vaccination. After 28th day, the vaccine showed high defense antibody titers of 512 and 2048, And 4096 in all of them.

Therefore, considering that the protective antibody titer is clinically determined to be more than 32, it is confirmed that the plant-derived porcine heat antigen protein pmE2 of the present invention is excellent in the ability to protect against swine fever virus in pigs.

Hereinafter, formulation examples of the pharmaceutical composition and the feed composition of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  1. Preparation of pharmaceutical compositions

1-1. Sanje  Produce

Plant-derived pig fever antigen protein pmE2 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

1-2. Manufacture of tablets

Plant derived pig fever antigen protein pmE2 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-3. Preparation of capsules

Plant derived pig fever antigen protein pmE2 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1-4. Injection preparation

Plant derived pig fever antigen protein pmE2 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

1-5. Liquid  Produce

Plant-derived pig fever antigen protein pmE2 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  2. Preparation of feed composition

Plant derived pig fever antigen protein pmE2 100 mg

Vitamin E 0.7 mg

L-carnitine 0.7 mg

The ingredients are mixed according to a conventional method for producing a feed to produce a feed.

<110> Animal and plant quaratine agency          BioApplications Inc. <120> Vaccine composition for classical swine fever from plant and          manufacturing method thereof <130> 1-61 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 1026 <212> DNA <213> DNA sequence for gp55 of Classical swin fever virus <400> 1 aacggctagc ctgcaaggaa gattacaggt acgcaatatc atcaaccaat gagatagggc 60 tactcggggc cggaggtctc accaccacct ggaaagaata caaccacgat ttgcaactga 120 atgacgggac cgttaaggcc atttgcgtgg caggttcctt taaagtcaca gcacttaatg 180 tggtcagtag gaggtatttg gcatcattgc ataaggaggc tttacccact tccgtgacat 240 tcgagctcct gttcgacggg accaacccat caactgagga aatgggagat gacttcgggt 300 tcgggctgtg cccgtttgat acgagtcctg ttgtcaaagg aaagtacaat acaaccttgt 360 tgaacggtag tgctttctat cttgtctgtc caatagggtg gacgggtgtt atagagtgca 420 cagcagtgag cccaacaact ctgagaacag aagtggtaaa gaccttcagg agggacaagc 480 cctttccgca cagaatggat tgtgtgacca caacagtgga aaatgaagat ttattctact 540 gtaagttggg gggcaactgg acatgtgtga aaggtgaacc agtggtctac acgggggggc 600 tagtaaaaca atgcagatgg tgtggctttg acttcaatga gcctgacgga ctcccacact 660 accccatagg taagtgcatt ttggcaaatg agacaggtta cagaatagtg gattcaacag 720 actgtaacag agatggtgtt gtaatcagca cagaggggag tcatgagtgc ttgatcggta 780 acacgactgt caaggtgcat gcatcagatg aaagactggg ccccatgcca tgcagaccta 840 aagagatcgt ctctagtgca ggacctgtaa ggaaaacttc ctgtacattc aactacgcaa 900 aaactttgaa gaacaagtac tatgagccca gggacagcta cttccagcaa tatatgctta 960 agggcgagta tcagtactgg tttgacctgg acgtgactga ccgccactca gattacttcg 1020 cagaag 1026 <210> 2 <211> 429 <212> DNA <213> DNA sequence for cellulose-binding domain <400> 2 tttcgaagtt caccagtgcc tgcacctggt gataacacaa gagacgcata ttctatcatt 60 caggccgagg attatgacag cagttatggt cccaaccttc aaatctttag cttaccaggt 120 ggtggcagcg ccattggcta tattgaaaat ggttattcca ctacctataa aaatattgat 180 tttggtgacg gcgcaacgtc cgtaacagca agagtagcta cccagaatgc tactaccatt 240 caggtaagat tgggaagtcc atcgggtaca ttacttggaa caatttacgt ggggtccaca 300 ggaagctttg atacttatag ggatgtatcc gctaccatta gtaatactgc gggtgtaaaa 360 gatattgttc ttgtattctc aggtcctgtt aatgttgact ggtttgtatt ctcaaaatca 420 ggaacttct 429 <210> 3 <211> 21 <212> DNA <213> 5'UTR of M17 gene <400> 3 ggcgtgtgtg tgtgttaaag a 21 <210> 4 <211> 273 <212> DNA <213> DNA sequence for chapherone binding protein (BiP) <400> 4 atggctcgct cgtttggagc taacagtacc gttgtgttgg cgatcatctt cttcggtgag 60 tgattttccg atcttcttct ccgatttaga tctcctctac attgttgctt aatctcagaa 120 ccttttttcg ttgttcctgg atctgaatgt gtttgtttgc aatttcacga tcttaaaagg 180 ttagatctcg attggtattg acgattggaa tctttacgat ttcaggatgt ttatttgcgt 240 tgtcctctgc aatagaagag gctacgaagt taa 273 <210> 5 <211> 1862 <212> DNA <213> Artificial Sequence <220> <223> particle sequence of recombinant vector <400> 5 tctagaggcg tgtgtgtgtg ttaaagaatg gctcgctcgt ttggagctaa cagtaccgtt 60 gtgttggcga tcatcttctt cggtgagtga ttttccgatc ttcttctccg atttagatct 120 cctctacatt gttgcttaat ctcagaacct tttttcgttg ttcctggatc tgaatgtgtt 180 tgtttgcaat ttcacgatct taaaaggtta gatctcgatt ggtattgacg attggaatct 240 ttacgatttc aggatgttta tttgcgttgt cctctgcaat agaagaggct acgaagttaa 300 ggatccaacg gctagcctgc aaggaagatt acaggtacgc aatatcatca accaatgaga 360 tagggctact cggggccgga ggtctcacca ccacctggaa agaatacaac cacgatttgc 420 aactgaatga cgggaccgtt aaggccattt gcgtggcagg ttcctttaaa gtcacagcac 480 ttaatgtggt cagtaggagg tatttggcat cattgcataa ggaggcttta cccacttccg 540 tgacattcga gctcctgttc gacgggacca acccatcaac tgaggaaatg ggagatgact 600 tcgggttcgg gctgtgcccg tttgatacga gtcctgttgt caaaggaaag tacaatacaa 660 ccttgttgaa cggtagtgct ttctatcttg tctgtccaat agggtggacg ggtgttatag 720 agtgcacagc agtgagccca acaactctga gaacagaagt ggtaaagacc ttcaggaggg 780 acaagccctt tccgcacaga atggattgtg tgaccacaac agtggaaaat gaagatttat 840 tctactgtaa gttggggggc aactggacat gtgtgaaagg tgaaccagtg gtctacacgg 900 gggggctagt aaaacaatgc agatggtgtg gctttgactt caatgagcct gacggactcc 960 cacactaccc cataggtaag tgcattttgg caaatgagac aggttacaga atagtggatt 1020 caacagactg taacagagat ggtgttgtaa tcagcacaga ggggagtcat gagtgcttga 1080 tcggtaacac gactgtcaag gtgcatgcat cagatgaaag actgggcccc atgccatgca 1140 gacctaaaga gatcgtctct agtgcaggac ctgtaaggaa aacttcctgt acattcaact 1200 acgcaaaaac tttgaagaac aagtactatg agcccaggga cagctacttc cagcaatata 1260 tgcttaaggg cgagtatcag tactggtttg acctggacgt gactgaccgc cactcagatt 1320 acttcgcaga atcccgggca gattacgaca ttccaacaac tgatgcagag aatttgtatt 1380 ttcagggtcg ggcacaccac caccaccacc actttcgaag ttcaccagtg cctgcacctg 1440 gtgataacac aagagacgca tattctatca ttcaggccga ggattatgac agcagttatg 1500 gtcccaacct tcaaatcttt agcttaccag gtggtggcag cgccattggc tatattgaaa 1560 atggttattc cactacctat aaaaatattg attttggtga cggcgcaacg tccgtaacag 1620 caagagtagc tacccagaat gctactacca ttcaggtaag attgggaagt ccatcgggta 1680 cattacttgg aacaatttac gtggggtcca caggaagctt tgatacttat agggatgtat 1740 ccgctaccat tagtaatact gcgggtgtaa aagatattgt tcttgtattc tcaggtcctg 1800 ttaatgttga ctggtttgta ttctcaaaat caggaacttc tcacgatgag ctctagctcg 1860 ag 1862

Claims (25)

CaMV 35S promoter gene; 5 'UTR (untranslational region) site gene of M17; A polynucleotide encoding a BiP (chaperone binding protein) protein; A polynucleotide of SEQ ID NO: 1 encoding E2 protein of swine fever virus; A polynucleotide represented by SEQ. ID. NO: 2 encoding a cellulosic binding domain (CBD) protein; And a polynucleotide encoding a HDEL (His-Asp-Glu-Leu) protein, which is a fusion protein comprising a E2 protein and a CBD protein, wherein the recombinant vector is a plant-derived porcine fever antigen pmE2 .
delete delete delete The method according to claim 1,
A recombinant vector for producing plant-derived porcine heat antigen pME2 protein, which is a fusion protein comprising an E2 protein and a CBD protein, wherein the 5 'UTR site gene of M17 is composed of the nucleotide sequence shown in SEQ ID NO: 3. The method according to claim 1,
And a polynucleotide encoding said BiP protein is a fusion protein comprising E2 protein and CBD protein, characterized in that the polynucleotide encoding said BiP protein is represented by SEQ ID NO: 4. 4. A recombinant vector for producing a plant-derived porcine fever antigen pmE2 protein, The method according to claim 1,
Wherein the recombinant vector comprises the nucleotide sequence shown in SEQ ID NO: 5. 2. The recombinant vector according to claim 1, wherein the recombinant vector is a fusion protein comprising the E2 protein and the CBD protein. The method according to claim 1,
A recombinant vector for producing a plant-derived porcine fever antigen pmE2 protein, which is a fusion protein comprising an E2 protein and a CBD protein, wherein said recombinant vector is represented by the following vector map.
[Degree]

A transgenic plant for producing plant-derived porcine fever antigen pmE2 protein, which is transformed with a recombinant vector for producing plant-derived porcine fever antigen pmE2 protein, which is the fusion protein comprising the E2 protein and the CBD protein of Claim 1. A plant-derived porcine fever antigen pmE2 protein, expressed as a fusion protein comprising an E2 protein and a CBD protein expressed in a transgenic plant for producing the plant-derived porcine fever antigen pmE2 protein of Claim 9. delete (a) transforming Agrobacterium with a recombinant vector for producing a plant-derived porcine fever antigen pmE2 protein, which is a fusion protein comprising the E2 protein and the CBD protein of claim 1;
(b) introducing the transformed Agrobacterium into a plant; And
(c) isolating and purifying the expressed plant-derived porcine fever antigen pmE2 protein in said plant. 13. The method of claim 12,
The plant may be selected from the group consisting of Arabidopsis, wheat, barley, corn, beans, potatoes, red beans, oats, sorghum, rice, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, Ginseng, tobacco, cotton, sesame seeds, sugar beet, perilla, peanut, rapeseed, apple tree, pear, jujube tree, peach, sheep, grape, citrus, persimmon, apricot, banana, rose, gladiolus, gerbera Wherein the plant-derived pig fever antigen pmE2 protein is characterized by being at least one plant selected from the group consisting of carnation, chrysanthemum, lily, tulip, raglass, red clover, orchardgrass, alfapa, tall fescue, Way. A vaccine composition for preventing swine fever comprising the plant-derived swine fever antigen pmE2 protein as a fusion protein comprising the E2 protein and the CBD protein of claim 10 as an active ingredient. A pharmaceutical composition for preventing swine fever comprising the plant-derived porcine fever antigen pmE2 protein as a fusion protein comprising the E2 protein and the CBD protein of claim 10 as an active ingredient. A feed composition for preventing swine fever comprising the plant-derived swine fever antigen pmE2 protein as a fusion protein comprising the E2 protein and the CBD protein of claim 10 as an active ingredient. A composition for diagnosing swine fever virus comprising the plant-derived swine fever antigen pmE2 protein, which is the fusion protein comprising the E2 protein and the CBD protein of claim 10. 12. A pig fever virus diagnostic kit comprising a plant-derived porcine fever antigen pmE2 protein which is a fusion protein comprising the E2 protein and the CBD protein of claim 10. A method for preventing swine fever by administering the vaccine composition for preventing swine fever according to claim 14 to an animal other than a human. A method for detecting swine fever virus in a sample through an antigen-antibody reaction using a plant-derived swine fever antigen pmE2 protein, which is a fusion protein comprising the E2 protein and the CBD protein of claim 10. 21. The method of claim 20,
The antigen-antibody reaction can be assayed by immunohistochemical staining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), Western blotting, immunoprecipitation assays, immunodiffusion assays, Wherein the detection is carried out using at least one method selected from the group consisting of Complement Fixation Assay, Fluorescence-activated cell sorter (FACS) and protein chip analysis. 21. The method of claim 20,
Wherein the sample is at least one selected from the group consisting of cells, blood, urine, saliva and tissues. 1) CaMV 35S promoter gene; 5 'UTR (untranslational region) site gene of M17; A polynucleotide encoding a BiP (chaperone binding protein) protein; A polynucleotide of SEQ ID NO: 1 encoding E2 protein of swine fever virus; A polynucleotide represented by SEQ. ID. NO: 2 encoding a cellulosic binding domain (CBD) protein; And a polynucleotide encoding a HDEL (His-Asp-Glu-Leu) protein, which is a fusion protein comprising a E2 protein and a CBD protein, wherein the recombinant vector is a plant-derived porcine fever antigen pmE2 A vaccine composition for preventing swine fever, comprising the plant-derived swine fever antigen pmE2 protein expressed in the transgenic plant as an active ingredient. Inoculation Pig or the vaccine composition for preventing swine fever Pig fever virus infection Collecting blood from the pig;
2) separating the serum from the blood collected in the step 1); And
3) treating the swine fever antigen pME2 protein of claim 10 with the antigen to the serum isolated in step 2), and reacting the serum. 24. The method of claim 23,
When the antibody against the E2 protein of the swine fever virus and the antibody against the cellulose-binding domain protein is detected together in the reaction of step 3), the antibody formed in the specimen is produced by administration of the vaccine composition for preventing swine fever in step 1) Wherein the pathogen is a pheosis virus. 24. The method of claim 23,
When only the antibody against the E2 protein of the swine fever virus is detected in the step 3), the antibody formed in the specimen is not administered by the vaccine composition for preventing swine fever in the step 1) but by the natural infection of the swine fever virus And determining that the virus has been generated.

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