KR101796104B1 - Recombinant vector for mass-producing N protein from PRRSV in plant cell organelle and uses thereof - Google Patents
Recombinant vector for mass-producing N protein from PRRSV in plant cell organelle and uses thereof Download PDFInfo
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Abstract
The present invention relates to a recombinant vector for mass production of N protein derived from PRRSV in a plant cell organelle and its use, and relates to a plant cell organelle-specific targeting vector system of the present invention, which is capable of expressing N (nucleocapsid) Since the N protein can be mass-produced by preparing a transgenic plant, the recombinant protein can be produced at a low cost and can be usefully used for mass production of an industrially expensive protein. Based on the technology of the present invention, it is possible to develop a plant subunit vaccine capable of effectively preventing and controlling PRRSV by introducing various antigen proteins derived from PRRSV, and this technology will contribute to the development of molecular agricultural technology in Korea do.
Description
The present invention relates to a recombinant vector for mass production of N protein derived from PRRSV and its use in plant organelles.
The porcine reproductive and respiratory syndrome (PRRS) virus is currently the causative organ of swine disease called the reproductive and respiratory syndrome (PRRS) of pigs. The entire genome size of PRRSV is 15 kb and has nine open reading frame (ORF, ORF 1a, 1b, 2a, 2b, 3-7) in which the lower genomic mRNAs overlap each other. ORFs 1a and 1b are replication related nonstructural proteins and ORF 3-7 constitutes structural proteins as GP2a, GP2b, GP3, GP4, GP5, membrane / matrix (M) protein and nucleocapsid (N) protein. The major structural proteins are the N protein, the M protein, and the GP5, the primary envelope glycoprotein. The virus is found in approximately 1987 in the United States and after 1990 in Europe and is a causative organism of swine diseases known in the early years as a wide variety of names such as mysterious swine disease, swine infertility and respiratory syndrome. PRRS viruses are now endemic in almost all swine countries and are considered to be one of the most economically important diseases affecting the world's pig production. Currently commercially available vaccines against PRRS include modified live vaccines and killed (inactivated) vaccines. The death vaccine has been criticized for failing to induce strong immunity against heterologous PRRS virus strains. The modified live vaccine is attenuated by continuous passage in cell culture until the loss of autotrophy. Modified live vaccines cause more widespread protection than death vaccines, but can lead to a number of safety issues including residual toxicity, spread to non-vaccinated pigs, and genetic return to toxic conditions. Due to the antigenic changes that occur during the attenuation process, the vaccine may lose some of its ability to protect against the toxic field strain of PRRS virus. Therefore, new and improved modified live vaccines that are able to protect against PRRS are strongly desired.
On the other hand, plants generally have a great potential for the production of biopharmaceutical proteins and peptides because they are easy to transform and economically inexpensive as protein materials. On the other hand, to date, most biologics have typically been produced by transforming cultured mammalian cells, bacteria, fungi, and the like. However, the production of therapeutic proteins in plants as compared to these mammalian cells, bacteria and fungi can result in reduced risk of contamination by pathogens and high yields of production, as well as economical aspects such as production in seeds or other storage facilities There are several advantages in terms of quality. In addition, plants can be an inexpensive target for potentially producing recombinant products, and the cultivation, harvesting, storage, and processing of transformed grains can also use current infrastructure and require only relatively little capital investment This allows for a very large prospect in the commercial production of biologics.
Accordingly, the development of plant expression systems for obtaining the desired useful protein with high efficiency by transforming plant cells has been receiving a great deal of attention. Such plant expression systems have been used for a variety of plant expression systems in which expression levels of recombinant proteins are expressed in plants, It is attractive in that it can be improved by using enemy classification and targeting mechanisms and has the advantage of being able to easily produce large quantities of plant-derived biopharmaceuticals. However, production of the target protein by using the plant may be an important factor determining the plant-based production potential, such as plant selection, tissue selection, expression and recovery strategy, and post-translational processing. Recently, a method for transforming nuclei has been studied in a method for producing a useful protein using plant cells.
However, since the genes introduced into the nuclei are usually introduced into one or a few genes, there is a limit to mass production of useful proteins. In addition, when a foreign gene is inserted into a heterochromatin region on a chromosome, there is a problem that the degree of transformation varies depending on the site to which the foreign gene is introduced, even if it is transformed. Recently, attempts have been made to induce large-scale expression of genes by introducing genes into mitochondria, chloroplasts or chromosomes.
Korean Patent No. 1545261 discloses a method for producing a transgenic plant that mass-produces a target protein in a chloroplast, and a plant according to the method. Korean Published Patent Application No. 2012-0080448 discloses a method for isolating a PRRSV antigen including North American and European PRRSV ≪ / RTI > proteins and their uses. However, the recombinant vector for mass production of N protein derived from PRRSV in plant cell organelles of the present invention and its use has not yet been disclosed.
In accordance with the present invention, the present invention provides a novel vector system for chloroplast or endoplasmic reticulum-specific targeting of plant cell organelles, wherein the N (nucleocapsid) protein high expression from porcine reproductive and respiratory syndrome virus (PRRSV) The present inventors completed the present invention by confirming that a transgenic plant can be produced and mass-produced the N protein.
In order to solve the above-mentioned problems, the present invention provides a plant cell, which comprises a promoter, a signal sequence coding gene for plant cell organelle-specific targeting, and a plant cell in which an N (nucleocapsid) protein coding gene derived from porcine reproductive and respiratory syndrome virus (PRRSV) Provides a recombinant vector for mass production of N (nucleocapsid) protein from PRRSV in the organelle.
The present invention also provides a plant cell transformed with said recombinant vector.
The present invention also relates to a method for mass production of PRRSV-derived N protein in a plant cell organelle including the step of overexpressing N (nucleocapsid) protein derived from porcine reproductive and respiratory syndrome virus (PRRSV) by transforming plant cells with the recombinant vector . ≪ / RTI >
In addition, the present invention relates to a method for producing N (nucleocapsid) protein derived from porcine reproductive and respiratory syndrome virus (PRRSV), which comprises transforming a plant cell with the recombinant vector and regenerating a plant from the transformed plant cell The present invention provides a method for producing a transgenic plant which is mass produced in a cell organelle.
In addition, the present invention provides transgenic plants and seeds thereof which mass-produce N (nucleocapsid) proteins derived from porcine reproductive and respiratory syndrome virus (PRRSV) produced by the above method in plant organelles.
The present invention also relates to a promoter; A signal sequence coding gene for chloroplast-specific targeting consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of SEQ ID NO: 2 and an endoplasmic reticulum retention signal KDEL (KDEL) ; Mass production of PRRSV-derived nucleocapsid protein in a chloroplast or endoplasmic reticulum of a plant containing, as an active ingredient, a plant recombinant vector in which a nucleocapsid (N) protein coding gene derived from porcine reproductive and respiratory syndrome virus (PRRSV) ≪ / RTI >
The present invention also provides a nucleocapsid (N) protein or a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine composition comprising the plant as an active ingredient.
The present invention relates to a recombinant vector for mass production of N protein derived from PRRSV in plant cell organelles and to a method for producing NRS from PRRSV through a vector system for chloroplast or endoplasmic specific targeting, nucleocapsid protein can be produced by mass-producing the transgenic plant. Therefore, the recombinant protein can be produced at a low cost and can be usefully used for mass production of industrially expensive protein. Based on the technology of the present invention, it is possible to develop a plant subunit vaccine capable of effectively preventing and controlling PRRSV by introducing various antigen proteins derived from PRRSV, and this technology will contribute to the development of molecular agricultural technology in Korea do.
Figure 1 shows signal amino acid sequences for plant cell organellular (endoplasmic reticulum, vacuole and chloroplast) specific targeting. A is an amino acid sequence for endoplasmic reticulum and vacuole-specific targeting; B is an amino acid sequence for chloroplast specific targeting; C is the KDEL sequence, an endoplasmic reticulum retention signal.
Figure 2 shows the results of transient expression analysis (B) of reporter protein mYFP in tobacco plants through the production of a plant-cell organelle-specific targeting vector (A) and Agrobacterium-infiltration (Agrobacterium-infiltration).
Figure 3 shows the results of confocal microscopy analysis of the localization of reporter protein mYFP in agrobacterium-infiltrated tobacco leaves.
Figure 4 shows the production of a plant cell organelle-specific targeting vector (PRRSV) with subunit N gene introduced (A) and N (nucleocapsid) of tobacco plants through Agrobacterium-infiltration ) ≪ / RTI > protein (B). ← indicates the size of the N protein (15 kDa).
5 shows the results of Western blot analysis of the expression of N protein introduced into a vector for chloroplast specific targeting in transgenic tobacco (A) and transformed Arabidopsis thaliana (B) plants. ← indicates the size of the N protein (15 kDa).
FIG. 6 shows the results of Western blot analysis of the expression of N protein introduced into a vector for endoplasmic reticulum-specific targeting in transgenic Arabidopsis plants. ← indicates the size of the N protein (15 kDa).
In order to accomplish the above object, the present invention provides a plant cell system comprising a promoter, a signal sequence coding gene for plant cell organelle-specific targeting, and a plant cell in which an N (nucleocapsid) protein coding gene derived from porcine reproductive and respiratory syndrome virus (PRRSV) Provides a recombinant vector for mass production of N (nucleocapsid) protein from PRRSV in the organelle.
In the recombinant vector according to an embodiment of the present invention, the signal sequence for plant cell organelle-specific targeting may be a signal sequence for chloroplast-specific targeting or a signal sequence for endoplasmic reticulum-specific targeting, but is not limited thereto .
In the recombinant vector according to an embodiment of the present invention, the signal sequence for chloroplast-specific targeting may be an amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
In the recombinant vector according to an embodiment of the present invention, the signal sequence for the endoplasmic reticulum-specific targeting may consist of the amino acid sequence of SEQ ID NO: 2 and the endoplasmic reticulum retention signal KDEL (KDEL) sequence. It does not.
The recombinant vector according to an embodiment of the present invention may have a promoter sequence on the 5 'side and a terminator sequence on the 3' side, but the present invention is not limited thereto.
In the present invention, "operably linked" refers to a component of an expression cassette that functions as a unit for expressing a heterologous protein. For example, a promoter operably linked to a heterologous DNA encoding a protein promotes the production of a functional mRNA corresponding to a heterologous DNA.
The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means. The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ".
The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known. A preferred example of a recombinant vector is a Ti-plasmid vector which is capable of transferring a so-called T-region to a plant cell when it is present in a suitable host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838.
Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.
The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as Kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, Resistant genes, but are not limited thereto.
In the recombinant vector of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS, or histone promoter, but is not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.
In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) Octopine gene terminator, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.
The present invention also provides a plant cell transformed with said recombinant vector.
Methods of delivering the vectors of the present invention into plant host cells include microinjection (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology, 52: 456 (Wong, TK et al., Gene, 10: 87 (1980)), DEAE (1, (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) and dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 ), And the like.
The present invention also relates to a method for mass production of PRRSV-derived N protein in a plant cell organelle including the step of overexpressing N (nucleocapsid) protein derived from porcine reproductive and respiratory syndrome virus (PRRSV) by transforming plant cells with the recombinant vector . ≪ / RTI >
The method for introducing the recombinant vector into plant tissue according to an embodiment of the present invention includes agro-infiltration ( Agrobacterium tumefaciens ) transformed with a recombinant vector into which a target gene is inserted and Agrobacterium tumefaciens ) Method, and the like can be used, but the present invention is not limited thereto.
The term " Agrobacterium-infiltration " used in the present invention means a method for introducing a gene into a plant and expressing it or producing a desired protein in a plant. A suspension of Agrobacterium having the gene to be transferred is injected into the leaves of a plant by using a needle-free syringe to transfer the desired gene to the plant cell. This has the advantage of being quick and convenient compared to traditional plant transformation.
In addition, the present invention relates to a method for producing N (nucleocapsid) protein derived from porcine reproductive and respiratory syndrome virus (PRRSV), which comprises transforming a plant cell with the recombinant vector and regenerating a plant from the transformed plant cell The present invention provides a method for producing a transgenic plant which is mass produced in a cell organelle.
The method of the invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformant is, for example, Agrobacterium tyumeo Pacific Enschede may be mediated by (Agrobacterium tumefiaciens). In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.
Transformed plant cells must be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989, Momillan, N. Y.).
In addition, the present invention provides transgenic plants and seeds thereof which mass-produce N (nucleocapsid) proteins derived from porcine reproductive and respiratory syndrome virus (PRRSV) produced by the above method in plant organelles. The plant may be selected from the group consisting of Arabidopsis thaliana, eggplant, tobacco, red pepper, tomato, burdock, lettuce, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, parsley, parsley, cabbage, cabbage, , Strawberry, soybean, mung bean, kidney bean and pea, or monocotyledonous plants such as rice, barley, wheat, rye, maize, sorghum, oats and onion, Preferably tobacco ( Nicotiana tabacum ) or Arabidopsis ( Arabidopsis thaliana , but are not limited thereto.
The present invention also relates to a promoter; A signal sequence coding gene for chloroplast-specific targeting consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of SEQ ID NO: 2 and an endoplasmic reticulum retention signal KDEL (KDEL) ; And a composition for mass production of PRRSV-derived N protein in a chloroplast or an endoplasmic reticulum of a plant containing, as an active ingredient, a plant recombinant vector in which a nucleocapsid (N) protein coding gene derived from porcine reproductive and respiratory syndrome virus (PRRSV) to provide.
The composition for mass production of N protein derived from porcine reproductive and respiratory syndrome virus (PRRSV) of the present invention comprises the promoter of the present invention as an active ingredient; A signal sequence coding gene for chloroplast-specific targeting consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of SEQ ID NO: 2 and an endoplasmic reticulum retention signal KDEL (KDEL) ; And an N (nucleocapsid) protein coding gene derived from porcine reproductive and respiratory syndrome virus (PRRSV); and transfecting the recombinant vector into a plant to mass-produce N protein from PRRSV It is.
The present invention also provides a nucleocapsid (N) protein or a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine composition comprising the plant as an active ingredient.
The vaccine composition may be in the form of an N protein or a concentrate thereof produced by the transgenic plant of the present invention, or in the form of an extract or a dry powder of the transgenic plant itself or a transgenic plant. It can also be used with other food or food ingredients and can be suitably used according to conventional methods. The amount of the plant as an active ingredient can be suitably determined according to the intended use (preventive, health or therapeutic treatment), and the plant, which is an active ingredient, usually has no safety problems, Can be used.
The vaccine composition may further comprise one or more excipients selected from the group consisting of stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylene, bacterial cell walls, An animal phoxvirus protein, a subviral particle adjuvant, a cholera toxin, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, dimethyl Dioctadecylammonium bromide, and mixtures thereof. The second auxiliary agent may further include at least one second auxiliary agent selected from the group consisting of benzoic acid, dioctadecyl-ammonium bromide, and mixtures thereof.
In addition, the vaccine composition may comprise a veterinarily acceptable carrier. The term "veterinarily acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, Examples of the carrier, excipient and diluent which can be contained in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
In addition, the composition for vaccine may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol and the like, and sterilized injection solution according to a conventional method. In the case of formulation, it may 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, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. As the liquid preparation for oral administration, suspensions, solutions, emulsions, syrups and the like may be used. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, and freeze-drying agents. Examples of the suspending agent include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.
Example 1. Plant cell organelle specific Targeting Signal sequence production
In the first embodiment, Specific Signal sequences for targeting were designed and genes were synthesized. Figure 1 A is a transit peptide of Preprosporamine A isolated from sweet potato. The front 21 amino acids are targeted to the ER (endoplasmic reticulum) and the rear 16 amino acids are targeted to the vacuole. Fig. 1B is a Cu / Zn superoxide dismutase II (SOD II) transit peptide isolated from pea and is targeted to chloroplast. Figure 1, C, is the KDEL sequence and is well known as the endoplasmic reticulum retention signal and is inserted at the 3 'side. A in FIG. 1 is designated as VTS, B as CPT, and C as K, respectively.
Example 2. Plant-cell organelle-specific Targeting Vector production
The plant cell organelle of Example 1 Specific Plant cell organelles targeted to vacuums, vesicles, chloroplasts, etc. using targeting signals Specific A targeting vector was produced (Fig. 2A). We used pGreenII0229 with BASTA resistance as a plant expression vector and inserted the duplicated 35S promoter and translation leader sequence TMV (Ω) and flag tag. MYFP was used as a reporter protein. Each organelle targeting signal sequence (VTS and CPT) was inserted into the cytosol targeting vector in front of the mYFP, the flag tag moved to the 3 'side, and the ER targeting signal (KDEL) '. The plant-cell organelle-specific targeting vectors thus constructed were named pGR-cytosol, pGR-VTS, pGR-VTS-ER and pGR-CPT, respectively.
Example 3. Plant-cell organelle-specific Targeting Reporter protein in vector mYFP Transient expression analysis
The pGR-cytosol, pGR-VTS, pGR-VTS-ER and pGR-CPT vectors prepared in Example 2 were suspended in Agrobacterium tumefeciens ) GV3101 strain using the freeze-thaw method. Transgenic Agrobacterium was used to transform tobacco ( Nicotiana Benthamiana) in-fill agent migration (infiltration) in the leaves was a transient expression of
Example 4. Agrobacterium-infiltration ( Agrobacterium - infiltration Transient Expression Analysis of N (nucleocapsid) Protein in Tobacco Leaves
A gene coding for the N protein (nucleocapsid) of porcine reproductive and respiratory syndrome virus (PRRS) was introduced into the pGR-cytosol, pGR-VTS, pGR-VTS-ER and pGR-CPT vectors prepared in Example 2 4A). Transient expression of the N gene on the 3 and 5 days of tobacco leaf was analyzed by Agrobacterium-infiltration in the same manner as in Example 3 above. N protein was isolated to a size of 15 kDa, and bands of about 2, 3, and 4 times of size were considered oligomer patterns of N protein. N protein expression was highest when targeted to chloroplast (CPT), followed by vesicle (VTS-ER), vacuolar (VTS) and cytosol (FIG. 4B).
Example 5. Chloroplasts ( CPT ) Targeting Expression of N Proteins in Tobacco Transgenic Tobacco Plants and Arabidopsis Plants
Two transgenic plants (tobacco and Arabidopsis thaliana) were prepared by introducing the chloroplast (CPT) targeting vector, which was the most expressed in Example 4, and using the Agrobacterium-mediated transformation method. Transgenic plants (tobacco and Arabidopsis thaliana) were selected using an antibiotic marker, BASTA. Transgenic tobacco plants were selected for five T0 generation lines and transgenic Arabidopsis plants were selected for T1 line. Expression of the N protein in the transgenic plant leaves was analyzed in the same manner as in Example 3 above. Transgenic tobacco plants showed high expression of N protein (15 kDa) in all five lines, and multimeric pattern bands with N protein sizes of 2, 3 and 4 were also observed (Fig. 5A). Transgenic Arabidopsis plants showed N protein expression in five out of twelve lines selected, and a band of a dimeric pattern was also observed (FIG. 5B). The higher the expression level of N protein, the more multimeric band is observed.
Example 6. Endoplasmic reticulum ( VTS - ER ) Targeting Analysis of N Protein Expression in Vector Transgenic Arabidopsis Plants
Transformed Arabidopsis thaliana plants were prepared by introducing the second highest expression vector (VTS-ER) targeting vector in Example 4 and transforming through Agrobacterium-mediated transformation. Twelve lines of transgenic Arabidopsis thaliana (T0) were selected using BASTA, an antibiotic marker. Expression of the N protein in the transgenic plant leaves was analyzed in the same manner as in Example 3 above. Transgenic Arabidopsis plants showed N protein (15 kDa) expressed in 8 out of 12 lines and multimeric pattern bands with N protein sizes of 2, 3 and 4 were also observed (Figure 6) .
<110> Korea Research Institute of Bioscience and Biotechnology
<120> Recombinant vector for mass-producing N protein from PRRSV in
plant cell organelle and uses thereof
<130> PN15358
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<170> Kopatentin 2.0
<210> 1
<211> 48
<212> PRT
<213> Pisum sativum
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Met Ala Ser Gln Thr Leu Val Ser Ser Ser Ser Le Ser Ser His
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