WO2023277286A1 - High frequency-specific expression promoter - Google Patents

High frequency-specific expression promoter Download PDF

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WO2023277286A1
WO2023277286A1 PCT/KR2021/019917 KR2021019917W WO2023277286A1 WO 2023277286 A1 WO2023277286 A1 WO 2023277286A1 KR 2021019917 W KR2021019917 W KR 2021019917W WO 2023277286 A1 WO2023277286 A1 WO 2023277286A1
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gene
cancer
promoter
virus
canine
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French (fr)
Korean (ko)
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모상현
이정훈
서효현
김영순
김수윤
백승혜
김지연
배윤진
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주식회사 바이오에프디엔씨
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Publication of WO2023277286A1 publication Critical patent/WO2023277286A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1225—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold or salt resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the present invention relates to a high-frequency specific expression promoter.
  • a cis-acting element is defined as a DNA sequence adjacent to a gene that directly or indirectly regulates the expression of a gene by regulating the binding of trans-acting elements called various transcription elements.
  • the promoter is one of the most important cis-acting elements and is generally considered a region of nucleotide sequence located 5 terminally upstream of the transcription initiation site of a gene.
  • a promoter contains a binding site for RNA polymerase II and initiates transcription of a gene.
  • the promoter contains several well-conserved sequence elements, such as the CAAT box around base position -70 and the TATA box around base position -30, which are related to the transcription initiation site (+1) (Joshi, 1987).
  • the TATA box is important for correctly locating the initiation of transcription.
  • the CAAT box is not present in all promoters, but in some promoters where GC-rich regions have been replaced.
  • promoters also contain additional sequences that respond to environmental cues and physiological conditions such as dry stress, heat shock, cold stress, nutrient availability, light and ionic strength.
  • some promoters contain sequence elements that direct expression of tissue-specific genes or expression of cell-specific genes.
  • promoters are important elements in the genetic engineering of eukaryotic cells by regulating gene expression by conditions such as expression level, tissue specificity or cell-specificity, developmental stage dependence, and responsiveness to specific environmental stimuli.
  • High frequency means an electromagnetic wave with a high frequency, and is an electromagnetic wave corresponding to a low frequency.
  • a commercial frequency of 50 to 60 Hz is referred to as a low frequency and a frequency higher than that is referred to as a high frequency, and in most fields, an electromagnetic wave having a frequency higher than the commercial frequency is referred to as a high frequency.
  • High frequency waves are electromagnetic waves of a waveform that are easy to radiate, and are characterized by being easy to induce disturbance or stress to objects such as cells.
  • salt-inducible promoters (Republic of Korea Patent No. 10-2208028), oxygen-inducible promoters (Republic of Korea Patent No. 10-0173845), plant hormone-inducible promoters that can regulate the expression of promoters under specific conditions ( Korean Registered Patent No. 10-1985653) and environmental stress inducible promoters (Korean Registered Patent No. 10-1151238) have been developed, but promoters whose expression is regulated under high-frequency conditions have not been studied.
  • An object of the present invention is to provide a high-frequency specific expression promoter.
  • Another object of the present invention is to provide a high-frequency specific recombinant expression vector containing the above promoter.
  • Another object of the present invention is to provide a transformant transformed with the above high-frequency specific expression vector.
  • an object of the present invention is to provide a high-frequency specific expression promoter.
  • the present invention provides a high-frequency specific expression recombinant vector containing the above promoter.
  • the present invention provides a transformant transformed with the above high-frequency specific expression vector.
  • the present invention relates to a high-frequency specific expression promoter, wherein the promoter of the present invention increases the expression of a foreign gene under a high-frequency condition, compared to a non-high-frequency condition.
  • the effect of increasing the content of aromatic amino acids such as tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) by the high frequency in the cell is excellent. Accordingly, it has the effect of maximizing the production of phenylpropanoid pathway related genes and phytochemicals, and can be usefully used in related industries.
  • FIG. 1 is a diagram showing a schematic diagram of a rose cell metabolic network changed by radiofrequency treatment to discover a radiofrequency-specific expression vector of the present invention.
  • Figure 2 is a diagram confirming the tandem repeat of the Tryptophan synthase alpha chain-like gene for discovering the high-frequency specific expression vector of the present invention.
  • Figure 3 shows a schematic diagram of inserting the high-frequency specific expression promoter of the present invention into a vector.
  • FIG. 4 is a diagram showing a method for preparing a high-frequency specific expression vector of the present invention as a vector map.
  • Figure 5 is a diagram confirming the fluorescence expression in rose cells transformed with the high-frequency specific expression vector of the present invention (A: negative control, B: positive control, C: Group 1 promoter group, D: Group 2 promoter group) .
  • FIG. 6 is a diagram quantifying fluorescence expression in rose cells transformed with the high-frequency specific expression vector of the present invention.
  • Figure 7 is a diagram confirming the induction of high frequency-specific EGF expression by Western blot with the high frequency-specific expression vector of the present invention.
  • FIG. 8 is a diagram confirming the induction of expression of high frequency-specific bFGF by Western blot with the high frequency-specific expression vector of the present invention.
  • the present invention provides a promoter specifically expressed at a high frequency.
  • “specific” means an increase in expression in some conditions of a transformant, including both high expression and specificity by the promoter according to the present invention. For example, it means an increase in expression of 50% or more, 100% or more, 200% or more, 5-fold or more or more compared to a transformant lacking the promoter according to the present invention.
  • the "functionally equivalent segment" to the promoter of the present invention means a fragment or part of a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1, which exhibits substantially equivalent effects to the promoter of the present invention.
  • These nucleic acid fragments are 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, It has 96%, 97%, 98%, 99% or more sequence homology, and such nucleic acid fragments can be easily prepared by molecular biological methods well known in the art.
  • the promoter represented by SEQ ID NO: 1 and part of the gene region of the present invention is a sequence including the promoter region of the Tryptophan synthase alpha chain-like gene and part of the gene on the published rose genome sequence, which is represented by SEQ ID NO: 1 Even if some of the nucleotide sequences are substituted, inserted, or deleted, as long as homology to the promoter of the tryptophan synthase alpha chain-like gene and sequences located in some gene regions are maintained and substantially equivalent effects are exhibited, it may be included in the scope of the present invention.
  • TS Tryptopane synthase
  • Trp Tryptophane
  • the activation of the aromatic amino acid (AA) biosynthesis pathway and the phenylpropanoid pathway can increase the content of aromatic compounds or flavonoids such as Caffoylquinic acids, Kaempferol, Quercetin, and Catechin.
  • the promoter may include the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the high frequency may be processed under conditions of 100 to 1200 khz, preferably 500 to 1200 khz, and more preferably 900 to 1200 khz, but is not limited thereto.
  • the high frequency may be processed under conditions of 1 to 60 minutes, preferably 10 to 50 minutes, and more preferably 20 to 30 minutes, but is not limited thereto.
  • the high frequency may be processed under a condition of 1 to 10 times/day, but may be preferably 1 to 7 times/day, more preferably 1 to 5 times. It may be processed under the condition of /day, but is not limited thereto.
  • the high frequency may be processed under conditions of 1 to 10 days.
  • the promoter may increase the content of aromatic amino acids such as tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr).
  • aromatic amino acids such as tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr).
  • the activity of the Phenylpropanoid pathway metabolism of plant metabolism and the gene of the metabolism may be enhanced by increasing the content of the aromatic amino acids, and the activity of the Phenylpropanoid pathway metabolism and the above Genetic enhancement of metabolism is a phytochemical selected from the group consisting of Caffeoylquinic acids, Quercetin, Kaempferol, Catechin, Benzaldehyde and Phenylacetic acid It may be to increase the production of (phytochemical).
  • the promoter is a cancer cell death gene, a fluorescent protein gene, a cancer cell suppressor gene, a pathogenic factor suppressor gene, an antigenic gene, a cytotoxic gene, a cell proliferation suppressor gene, a cytokine gene, a pro-cell
  • a pro-cell One or more genes selected from the group consisting of a pro-apoptotic gene and an anti-angiogenic gene may be overexpressed in a high-frequency specific manner.
  • the cancer cell death gene may be a BCL-2 lineage pro-apoptotic gene or a death receptor/ligand gene.
  • the fluorescent protein gene is luciferase, enhanced green fluorescent protein (EGFP), green fluorescent protein (GFP), yellow fluorescent protein (Yellow It may be one or more selected from the group consisting of Fluorescent Protein (YFP), Red Fluorescent Protein (RFP), and Cyan Fluorescent Protein (CFP), preferably green fluorescent protein, but not limited thereto. does not
  • the cancer cell suppressor gene is p53 gene, APC gene, DPC-4/Smad4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, MMAC-1 gene, MMSC- 2 gene, NF-1 gene, MTS1 gene, CDK4 gene, NF-1 gene, NF-2 gene, and may be at least one selected from the group consisting of the VHL gene.
  • the cancer is lung cancer, blood cancer, colorectal cancer, rectal cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, fallopian tube carcinoma, ovarian cancer, vaginal carcinoma, vulvar carcinoma, liver cancer, stomach cancer, esophageal cancer , small bowel cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, urethral cancer, penile cancer, prostate cancer, testicular cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, non-small cell lung cancer, bone cancer, skin cancer, head or neck cancer, skin or It may be at least one selected from the group consisting of intraocular melanoma, Hodgkin's disease, endocrine adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, central nervous system tumor, spinal cord tumor, brainstem glioma, and pituitary adenoma.
  • intraocular melanoma Hodgkin's
  • pathogenic factor suppression gene refers to a property or substance that causes a disease by infecting a host with a pathogen such as an antigen, virus, bacterium, or fungus having pathogenicity
  • pathogenic factor suppression gene means a gene that inhibits the increase, activity, and expression of the pathogenic factor.
  • Chemicals, natural compounds, extracts derived from natural materials, nucleic acids such as siRNA, micro RNA, or recombinant DNA, peptides, and proteins containing the gene or amino acids encoded by the gene may contain one or more.
  • the gene for inhibiting the pathogenic factor refers to a gene of an antigen or an antigen-binding fragment, and includes at least two heavy (heavy (H) chains) linked to each other by disulfide bonds acting with the antigen, and A protein containing two light (L) chains.
  • Each heavy chain (HC) is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of three domains, CH1, CH2 and CH3.
  • Each light chain (LC) is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions are further divided into highly variable (hypervariability) regions, so-called complementarity determining regions (CDRs), which are arranged in more conserved regions, so-called framework regions (FR) regions. can be further divided.
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that bind antigen.
  • the constant region of an antibody is a host tissue or several cells of the immune system (e.g., effector cells) and elements, including the first component (Clq), which is the classical complement system.
  • antibody includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies and chimeric antibodies. antibodies).
  • An antibody may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or Both light and heavy chains can be divided into regions of structural and functional homology.
  • Antigen-binding fragment means one or more portions of an antibody that retain the ability to specifically interact with an antigen (eg, by binding, sterically hindering, or stabilizing spatial distribution).
  • binding fragments include, but are not limited to, Fab fragments, monovalent fragments composed of VL, VH, CL and CH1 domains; a Fab' fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains and the N-terminal part of the hinge region of an immunoglobulin; F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked at the hinge region by a disulfide bridge; Fd fragment consisting of VH and CH1 domains; Fv fragment consisting of the VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., (1989) Nature 341:544-546), consisting of the VH domain; and an isolated complementarity determining region (CDR).
  • Fab fragments monovalent fragments
  • the two domains of the Fv fragment, VL and VH are encoded by separate genes, but, using recombinant methods, the VL and VH regions are monovalent molecules (single-chain Fv, scFv; Bird et al., (1988 ) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883) can be linked by a synthetic linker that allows them to be made as a single protein. there is.
  • Such single chain antibodies are also intended to be included within the term "antibody fragment".
  • antibody fragments can be obtained using conventional techniques known in the art, and these fragments are screened for use in the same way as intact antibodies.
  • Antibody fragments also include single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis- can be merged into scFv (bis-scFv) (Hollinger and Hudson, (2005) Nature Biotechnology 23: 1126-1136). Antibody fragments can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (U.S. Pat. No. 6,703,199).
  • Fn3 Fibronectin type III
  • Antibody fragments can be merged into single chain molecules comprising a pair of side-by-side Fv fragments (VH-CH1-VH-CH1) that form a pair of antigen-binding sites, together with a complementary light chain polypeptide (Zapata et al. ., (1995) Protein Eng. 8:1057-1062; and U.S. Pat. No. 5,641,870).
  • the pathogenic factor suppression gene may be a gene that suppresses a pathogenic factor causing a disease in mammals, preferably an antibody, a heavy chain variable region, a light chain variable region, and an antigen-binding fragment. , more preferably an antibody, but is not limited thereto.
  • the mammal may be an animal selected from the group consisting of dogs, pigs, cows, cats, monkeys, foxes, raccoons, bats, coyotes and weasels, preferably dogs, pigs, cats or , but is not limited thereto.
  • the pathogenicity is rabies virus or Rabies lyssavirus, Porcine circovirus, Feline calicivirus, Chlamydophila felis ( Chlamydophila felis ), feline leukemia virus, feline panleukopenia virus, feline bronchitis virus, feline immunodeficiency virus, feline infectious peritonitis virus, Ehrlichia canis , canine parvovirus, canine distemper, canine parainfluenza It may be caused by a virus selected from the group consisting of virus, canine type II adenovirus, canine adenovirus and canine coronavirus, preferably rabies virus, porcine circovirus or feline calicivirus (Feline calicivirus), but is not limited thereto.
  • the "antigenic gene” is a factor capable of inducing an immune response by acting as an antigen in the body, and refers to a viral coat protein, viral DNA or RNA, etc., and may encode the amino acid sequence constituting it. .
  • the encoded envelope protein, DNA or RNA may be a viral construct, a fragment having antigenicity, or a gene that induces the expression of the envelope protein or may be used as a vaccine composition as such.
  • the vaccine composition according to the present invention may be prepared in the form of incorporating the active ingredient into a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coating agents, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like.
  • Pharmaceutically acceptable carriers that can be used in the vaccine composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the vaccine composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. .
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin in addition to active ingredients. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.
  • Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc.
  • compositions for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
  • a base for suppositories witepsol, tween 61, cacao paper, laurin paper, glycerogelatin, and the like may be used.
  • the excipient is also called an adjuvant, and is a substance that non-specifically promotes an immune response to an antigen in the initial activation process of immune cells. It is not an immunogen to the host, but enhances immunity by increasing the activity of cells of the immune system. Means an agent, molecule, etc. The selection of excipients in the production of inactivated vaccines is an important factor in vaccine efficacy.
  • the vaccine composition may include excipients known in the art without limitation, preferably oil and gel, more preferably oil and aluminum hydroxide gel.
  • the mixing ratio of the oil and gel is characterized in that, as the content of the gel increases, the properties of the excipient are stabilized, vaccine side effects are reduced, and cell aggregation is weak.
  • the oil and aluminum hydroxide gel may preferably additionally contain oil and aluminum hydroxide gel in a volume ratio of 1: 3 to 5, more preferably oil and aluminum hydroxide gel in a volume ratio of 1: 4. can do.
  • the vaccine composition according to the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.
  • the dose of the vaccine composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the subject. It is obvious that the concentration of the active ingredient included in the vaccine composition can be variously selected according to the subject, and is preferably included in the vaccine composition at a concentration of 0.01 to 5,000 ⁇ g/ml. If the concentration is less than 0.01 ⁇ g/ml, pharmacological activity may not be exhibited, and if the concentration exceeds 5,000 ⁇ g/ml, it may exhibit toxicity to the subject.
  • the promoter may be a promoter derived from plant cells.
  • the present invention provides a high-frequency specific expression recombinant vector containing the above promoter.
  • vector refers to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell, capable of replicating DNA and independently reproducing in a host cell.
  • "Expression vector” means a recombinant DNA molecule comprising a coding sequence of interest and appropriate nucleic acid sequences necessary to express an operably linked coding sequence in a particular host organism.
  • Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • expression vector refers to a recombinant DNA or RNA construct such as a plasmid, phage, recombinant virus or other vector that, upon introduction into a suitable host cell, results in the expression of the cloned DNA.
  • Suitable expression vectors are well known to those skilled in the art, and include those replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or integrate into the host cell genome.
  • the conventional vector may be any one as long as it can introduce the promoter of the present invention, but preferably pCAMBIA series, pGA series, pGWB series such as pGA3519, pCAMBIA3301, pCAMBIA3300, pGA3426, pGA3780, pGWB12, pGWB14 It may be any one selected from the group consisting of Ti-plasmid and vectors derived therefrom.
  • the present invention provides a transformant transformed with the above recombinant expression vector.
  • the expression vector may be one in which the promoter of the present invention is introduced into a conventional vector, and thus preparing an expression vector by introducing the promoter of the present invention is easily performed according to a method known to those skilled in the art to which the present invention belongs. It is possible.
  • the above expression vectors can be inserted into cultured host cells using well known techniques such as infection, transduction, transfection, electroporation and transformation.
  • Representative examples of hosts are bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium or Agrobacterium tumefaciens; Or plant cells, for example monocotyledonous plants such as corn, barley, wheat, rice, oats, rye or sugar cane, or roses, carrots, tea trees, camellia trees, apple trees, It may be a cell of a dicotyledonous plant such as Arabidopsis, potato, eggplant, tobacco, red pepper, lettuce, Chinese cabbage, cabbage, watermelon, or melon.
  • Plant cell used for plant transformation.
  • the plant cell may be any type of cultured cell, cultured tissue, cultured organ or whole plant, preferably a cultured cell, cultured tissue or cultured organ and more preferably a cultured cell.
  • Plant tissue refers to differentiated or undifferentiated plant tissues such as, but not limited to, stems, leaves, cancer tissues, and various types of cells used in culture, such as single cells, protoplasts, shoots and Contains callus tissue.
  • genes with significant changes were classified by pathway according to the TPM (transcripts per million) analysis value, and the expression of genes related to the phenylpropanoid pathway was strongly increased by radiofrequency treatment, and genes related to the phenylpropanoid pathway were selected. (Fig. 1).
  • TFBS transcription factor binding site
  • the pCAMBIA1302 vector was used as a backbone, and the mGFP5 gene was used as a gene to induce expression (FIG. 3).
  • the mGFP5 gene was used as a gene to induce expression (FIG. 3).
  • BstXI and NcoI were removed by treatment, and a candidate promoter was inserted into the site to construct a vector.
  • the vector construction method is shown in FIG. 4 .
  • the pCAMBIA1302 vector in which the candidate promoters of Group 1 and Group 2 of Example 3 were cloned, was inserted into rose cells, and the rose cells into which the vectors were inserted were radiofrequency treated.
  • As a negative control non-transformed rose cells were used, and as a positive control, a vector constructed using the CaMV 35S promoter was transformed.
  • the transformed rose cells were cultured, and after treatment with high frequency once/day for 20 minutes, fluorescence expression of mGFP5 was confirmed through confocal microscopy.
  • EGF epidermal growth factor
  • Example 4-1 which is the promoter selected in Example 4-1, it was attempted to confirm whether the expression of the foreign transgene can be increased in a high-frequency specific manner.
  • an expression vector was prepared in the same manner as in Example 4-1, and an epidermal growth factor (EGF) as a foreign gene was inserted into the vector to prepare a high-frequency specific expression vector.
  • EGF epidermal growth factor
  • the prepared vector was transformed into rose cells, treated with high frequency and cultured for 24 hours, and then the expression of EGF was confirmed by polyacrylamide gel electrophoresis (sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE).
  • EGF expressed in Escherichia coli and purified was used.
  • EGF expressed in Escherichia coli and purified was used.
  • non-transformed rose cells and rose cells treated with Methyl jasmontae high expression of EGF due to the promoter of the present invention was confirmed.
  • EGF expressed in E. coli had a size of 6 kDa, compared to the positive control, rose cells and rose cells treated with Methyl jasmontae, transformed with a high-frequency specific promoter It was confirmed that the expression of EGF was strongly induced in rose cells.
  • Basic fibroblast growth factor (basic fibroblast growth factor ( Expression of basic fibroblast growth factor (bFGF) was confirmed by SDS-PAGE.
  • the high frequency-specific promoter derived from the gene related to the phenylpropanoid pathway of the present invention was specifically expressed when the transformed plant was treated with high frequency, and it was confirmed that the expression of the foreign gene was specifically increased at high frequency. .

Abstract

The present invention relates to a high frequency-specific expression promoter. The promoter of the present invention increases the expression of a foreign gene under a high frequency condition as compared to under a non-high frequency condition. In addition, the promoter has an excellent effect of increasing the intracellular content of the aromatic amino acids tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr) by means of the high frequency. Accordingly, the promoter has an effect of maximizing the production of a phenylpropanoid pathway-related gene and a phytochemical and thus may be usefully employed in relevant industries.

Description

고주파 특이적 발현 프로모터High-frequency specific expression promoter
본 발명은, 고주파 특이적 발현 프로모터에 관한 것이다.The present invention relates to a high-frequency specific expression promoter.
진핵세포의 시스템에서 유전자 발현은 다양한 시스-작용 및 트랜스-작용 조절 요소들 사이에서의 복잡한 상호작용을 통해 조절된다. 시스-작용 요소는 다양한 전사 요소라 불리는 트랜스-작용 요소의 결합을 조절함으로써 유전자의 발현을 직접 또는 간접적으로 조절하는 유전자에 인접한 DNA 서열로 정의된다. 프로모터는 가장 중요한 시스-작용 요소 중 하나이며 일반적으로 유전자의 전사 개시 부위의 5 말단 상류에 위치한 뉴클레오티드 서열 영역으로 간주된다. 프로모터는 RNA 중합효소 Ⅱ에 대한 결합 부위를 포함하며 유전자의 전사를 개시한다. 또한, 프로모터는 전사 개시 부위(+1)와 관련되어 있는 염기 위치 -70 부근의 CAAT 박스 및 염기 위치 -30 부근의 TATA 박스와 같이 잘 보전된 여리 개의 서열 요소를 포함한다(Joshi, 1987). TATA 박스는 전사의 개시를 정확하게 위치화하는데 중요하다. CAAT 박스는 모든 프로모터에 존재하지는 않으며, GC가 풍부한 영역이 대체된 몇몇 프로모터에 존재한다. 이러한 염기 요소들뿐 아니라, 프로모터는 또한 건조 스트레스, 열 쇼크, 추위 스트레스, 영양소의 유용성, 광 및 이온 강도와 같은 생리적인 조건과 환경 신호에 반응하는 부가적 염기서열을 포함한다. 또한, 어떤 프로모터는 조직-특이적 유전자 발현 또는 세포-특이적 유전자의 발현을 유도하는 서열 요소를 포함한다. 또한, 프로모터는 발현 수준, 조직 특이성 또는 세포-특이성, 발달 단계 의존성 및 특이적 환경 자극에 대한 반응성 등의 조건에 의해 유전자 발현을 조절함으로써, 진핵세포의 유전공학에서 중요한 요소이다.Gene expression in eukaryotic systems is regulated through complex interactions between various cis-acting and trans-acting regulatory elements. A cis-acting element is defined as a DNA sequence adjacent to a gene that directly or indirectly regulates the expression of a gene by regulating the binding of trans-acting elements called various transcription elements. The promoter is one of the most important cis-acting elements and is generally considered a region of nucleotide sequence located 5 terminally upstream of the transcription initiation site of a gene. A promoter contains a binding site for RNA polymerase II and initiates transcription of a gene. In addition, the promoter contains several well-conserved sequence elements, such as the CAAT box around base position -70 and the TATA box around base position -30, which are related to the transcription initiation site (+1) (Joshi, 1987). The TATA box is important for correctly locating the initiation of transcription. The CAAT box is not present in all promoters, but in some promoters where GC-rich regions have been replaced. In addition to these base elements, promoters also contain additional sequences that respond to environmental cues and physiological conditions such as dry stress, heat shock, cold stress, nutrient availability, light and ionic strength. In addition, some promoters contain sequence elements that direct expression of tissue-specific genes or expression of cell-specific genes. In addition, promoters are important elements in the genetic engineering of eukaryotic cells by regulating gene expression by conditions such as expression level, tissue specificity or cell-specificity, developmental stage dependence, and responsiveness to specific environmental stimuli.
고주파(High frequency)는 높은 주파수를 가진 전자파를 뜻하는 것으로, 저주파에 대응하는 전자파이다. 전력계에서는 상용 주파수인 50~60Hz를 저주파, 그 이상을 고주파라고 하며, 대부분의 분야에서 상용 주파수 이상의 진동수를 가지는 전자파를 고주파라 칭한다. 고주파는 전자파를 방사하기 쉬운 파형의 전자파로서, 세포와 같은 대상에 방해 또는 스트레스를 유도하기 쉽다는 특징이 있다.High frequency means an electromagnetic wave with a high frequency, and is an electromagnetic wave corresponding to a low frequency. In the power system, a commercial frequency of 50 to 60 Hz is referred to as a low frequency and a frequency higher than that is referred to as a high frequency, and in most fields, an electromagnetic wave having a frequency higher than the commercial frequency is referred to as a high frequency. High frequency waves are electromagnetic waves of a waveform that are easy to radiate, and are characterized by being easy to induce disturbance or stress to objects such as cells.
한편, 특정 조건 하에서, 프로모터의 발현을 조절할 수 있는, 염 유도성 프로모터(대한민국 등록특허 제10-2208028호), 산소 유도성 프로모터(대한민국 등록특허 제10-0173845호), 식물 호르몬 유도성 프로모터(대한민국 등록특허 제10-1985653 호), 환경 스트레스 유도성 프로모터(대한민국 등록특허 제10-1151238호)가 개발되어 왔으나, 고주파 조건하에서, 발현이 조절되는 프로모터에 대해서는 연구된 바가 없다.On the other hand, salt-inducible promoters (Republic of Korea Patent No. 10-2208028), oxygen-inducible promoters (Republic of Korea Patent No. 10-0173845), plant hormone-inducible promoters that can regulate the expression of promoters under specific conditions ( Korean Registered Patent No. 10-1985653) and environmental stress inducible promoters (Korean Registered Patent No. 10-1151238) have been developed, but promoters whose expression is regulated under high-frequency conditions have not been studied.
본 발명의 목적은 고주파 특이적 발현 프로모터를 제공하는 것이다.An object of the present invention is to provide a high-frequency specific expression promoter.
본 발명의 다른 목적은 상기의 프로모터를 포함하는 고주파 특이적 재조합 발현 벡터를 제공하는 것이다.Another object of the present invention is to provide a high-frequency specific recombinant expression vector containing the above promoter.
본 발명의 또 다른 목적은 상기의 고주파 특이적 발현 벡터로 형질전환된 형질전환체를 제공하는 것이다.Another object of the present invention is to provide a transformant transformed with the above high-frequency specific expression vector.
상기 목적을 달성하기 위하여, 본 발명의 목적은 고주파 특이적 발현 프로모터를 제공한다.In order to achieve the above object, an object of the present invention is to provide a high-frequency specific expression promoter.
또한, 본 발명은 상기의 프로모터를 포함하는 고주파 특이적 발현 재조합 벡터를 제공한다.In addition, the present invention provides a high-frequency specific expression recombinant vector containing the above promoter.
또한, 본 발명은 상기의 고주파 특이적 발현 벡터로 형질전환된 형질전환체를 제공한다.In addition, the present invention provides a transformant transformed with the above high-frequency specific expression vector.
본 발명은 고주파 특이적 발현 프로모터에 관한 것으로서, 본 발명의 프로모터는 비-고주파 조건과 비교하여, 고주파 조건 시 외래 유전자의 발현을 증가시킨다. 또한, 세포 내 상기 고주파에 의한 트립토판(Trp), 페닐알라닌(Phe), 티로신(Tyr)의 방향족 아미노산(Aromatic amino acids)의 함량을 증가시키는 효과가 우수하다. 이에 따라 phenylpropanoid pathway 관련 유전자 및 파이토케미컬(Phytochemical)의 생산을 극대화하는 효과가 있어, 관련 산업에 유용하게 이용할 수 있다.The present invention relates to a high-frequency specific expression promoter, wherein the promoter of the present invention increases the expression of a foreign gene under a high-frequency condition, compared to a non-high-frequency condition. In addition, the effect of increasing the content of aromatic amino acids such as tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) by the high frequency in the cell is excellent. Accordingly, it has the effect of maximizing the production of phenylpropanoid pathway related genes and phytochemicals, and can be usefully used in related industries.
도 1은 본 발명의 고주파 특이적 발현 벡터를 발굴하기 위한, 고주파 처리에 의하여 변화되는 장미 세포 대사 네트워크의 모식도를 나타낸 도이다.1 is a diagram showing a schematic diagram of a rose cell metabolic network changed by radiofrequency treatment to discover a radiofrequency-specific expression vector of the present invention.
도 2는 본 발명의 고주파 특이적 발현 벡터를 발굴하기 위한 Tryptophan synthase alpha chain-like 유전자의 Tandem repeat를 확인한 도이다.Figure 2 is a diagram confirming the tandem repeat of the Tryptophan synthase alpha chain-like gene for discovering the high-frequency specific expression vector of the present invention.
도 3은 본 발명의 고주파 특이적 발현 프로모터를 벡터에 삽입하는 모식도를 나타낸 것이다.Figure 3 shows a schematic diagram of inserting the high-frequency specific expression promoter of the present invention into a vector.
도 4는 본 발명의 고주파 특이적 발현 벡터를 제작하는 방법을 벡터맵으로 나타낸 도이다.4 is a diagram showing a method for preparing a high-frequency specific expression vector of the present invention as a vector map.
도 5는 본 발명의 고주파 특이적 발현 벡터로 형질전환된 장미 세포에서의 형광 발현을 확인한 도이다(A: 음성 대조군, B: 양성 대조군, C: Group 1 프로모터 군, D: Group 2 프로모터 군).Figure 5 is a diagram confirming the fluorescence expression in rose cells transformed with the high-frequency specific expression vector of the present invention (A: negative control, B: positive control, C: Group 1 promoter group, D: Group 2 promoter group) .
도 6은 본 발명의 고주파 특이적 발현 벡터로 형질전환된 장미 세포에서의 형광 발현을 정량화한 도이다.6 is a diagram quantifying fluorescence expression in rose cells transformed with the high-frequency specific expression vector of the present invention.
도 7은 본 발명의 고주파 특이적 발현 벡터로, 고주파 특이적 EGF의 발현 유도를 웨스턴 블랏으로 확인한 도이다.Figure 7 is a diagram confirming the induction of high frequency-specific EGF expression by Western blot with the high frequency-specific expression vector of the present invention.
도 8은 본 발명의 고주파 특이적 발현 벡터로, 고주파 특이적 bFGF의 발현 유도를 웨스턴 블랏으로 확인한 도이다.8 is a diagram confirming the induction of expression of high frequency-specific bFGF by Western blot with the high frequency-specific expression vector of the present invention.
본 발명은 고주파(high frequency)에 특이적으로 발현하는 프로모터를 제공한다.The present invention provides a promoter specifically expressed at a high frequency.
본 발명에 있어서 "특이적"이란, 본 발명에 따른 프로모터에 의한 고발현성, 특이성 등을 모두 포함하여 형질전환체 일부 조건에서 발현 증가를 의미한다. 예컨대 본 발명에 따른 프로모터가 결여된 형질전환체와 대비하여 50% 이상, 100% 이상, 200% 이상, 5배 이상 또는 그 이상의 발현 증가를 의미한다.In the present invention, "specific" means an increase in expression in some conditions of a transformant, including both high expression and specificity by the promoter according to the present invention. For example, it means an increase in expression of 50% or more, 100% or more, 200% or more, 5-fold or more or more compared to a transformant lacking the promoter according to the present invention.
본 발명의 프로모터와 "기능적으로 동등한 절편"은 본 발명의 프로모터와 실질적으로 동등한 효과를 나타내는, 서열번호 1로 표시되는 염기서열로 이루어진 핵산의 조각 또는 일부분을 의미한다. 이러한 핵산 절편은 서열번호 1에 기재된 염기서열과 비교하여 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 또는 그 이상의 서열 상동성을 가지며, 이러한 핵산 절편은 당업계에 널리 알려진 분자생물학적 방법에 의하여 용이하게 제작될 수 있다. 또한, 본 발명의 서열번호 1로 표시되는 프로모터와 일부 유전자 영역은 공개된 장미 게놈 서열 상, Tryptophan synthase alpha chain-like 유전자의 프로모터 영역과 유전자의 일부를 포함하는 서열로, 서열번호 1로 표시되는 염기 서열 중 일부가 치환, 삽입 또는 삭제되더라도 Tryptophan synthase alpha chain-like 유전자의 프로모터와 일부 유전자 영역에 위치한 서열과 상동성이 유지되어 실질적으로 동등한 효과를 나타낸다면 본 발명의 범주에 포함될 수 있다.The "functionally equivalent segment" to the promoter of the present invention means a fragment or part of a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1, which exhibits substantially equivalent effects to the promoter of the present invention. These nucleic acid fragments are 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, It has 96%, 97%, 98%, 99% or more sequence homology, and such nucleic acid fragments can be easily prepared by molecular biological methods well known in the art. In addition, the promoter represented by SEQ ID NO: 1 and part of the gene region of the present invention is a sequence including the promoter region of the Tryptophan synthase alpha chain-like gene and part of the gene on the published rose genome sequence, which is represented by SEQ ID NO: 1 Even if some of the nucleotide sequences are substituted, inserted, or deleted, as long as homology to the promoter of the tryptophan synthase alpha chain-like gene and sequences located in some gene regions are maintained and substantially equivalent effects are exhibited, it may be included in the scope of the present invention.
본 발명의 일실시예에 따르면, 식물에서는 해당과정(Glycolysis), 펜토스인산대사경로(Pentose Phosphate pathway), 시키믹산대사경로(Shikimate pathway), 방향족 아미노산 생합성경로(Aromatic amino acid(AA) Biosynthesis), 페닐프로파노이드대사경로(Phenylpropanoid pathway)가 존재하고, 고주파 파형에 반응하여 Anthranilate에서 Tryptophane(Trp)으로 될 때 관여하는 효소인 Tryptopane synthase (TS) 암호화되고 있는 유전자발현이 증가 되는 것을 확인하였으며, 이러한 방향족 아미노산 생합성경로(Aromatic amino acid(AA) Biosynthesis), 페닐프로파노이드대사경로(Phenylpropanoid pathway) 활성화는, 방향족 화합물 혹은 Caffoylquinic acids, Kaempferol, Quercetin, Catechin 등의 플라보노이드(Flavonoids)의 함량을 증가시킬 수 있다.According to one embodiment of the present invention, in plants, Glycolysis, Pentose Phosphate pathway, Shikimate pathway, Aromatic amino acid (AA) Biosynthesis pathway , It was confirmed that the Phenylpropanoid pathway exists and the expression of the gene encoding Tryptopane synthase (TS), an enzyme involved in converting anthranilate into Tryptophane (Trp) in response to high-frequency waves, increased, The activation of the aromatic amino acid (AA) biosynthesis pathway and the phenylpropanoid pathway can increase the content of aromatic compounds or flavonoids such as Caffoylquinic acids, Kaempferol, Quercetin, and Catechin. can
본 발명의 일실시예에 따르면, 상기 프로모터는 서열번호 1의 염기서열 또는 서열번호 2의 염기서열을 포함하는 것일 수 있다.According to one embodiment of the present invention, the promoter may include the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
본 발명의 일실시예에 따르면, 상기 고주파는 100 내지 1200khz 조건으로 처리되는 것일 수 있으며, 바람직하게는 500 내지 1200khz일 수 있고, 더욱 바람직하게는 900 내지 1200khz일 수 있으나, 이에 제한되지는 않는다.According to one embodiment of the present invention, the high frequency may be processed under conditions of 100 to 1200 khz, preferably 500 to 1200 khz, and more preferably 900 to 1200 khz, but is not limited thereto.
본 발명의 일실시예에 따르면, 상기 고주파는 1 내지 60분의 조건으로 처리되는 것일 수 있고, 바람직하게는 10 내지 50분일 수 있고, 더욱 바람직하게는 20 내지 30분일 수 있으나, 이에 제한되지는 않는다.According to one embodiment of the present invention, the high frequency may be processed under conditions of 1 to 60 minutes, preferably 10 to 50 minutes, and more preferably 20 to 30 minutes, but is not limited thereto. don't
본 발명의 일실시예에 따르면, 상기 고주파는 1 내지 10회/day의 조건으로 처리되는 것일 수 있으나, 바람직하게는 1 내지 7회/day의 조건일 수 있고, 더욱 바람직하게는 1 내지 5회/day의 조건으로 처리될 수 있으나, 이에 제한되지는 않는다.According to one embodiment of the present invention, the high frequency may be processed under a condition of 1 to 10 times/day, but may be preferably 1 to 7 times/day, more preferably 1 to 5 times. It may be processed under the condition of /day, but is not limited thereto.
본 발명의 일실시예에 따르면, 상기 고주파는 1 내지 10일 조건으로 처리되는 것일 수 있다.According to one embodiment of the present invention, the high frequency may be processed under conditions of 1 to 10 days.
본 발명의 일실시예에 따르면, 상기 프로모터는 트립토판(Trp), 페닐알라닌(Phe), 티로신(Tyr)의 방향족 아미노산(Aromatic amino acids)의 함량을 증가시키는 것일 수 있다.According to one embodiment of the present invention, the promoter may increase the content of aromatic amino acids such as tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr).
본 발명의 일실시예에 따르면, 상기 방향족 아미노산(Aromatic amino acids)의 함량을 증진시켜 식물 대사의 Phenylpropanoid pathway 대사의 활성 및 상기 대사의 유전자를 증진시키는 것일 수 있으며, 상기 Phenylpropanoid pathway 대사의 활성 및 상기 대사의 유전자 증진은 카페오일퀴닉 산(Caffeoylquinic acids), 퀘르세틴(Quercetin), 캠페롤(Kaempferol), 카테킨(Catechin), 벤즈알데하이드(Benzaldehyde) 및 페닐아세트 산(Phenylacetic acid)으로 이루어진 군에서 선택된 파이토케미컬(Phytochemical)의 생산을 증가시키는 것일 수 있다.According to one embodiment of the present invention, the activity of the Phenylpropanoid pathway metabolism of plant metabolism and the gene of the metabolism may be enhanced by increasing the content of the aromatic amino acids, and the activity of the Phenylpropanoid pathway metabolism and the above Genetic enhancement of metabolism is a phytochemical selected from the group consisting of Caffeoylquinic acids, Quercetin, Kaempferol, Catechin, Benzaldehyde and Phenylacetic acid It may be to increase the production of (phytochemical).
본 발명의 일실시예에 따르면, 상기 프로모터는 암세포 사멸 유전자, 형광 단백질 유전자, 암세포 억제인자 유전자, 병원성 인자 억제 유전자, 항원성 유전자, 세포 독성 유전자, 세포 증식 억제 유전자, 사이토카인 유전자, 친-세포사멸 유전자(pro-apoptotic gene) 및 항-신생혈관생성 유전자(anti-angiogenic gene)로 이루어진 군에서 선택된 1종 이상의 유전자를 고주파 특이적으로 과발현시키는 것일 수 있다.According to one embodiment of the present invention, the promoter is a cancer cell death gene, a fluorescent protein gene, a cancer cell suppressor gene, a pathogenic factor suppressor gene, an antigenic gene, a cytotoxic gene, a cell proliferation suppressor gene, a cytokine gene, a pro-cell One or more genes selected from the group consisting of a pro-apoptotic gene and an anti-angiogenic gene may be overexpressed in a high-frequency specific manner.
본 발명의 일실시예에 따르면, 상기 암세포 사멸 유전자는 BCL-2 계통 세포사멸 촉진(pro-apoptotic) 유전자 또는 자살 수용체/리간드(death receptor/ligand) 유전자인 것일 수 있다.According to one embodiment of the present invention, the cancer cell death gene may be a BCL-2 lineage pro-apoptotic gene or a death receptor/ligand gene.
본 발명의 일실시예에 따르면, 상기 형광 단백질 유전자는 루시퍼라아제(luciferase), 증강 녹색 형광 단백질(enhanced green fluorescent protein, EGFP), 녹색 형광 단백질(green fluorescent protein, GFP), 노랑 형광 단백질 (Yellow Fluorescent Protein, YFP), 적색 형광 단백질 (Red Fluorescent Protein, RFP)및 청색 형광 단백질(cyan fluorescent protein, CFP)으로 이루어진 군에서 선택된 1종 이상인 것일 수 있으며, 바람직하게는 녹색 형광 단백질이나, 이에 제한되지는 않는다.According to one embodiment of the present invention, the fluorescent protein gene is luciferase, enhanced green fluorescent protein (EGFP), green fluorescent protein (GFP), yellow fluorescent protein (Yellow It may be one or more selected from the group consisting of Fluorescent Protein (YFP), Red Fluorescent Protein (RFP), and Cyan Fluorescent Protein (CFP), preferably green fluorescent protein, but not limited thereto. does not
본 발명의 일실시예에 따르면, 상기 암세포 억제인자 유전자는 p53 유전자, APC 유전자, DPC-4/Smad4 유전자, BRCA-1 유전자, BRCA-2 유전자, WT-1유전자, MMAC-1 유전자, MMSC-2 유전자, NF-1 유전자, MTS1 유전자, CDK4 유전자, NF-1 유전자, NF-2 유전자 및 VHL 유전자로 이루어진 군에서 선택된 1종 이상인 것일 수 있다.According to one embodiment of the present invention, the cancer cell suppressor gene is p53 gene, APC gene, DPC-4/Smad4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, MMAC-1 gene, MMSC- 2 gene, NF-1 gene, MTS1 gene, CDK4 gene, NF-1 gene, NF-2 gene, and may be at least one selected from the group consisting of the VHL gene.
본 발명의 일실시예에 따르면, 상기 암은 폐암, 혈액암, 대장암, 직장암, 결장암, 유방암, 자궁경부암, 자궁내막암, 나팔관암종, 난소암, 질암종, 음문암종, 간암, 위암, 식도암, 소장암, 췌장암, 담낭암, 신장암, 방광암, 요도암, 음경암, 전립선암, 고환암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 비소세포성폐암, 골암, 피부암, 두부 또는 경부암, 피부 또는 안구 내 흑색종, 호지킨병, 내분비선암, 만성 또는 급성 백혈병, 림프구 림프종, 중추 신경계 종양, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군에서 선택된 1종 이상인 것일 수 있다.According to one embodiment of the present invention, the cancer is lung cancer, blood cancer, colorectal cancer, rectal cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, fallopian tube carcinoma, ovarian cancer, vaginal carcinoma, vulvar carcinoma, liver cancer, stomach cancer, esophageal cancer , small bowel cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, urethral cancer, penile cancer, prostate cancer, testicular cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, non-small cell lung cancer, bone cancer, skin cancer, head or neck cancer, skin or It may be at least one selected from the group consisting of intraocular melanoma, Hodgkin's disease, endocrine adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, central nervous system tumor, spinal cord tumor, brainstem glioma, and pituitary adenoma.
본 발명에서 사용하는 용어 "병원성 인자 억제 유전자"에서, "병원성 인자"는 병원성을 가지는 항원, 바이러스, 세균 또는 진균 등과 같은 병원체가 숙주에 감염하여 병을 일으키는 원인이 되는 성질, 물질을 의미하며, "병원성 인자 억제 유전자"는 상기 병원성 인자의 증가, 활성 및 발현을 억제하는 유전자를 의미한다. In the term "pathogenic factor suppression gene" used in the present invention, "pathogenic factor" refers to a property or substance that causes a disease by infecting a host with a pathogen such as an antigen, virus, bacterium, or fungus having pathogenicity, "Pathogenic factor suppression gene" means a gene that inhibits the increase, activity, and expression of the pathogenic factor.
상기 유전자 또는 이에 의해 코딩되는 아미노산을 포함하는 화합물(chemicals), 천연물(natural compounds), 천연소재 유래 추출물, siRNA, micro RNA 또는 recombinant DNA 등의 핵산(nucleic acids), 펩타이드 및 단백질로 구성된 군에서 선택된 하나 이상을 포함할 수 있다. Chemicals, natural compounds, extracts derived from natural materials, nucleic acids such as siRNA, micro RNA, or recombinant DNA, peptides, and proteins containing the gene or amino acids encoded by the gene may contain one or more.
상기 병원성 인자를 억제하기 위한 유전자는 항원 또는 항원 결합 단편의 유전자을 의미하며, 항원과 작용하는 이황화 결합(disulfide bonds)에 의해 서로-연결되어 있는 적어도 두 개의 중쇄((heavy (H) chains)) 및 두 개의 경쇄((light (L) chains))를 포함하는 단백질을 의미한다. 각 중쇄(heavy chain, HC) 는 중쇄 가변 부위(heavy chain variable region, VH) 및 중쇄 불변 부위(heavy chain constant region, CH)로 구성된다. 중쇄 불변 부위는 세 도메인, CH1, CH2 및 CH3으로 구성된다. 각 경쇄(light chain, LC)는 경쇄 가변 부위(light chain variable region, VL) 및 경쇄 불변 부위(light chain constant region, CL)로 구성된다. 경쇄 불변 부위는 하나의 도메인, CL로 구성된다. VH 및 VL 부위는 더 나아가, 소위 프레임워크(framework regions, FR) 부위라고 불리는 좀 더 보존적인 부위로 배치되어 있는 소위 보완성 결정 부위(complementarity determining regions, CDR)라는 고도로 가변인(hypervariability) 부위로 더 나누어질 수 있다. 각 VH 및 VL은 아미노-말단에서 카복시-말단으로 세 개의 CDRs 및 네 개의 FR로 다음의 순서로 배열로 구성된다: FR1, CDR1, FR2, CDR2, FR3, CDR3, 및 FR4. 중쇄 및 경쇄의 가변 부위는 항원과 결합하는 결합 도메인을 포함한다. 항체의 불변 부위는 면역글로블린이 숙주 조직 또는 면역 시스템의 여러 세포(예를 들어, 주효 세포(effector cell)) 및 고전적인 보완 시스템인 첫 번째 성분 (the first component, Clq) 을 포함하는, 인자들과 결합하는 것을 매개할 수 있다. "항체 (antobody)" 라는 용어는 예를 들어, 단일 클론 항체(monoclonal antibodies), 인간 항체(human antibodies), 인간화된 항체(humanized antibodies), 카멜화된 항체(camelised antibodies) 및 키메릭 항체(chimeric antibodies)를 포함한다. 항체는 어느 아이소타입(isotype) (예를 들어, IgG, IgE, IgM, IgD, IgA 및 IgY), 부류 (class) ((예를 들어, IgG1, IgG2, IgG3, IgG4, IgA1 및 IgA2), 또는 아류 (sublass) 가 될 수 있다. 경쇄 및 중쇄는 둘 다 구조적 및 기능적인 상동성 부위로 나뉠 수 있다.The gene for inhibiting the pathogenic factor refers to a gene of an antigen or an antigen-binding fragment, and includes at least two heavy (heavy (H) chains) linked to each other by disulfide bonds acting with the antigen, and A protein containing two light (L) chains. Each heavy chain (HC) is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each light chain (LC) is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The VH and VL regions are further divided into highly variable (hypervariability) regions, so-called complementarity determining regions (CDRs), which are arranged in more conserved regions, so-called framework regions (FR) regions. can be further divided. Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that bind antigen. The constant region of an antibody is a host tissue or several cells of the immune system (e.g., effector cells) and elements, including the first component (Clq), which is the classical complement system. It can mediate bonding with. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies and chimeric antibodies. antibodies). An antibody may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or Both light and heavy chains can be divided into regions of structural and functional homology.
"항원 결합 단편"은 항원과 특이적으로 상호 작용하는 능력을 유지하고 있는 (예를 들어, 결합, 입체적 방해, 공간적인 분포의 안정화에 의해) 하나 또는 그 이상의 항체 부분을 의미한다. 결합하는 단편의 예에는, 이것에만 국한하지 않으나, Fab 단편, VL, VH, CL 및 CH1 도메인으로 구성된 단일가 단편; Fab' 단편, VL, VH, CL 및 CH1 도메인 및 면역글로블린의 힌지 부위의 N-말단 부분으로 구성된 단일가 단편; F(ab)2 단편, 힌지 부위에 이황화 브리지에 의해 연결된 두 개의 Fab 단편을 포함하는 2가 단편; VH 및 CH1 도메인으로 구성된 Fd 단편; 항체의 단일 팔 (single arm)의 VL 및 VH 도메인으로 구성된 Fv단편; VH 도메인으로 구성된, dAb 단편 (Ward et al., (1989) Nature 341:544-546); 및 분리된 상보성 결정 부위 (complementarity determining region, CDR) 를 포함한다. 더 나아가, Fv 단편의 두 도메인, VL 및 VH, 이별개의 유전자에 의해 암호화되지만, 이들은, 재조합 방법을 사용하여, VL 및 VH 부위가 단가 분자(단일 체인 Fv, scFv; Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883)를 형성하도록 단일 단백질로서 만들어질 수 있게 하는 합성적 링커에 의해 연결시킬 수 있다. 그러한 단일 체인 항체도 또한 "항체 단편(antibody fragment)" 이란 용어 내에 포함하려는 의도가 있다. 이러한 항체 단편은 이 분야 기술에서 알려진 전통적인 기술을 사용하여 얻어질 수 있으며, 및 이 단편들은 온전한 항체와 같은 방식으로 그 용도에 대해 스크린 된다. 항체 단편은 또한 단일 도메인 항체, 맥시바디(maxibodies), 미니바디(minibodies), 인트라바디(intrabodies), 디아바디(diabodies), 트리아바디(triabodies), 테트라바디(tetrabodies), v-NAR 및 비스-scFv(bis-scFv)로 병합될 수 있다(Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136). 항체 단편은 피브로넥틴 타입 III (Fibronectin type III, Fn3)와 같은 폴리펩티드에 근거한 스카폴드(scaffolds)로 이식될 수 있다(U.S. Pat. No. 6,703,199). 항체 단편은 상보성 경쇄(complementary light chain) 폴리펩티드와 함께, 항원-결합 부위 쌍을 형성하는 나란한 Fv 조각 쌍(VH-CH1-VH-CH1)을 포함하는 단일 체인 분자로 병합될 수 있다(Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Pat. No. 5,641,870)."Antigen-binding fragment" means one or more portions of an antibody that retain the ability to specifically interact with an antigen (eg, by binding, sterically hindering, or stabilizing spatial distribution). Examples of binding fragments include, but are not limited to, Fab fragments, monovalent fragments composed of VL, VH, CL and CH1 domains; a Fab' fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains and the N-terminal part of the hinge region of an immunoglobulin; F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked at the hinge region by a disulfide bridge; Fd fragment consisting of VH and CH1 domains; Fv fragment consisting of the VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., (1989) Nature 341:544-546), consisting of the VH domain; and an isolated complementarity determining region (CDR). Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but, using recombinant methods, the VL and VH regions are monovalent molecules (single-chain Fv, scFv; Bird et al., (1988 ) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883) can be linked by a synthetic linker that allows them to be made as a single protein. there is. Such single chain antibodies are also intended to be included within the term "antibody fragment". Such antibody fragments can be obtained using conventional techniques known in the art, and these fragments are screened for use in the same way as intact antibodies. Antibody fragments also include single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis- can be merged into scFv (bis-scFv) (Hollinger and Hudson, (2005) Nature Biotechnology 23: 1126-1136). Antibody fragments can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (U.S. Pat. No. 6,703,199). Antibody fragments can be merged into single chain molecules comprising a pair of side-by-side Fv fragments (VH-CH1-VH-CH1) that form a pair of antigen-binding sites, together with a complementary light chain polypeptide (Zapata et al. ., (1995) Protein Eng. 8:1057-1062; and U.S. Pat. No. 5,641,870).
본 발명의 일실시예에 따르면, 상기 병원성 인자 억제 유전자는 포유류에 질환을 일으키는 병원성 인자를 억제시키는 유전자인 것일 수 있으며, 바람직하게는 항체, 중쇄 가변영역, 경쇄 가변영역 및 항원 결합 단편일 수 있고, 더욱 바람직하게는 항체이나, 이에 제한되지는 않는다.According to one embodiment of the present invention, the pathogenic factor suppression gene may be a gene that suppresses a pathogenic factor causing a disease in mammals, preferably an antibody, a heavy chain variable region, a light chain variable region, and an antigen-binding fragment. , more preferably an antibody, but is not limited thereto.
본 발명의 일실시예에 따르면, 포유류는 개, 돼지, 소, 고양이, 원숭이, 여우, 너구리, 박쥐, 코요테 및 족제비로 이루어진 군에서 선택된 동물인 것일 수 있으며, 바람직하게는 개, 돼지, 고양이이나, 이에 제한되지는 않는다.According to one embodiment of the present invention, the mammal may be an animal selected from the group consisting of dogs, pigs, cows, cats, monkeys, foxes, raccoons, bats, coyotes and weasels, preferably dogs, pigs, cats or , but is not limited thereto.
본 발명의 일실시예에 따르면, 상기 병원성은 라비스 바이러스(rabies virus) 또는 라비스 리사바이러스(Rabies lyssavirus), 돼지 써코바이러스(Porcine circovirus), 고양이 칼리시바이러스(Feline calicivirus), 클라미도필라 펠리스(Chlamydophila felis), 고양이 백혈병 바이러스, 고양이 범백혈구감소증 바이러스, 고양이 코기관지염 바이러스, 고양이 면역결핍 바이러스, 고양이 전염성 복막염 바이러스, 에를리히아 카니스(Ehrlichia canis), 개 파르보바이러스, 개 디스템퍼, 개 파라인플루엔자 바이러스, 개 II형 아데노바이러스, 개 아데노바이러스 및 개 코로나바이러스로 이루어진 군에서 선택된 바이러스에 의한 것일 수 있고, 바람직하게는 라비스 바이러스(rabies virus), 돼지 써코바이러스(Porcine circovirus) 또는 고양이 칼리시바이러스(Feline calicivirus)이나, 이에 제한되지는 않는다.According to one embodiment of the present invention, the pathogenicity is rabies virus or Rabies lyssavirus, Porcine circovirus, Feline calicivirus, Chlamydophila felis ( Chlamydophila felis ), feline leukemia virus, feline panleukopenia virus, feline bronchitis virus, feline immunodeficiency virus, feline infectious peritonitis virus, Ehrlichia canis , canine parvovirus, canine distemper, canine parainfluenza It may be caused by a virus selected from the group consisting of virus, canine type II adenovirus, canine adenovirus and canine coronavirus, preferably rabies virus, porcine circovirus or feline calicivirus (Feline calicivirus), but is not limited thereto.
상기 "항원성 유전자"는 체내에서 항원으로 작용하여, 면역반응을 유도할 수 있는 인자로서, 바이러스의 외피 단백질, 바이러스의 DNA 또는 RNA등을 의미하며, 이를 구성하는 아미노산 서열을 코딩하는 것일 수 있다. 상기 코딩된 외피 단백질, DNA 또는 RNA는 바이러스의 구조체로서, 항원력을 가지는 단편일 수 있고, 외피 단백질의 발현을 유도하거나, 그 자체로서, 백신 조성물로서 이용가능한 유전자 일 수 있다.The "antigenic gene" is a factor capable of inducing an immune response by acting as an antigen in the body, and refers to a viral coat protein, viral DNA or RNA, etc., and may encode the amino acid sequence constituting it. . The encoded envelope protein, DNA or RNA may be a viral construct, a fragment having antigenicity, or a gene that induces the expression of the envelope protein or may be used as a vaccine composition as such.
본 발명에 따른 백신 조성물은 유효성분을 약학적으로 허용된 담체에 혼입시킨 형태로 제조될 수 있다. 여기서, 약학적으로 허용된 담체는 제약 분야에서 통상 사용되는 담체, 부형제 및 희석제를 포함한다. 본 발명에서 용어 "약학적으로 허용 가능한 담체"란 임의의 및 모든 용매, 분산 매질, 코팅제, 항원 보강제, 안정제, 희석제, 보존제, 항균제 및 항진균제, 등장성 작용제, 흡착 지연제 등을 포함한다. 본 발명의 백신 조성물에 이용할 수 있는 약학적으로 허용된 담체는 이들로 제한되는 것은 아니지만, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The vaccine composition according to the present invention may be prepared in the form of 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. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating agents, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Pharmaceutically acceptable carriers that can be used in the vaccine composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 백신 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀전, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The vaccine composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. .
제제화할 경우에는 통상 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 그러한 고형 제제는 유효성분에 적어도 하나 이상의 부형제, 예를 들면 전분, 칼슘 카르보네이트, 수크로스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 일반적으로 사용되는 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수용성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수용성용제, 현탁제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브유와 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin in addition to active ingredients. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, tween 61, cacao paper, laurin paper, glycerogelatin, and the like may be used.
또한, 상기 부형제는 애쥬번트(adjuvant)라고도 불리며, 면역세포의 초기 활성화 과정에서 비특이적으로 항원에 대한 면역 반응을 촉진하는 물질로, 숙주에게 면역원은 아니지만 면역계의 세포의 활성을 증대시킴으로써 면역을 강화하는 제제, 분자 등을 의미한다. 불활화 백신의 제작시 부형제의 선발은 백신 효능의 중요한 원인이 된다.In addition, the excipient is also called an adjuvant, and is a substance that non-specifically promotes an immune response to an antigen in the initial activation process of immune cells. It is not an immunogen to the host, but enhances immunity by increasing the activity of cells of the immune system. Means an agent, molecule, etc. The selection of excipients in the production of inactivated vaccines is an important factor in vaccine efficacy.
일 구현예에서, 상기 백신 조성물은 당분야에 공지된 부형제를 제한없이 포함할 수 있고, 바람직하게는 오일 및 겔을 포함할 수 있으며, 더욱 바람직하게는 오일 및 수산화 알루미늄 겔을 포함할 수 있다. 상기 오일과 겔의 혼합 비율은 겔의 함유량이 많을 수록 부형제의 성상이 안정되고, 백신 부작용이 적어지며, 세포 응집도 약하게 나타나는 것을 특징으로 한다. 상기 오일과 수산화 알루미늄 겔은 바람직하게는 오일 및 수산화 알루미늄 겔을 1 : 3 내지 5의 부피비로 하여 추가적으로 포함할 수 있으며, 더욱 바람직하게는 오일 및 수산화 알루미늄 겔을 1 : 4의 부피비로 하여 추가적으로 포함할 수 있다.In one embodiment, the vaccine composition may include excipients known in the art without limitation, preferably oil and gel, more preferably oil and aluminum hydroxide gel. The mixing ratio of the oil and gel is characterized in that, as the content of the gel increases, the properties of the excipient are stabilized, vaccine side effects are reduced, and cell aggregation is weak. The oil and aluminum hydroxide gel may preferably additionally contain oil and aluminum hydroxide gel in a volume ratio of 1: 3 to 5, more preferably oil and aluminum hydroxide gel in a volume ratio of 1: 4. can do.
본 발명에 따른 백신 조성물은 개체에 다양한 경로로 투여될 수 있다. 투여의 모든 방식이 예상될 수 있는데, 예를 들면 경구, 정맥, 근육, 피하, 복강내 주사에 의해 투여될 수 있다.The vaccine composition according to the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.
본 발명에 따른 백신 조성물의 투여량은 개체의 연령, 체중, 성별, 신체 상태 등을 고려하여 선택된다. 상기 백신 조성물 중 포함되는 유효성분의 농도는 대상에 따라 다양하게 선택할 수 있음은 자명하며, 바람직하게는 백신 조성물에0.01 ~ 5,000 ㎍/ml의 농도로 포함되는 것이다. 그 농도가 0.01 ㎍/ml 미만일 경우에는 약학 활성이 나타나지 않을 수 있고, 5,000 ㎍/ml를 초과할 경우에는 개체에 독성을 나타낼 수 있다.The dose of the vaccine composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the subject. It is obvious that the concentration of the active ingredient included in the vaccine composition can be variously selected according to the subject, and is preferably included in the vaccine composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmacological activity may not be exhibited, and if the concentration exceeds 5,000 μg/ml, it may exhibit toxicity to the subject.
본 발명의 일실시예에 따르면, 상기 프로모터는 식물 세포로 부터 유래된 프로모터인 것일 수 있다.According to one embodiment of the present invention, the promoter may be a promoter derived from plant cells.
또한, 본 발명은 상기의 프로모터를 포함하는 고주파 특이적 발현 재조합 벡터를 제공한다.In addition, the present invention provides a high-frequency specific expression recombinant vector containing the above promoter.
용어 "벡터"는 세포 내로 전달하는 DNA 단편(들), 핵산 분자를 의미하며, DNA를 복제하고, 숙주세포에서 독립적으로 재생산될 수 있다. "발현 벡터"는 목적한 코딩 서열과, 특정 숙주 생물에서 작동가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 발현 벡터는 일반적으로 플라스미드 또는 바이러스 DNA로부터 유래하거나, 둘 다의 요소를 포함할 수 있다. 따라서, 발현 벡터는 재조합 DNA 또는 RNA 구축물, 예컨대, 플라스미드, 파지, 재조합 바이러스 또는 적절한 숙주 세포 내 도입 시, 클로닝된 DNA의 발현을 초래하는 다른 벡터를 의미한다. 적절한 발현 벡터는 당업자에게 잘 알려져 있으며, 진핵세포 및/또는 원핵세포 내에서 복제가능한 것들 및 에피솜으로 남는 것들 또는 숙주 세포 게놈 내에 통합되는 것들을 포함한다.The term “vector” refers to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell, capable of replicating DNA and independently reproducing in a host cell. "Expression vector" means a recombinant DNA molecule comprising a coding sequence of interest and appropriate nucleic acid sequences necessary to express an operably linked coding sequence in a particular host organism. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Thus, expression vector refers to a recombinant DNA or RNA construct such as a plasmid, phage, recombinant virus or other vector that, upon introduction into a suitable host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include those replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or integrate into the host cell genome.
상기 통상의 벡터는 본 발명의 프로모터를 도입할 수 있는 것이면 어떠한 것이든 무방하나, 바람직하게는 pCAMBIA계열, pGA계열, pGWB계열, 예컨대, pGA3519, pCAMBIA3301, pCAMBIA3300, pGA3426, pGA3780, pGWB12, pGWB14와 같은 Ti-plasmid 및 이에 파생된 벡터로 이루어진 군에서 선택된 어느 하나일 수 있다.The conventional vector may be any one as long as it can introduce the promoter of the present invention, but preferably pCAMBIA series, pGA series, pGWB series such as pGA3519, pCAMBIA3301, pCAMBIA3300, pGA3426, pGA3780, pGWB12, pGWB14 It may be any one selected from the group consisting of Ti-plasmid and vectors derived therefrom.
또한, 본 발명은 상기의 재조합 발현 벡터로 형질전환된 형질전환체를 제공한다.In addition, the present invention provides a transformant transformed with the above recombinant expression vector.
상기 발현 벡터는 통상의 벡터에 본 발명의 프로모터를 도입한 것일 수 있으며, 이처럼 본 발명의 프로모터를 도입하여 발현벡터를 제조하는 것은 본 발명이 속하는 기술분야의 당업자라면 공지의 방법에 따라 용이하게 실시 가능하다.The expression vector may be one in which the promoter of the present invention is introduced into a conventional vector, and thus preparing an expression vector by introducing the promoter of the present invention is easily performed according to a method known to those skilled in the art to which the present invention belongs. It is possible.
상기한 발현 벡터는 감염, 형질도입, 트랜스펙션, 전기천공 및 형질전환과 같은 주지된 기술을 이용하여 배양된 숙주 세포 내로 삽입될 수 있다. 숙주의 대표적인 예는 박테리아 세포, 예를 들면, 대장균, 스트렙토마이세스, 살모넬라 티피무리움 또는 아그로박테리움 투메파시엔스; 또는 식물 세포, 예를 들면, 옥수수, 보리, 밀, 벼, 귀리, 호밀 또는 사탕수수 등의 단자엽 식물 또는, 장미, 당근, 차나무, 동백나무,사과나무, 애기장대, 감자, 가지, 담배, 고추, 상추, 배추, 양배추, 수박, 참외 등의 쌍자엽 식물의 세포일 수 있다.The above expression vectors can be inserted into cultured host cells using well known techniques such as infection, transduction, transfection, electroporation and transformation. Representative examples of hosts are bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium or Agrobacterium tumefaciens; Or plant cells, for example monocotyledonous plants such as corn, barley, wheat, rice, oats, rye or sugar cane, or roses, carrots, tea trees, camellia trees, apple trees, It may be a cell of a dicotyledonous plant such as Arabidopsis, potato, eggplant, tobacco, red pepper, lettuce, Chinese cabbage, cabbage, watermelon, or melon.
식물의 형질전환에 이용되는 "식물 세포"는 임의의 식물 세포가 이용 가능하다. 식물 세포는 배양 세포, 배양 조직, 배양기관 또는 전체 식물, 바람직하게는 배양 세포, 배양 조직 또는 배양 기관 및 더욱 바람직하게는 배양 세포의 어떤 형태도 가능하다. "식물 조직"은 분화된 또는 미분화된 식물의 조직, 예를 들면 이에 한정되진 않으나, 줄기, 잎, 암 조직 및 배양에 이용되는 다양한 형태의 세포들, 즉 단일 세포, 원형질체(protoplast), 싹 및 캘러스 조직을 포함한다.Any plant cell may be used as the "plant cell" used for plant transformation. The plant cell may be any type of cultured cell, cultured tissue, cultured organ or whole plant, preferably a cultured cell, cultured tissue or cultured organ and more preferably a cultured cell. "Plant tissue" refers to differentiated or undifferentiated plant tissues such as, but not limited to, stems, leaves, cancer tissues, and various types of cells used in culture, such as single cells, protoplasts, shoots and Contains callus tissue.
이하, 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail by examples. These examples are merely for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
<실시예 1> 고주파 특이적 발현 유전자 확인<Example 1> Confirmation of high-frequency specific expression genes
본 발명의 고주파 특이적 발현 프로모터를 발굴하기 위하여, 장미 세포를 배양하면서, 고주파를 처리하였다. 구체적으로는, 3 L의 MS 기본배지(Duchefa 社, Cat No. M0221)에 식물성장조절제(Plant Growth Regulator)인 1 mg/L Zeatin, 3 % Sucrose가 포함된 배지에 장미 세포를 접종하고, 20분의 고주파 처리를 하루 2회(twice/day)처리하여 3일간 배양하였다. 대조군으로는 고주파의 처리 전 배양된 장미 세포를 이용하였다. 배양 후 장미 세포를 모두 수득하고, 고주파 처리전 장미세포와 고주파 처리 후 장미세포를 체세대 염기서열 분석법(Next Generation Sequencing, NGS)을 이용하여, 전세체 분석(RNAseq)을 실시하였다. 그 후 분석 결과에서 adjusted p-value가 0.05보다 작은 의미를 갖는 유전자를 기준으로 선발하고, Log2FC가 1 보다 큰 유전자를 유의미한 변화가 있는 것으로 분석하여 선발하였다.In order to discover the high-frequency specific expression promoter of the present invention, while culturing rose cells, high-frequency was treated. Specifically, rose cells were inoculated into 3 L of MS basal medium (Duchefa, Cat No. M0221) containing 1 mg/L Zeatin and 3% Sucrose, a plant growth regulator, and 20 The high-frequency treatment was performed twice a day (twice/day) and cultured for 3 days. As a control group, rose cells cultured before radiofrequency treatment were used. After culturing, all rose cells were obtained, and whole body analysis (RNAseq) was performed on the rose cells before radiofrequency treatment and after radiofrequency treatment using Next Generation Sequencing (NGS). Then, in the analysis results, genes with an adjusted p-value of less than 0.05 were selected based on the criteria, and genes with a Log2FC greater than 1 were analyzed and selected as having significant changes.
그 결과, TPM(transcripts per million) 분석 값에 따라 유의적인 변화가 있는 유전자를 pathway별로 분류하고 phenylpropanoid pathway에 관련된 유전자가 고주파 처리에 따라 강하게 발현이 증가하는 것을 확인하여, phenylpropanoid pathway관련 유전자가 선별 되었다(도 1).As a result, genes with significant changes were classified by pathway according to the TPM (transcripts per million) analysis value, and the expression of genes related to the phenylpropanoid pathway was strongly increased by radiofrequency treatment, and genes related to the phenylpropanoid pathway were selected. (Fig. 1).
<실시예 2> 고주파 특이적 프로모터 발굴<Example 2> Discovery of high-frequency specific promoters
상기 실시예 1에서 선별된 유전자인 Tryptophan synthase alpha chain-like 유전자를 생물정보학 분석(Bioinformatics analysis)을 시행하였다. Tryptophan synthase alpha chain-like 유전자의 transcription factor binding site(TFBS)를 확인하였으며, 확인된 TFBS의 서열을 기준으로 유사서열을 검색하여 분석하였다. 그 후 Tandem repeat를 기준으로 크게 2개의 Group으로 분류 하였다(도 2). 분류된 Group 1 및 Group 2 유전자 단편 중 각각의 주요 TFBS는 하기 표 1에 나타내었으며, Group 1 및 Group 2의 프로모터를 발굴하였다.Bioinformatics analysis was performed on the Tryptophan synthase alpha chain-like gene selected in Example 1 above. The transcription factor binding site (TFBS) of the tryptophan synthase alpha chain-like gene was identified, and similar sequences were searched for and analyzed based on the sequence of the identified TFBS. After that, it was classified into two groups based on the tandem repeat (Fig. 2). Each of the major TFBSs among the classified Group 1 and Group 2 gene fragments are shown in Table 1 below, and Group 1 and Group 2 promoters were discovered.
[표 1][Table 1]
Figure PCTKR2021019917-appb-img-000001
Figure PCTKR2021019917-appb-img-000001
<실시예 3> 프로모터 발현 벡터 제작<Example 3> Construction of promoter expression vector
상기 발굴된 고주파 특이적 프로모터의 활성을 확인하기 위하여, pCAMBIA1302 벡터를 backbone으로 사용하였으며, 발현을 유도하고자 하는 유전자로서 mGFP5 유전자를 이용하였다(도 3). 구체적으로, 벡터의 mGFP5앞에 위치한 CaMV35S 프로모터를 제거하기 위하여, BstXI 및 NcoI를 처리하여 제거하고, 그 위치에, 후보 프로모터(promoter candidate)를 삽입하여, 벡터를 제작하였다. 벡터 제작 방법은 도 4에 나타내었다.In order to confirm the activity of the high-frequency specific promoter discovered above, the pCAMBIA1302 vector was used as a backbone, and the mGFP5 gene was used as a gene to induce expression (FIG. 3). Specifically, in order to remove the CaMV35S promoter located in front of mGFP5 of the vector, BstXI and NcoI were removed by treatment, and a candidate promoter was inserted into the site to construct a vector. The vector construction method is shown in FIG. 4 .
<실시예 4> 고주파 특이적 발현 확인<Example 4> Confirmation of high-frequency specific expression
<4-1> 녹색 형광 단백질 발현 확인<4-1> Verification of expression of green fluorescent protein
상기 실시예 3의 Group 1 및 Group 2의 후보 프로모터가 클로닝된 pCAMBIA1302벡터를 장미세포에 삽입하고, 벡터가 삽입된 장미세포를 고주파 처리 하였다. 음성 대조군으로는 형질전환 하지 않은 장미세포를 이용하였으며, 양성 대조군으로는 CaMV 35S 프로모터를 이용하여 제작된 벡터로 형질전환하였다. 고주파에 특이적인 발현 프로모터를 확인하기 위하여, 형질전환 된 장미세포를 배양하고, 고주파 처리는 1회/1일, 20분간 처리한 후 mGFP5의 형광 발현을 confocal microscopy를 통해 확인하였다.The pCAMBIA1302 vector, in which the candidate promoters of Group 1 and Group 2 of Example 3 were cloned, was inserted into rose cells, and the rose cells into which the vectors were inserted were radiofrequency treated. As a negative control, non-transformed rose cells were used, and as a positive control, a vector constructed using the CaMV 35S promoter was transformed. In order to confirm the expression promoter specific to high frequency, the transformed rose cells were cultured, and after treatment with high frequency once/day for 20 minutes, fluorescence expression of mGFP5 was confirmed through confocal microscopy.
그 결과, 음성 대조군과 비교하여, 후보 프로모터로 형질전환된 장미세포에서는 형광이 발현하는 것을 확인하였으며, 후보 프로모터 중에서, Group 1의 형광 발현은 고주파 처리 전보다 59.65% 증가하는 것을 확인하였으며, Group 2의 형광 발현은 고주파 처리 전보다 78.06% 증가하는 것을 확인하였다(도 5, 도 6 및 표 2).As a result, compared to the negative control group, it was confirmed that fluorescence was expressed in rose cells transformed with the candidate promoter. It was confirmed that fluorescence expression was increased by 78.06% compared to before radiofrequency treatment (FIG. 5, FIG. 6 and Table 2).
[표 2][Table 2]
Figure PCTKR2021019917-appb-img-000002
Figure PCTKR2021019917-appb-img-000002
<4-2> 외래 도입 유전자의 발현 확인(epidermal growth factor, EGF)<4-2> Verification of expression of foreign transgene (epidermal growth factor, EGF)
상기 실시예 4-1에서 선별된 프로모터인 Group 2 프로모터를 이용하여, 고주파 특이적으로 외래 도입 유전자의 발현을 증가시킬 수 있는지 확인하고자 하였다. 구체적으로, 상기 실시예 4-1과 동일하게 발현 벡터를 제작하고, 외래 유전자로서 표피증식인자(epidermal growth factor, EGF)를 벡터에 삽입하여, 고주파 특이적 발현 벡터를 제작하였다. 제작된 벡터를 장미세포에 형질전환시키고, 고주파를 처리하여 24시간 배양한 후, 폴리아크릴아마이드 겔 전기 영동법(sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE)으로, EGF의 발현을 확인하였다. 음성 대조군으로는 아무것도 처리하지 않은 Vehicle을 사용하였으며, 양성 대조군으로는 대장균에서 발현되어, 정제된 EGF를 이용하였다. 또한, 형질전환하지 않은 장미세포 및 Methyl jasmontae를 처리한 장미세포를 이용하여, 본 발명의 프로모터로인한 EGF의 고발현을 확인하고자 하였다.Using the Group 2 promoter, which is the promoter selected in Example 4-1, it was attempted to confirm whether the expression of the foreign transgene can be increased in a high-frequency specific manner. Specifically, an expression vector was prepared in the same manner as in Example 4-1, and an epidermal growth factor (EGF) as a foreign gene was inserted into the vector to prepare a high-frequency specific expression vector. The prepared vector was transformed into rose cells, treated with high frequency and cultured for 24 hours, and then the expression of EGF was confirmed by polyacrylamide gel electrophoresis (sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE). As a negative control, an untreated vehicle was used, and as a positive control, EGF expressed in Escherichia coli and purified was used. In addition, using non-transformed rose cells and rose cells treated with Methyl jasmontae, high expression of EGF due to the promoter of the present invention was confirmed.
그 결과 도 7에 나타낸 바와 같이, 대장균에서 발현된 EGF가 6 kDa의 크기를 가지는 것을 확인하였으며, 양성대조군, 장미세포 및 Methyl jasmontae를 처리한 장미세포와 비교하여, 고주파 특이적 프로모터로 형질전환된 장미세포에서 EGF의 발현이 강하게 유도된 것을 확인하였다.As a result, as shown in Figure 7, it was confirmed that EGF expressed in E. coli had a size of 6 kDa, compared to the positive control, rose cells and rose cells treated with Methyl jasmontae, transformed with a high-frequency specific promoter It was confirmed that the expression of EGF was strongly induced in rose cells.
<4-3> 외래 도입 유전자의 발현 확인(Basic fibroblast growth factor, bFGF)<4-3> Verification of expression of foreign transgene (Basic fibroblast growth factor, bFGF)
상기 실시예 4-1에서 선별된 프로모터인 Group 2 프로모터를 이용하여, 고주파 특이적으로 외래 도입 유전자의 발현을 증가시킬 수 있는지 확인하고자 상기 실시예 4-2와 동일한 방법으로 염기성 섬유아세포 성장인자(Basic fibroblast growth factor, bFGF)의 발현을 SDS-PAGE로 확인하였다.Basic fibroblast growth factor (basic fibroblast growth factor ( Expression of basic fibroblast growth factor (bFGF) was confirmed by SDS-PAGE.
그 결과 도 8에 나타낸 바와 같이, 대장균에서 발현된 bFGF가 17kDa의 크기를 가지는 것을 확인하였으며, 양성대조군, 장미세포 및 Methyl jasmontae를 처리한 장미세포와 비교하여, 고주파 특이적 프로모터로 형질전환된 장미세포에서 bFGF의 발현이 강하게 유도된 것을 확인하였다.As a result, as shown in FIG. 8, it was confirmed that bFGF expressed in E. coli had a size of 17 kDa, and compared to the positive control, rose cells and rose cells treated with Methyl jasmontae, roses transformed with a high-frequency specific promoter It was confirmed that the expression of bFGF was strongly induced in the cells.
따라서, 본 발명의 phenylpropanoid pathway관련 유전자 유래의 고주파 특이적 프로모터는, 형질전환된 식물체에, 고주파가 처리되면 특이적으로 발현하는 것을 확인하였으며, 외래 유전자의 발현을 고주파 특이적으로 증가시키는 것을 확인하였다.Therefore, it was confirmed that the high frequency-specific promoter derived from the gene related to the phenylpropanoid pathway of the present invention was specifically expressed when the transformed plant was treated with high frequency, and it was confirmed that the expression of the foreign gene was specifically increased at high frequency. .

Claims (21)

  1. 고주파(high frequency)에 특이적으로 발현하는 프로모터.A promoter specifically expressed at a high frequency.
  2. 제 1항에 있어서,According to claim 1,
    상기 프로모터는 서열번호 1의 염기서열 또는 서열번호 2의 염기서열을 포함하는 것인, 프로모터.Wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 2, the promoter.
  3. 제 1항에 있어서, 상기 고주파는 100 내지 1200khz 조건으로 처리되는 것인, 프로모터.The promoter according to claim 1, wherein the high frequency is processed under conditions of 100 to 1200 khz.
  4. 제 1항에 있어서, 상기 고주파는 1 내지 60분의 조건으로 처리되는 것인, 프로모터.The promoter according to claim 1, wherein the high frequency is processed under conditions of 1 to 60 minutes.
  5. 제 1항에 있어서, 상기 고주파는 1 내지 10회/day의 조건으로 처리되는 것인, 프로모터.The promoter according to claim 1, wherein the high frequency is applied under conditions of 1 to 10 times/day.
  6. 제 1항에 있어서, 상기 고주파는 1 내지 10일 조건으로 처리되는 것인, 프로모터.The promoter according to claim 1, wherein the high frequency treatment is performed under conditions of 1 to 10 days.
  7. 제 1항에 있어서, 상기 프로모터는 트립토판(Trp), 페닐알라닌(Phe), 티로신(Tyr)의 방향족 아미노산(Aromatic amino acids)의 함량을 증가시키는 것인, 프로모터.The promoter according to claim 1, wherein the promoter increases the content of aromatic amino acids such as tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr).
  8. 제 7항에 있어서, 상기 방향족 아미노산(Aromatic amino acids)의 함량을 증진시켜 식물 대사의 Phenylpropanoid pathway 대사의 활성 및 상기 대사의 유전자를 증진시키는 것인, 프로모터.The promoter according to claim 7, which promotes the activity of Phenylpropanoid pathway metabolism and the genes of the metabolism by enhancing the content of the aromatic amino acids.
  9. 제 8항에 있어서,According to claim 8,
    상기 Phenylpropanoid pathway 대사의 활성 및 상기 대사 유전자의 증진은 카페오일퀴닉 산(Caffeoylquinic acids), 퀘르세틴(Quercetin), 캠페롤(Kaempferol), 카테킨(Catechin), 벤즈알데하이드(Benzaldehyde) 및 페닐아세트 산(Phenylacetic acid)으로 이루어진 군에서 선택된 파이토케미컬(Phytochemical)의 생산을 증가시키는 것을 특징으로 하는 프로모터.The activation of the Phenylpropanoid pathway metabolism and the enhancement of the metabolic genes are Caffeoylquinic acids, Quercetin, Kaempferol, Catechin, Benzaldehyde and Phenylacetic acid ) A promoter characterized in that it increases the production of a phytochemical selected from the group consisting of.
  10. 제 1항에 있어서, According to claim 1,
    상기 프로모터는 암세포 사멸 유전자, 형광 단백질 유전자, 암세포 억제인자 유전자, 병원성 인자 억제 유전자, 항원성 유전자, 세포 독성 유전자, 세포 증식 억제 유전자, 사이토카인 유전자, 친-세포사멸 유전자(pro-apoptotic gene) 및 항-신생혈관생성 유전자(anti-angiogenic gene)로 이루어진 군에서 선택된 1종 이상의 유전자를 고주파 특이적으로 과발현시키는 것인, 프로모터.The promoter is a cancer cell death gene, a fluorescent protein gene, a cancer cell suppressor gene, a pathogenic factor suppressor gene, an antigenic gene, a cytotoxic gene, a cell proliferation suppressor gene, a cytokine gene, a pro-apoptotic gene, and An anti-angiogenic gene (anti-angiogenic gene) to overexpress one or more genes selected from the group consisting of high-frequency specificity, the promoter.
  11. 제 10항에 있어서, According to claim 10,
    상기 암세포 사멸 유전자는 BCL-2 계통 세포사멸 촉진(pro-apoptotic) 유전자 또는 자살 수용체/리간드(death receptor/ligand) 유전자인 것인, 프로모터.Wherein the cancer cell death gene is a BCL-2 lineage pro-apoptotic gene or a suicide receptor/ligand gene, the promoter.
  12. 제 10항에 있어서, 상기 형광 단백질 유전자는 루시퍼라아제(luciferase), 증강 녹색 형광 단백질(enhanced green fluorescent protein, EGFP), 녹색 형광 단백질(green fluorescent protein, GFP), 노랑 형광 단백질 (Yellow Fluorescent Protein, YFP), 적색 형광 단백질 (Red Fluorescent Protein, RFP)및 청색 형광 단백질(cyan fluorescent protein, CFP)으로 이루어진 군에서 선택된 1종 이상인 것인, 프로모터.11. The method of claim 10, wherein the fluorescent protein gene is luciferase, enhanced green fluorescent protein (EGFP), green fluorescent protein (GFP), yellow fluorescent protein (Yellow Fluorescent Protein, YFP), at least one selected from the group consisting of red fluorescent protein (RFP) and blue fluorescent protein (cyan fluorescent protein, CFP), the promoter.
  13. 제 10항에 있어서, 상기 암세포 억제인자 유전자는 p53 유전자, APC 유전자, DPC-4/Smad4 유전자, BRCA-1 유전자, BRCA-2 유전자, WT-1유전자, MMAC-1 유전자, MMSC-2 유전자, NF-1 유전자, MTS1 유전자, CDK4 유전자, NF-1 유전자, NF-2 유전자 및 VHL 유전자로 이루어진 군에서 선택된 1종 이상인 것인, 프로모터.11. The method of claim 10, wherein the cancer cell suppressor gene is p53 gene, APC gene, DPC-4/Smad4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, MMAC-1 gene, MMSC-2 gene, At least one selected from the group consisting of NF-1 gene, MTS1 gene, CDK4 gene, NF-1 gene, NF-2 gene and VHL gene, the promoter.
  14. 제 10항에 있어서, 상기 암은 폐암, 혈액암, 대장암, 직장암, 결장암, 유방암, 자궁경부암, 자궁내막암, 나팔관암종, 난소암, 질암종, 음문암종, 간암, 위암, 식도암, 소장암, 췌장암, 담낭암, 신장암, 방광암, 요도암, 음경암, 전립선암, 고환암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 비소세포성폐암, 골암, 피부암, 두부 또는 경부암, 피부 또는 안구 내 흑색종, 호지킨병, 내분비선암, 만성 또는 급성 백혈병, 림프구 림프종, 중추 신경계 종양, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군에서 선택된 1종 이상인 것을 특징으로 하는, 프로모터.The method of claim 10, wherein the cancer is lung cancer, blood cancer, colorectal cancer, rectal cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, fallopian tube carcinoma, ovarian cancer, vaginal carcinoma, vulvar carcinoma, liver cancer, stomach cancer, esophageal cancer, small intestine cancer , pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, urethral cancer, penile cancer, prostate cancer, testicular cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, non-small cell lung cancer, bone cancer, skin cancer, head or neck cancer, skin or intraocular blackness Species, Hodgkin's disease, endocrine adenocarcinoma, chronic or acute leukemia, lymphocytic lymphoma, central nervous system tumors, spinal cord tumors, characterized in that at least one selected from the group consisting of brainstem glioma and pituitary adenoma, promoter.
  15. 제 10항에 있어서,According to claim 10,
    상기 병원성 인자 억제 유전자는 포유류에 질환을 일으키는 병원성 인자를 억제시키는 유전자인 것인, 프로모터.The pathogenic factor suppression gene is a gene that suppresses a pathogenic factor causing a disease in a mammal, a promoter.
  16. 제 15항에 있어서,According to claim 15,
    상기 포유류는 개, 돼지, 소, 고양이, 원숭이, 여우, 너구리, 박쥐, 코요테 및 족제비로 이루어진 군에서 선택된 동물인 것인, 프로모터.Wherein the mammal is an animal selected from the group consisting of dogs, pigs, cows, cats, monkeys, foxes, raccoons, bats, coyotes and weasels.
  17. 제 15항에 있어서,According to claim 15,
    상기 병원성은 라비스 바이러스(rabies virus) 또는 라비스 리사바이러스(Rabies lyssavirus), 돼지 써코바이러스(Porcine circovirus), 고양이 칼리시바이러스(Feline calicivirus), 클라미도필라 펠리스(Chlamydophila felis), 고양이 백혈병 바이러스, 고양이 범백혈구감소증 바이러스, 고양이 코기관지염 바이러스, 고양이 면역결핍 바이러스, 고양이 전염성 복막염 바이러스, 에를리히아 카니스(Ehrlichia canis), 개 파르보바이러스, 개 디스템퍼, 개 파라인플루엔자 바이러스, 개 II형 아데노바이러스, 개 아데노바이러스 및 개 코로나바이러스로 이루어진 군에서 선택된 바이러스에 의한 것인, 프로모터.The pathogenicity is rabies virus or rabies lyssavirus , porcine circovirus, feline calicivirus, chlamydophila felis, feline leukemia virus, Feline panleukopenia virus, feline rhinotracheitis virus, feline immunodeficiency virus, feline infectious peritonitis virus, Ehrlichia canis, canine parvovirus, canine distemper, canine parainfluenza virus, canine type II adenovirus, canine Promoter, by a virus selected from the group consisting of adenovirus and canine coronavirus.
  18. 제 10항에 있어서,According to claim 10,
    상기 항원성 유전자는 라비스 바이러스(rabies virus) 또는 라비스 리사바이러스(Rabies lyssavirus), 돼지 써코바이러스(Porcine circovirus), 고양이 칼리시바이러스(Feline calicivirus), 클라미도필라 펠리스(Chlamydophila felis), 고양이 백혈병 바이러스, 고양이 범백혈구감소증 바이러스, 고양이 코기관지염 바이러스, 고양이 면역결핍 바이러스, 고양이 전염성 복막염 바이러스, 에를리히아 카니스(Ehrlichia canis), 개 파르보바이러스, 개 디스템퍼, 개 파라인플루엔자 바이러스, 개 II형 아데노바이러스, 개 아데노바이러스 및 개 코로나바이러스로 이루어진 군에서 선택된 바이러스에서 유래된 것인, 프로모터.The antigenic gene is Rabies virus or Rabies lyssavirus , Porcine circovirus, Feline calicivirus, Chlamydophila felis, Feline leukemia Viruses, feline panleukopenia virus, feline rhinotracheitis virus, feline immunodeficiency virus, feline infectious peritonitis virus, Ehrlichia canis, canine parvovirus, canine distemper, canine parainfluenza virus, canine type II adenovirus , a promoter derived from a virus selected from the group consisting of canine adenovirus and canine coronavirus.
  19. 제 1항에 있어서, 상기 프로모터는 식물 세포로부터 유래된 프로모터인 것인, 프로모터.The promoter according to claim 1, wherein the promoter is a promoter derived from plant cells.
  20. 제 1항의 프로모터를 포함하는 고주파 특이적 발현 재조합 벡터.A high-frequency specific expression recombinant vector comprising the promoter of claim 1.
  21. 제 20항의 재조합 발현 벡터로 형질전환된 형질전환체.A transformant transformed with the recombinant expression vector of claim 20.
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US6018104A (en) * 1991-10-04 2000-01-25 Novartis Finance Corporation Nucleic acid promoter fragment isolated from a plant tryptophan synthase alpha subunit (trpA) gene
KR20100031523A (en) * 2007-05-30 2010-03-22 와이어쓰 엘엘씨 Raccoon poxvirus expressing rabies glycoproteins
JP2017504329A (en) * 2014-01-16 2017-02-09 キャリスタ， インコーポレイテッド Microorganisms and related methods for enhanced production of amino acids
KR20170142222A (en) * 2016-06-16 2017-12-28 주식회사 바이오에프디엔씨 Recombinant Protein Expression Vector in Plant cell and the Method for Preparing the Protein
KR20190019291A (en) * 2017-08-17 2019-02-27 (주)큐리진 Promoter of protein regulator of cytokinesis 1 for regulating cancer cell-specific gene expression and recombinant vector comprising the same
KR20200042572A (en) * 2018-10-15 2020-04-24 동국대학교 산학협력단 A method for overexpressing target gene using electromagnetic inducible promoter

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6018104A (en) * 1991-10-04 2000-01-25 Novartis Finance Corporation Nucleic acid promoter fragment isolated from a plant tryptophan synthase alpha subunit (trpA) gene
KR20100031523A (en) * 2007-05-30 2010-03-22 와이어쓰 엘엘씨 Raccoon poxvirus expressing rabies glycoproteins
JP2017504329A (en) * 2014-01-16 2017-02-09 キャリスタ， インコーポレイテッド Microorganisms and related methods for enhanced production of amino acids
KR20170142222A (en) * 2016-06-16 2017-12-28 주식회사 바이오에프디엔씨 Recombinant Protein Expression Vector in Plant cell and the Method for Preparing the Protein
KR20190019291A (en) * 2017-08-17 2019-02-27 (주)큐리진 Promoter of protein regulator of cytokinesis 1 for regulating cancer cell-specific gene expression and recombinant vector comprising the same
KR20200042572A (en) * 2018-10-15 2020-04-24 동국대학교 산학협력단 A method for overexpressing target gene using electromagnetic inducible promoter

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