KR101793269B1 - Pharmaceutical composition for preventing or treating metabolic syndrome - Google Patents
Pharmaceutical composition for preventing or treating metabolic syndrome Download PDFInfo
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Abstract
The present invention relates to a polypeptide comprising a helicase domain and an RD domain of a retinoic acid-inducible gene-1 (DDX58) protein, a polynucleotide encoding the polypeptide, a recombinant protein comprising the polynucleotide Or a composition for preventing and / or treating diseases related to the metabolic syndrome containing said polypeptide, polynucleotide, recombinant vector or a mixture thereof, a pharmaceutical preparation containing said composition as an active ingredient, and And a method for treating or preventing a disease associated with metabolic syndrome using the composition.
Description
The present invention relates to a polypeptide comprising a helicase domain and an RD domain of a retinoic acid-inducible gene-1 (DDX58) protein, a polynucleotide encoding the polypeptide, a recombinant protein comprising the polynucleotide Or a composition for preventing and / or treating diseases related to the metabolic syndrome containing said polypeptide, polynucleotide, recombinant vector or a mixture thereof, a pharmaceutical preparation containing said composition as an active ingredient, and And a method for treating or preventing a disease associated with metabolic syndrome using the composition.
Mitochondria are intracellular organelles that are present in most eukaryotic cells and have mitochondrial DNA (mtDNA), which is its own DNA that is separated from nuclear DNA. These mitochondria are involved in the energy metabolism process. They are responsible for the production of ATP (adenosine triphosphate) type energy through oxidation of pyruvic acid and NADPH derived from glucose. Mitochondria are involved in intracellular signaling through active oxygen, Regulation of lipid, amino acid, regulation of nucleic acid metabolism, and cell death control.
These mitochondria undergo biogenesis through circular DNA replication, and their amounts and activities are regulated through fission and fusion of mitochondrial membranes and autophagy. The activity of mitochondria can be regulated by intracellular conditions and stimuli.
Mitochondrial biosynthesis is induced by extracellular stimuli such as exercise, oxidative stress, cell division, and differentiation, resulting in an increase in the number of mitochondria and the number of copies of mitochondrial DNA. Mitochondrial biosynthesis is regulated by proteins such as NRF1, TFAM, and PGC1α encoded in nuclear genes. PGC1α is a co-activator that binds to transcription factors such as NRF1, ERRs, and PPARs to induce the expression of genes involved in mitochondrial activity. These genes encode proteins involved in mitochondrial DNA replication and transcription, fatty acid oxidation, translation, and mitochondrial respiratory chain.
Mitochondria can be damaged by stimuli such as inflammation, aging, dietary metabolism, and stress. If the damaged mitochondria are not replaced by new mitochondria, they cause dysfunction. However, mitochondrial DNA (mtDNA), unlike nuclear DNA in cells, does not have its own repair mechanism and is relatively prone to damage because there is no histone protein that protects DNA.
Mitochondrial DNA (mtDNA) damage is known to be closely related to the development of mitochondrial diseases. Mitochondrial dysfunction causes ATP synthesis, which is an energy source for cell activity, to be reduced, and not only a variety of genetic diseases, Diabetes, obesity and other neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease.
The present inventors confirmed that retinoic acid-inducible gene-1 (DDX58) protein increases mitochondrial biosynthesis and activity, thereby increasing intracellular ATP levels and reducing accumulation of lipids in the cytoplasm , Particularly the RIG-I protein, particularly the helicase domain and the RD domain of the RIG-I protein, by confirming that the polypeptide comprising the helicase domain of the RIG-I protein is important for this function Of the polypeptides can be used to treat various diseases caused by mitochondrial dysfunction.
Accordingly, one example of the present invention relates to a polypeptide comprising the helicase domain and the RD domain of the RIG-I protein (Retinoic-acid inducible gene-1, DDX58).
Another example of the present invention relates to a polynucleotide encoding a polypeptide comprising a helicase domain and a RD domain of a retinoic acid-inducible gene-1 (DDX58).
Another example of the present invention relates to a recombinant vector comprising the polynucleotide.
Another example of the present invention relates to a cell transformed with the recombinant vector.
Another example of the present invention relates to a method for producing the polypeptide comprising the step of expressing the polynucleotide or a recombinant vector comprising the polynucleotide in a cell.
Another example of the present invention is a polypeptide comprising the helicase domain and the RD domain of a retinoic acid-inducible gene-1 (DDX58) protein, a polypeptide comprising the RIG-I protein or polypeptide encoding the polypeptide A recombinant vector comprising the polynucleotide, a recombinant cell comprising the recombinant vector, or a mixture thereof, as an active ingredient. The present invention also relates to a pharmaceutical composition for preventing or treating diseases related to metabolic syndrome.
Another example of the present invention relates to a pharmaceutical preparation containing the above composition as an active ingredient.
Another example of the present invention relates to a method of treating or preventing a metabolic syndrome related disease comprising administering a pharmaceutically effective amount of the composition.
Another example of the present invention is a polypeptide comprising the helicase domain and the RD domain of a retinoic acid-inducible gene-1 (DDX58) protein, a polypeptide comprising the RIG-I protein or polypeptide encoding the polypeptide A recombinant vector comprising the polynucleotide, a recombinant cell comprising the recombinant vector, or a mixture thereof, for the treatment or prevention of diseases associated with the metabolic syndrome.
The RIG-I (Retinoic-acid inducible gene-1, DDX58) protein is a cytoplasmic receptor that recognizes non-magnetic RNAs derived from viruses, and expresses the antiviral cytokines interferon alpha and beta through signal transduction . These RIG-I proteins include a helicase that recognizes the viral RNA, a caspase recruitment domain (CARD), a repressor domain, and a C-terminal domain that are important for signal transduction. CTD) and the like.
Various pathways are known for secretion of cytoplasmic proteins out of the cell. If the peptide has a signal peptide at the N-terminal or intermediate part of the protein, it is secreted out of the cell via the endoplasmic reticulum by a protein recognizing it.
On the other hand, proteins that do not have a clearly defined signal peptide are secreted out of the cell without going through the endoplasmic reticulum. It has been reported that protein secretion occurs through caspase-1 (Caspase-1) enzyme activity under stress conditions such as UV (Ultraviolet) exposure or Lipopolysaccharide (LPS) treatment of gram-negative bacterial cell membrane component. Specifically, the caspase-1 protein binds to proteins such as IL-1 alpha (Interleukin-1 alpha) and FGF2 (Fibroblast Growth Factor 2) and moves out of the cell together with the binding protein in an activity-dependent manner in caspase-1. When these proteins are analyzed by function, proteins involved in inflammation, cytoprotection, and tissue regeneration are included.
The RIG-I protein is also released outside the cell, which is dependent on caspase-1 enzyme activity, and the extracellular release of the RIG-I protein leads to the release of caspase- poly-I: C, polyinosinic-polycytidylic acid) or LPS and ATP (Adenosine triphosphate) together. In this case, the function of RIG-I protein in the cytoplasm is decreased.
The present inventors have found that when the recombinant RIG-I protein is treated with a cell, the mitochondrial biosynthesis and activity of the cell are increased, and that it also increases intracellular ATP level and reduces the amount of lipids in the cell, A polypeptide comprising a helicase domain of RIG-I protein, particularly retinoic acid-inducible gene-1 (DDX58) protein, is used as a preventive and therapeutic agent for restoring mitochondrial function, Prophylactic and therapeutic agents, and a prophylactic and therapeutic agent for reducing the level of intracellular lipids.
Accordingly, the present invention relates to a polypeptide comprising a helicase domain and an RD domain of a retinoic acid-inducible gene-1 (DDX58) protein, a polynucleotide encoding the polypeptide, and a polynucleotide Or a composition for preventing and / or treating a disease associated with a metabolic syndrome containing the polypeptide, polynucleotide, recombinant vector or a mixture thereof, a pharmaceutical preparation containing the composition as an active ingredient And a method of treating or preventing a disease associated with the metabolic syndrome using the composition.
Hereinafter, the present invention will be described in more detail
One example of the present invention relates to a polypeptide comprising the helicase domain and the RD domain of RIG-1 (retinoic acid-inducible gene-1, DDX58) protein. The polypeptide may be a fragment of the RIG-1 protein, or a variant thereof.
The RIG-I protein may be derived from a mammal such as a primate such as a human, a rodent such as a mouse, or may be a natural protein or a synthetic or recombinantly produced one. Human (human, Homo I protein (e.g., NCBI Accession No. NP_766277 (SEQ ID NO: 12) derived from sapiens (for example NCBI Accession No. NP_055129 (SEQ ID NO: 1)) and mouse (house mouse, Mus musculus ) ) Sequences and the amino acid sequences of the respective domains constituting the sequences are shown in Tables 1 to 4 (H: human, M: mouse).
(H)
(H)
(H)
(H)
(H)
(H)
Domain
(H)
(H)
Domain
(H)
Domain + RD domain
(H)
GSKCFLLTSNAGVIEKEQINMYKEKMMNDSILRLQTWDEAVFREKILHIQTHEKFIRDSQEKPKPVPDKENKKLLCRKCKALACYTADVRVIEECHYTVLGDAFKECFVSRPHPKPKQFSSFEKRAKIFCARQNCSHDWGIHVKYKTFEIPVIKIESFVVEDIATGVQTLYSKWKDFHFEKIPFDPAEMSK
(H) +
Helicase
Domain + RD domain
(H)
LPAMKGKNTIICAPTGCGKTFVSLLICEHHLKKFPQGQKGKVVFFANQIPVYEQQKSVFSKYFERHGYRVTGISGATAENVPVEQIVENNDIIILTPQILVNNLKKGTIPSLSIFTLMIFDECHNTSKQHPYNMIMFNYLDQKLGGSSGPLPQVIGLTASVGVGDAKNTDEALDYICKLCASLDASVIATVKHNLEELEQVVYKPQKFFRKVESRISDKFKYIIAQLMRDTESLAKRICKDLENLSQIQNREFGTQKYEQWIVTVQKACMVFQMPDKDEESRICKALFLYTSHLRKYNDALIISEHARMKDALDYLKDFFSNVRAAGFDEIEQDLTQRFEEKLQELESVSRDPSNENPKLEDLCFILQEEYHLNPETITILFVKTRALVDALKNWIEGNPKLSFLKPGILTGRGKTNQNTGMTLPAQKCILDAFKASGDHNILIATSVADEGIDIAQCNLVILYEYVGNVIKMIQTRGRGRAR
GSKCFLLTSNAGVIEKEQINMYKEKMMNDSILRLQTWDEAVFREKILHIQTHEKFIRDSQEKPKPVPDKENKKLLCRKCKALACYTADVRVIEECHYTVLGDAFKECFVSRPHPKPKQFSSFEKRAKIFCARQNCSHDWGIHVKYKTFEIPVIKIESFVVEDIATGVQTLYSKWKDFHFEKIPFDPAEMSK
(M)
(M)
(M)
(M)
(M)
(M)
Domain
(M)
(M)
Domain
(M)
Domain + RD domain
(M)
DSKCFLLTSSADVIEKEKANMIKEKIMNESILRLQTWDEMKFGKTVHRIQVNEKLLRDSQHKPQPVPDKENKKLLCGKCKNFACYTADIRVVETSHYTVLGDAFKERFVCKPHPKPKIYDNFEKKAKIFCAKQNCSHDWGIFVRYKTFEIPVIKIESFVVEDIVSGVQNRHSKWKDFHFERIQFDPAEMSV
(M) +
Helicase
Domain + RD domain
(M)
LPAKKGKNTIICAPTGCGKTFVSLLICEHHLKKFPCGQKGKVVFFANQIPVYEQQATVFSRYFERLGYNIASISGATSDSVSVQHIIEDNDIIILTPQILVNNLNNGAIPSLSVFTLMIFDECHNTSKNHPYNQIMFRYLDHKLGESRDPLPQVVGLTASVGVGDAKTAEEAMQHICKLCAALDASVIATVRDNVAELEQVVYKPQKISRKVASRTSNTFKCIISQLMKETEKLAKDVSEELGKLFQIQNREFGTQKYEQWIVGVHKACSVFQMADKEEESRVCKALFLYTSHLRKYNDALIISEDAQMTDALNYLKAFFHDVREAAFDETERELTRRFEEKLEELEKVSRDPSNENPKLRDLYLVLQEEYHLKPETKTILFVKTRALVDALKKWIEENPALSFLKPGILTGRGRTNRATGMTLPAQKCVLEAFRASGDNNILIATSVADEGIDIAECNLVILYEYVGNVIKMIQTRGRGRAR
DSKCFLLTSSADVIEKEKANMIKEKIMNESILRLQTWDEMKFGKTVHRIQVNEKLLRDSQHKPQPVPDKENKKLLCGKCKNFACYTADIRVVETSHYTVLGDAFKERFVCKPHPKPKIYDNFEKKAKIFCAKQNCSHDWGIFVRYKTFEIPVIKIESFVVEDIVSGVQNRHSKWKDFHFERIQFDPAEMSV
The polypeptide was purified from a bacterial or Drosophila cell line expressing a polypeptide comprising the helicase domain and the RD domain of the RIG-I protein (Retinoic acid inducible gene-1, DDX58) to obtain HepG2, Huh-7 , Treatment of THP-1 cells increases mitochondrial activity and increases the number of mitochondrial DNA copies. In addition, the level of ATP produced by mitochondrial activity increases and the level of lipid consumed by the beta-oxidation process of mitochondria decreases.
In addition, it was confirmed that expression of genes (PGC1 alpha, NRF1, TFAM) that regulate activity and biosynthesis when the polypeptide was treated increased. When PGC1α involved in the expression of genes important for mitochondrial activity and biosynthesis was knocked down, there was no increase in mitochondrial activity by the polypeptide treatment. This shows that the polypeptide treated with the cells regulates the biosynthesis and activity of mitochondria through the activation of PGC1 < alpha > transcription factor.
However, there is no change in mitochondrial activity when the polypeptide is heat-treated and processed into cells. This shows that the structure due to peptide folding is more important for the regulation of mitochondrial activity by the polypeptide than the sequence itself of the protein.
The polypeptide comprises two CARDs, a helicase domain, and an RD domain. In order to identify a domain that regulates mitochondrial activity and biosynthesis, the polypeptide has only two CARD domains, or a helicase domain and a RD The protein having the domain was treated with the cells.
As a result, there was no change in the mitochondrial activity when the protein consisting of the CARD domain was treated. However, when the protein containing the helicase domain and the RD domain was treated, mitochondrial activity was increased, Domain is needed.
In addition, the polypeptide may be selected from peptides having a length of 484 to 708 amino acids including a helicase domain and an RD domain.
In one example, the polypeptide may be a polypeptide comprising the helicase domain consisting of the amino acid sequence of SEQ ID NO: 7 or 18 and the RD domain consisting of the amino acid sequence of SEQ ID NO: 8 or 19, Or < RTI ID = 0.0 > 21. ≪ / RTI >
In addition, the polypeptide may further comprise a linker consisting of 30 to 40, 30 to 35, for example 34 amino acids. The linker may be composed of the amino acid sequence of SEQ ID NO: 6 or 17, but is not limited thereto. In addition, when the polypeptide further comprises a linker, it may include the amino acid sequence of SEQ ID NO: 11 or 22.
The term "polypeptide" as used herein refers to a molecule comprising a polymer of amino acids linked together by a peptide bond (s). The polypeptide may be one comprising two or more amino acids.
Another example of the present invention relates to a polynucleotide encoding a polypeptide comprising a helicase domain and a RD domain of a retinoic acid-inducible gene-1 (DDX58) protein.
The nucleotide sequences coding for each domain constituting the human-derived RIG-I protein and the mouse-derived RIG-I protein are shown in Tables 5 to 14 (H: human (NM_014314.3), M: mouse NM_172689.3)).
(H)
(H)
(H)
(H)
(H)
(H)
Domain
(H)
(H)
Domain
(H)
Domain + RD domain
(H)
(H) +
Helicase
Domain + RD domain
(H)
(M)
(M)
(M)
(M)
(M)
(M)
Domain
(M)
(M)
Domain
(M)
Domain + RD domain
(M)
(M) +
Helicase
Domain + RD domain
(M)
In one example, when the helicase domain comprises the amino acid sequence of SEQ ID NO: 7 or 18, the polynucleotide sequence encoding the helicase domain may comprise the nucleotide sequence of SEQ ID NO: 29 or 40 , But is not limited thereto.
In addition, when the RD domain comprises the amino acid sequence of SEQ ID NO: 8 or 19, the polynucleotide encoding the RD domain may include the nucleotide sequence of SEQ ID NO: 30 or 41, but is not limited thereto.
The polynucleotide encoding the polypeptide comprising the helicase domain and RD domain of the RIG-I protein (Retinoic acid inducible gene-1, DDX58) may comprise the nucleotide sequence of SEQ ID NO: 32 or 43 However, the present invention is not limited thereto.
In addition, the polynucleotide may further comprise a nucleotide sequence encoding a linker consisting of 30 to 40, 30 to 35, for example 34 amino acids. The linker may be composed of the amino acid sequence of SEQ ID NO: 6 or 17, and the nucleotide sequence coding for the linker may comprise the nucleotide sequence of SEQ ID NO: 28 or 39, but is not limited thereto.
The polynucleotide further comprising the nucleotide sequence encoding the linker may include, but is not limited to, the nucleotide sequence of SEQ ID NO: 33 or 44.
Another example of the present invention relates to a recombinant vector comprising a polynucleotide encoding a polypeptide comprising a helicase domain and a RD domain of a retinoic acid-inducible gene-1 (DDX58) protein.
The polypeptide comprising the helicase domain and the RD domain of the RIG-1 (retinoic acid-inducible gene-1, DDX58) protein and the polynucleotide encoding the polypeptide are the same as described above.
The recombination vector may have a cleavage map as shown in Fig.
The recombinant vector may be a viral or non-viral vector.
The viral vector may be, but is not limited to, adenovirus, adeno-associated virus, helper-dependent adenovirus, retroviral vector, and the like.
The term "vector" means means for expressing a gene of interest in a host cell. The vector includes elements for expression of a target gene and may include a replication origin, a promoter, an operator, a transcription termination terminator, and the like into the genome of the host cell. Ribosome binding sites (RBS) for translation into selectable markers and / or proteins to confirm successful introduction into the host cell and / or appropriate enzyme sites for introduction of the IRES (e.g., IRES (Internal Ribosome Entry Site), and the like. The vector may be engineered in a conventional genetic engineering manner to have the fusion polynucleotide (fusion promoter) described above as a promoter. The vector may further comprise a transcription control sequence other than the promoter (e.g., an enhancer, etc.).
The recombinant vector may be constructed by a variety of methods known in the art.
The transfer (introduction) of the recombinant vector into a cell can be carried out using a carrier method well known in the art. For example, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, gene bombardment, and the like can be used as the delivery method. For example, liposome-mediated transfection (Lipofector reagent). < / RTI >
Another example of the present invention provides a recombinant cell comprising the polynucleotide or a recombinant vector. The recombinant cell may be a recombinant cell obtained by transforming a host cell with the recombinant vector.
The host cell may be selected from the group consisting of viruses, bacteria, plant cells, insect cells, mammalian cells, for example, mammalian cells other than human, but is not limited to, Lt; / RTI > cells.
Another example of the present invention is a polypeptide comprising a helicase domain and an RD domain of a retinoic acid-inducible gene-1 (DDX58) protein, a polynucleotide encoding the polypeptide, a polynucleotide comprising the polynucleotide A recombinant vector comprising the recombinant vector, or a mixture thereof as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating a disease associated with a metabolic syndrome.
A polypeptide comprising the helicase domain and the RD domain of the retinoic acid-inducible gene-1 (DDX58) protein, a polynucleotide encoding the polypeptide, a recombinant vector comprising the polynucleotide, And the recombinant cell comprising the recombinant vector are the same as described above.
The disease associated with the metabolic syndrome may be, but is not limited to, type 2 diabetes, obesity, cardiovascular disease, hypertension, hyperglycemia, hyperlipidemia, obesity, atherosclerosis, ≪ / RTI >
In the above composition, the active ingredient may be contained in a pharmaceutically effective amount capable of exhibiting a desired preventive and / or therapeutic effect.
Another example of the present invention provides a pharmaceutical preparation containing the composition as an active ingredient.
The pharmaceutical preparations according to the present invention may be formulated into oral preparations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups and aerosols or percutaneous preparations such as suppositories and sterilized injection solutions Parenteral formulations, and the like.
The pharmaceutical preparations of the present invention may be pharmaceutically acceptable and additionally contain adjuvants such as physiologically acceptable carriers, excipients and diluents. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used can be used
When the pharmaceutical preparation according to the present invention is used for parenteral administration, for example, a topical administration such as a liquid preparation, a gel preparation, a cleaning composition, a tablet for insertion, a suppository form, cream, ointment, dressing solution, spray, It may be a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, a suppository, a cream, an ointment, a jelly, a foam, a cleanser or an insert, May be a skin external agent such as a liquid preparation, a gel preparation, a cleaning composition, and a tablet for insertion. The formulation can be prepared, for example, by adding a solubilizer, an emulsifying agent, a buffering agent for pH control, etc. to the sterilized water. Examples of the non-aqueous solvent or suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.
More specifically, the pharmaceutical preparation may comprise a carrier for additionally formulating the composition. The carrier may be a binder, a lubricant, a suspending agent, a solubilizer, a buffer, a preservative, a lubricant, an isotonic agent, an excipient, a stabilizer, a dispersant, a suspending agent,
In embodiments where the pharmaceutical preparations of the present invention are applied to humans, the pharmaceutical preparations of the present invention may be administered alone, but are generally administered in a pharmacological manner selected in consideration of the mode of administration and standard phamaceutical practice May be administered in admixture with a carrier. For example, the pharmaceutical preparations of the present invention may be in the form of tablets containing starch or lactose, or in capsules containing the active ingredient alone or as an excipient, or as an elixir or a suspending agent containing a chemical that tastes or colors 0.0 > oral, < / RTI > intraoral, or sublingually.
The dose of the pharmaceutical preparation of the present invention may be varied depending on the age, body weight, sex, dosage form, health condition and disease severity of the patient, and may be determined once or several times per day It may be administered in divided doses. For example, the daily dose may be 0.001 to 10000 mg / kg, 0.01 to 10000 mg / kg, 0.1 to 10000 mg / kg, or the like, based on the content of the active ingredient (i.e., RIG-I protein, its coding polynucleotide, Kg, 0.1 to 1000 mg / kg, 0.001 to 500 mg / kg, 0.01 to 500 mg / kg, 0.1 to 1000 mg / kg, Kg to 500 mg / kg, 0.5 to 500 mg / kg, 0.001 to 300 mg / kg, 0.01 to 300 mg / kg, 0.1 to 300 mg / kg, or 0.5 to 300 mg / kg. The above-mentioned dosage is an average case, and the dose may be high or low depending on individual differences.
If the daily dose of the pharmaceutical preparation of the present invention is less than the above dose, no significant effect can be obtained, and if it exceeds the above range, it is not only economical but also causes an undesirable side effect due to deviation from the commercial dose range It is preferable to set the above range.
Another embodiment of the present invention provides a method of treating or preventing a disease associated with a metabolic syndrome comprising administering a pharmaceutically effective amount of the composition. The treatment or prevention method may be performed in vitro or may be performed on an animal other than human.
The composition used in the method for treating or preventing the disease associated with the metabolic syndrome is the same as described above. The disease related to the metabolic syndrome may be, but is not limited to, type 2 diabetes, obesity, cardiovascular disease, hypertension, hyperglycemia, hyperlipidemia, obesity, atherosclerosis, It can be any disease caused by dysfunction.
Another example of the present invention is a polynucleotide comprising a helicase domain of a retinoic acid-inducible gene-1 (DDX58) protein, a polynucleotide encoding the RIG-I protein or polypeptide , A recombinant vector comprising the polynucleotide, a recombinant cell comprising the recombinant vector, or a mixture thereof, for the treatment or prevention of a disease associated with the metabolic syndrome.
A polypeptide comprising the helicase domain and the RD domain of the retinoic acid-inducible gene-1 (DDX58) protein, a polynucleotide encoding the polypeptide, a recombinant vector comprising the polynucleotide, And the recombinant cell comprising the recombinant vector are the same as described above.
The disease associated with the metabolic syndrome may be, but is not limited to, type 2 diabetes, obesity, cardiovascular disease, hypertension, hyperglycemia, hyperlipidemia, obesity, atherosclerosis, ≪ / RTI >
The present invention relates to a polypeptide comprising a helicase domain and an RD domain of a retinoic acid-inducible gene-1 (DDX58) protein, a polynucleotide encoding the polypeptide, a recombinant protein comprising the polynucleotide Or a composition for preventing and / or treating diseases related to the metabolic syndrome containing said polypeptide, polynucleotide, recombinant vector or a mixture thereof, a pharmaceutical preparation containing said composition as an active ingredient, and The present invention relates to a method for treating or preventing diseases associated with metabolic syndrome using the above composition, wherein the expression of mitochondrial biosynthesis and activity-related proteins (PGC1 alpha, NRF1, TFAM) is increased to regulate biosynthesis of mitochondria, And vaginal use for activity studies or due to mitochondrial dysfunction Of it can be usefully employed in the treatment.
FIG. 1 is a graph showing MTT assays for increasing mitochondrial activity according to treatment of recombinant rhRIG-I protein according to an embodiment of the present invention.
FIG. 2 is a graph showing the effect of increasing the mitochondrial activity according to the recombinant rmRIG-I protein treatment time according to an embodiment of the present invention through MTT assay.
FIG. 3 is a graph showing changes in the number of HepG2 cells by the time-course recombinant rhRIG-I protein treatment according to an embodiment of the present invention.
FIG. 4 is a graph showing the amount of intracellular mitochondrial DNA according to the recombinant rhRIG-I protein treatment according to an embodiment of the present invention relative to genomic DNA.
FIG. 5 is a graph showing the STAT1 / STAT2 activity according to the recombinant rhRIG-I protein treatment according to an embodiment of the present invention through an interferon stimulated response element (ISRE) luciferase assay.
FIG. 6 is a graph showing the activity of IRF3 according to the recombinant rhRIG-I protein treatment according to one embodiment of the present invention through the PRDIII-I luciferase assay.
FIG. 7 is a photograph showing NDV proliferation according to the recombinant rmRIG-I protein treatment according to an embodiment of the present invention.
FIG. 8 is a graph showing changes in the amount of intracellular ATP according to the treatment of recombinant rhRIG-I or heat-treated recombinant rhRIG-I protein according to an embodiment of the present invention.
FIG. 9 is a graph showing changes in intracellular lipid levels according to the treatment of recombinant rhRIG-I or heat-treated recombinant rhRIG-I protein according to an embodiment of the present invention.
FIG. 10 is a photograph showing lipid accumulation induced by olive fatty acid in HepG2 and Huh-7 cell lines according to the present invention, according to the presence or absence of recombinant rhRIG-I protein treatment. FIG.
FIG. 11 is a graph showing quantitation of lipid levels in cells without or with the treatment of olive fatty acid according to the presence or absence of recombinant rhRIG-I protein treatment in HepG2 cell line according to an embodiment of the present invention.
FIG. 12 is a graph showing the level of intracellular lipids in a THP-1 cell line treated with or without the treatment of recombinant rhRIG-I protein, in the absence of treatment with olive oil.
FIG. 13 is a graph showing expression of ACADL and CPT1A genes associated with lipid accumulation according to time-dependent treatment of recombinant rhRIG-I protein according to an embodiment of the present invention.
FIG. 14 is a graph showing the expression of NRF1, PGC1α, and TFAM, which regulate mitochondrial biosynthesis and activity according to the treatment of recombinant rhRIG-I protein according to an embodiment of the present invention.
15 is a graph showing the effect of PGC1? Knockdown on the regulation of mitochondrial activity by the recombinant rhRIG-I protein according to an embodiment of the present invention.
Fig. 16 is a schematic diagram showing the domain structure of the human RIG-I protein used in the present invention and the constitutive domain of the deletion peptide.
FIG. 17 shows the activity of mitochondria when treated with recombinant rmRIG-I whole protein, CARD domain peptide (rhCard) of human RIG-I protein, and Helicase domain peptide (rhHelicase) of human RIG-I protein according to an embodiment of the present invention. As shown in FIG.
18 is a graph showing changes in the amount of intracellular ATP when the recombinant rhRIG-I protein, the recombinant rhCard peptide, and the recombinant rhHelicase peptide are treated according to an embodiment of the present invention.
19 is a graph showing changes in intracellular lipid levels when the recombinant rhRIG-I whole protein, recombinant rhCard peptide, and recombinant rhHelicase peptide are treated according to an embodiment of the present invention.
20 is a diagram showing a cleavage map of a recombination vector according to an embodiment of the present invention.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
Experimental Example 1. Plasmid preparation
Human RIG-I CDS or mouse RIG-I CDS was cloned into the pFastBac vector (Invitrogen) and expressed in the SF9 Drosophila cell line (ATCC) with the FLAG Tag and the 10XHIS-GST Tag co-expressed at the N-terminus. The plasmids used in the bacterial expression system were constructed as follows.
The nucleotide sequence encoding the human-derived RIG-I protein (SEQ ID NO: 1), mouse-derived RIG-I protein (SEQ ID NO: 4), and CARD domain peptide (SEQ ID NO: 3) was inserted into pProExHTa vector (Invitrogen) And the nucleotide sequence encoding the helicase domain peptide (SEQ ID NO: 2) was cloned into the pET22b-CPD (Invitrogen) vector for expression together with 10xHis-CPD tagging. The amino acid sequence of the RIG-I protein and the RIG-I protein fragment and the nucleotide sequence encoding the amino acid sequence are the same as in Tables 1 to 8 above.
Experimental Example 2 Purification of Protein
Each plasmid prepared in Example 1 was expressed in Sf9 cell line or BL21-pLysS bacteria, resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 1 M NaCl, 1 mM dithiothreitol) and sonicated And dissolved to obtain a lysate. Then, the lysate was centrifuged at 18,000 rpm for 30 minutes, and the supernatant was passed through a cobalt column (Thermo) to separate the human-derived RIG-I protein, the helicase domain peptide, and the CARD peptide .
Then, 10xHis-GST and 10xHis-CPD were removed by treatment with TEV (Tobacco Etch Virus nuclear-inclusion-a endopeptidase) or phytate, and a second cobalt column was passed through to remove untagged proteins . Purification of the recombinant protein was then performed by passing through Hitrap Q (GE Healthcare) and
Experimental Example 3. Cell Culture and Transfection
The HepG2 cell line (ATCC) was cultured in MEM (Welgene) containing 10% fetal bovine serum (FBS, Hyclone) and the THP1 cell line (ATCC) was cultured in Dulbecco's modified Eagle's medium (DMEM, Welgene).
5 μg / ml of poly I: C (Amersham Biosciences) was transfected with lipofectamine 2000 (Invitrogen) for intracellular poly I: C treatment. Lipofectamine 2000 was used for transfection of plasmid (total 2 ug of plasmids / 35 pi dish) or siRNA (100 pmol). Control siRNA and PGC1? SiRNA sequences are shown in Table 15 below.
Experimental Example 4 Luciferase assay
HepG2 cell lines were transformed with pISRE or pPRDIII-I Luciferase Reporter plasmid (pISRE Luciferase Reporter-Stratagene, pPRDIII-I Luciferase Reporter-Katherine A. Fitzgerald, University of Massachusetts). RIG-I protein (20 μg / ml) or poly I: C (5 μg / ml) was treated for 12 hours. After 48 hours, the cells were lysed and luciferase activity was measured using the Dual-luciferase assay kit (Promega).
Experimental Example 5. MTT Assay
MTT (Sigma) solution dissolved in PBS was treated with the cells at 37 ° C for 3 hours. After removal of the supernatant, DMSO dissolved in isopropanol was treated and the absorbance at 570 nm was measured using an ELISA plate reader (BioRad).
Experimental Example 6. Determination of intracellular ATP
Cells were treated with recombinant RIG-I protein for 24 hours, then the culture was removed and washed with PBS. Cells were lysed in lysis buffer (1% TCA, 4 mM EDTA, PMSF, Tris-HCl pH 7.8) and placed on ice for 10 min. The intracellular ATP was measured by ATP bioluminescent assay kit (Sigma).
Experimental Example 7. Oil red O staining and analysis
Cells were washed with PBS and fixed with 10% formalin for 10 min at room temperature. After fixation, the cells were washed with 10% formalin and treated with 60% isopropanol for 5 minutes. Isopropanol was removed and cells were stained with oil red (Sigma) for 3 hours at room temperature. After removing the staining solution, the cells were rinsed with 60% isopropanol, and the cell sample was rinsed again with water. Cell samples were dried, 100% isopropanol was added, and the absorbance at 500 nm was measured using an ELISA plate reader (Biorad).
Experimental Example 8. Mitochondrial DNA Analysis
Cells were resuspended in DNA lysis buffer (0.2 M NaCl, 10 mM EDTA, 10 mM Tris-HCl pH 8.0) and then allowed to shake for 2 h at 55 ° C with 200 μg / ml Proteinase K and 0.5% SDS. DNA is extracted using Phenol / Chloroform / IAA. The amounts of genomic DNA (18S ribosomal DNA, 18S rDNA) and mitochondrial DNA in the DNA were quantitated by PCR using the primers shown in Table 16 below.
Experimental Example 9. RNA Extraction and Analysis
RNA was extracted from cells using RNA isoplus (TAKARA Bio) according to the manufacturer's manual. 1 μg of RNA was subjected to RT-PCR using the ImProme-II® Reverse transcription system (Promega). For quantitative real-time PCR, the primers shown in Table 17 below were used.
Example 1. Confirmation of increase in mitochondrial activity
1-1. MTT assay
MTT assays were performed in which HepG2 cells (ATCC) were treated with recombinant rhRIG-
Specifically, MTT (Sigma) solution dissolved in PBS was treated with HepG2 cells at 37 占 폚 for 3 hours. After removal of the supernatant, DMSO dissolved in isopropanol was treated and the absorbance at 570 nm was measured using an ELISA plate reader (BioRad) to confirm the increase in mitochondrial activity. The results are shown in Tables 18 to 19 and Figs.
(Mean, n = 3)
(Standard Deviation)
As shown in Table 18 and FIG. 1, when the RIG-I protein was treated with the cells, the mitochondrial activity was increased as the treated dose was increased.
(Mean, n = 3)
(Standard Deviation)
As shown in Table 19 and FIG. 2, when the recombinant rmRIG-I protein (20 μg / ml) was treated with time and the activity of mitochondria was measured through MTT assay, it was increased after 3 hours and further increased until 12 hours Respectively.
1-2. Measurement of cell number change
HepG2 cells were treated with recombinant rhRIG-I protein (20 μg / ml) for 24, 36, and 48 hours, and the number of cells was counted and shown in Table 20 and FIG.
(Mean, n = 17)
(Standard Deviation)
As shown in Table 20 and FIG. 3, it was confirmed that the recombinant RIG-I protein treatment did not significantly change the cell number. In other words, MTT assays are affected not only by mitochondrial activity but also by cell number, confirming that recombinant RIG-I protein treatment does not affect cell numbers.
1-3. Measurement of DNA amount by real-time PCR
The relative levels of mitochondrial DNA (COX2, MTCO1, ND1) to genomic DNA (18S rDNA) were measured by real-time PCR using rhGIG-I (20 μg / ml) The results are shown in Table 21 and FIG.
(Mean, n = 4)
(Standard Deviation)
(4 sets of buffer treatment conditions to 1)
As shown in Table 21 and FIG. 4, it was confirmed that the amount of intracellular mitochondrial DNA was increased by recombinant RIG-I protein treatment. In other words, the change in mitochondrial activity confirmed above was attributed to an increase in mitochondrial biosynthesis expressed by the amount of mitochondrial DNA.
Example 2. Confirmation of change in antiviral signal activity
2-1. Lucifer Reyes Assay
After incubation for 12 hours with poly I: C (5 μg / ml) and rhRIG-I protein (20 μg / ml), luciferase assays were performed on the HepG2 cell line using the pISRE or pPRDIII-I luciferase reporter plasmid And the results are shown in Table 22, FIG. 5, and FIG.
(20 [mu] g / ml)
(5 [mu] g / ml)
(Average value, n = 3)
As shown in Table 22 and FIG. 5, it was confirmed that the ISRE luciferase activity increased by the poly I: C treatment was not changed when the RIG-I recombinant protein was treated to the cells. This means that the extracellular RIG-I protein does not affect the activity of the STAT1 / STAT2 transcription factor.
As shown in Table 16 and FIG. 6, it was confirmed that the PRDIII-I luciferase activity increased by the poly I: C treatment was not changed when the RIG-I recombinant protein was treated with the cells. This means that the extracellular RIG-I protein does not affect the activity of the IRF3 transcription factor.
2-2. GFP (green fluorescent protein) assay
The HepG2 cell line was infected with GFP-NDV (5MOI) for 3 hours, and the recombinant rmRIG-I protein (20 μg / ml) was treated for 0 or 24 hours and the intracellular GFP level was confirmed by fluorescence microscopy. The results are shown in Fig.
As can be seen in FIG. 7, it was confirmed that RIG-I protein treatment outside the cells did not affect GFP-NDV virus proliferation.
Example 3. Measurement of changes in intracellular ATP and lipid levels by heat treatment
3-1. Intracellular ATP Level Measurement
20 μg / ml of the recombinant rhRIG-I protein without heat treatment (95 ° C., 5 minutes) or without heat treatment was treated with HepG2 cells for 24 hours and the level of intracellular ATP was measured. The results are shown in Table 23 and FIG.
As shown in Table 23 and FIG. 8, it was confirmed that the treatment of recombinant RIG-I protein outside the cells increased the amount of intracellular ATP, but the heat-treated RIG-I protein did not affect the amount of ATP. This means that maintaining the structure of the RIG-I protein is necessary to increase the amount of intracellular ATP.
3-2. Intracellular lipid level measurement
The recombinant rhRIG-I protein (20 占 퐂 / ml) without heat treatment (95 占 폚, 5 minutes) or without heat treatment was treated with HepG2 cells for 24 hours and the amount of lipids in the cells was measured. The results are shown in Tables 24 and 9 Respectively.
As shown in Table 24 and FIG. 9, it was confirmed that the recombinant RIG-I protein treatment reduced intracellular lipid level, but the heat-treated RIG-I protein did not affect the intracellular lipid level. This means that maintaining the structure of the RIG-I protein is necessary to lower the lipid content in the cell.
Example 4. Measurement of changes in intracellular ATP and lipid level
Since the accumulation of intracellular lipids has been reported to be associated with diseases such as
As shown in FIG. 10, when the RIG-I protein (20 μg / ml) was treated, the amount of lipid in the cells was decreased in HepG2 cells and Huh7 cells.
After lipopolysaccharide (100mM) and rhRIG-I recombinant protein (20μg / ml) were treated with HepG2 cells or THP1 cells and stained with oil-red-o, the amount of lipids in the cells was measured with an ELISA reader (500nm) The results are shown in Table 25 and Figs. 11 and 12.
(Average value, n = 3)
As shown in Table 25 and FIGS. 11 and 12, when the accumulation amount of lipids in HepG2 cells and THP-1 cells was arbitrarily increased through the orate treatment, the amount of accumulated lipids by recombinant RIG-I protein treatment was 2 And it decreased in all cells.
Then, the rhRIG-I protein (20 μg / ml) was treated with HepG2 cells for 0, 6, 12, and 24 hours, and the intracellular RNA was extracted and the RNA level of hACADL and hCTP1 was subjected to real-time PCR And the graphs are shown in Table 26 and FIG.
As shown in Table 26 and FIG. 13, when RIG-I protein was treated, expression levels of hACADL and hCTP1, which are involved in lipid metabolism, were increased.
Example 5. Regulation of Mitochondrial Activity of RIG-I Recombinant Protein via PGC1α
After incubation with rhGIG-I recombinant protein (20 μg / ml) for 6 hours, RNA was extracted from the HepG2 cells and RT-PCR was performed. NRF1, PGC1α and TFAM mRNA levels were analyzed by real-time PCR As a graph are shown in Table 27 and Fig.
As shown in Table 27 and FIG. 14, expression of NRF1, PGC1α and TFAM RNA was increased by recombinant RIG-I protein treatment.
Control siRNA or PGC1α siRNA was expressed in HepG2 cells by liposome infusion and treated with rhRIG-I recombinant protein (20 μg / ml) for 24 hours. The results of analysis of mitochondrial activity through MTT assays and the results of PGC1? MRNA RT-qPCR are shown in Tables 28 to 29 and FIG.
As shown in Tables 28 to 29 and FIG. 15, mitochondrial activity increased by recombinant RIG-I protein treatment did not appear when PGC1α was knocked down, suggesting that regulation of mitochondrial activity by extracellular RIG- It can be seen that it happens.
Example 5. Identification of RIG-I Protein Domain Required for Mitochondrial Activity Control
The RIG-I protein consists of two CARD domains, a Helicase domain, and an RD domain, and a deletion protein was constructed as shown in FIG. 16 to determine which domain is required for mitochondrial activity regulation.
The results of MTT assay and intracellular ATP levels after treatment of 20 μg / ml of each of rmRIG-I recombinant protein, rhRIG-I recombinant protein, rhCard recombinant peptide and rhHelicase recombinant peptide in HepG2 cells for 24 hours Are shown in Table 30, FIG. 17, Table 31, and FIG. 18, respectively.
As shown in Table 30 and FIG. 17, the rhHelicase recombinant peptide increased the mitochondrial activity to a level similar to that of the rmRIG-I recombinant protein, but the rhCard recombinant peptide did not affect the mitochondrial activity. This implies that the function of the RIG-I protein, which controls intracellular mitochondrial activity, requires a domain containing Helicase.
In addition, as shown in Table 31 and FIG. 18, rhHelicase recombinant peptide treatment increased intracellular ATP level, but rhCard recombinant peptide treatment did not affect intracellular ATP level. This implies that the function of the RIG-I protein, which controls the amount of intracellular ATP, requires a domain containing Helicase.
After incubation for 24 hours with 20 μg / ml of rhuRIG-I recombinant protein or rhCIG-I recombinant peptide or rhHelicase recombinant peptide at 20 μg / ml concentration, the amount of lipid in the cells was stained with oil-red- 500 nm), and the graphs are shown in Table 32 and FIG.
(Average value, n = 3)
As shown in Table 32 and FIG. 19, when the helicase recombinant peptide was treated, the amount of lipid in the cells was decreased, but when the CARD recombinant peptide was treated, the amount of lipid in the cells was not changed. This implies that the function of the RIG-I protein, which regulates intracellular lipid levels, requires a domain containing Helicase.
<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Pharmaceutical composition for preventing or treating metabolic syndrome <130> DPP20155173KR <160> 62 <170> Kopatentin 2.0 <210> 1 <211> 925 <212> PRT <213> Artificial Sequence <220> ≪ 223 > RIG-I (H) <400> 1 Met Thr Thr Glu Gln Arg Arg Ser Leu Gln Ala Phe Gln Asp Tyr Ile 1 5 10 15 Arg Lys Thr Leu Asp Pro Thr Tyr Ile Leu Ser Tyr Met Ala Pro Trp 20 25 30 Phe Arg Glu Glu Glu Val Gln Tyr Ile Gln Ala Glu Lys Asn Asn Lys 35 40 45 Gly Pro Met Glu Ala Ala Thr Leu Phe Leu Lys Phe Leu Leu Glu Leu 50 55 60 Glu Glu Glu Gly Trp Phe Arg Gly Phe Leu Asp Ala Leu Asp His Ala 65 70 75 80 Gly Tyr Ser Gly Leu Tyr Glu Ala Ile Glu Ser Trp Asp Phe Lys Lys 85 90 95 Ile Glu Lys Leu Glu Glu Tyr Arg Leu Leu Leu Lys Arg Leu Gln Pro 100 105 110 Glu Phe Lys Thr Arg Ile Ile Pro Thr Asp Ile Ile Ser Asp Leu Ser 115 120 125 Glu Cys Leu Ile Asn Gln Glu Cys Glu Glu Ile Leu Gln Ile Cys Ser 130 135 140 Thr Lys Gly Met Met Ala Gly Ala Glu Lys Leu Val Glu Cys Leu Leu 145 150 155 160 Arg Ser Asp Lys Glu Asn Trp Pro Lys Thr Leu Lys Leu Ala Leu Glu 165 170 175 Lys Glu Arg Asn Lys Phe Ser Glu Leu Trp Ile Val Glu Lys Gly Ile 180 185 190 Lys Asp Val Glu Thr Glu Asp Leu Glu Asp Lys Met Glu Thr Ser Asp 195 200 205 Ile Gln Ile Phe Tyr Gln Glu Asp Pro Glu Cys Gln Asn Leu Ser Glu 210 215 220 Asn Ser Cys Pro Pro Ser Glu Val Ser Asp Thr Asn Leu Tyr Ser Pro 225 230 235 240 Phe Lys Pro Arg Asn Tyr Gln Leu Glu Leu Ala Leu Pro Ala Met Lys 245 250 255 Gly Lys Asn Thr Ile Ile Cys Ala Pro Thr Gly Cys Gly Lys Thr Phe 260 265 270 Val Ser Leu Leu Ile Cys Glu His His Leu Lys Lys Phe Pro Gln Gly 275 280 285 Gln Lys Gly Lys Val Val Phe Phe Ala Asn Gln Ile Pro Val Tyr Glu 290 295 300 Gln Gln Lys Ser Val Phe Ser Lys Tyr Phe Glu Arg His Gly Tyr Arg 305 310 315 320 Val Thr Gly Ile Ser Gly Ala Thr Gly Asn Val Val Glu Gln 325 330 335 Ile Val Glu Asn Asn Asp Ile Ile Leu Thr Pro Gln Ile Leu Val 340 345 350 Asn Asn Leu Lys Lys Gly Thr Ile Pro Ser Leu Ser Ile Phe Thr Leu 355 360 365 Met Ile Phe Asp Glu Cys His Asn Thr Ser Lys Gln His Pro Tyr Asn 370 375 380 Met Ile Met Phe Asn Tyr Leu Asp Gln Lys Leu Gly Gly Ser Ser Gly 385 390 395 400 Pro Leu Pro Gln Val Ile Gly Leu Thr Ala Ser Val Gly Val Gly Asp 405 410 415 Ala Lys Asn Thr Asp Glu Ala Leu Asp Tyr Ile Cys Lys Leu Cys Ala 420 425 430 Ser Leu Asp Ala Ser Val Ile Ala Thr Val Lys His Asn Leu Glu Glu 435 440 445 Leu Glu Gln Val Val Tyr Lys Pro Gln Lys Phe Phe Arg Lys Val Glu 450 455 460 Ser Arg Ile Ser Asp Lys Phe Lys Tyr Ile Ile Ala Gln Leu Met Arg 465 470 475 480 Asp Thr Glu Ser Leu Ala Lys Arg Ile Cys Lys Asp Leu Glu Asn Leu 485 490 495 Ser Gln Ile Gln Asn Arg Glu Phe Gly Thr Gln Lys Tyr Glu Gln Trp 500 505 510 Ile Val Thr Val Gln Lys Ala Cys Met Val Phe Gln Met Pro Asp Lys 515 520 525 Asp Glu Glu Ser Arg Ile Cys Lys Ala Leu Phe Leu Tyr Thr Ser His 530 535 540 Leu Arg Lys Tyr Asn Asp Ala Leu Ile Ile Ser Glu His Ala Arg Met 545 550 555 560 Lys Asp Ala Leu Asp Tyr Leu Lys Asp Phe Phe Ser Asn Val Arg Ala 565 570 575 Ala Gly Phe Asp Glu Ile Glu Gln Asp Leu Thr Gln Arg Phe Glu Glu 580 585 590 Lys Leu Gln Glu Leu Glu Ser Val Ser Arg Asp Pro Ser Asn Glu Asn 595 600 605 Pro Lys Leu Glu Asp Leu Cys Phe Ile Leu Gln Glu Glu Tyr His Leu 610 615 620 Asn Pro Glu Thr Ile Thr Ile Leu Phe Val Lys Thr Arg Ala Leu Val 625 630 635 640 Asp Ala Leu Lys Asn Trp Ile Glu Gly Asn Pro Lys Leu Ser Phe Leu 645 650 655 Lys Pro Gly Ile Leu Thr Gly Arg Gly Lys Thr Asn Gln Asn Thr Gly 660 665 670 Met Thr Leu Pro Ala Gln Lys Cys Ile Leu Asp Ala Phe Lys Ala Ser 675 680 685 Gly Asp His Asn Ile Leu Ile Ala Thr Ser Val Ala Asp Glu Gly Ile 690 695 700 Asp Ile Ala Gln Cys Asn Leu Val Ile Leu Tyr Glu Tyr Val Gly Asn 705 710 715 720 Val Ile Lys Met Ile Gln Thr Arg Gly Arg Gly Arg Ala Arg Gly Ser 725 730 735 Lys Cys Phe Leu Leu Thr Ser Asn Ala Gly Val Ile Glu Lys Glu Gln 740 745 750 Ile Asn Met Tyr Lys Glu Lys Met Met Asn Asp Ser Ile Leu Arg Leu 755 760 765 Gln Thr Trp Asp Glu Ala Val Phe Arg Glu Lys Ile Leu His Ile Gln 770 775 780 Thr His Glu Lys Phe Ile Arg Asp Ser Gln Glu Lys Pro Lys Pro Val 785 790 795 800 Pro Asp Lys Glu Asn Lys Lys Leu Leu Cys Arg Lys Cys Lys Ala Leu 805 810 815 Ala Cys Tyr Thr Ala Asp Val Arg Ile Glu Glu Cys His Tyr Thr 820 825 830 Val Leu Gly Asp Ala Phe Lys Glu Cys Phe Val Ser Arg Pro His Pro 835 840 845 Lys Pro Lys Gln Phe Ser Ser Phe Glu Lys Arg Ala Lys Ile Phe Cys 850 855 860 Ala Arg Gln Asn Cys Ser His Asp Trp Gly Ile His Val Lys Tyr Lys 865 870 875 880 Thr Phe Glu Ile Pro Val Ile Lys Ile Glu Ser Phe Val Val Glu Asp 885 890 895 Ile Ala Thr Gly Val Gln Thr Leu Tyr Ser Lys Trp Lys Asp Phe His 900 905 910 Phe Glu Lys Ile Pro Phe Asp Pro Ala Glu Met Ser Lys 915 920 925 <210> 2 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> CARD 1 (H) <400> 2 Met Thr Thr Glu Gln Arg Arg Ser Leu Gln Ala Phe Gln Asp Tyr Ile 1 5 10 15 Arg Lys Thr Leu Asp Pro Thr Tyr Ile Leu Ser Tyr Met Ala Pro Trp 20 25 30 Phe Arg Glu Glu Glu Val Gln Tyr Ile Gln Ala Glu Lys Asn Asn Lys 35 40 45 Gly Pro Met Glu Ala Ala Thr Leu Phe Leu Lys Phe Leu Leu Glu Leu 50 55 60 Glu Glu Glu Gly Trp Phe Arg Gly Phe Leu Asp Ala Leu Asp His Ala 65 70 75 80 Gly Tyr Ser Gly Leu Tyr Glu 85 <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Linker 1 (H) <400> 3 Ala Ile Glu Ser One <210> 4 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> CARD 2 (H) <400> 4 Trp Asp Phe Lys Lys Ile Glu Lys Leu Glu Glu Tyr Arg Leu Leu Leu 1 5 10 15 Lys Arg Leu Gln Pro Glu Phe Lys Thr Arg Ile Ile Pro Thr Asp Ile 20 25 30 Ile Ser Asp Leu Ser Glu Cys Leu Ile Asn Gln Glu Cys Glu Glu Ile 35 40 45 Leu Gln Ile Cys Ser Thr Lys Gly Met Met Ala Gly Ala Glu Lys Leu 50 55 60 Val Glu Cys Leu Leu Arg Ser Asp Lys Glu Asn Trp Pro Lys Thr Leu 65 70 75 80 Lys <210> 5 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Linker 2 (H) <400> 5 Leu Ala Leu Glu Lys Glu Arg Asn Lys Phe Ser Glu Leu Trp Ile Val 1 5 10 15 Glu Lys Gly Ile Lys Asp Val Glu Thr Glu Asp Leu Glu Asp Lys Met 20 25 30 Glu Thr Ser Asp Ile Gln Ile Phe Tyr Gln Glu Asp Pro 35 40 45 <210> 6 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Linker 3 (H) <400> 6 Glu Cys Gln Asn Leu Ser Glu Asn Ser Cys Pro Ser Ser Glu Val Ser 1 5 10 15 Asp Thr Asn Leu Tyr Ser Pro Phe Lys Pro Arg Asn Tyr Gln Leu Glu 20 25 30 Leu Ala <210> 7 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Helicase Domain (H) <400> 7 Leu Pro Ala Met Lys Gly Lys Asn Thr Ile Ile Cys Ala Pro Thr Gly 1 5 10 15 Cys Gly Lys Thr Phe Val Ser Leu Leu Ile Cys Glu His His Leu Lys 20 25 30 Lys Phe Pro Gln Gly Gln Lys Gly Lys Val Val Phe Phe Ala Asn Gln 35 40 45 Ile Pro Val Tyr Glu Gln Gln Lys Ser Val Phe Ser Lys Tyr Phe Glu 50 55 60 Arg His Gly Tyr Arg Val Thr Gly Ile Ser Gly Ala Thr Ala Glu Asn 65 70 75 80 Val Pro Glu Gln Ile Val Glu Asn Asn Asp Ile Ile Ile Leu Thr 85 90 95 Pro Gln Ile Leu Val Asn Asn Leu Lys Lys Gly Thr Ile Pro Ser Leu 100 105 110 Ser Ile Phe Thr Leu Met Ile Phe Asp Glu Cys His Asn Thr Ser Lys 115 120 125 Gln His Pro Tyr Asn Met Ile Met Phe Asn Tyr Leu Asp Gln Lys Leu 130 135 140 Gly Gly Ser Ser Gly Pro Leu Pro Gln Val Ile Gly Leu Thr Ala Ser 145 150 155 160 Val Gly Val Gly Asp Ala Lys Asn Thr Asp Glu Ala Leu Asp Tyr Ile 165 170 175 Cys Lys Leu Cys Ala Ser Leu Asp Ala Ser Val Ile Ala Thr Val Lys 180 185 190 His Asn Leu Glu Glu Leu Glu Gln Val Val Tyr Lys Pro Gln Lys Phe 195 200 205 Phe Arg Lys Val Glu Ser Arg Ile Ser Asp Lys Phe Lys Tyr Ile Ile 210 215 220 Ala Gln Leu Met Arg Asp Thr Glu Ser Leu Ala Lys Arg Ile Cys Lys 225 230 235 240 Asp Leu Glu Asn Leu Ser Gln Ile Gln Asn Arg Glu Phe Gly Thr Gln 245 250 255 Lys Tyr Glu Gln Trp Ile Val Thr Val Gln Lys Ala Cys Met Val Phe 260 265 270 Gln Met Pro Asp Lys Asp Glu Glu Ser Arg Ile Cys Lys Ala Leu Phe 275 280 285 Leu Tyr Thr Ser Leu Arg Lys Tyr Asn Asp Ala Leu Ile Ile Ser 290 295 300 Glu His Ala Arg Met Lys Asp Ala Leu Asp Tyr Leu Lys Asp Phe Phe 305 310 315 320 Ser Asn Val Arg Ala Ala Gly Phe Asp Glu Ile Glu Gln Asp Leu Thr 325 330 335 Gln Arg Phe Glu Glu Lys Leu Gln Glu Leu Glu Ser Val Ser Arg Asp 340 345 350 Pro Ser Asn Glu Asn Pro Lys Leu Glu Asp Leu Cys Phe Ile Leu Gln 355 360 365 Glu Glu Tyr His Leu Asn Pro Glu Thr Ile Thr Ile Leu Phe Val Lys 370 375 380 Thr Arg Ala Leu Val Asp Ala Leu Lys Asn Trp Ile Glu Gly Asn Pro 385 390 395 400 Lys Leu Ser Phe Leu Lys Pro Gly Ile Leu Thr Gly Arg Gly Lys Thr 405 410 415 Asn Gln Asn Thr Gly Met Thr Leu Pro Ala Gln Lys Cys Ile Leu Asp 420 425 430 Ala Phe Lys Ala Ser Gly Asp His Asn Ile Leu Ile Ala Thr Ser Val 435 440 445 Ala Asp Glu Gly Ile Asp Ile Ala Gln Cys Asn Leu Val Ile Leu Tyr 450 455 460 Glu Tyr Val Gly Asn Val Ile Lys Met Ile Gln Thr Arg Gly Arg Gly 465 470 475 480 Arg Ala Arg <210> 8 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> RD Domain (H) <400> 8 Gly Ser Lys Cys Phe Leu Leu Thr Ser Asn Ala Gly Val Ile Glu Lys 1 5 10 15 Glu Gln Ile Asn Met Tyr Lys Glu Lys Met Met Asn Asp Ser Ile Leu 20 25 30 Arg Leu Gln Thr Trp Asp Glu Ala Val Phe Arg Glu Lys Ile Leu His 35 40 45 Ile Gln Thr His Glu Lys Phe Ile Arg Asp Ser Gln Glu Lys Pro Lys 50 55 60 Pro Val Pro Asp Lys Glu Asn Lys Lys Leu Leu Cys Arg Lys Cys Lys 65 70 75 80 Ala Leu Ala Cys Tyr Thr Ala Asp Val Arg Ile Glu Glu Cys His 85 90 95 Tyr Thr Val Leu Gly Asp Ala Phe Lys Glu Cys Phe Val Ser Arg Pro 100 105 110 His Pro Lys Pro Lys Gln Phe Ser Ser Phe Glu Lys Arg Ala Lys Ile 115 120 125 Phe Cys Ala Arg Gln Asn Cys Ser His Asp Trp Gly Ile His Val Lys 130 135 140 Tyr Lys Thr Phe Glu Ile Pro Val Ile Lys Ile Glu Ser Phe Val Val 145 150 155 160 Glu Asp Ile Ala Thr Gly Val Gln Thr Leu Tyr Ser Lys Trp Lys Asp 165 170 175 Phe His Phe Glu Lys Ile Pro Phe Asp Pro Ala Glu Met Ser Lys 180 185 190 <210> 9 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> CARD Domain (H) <400> 9 Met Thr Thr Glu Gln Arg Arg Ser Leu Gln Ala Phe Gln Asp Tyr Ile 1 5 10 15 Arg Lys Thr Leu Asp Pro Thr Tyr Ile Leu Ser Tyr Met Ala Pro Trp 20 25 30 Phe Arg Glu Glu Glu Val Gln Tyr Ile Gln Ala Glu Lys Asn Asn Lys 35 40 45 Gly Pro Met Glu Ala Ala Thr Leu Phe Leu Lys Phe Leu Leu Glu Leu 50 55 60 Glu Glu Glu Gly Trp Phe Arg Gly Phe Leu Asp Ala Leu Asp His Ala 65 70 75 80 Gly Tyr Ser Gly Leu Tyr Glu Ala Ile Glu Ser Trp Asp Phe Lys Lys 85 90 95 Ile Glu Lys Leu Glu Glu Tyr Arg Leu Leu Leu Lys Arg Leu Gln Pro 100 105 110 Glu Phe Lys Thr Arg Ile Ile Pro Thr Asp Ile Ile Ser Asp Leu Ser 115 120 125 Glu Cys Leu Ile Asn Gln Glu Cys Glu Glu Ile Leu Gln Ile Cys Ser 130 135 140 Thr Lys Gly Met Met Ala Gly Ala Glu Lys Leu Val Glu Cys Leu Leu 145 150 155 160 Arg Ser Asp Lys Glu Asn Trp Pro Lys Thr Leu Lys Leu Ala Leu Glu 165 170 175 Lys Glu Arg Asn Lys Phe Ser Glu Leu Trp Ile Val Glu Lys Gly Ile 180 185 190 Lys Asp Val Glu Thr Glu Asp Leu Glu Asp Lys Met Glu Thr Ser Asp 195 200 205 Ile Gln Ile Phe Tyr Gln Glu Asp Pro 210 215 <210> 10 <211> 674 <212> PRT <213> Artificial Sequence <220> <223> Helicase Domain + RD domain (H) <400> 10 Leu Pro Ala Met Lys Gly Lys Asn Thr Ile Ile Cys Ala Pro Thr Gly 1 5 10 15 Cys Gly Lys Thr Phe Val Ser Leu Leu Ile Cys Glu His His Leu Lys 20 25 30 Lys Phe Pro Gln Gly Gln Lys Gly Lys Val Val Phe Phe Ala Asn Gln 35 40 45 Ile Pro Val Tyr Glu Gln Gln Lys Ser Val Phe Ser Lys Tyr Phe Glu 50 55 60 Arg His Gly Tyr Arg Val Thr Gly Ile Ser Gly Ala Thr Ala Glu Asn 65 70 75 80 Val Pro Glu Gln Ile Val Glu Asn Asn Asp Ile Ile Ile Leu Thr 85 90 95 Pro Gln Ile Leu Val Asn Asn Leu Lys Lys Gly Thr Ile Pro Ser Leu 100 105 110 Ser Ile Phe Thr Leu Met Ile Phe Asp Glu Cys His Asn Thr Ser Lys 115 120 125 Gln His Pro Tyr Asn Met Ile Met Phe Asn Tyr Leu Asp Gln Lys Leu 130 135 140 Gly Gly Ser Ser Gly Pro Leu Pro Gln Val Ile Gly Leu Thr Ala Ser 145 150 155 160 Val Gly Val Gly Asp Ala Lys Asn Thr Asp Glu Ala Leu Asp Tyr Ile 165 170 175 Cys Lys Leu Cys Ala Ser Leu Asp Ala Ser Val Ile Ala Thr Val Lys 180 185 190 His Asn Leu Glu Glu Leu Glu Gln Val Val Tyr Lys Pro Gln Lys Phe 195 200 205 Phe Arg Lys Val Glu Ser Arg Ile Ser Asp Lys Phe Lys Tyr Ile Ile 210 215 220 Ala Gln Leu Met Arg Asp Thr Glu Ser Leu Ala Lys Arg Ile Cys Lys 225 230 235 240 Asp Leu Glu Asn Leu Ser Gln Ile Gln Asn Arg Glu Phe Gly Thr Gln 245 250 255 Lys Tyr Glu Gln Trp Ile Val Thr Val Gln Lys Ala Cys Met Val Phe 260 265 270 Gln Met Pro Asp Lys Asp Glu Glu Ser Arg Ile Cys Lys Ala Leu Phe 275 280 285 Leu Tyr Thr Ser Leu Arg Lys Tyr Asn Asp Ala Leu Ile Ile Ser 290 295 300 Glu His Ala Arg Met Lys Asp Ala Leu Asp Tyr Leu Lys Asp Phe Phe 305 310 315 320 Ser Asn Val Arg Ala Ala Gly Phe Asp Glu Ile Glu Gln Asp Leu Thr 325 330 335 Gln Arg Phe Glu Glu Lys Leu Gln Glu Leu Glu Ser Val Ser Arg Asp 340 345 350 Pro Ser Asn Glu Asn Pro Lys Leu Glu Asp Leu Cys Phe Ile Leu Gln 355 360 365 Glu Glu Tyr His Leu Asn Pro Glu Thr Ile Thr Ile Leu Phe Val Lys 370 375 380 Thr Arg Ala Leu Val Asp Ala Leu Lys Asn Trp Ile Glu Gly Asn Pro 385 390 395 400 Lys Leu Ser Phe Leu Lys Pro Gly Ile Leu Thr Gly Arg Gly Lys Thr 405 410 415 Asn Gln Asn Thr Gly Met Thr Leu Pro Ala Gln Lys Cys Ile Leu Asp 420 425 430 Ala Phe Lys Ala Ser Gly Asp His Asn Ile Leu Ile Ala Thr Ser Val 435 440 445 Ala Asp Glu Gly Ile Asp Ile Ala Gln Cys Asn Leu Val Ile Leu Tyr 450 455 460 Glu Tyr Val Gly Asn Val Ile Lys Met Ile Gln Thr Arg Gly Arg Gly 465 470 475 480 Arg Ala Arg Gly Ser Lys Cys Phe Leu Leu Thr Ser Asn Ala Gly Val 485 490 495 Ile Glu Lys Glu Gln Ile Asn Met Tyr Lys Glu Lys Met Met Asn Asp 500 505 510 Ser Ile Leu Arg Leu Gln Thr Trp Asp Glu Ala Val Phe Arg Glu Lys 515 520 525 Ile Leu His Ile Gln Thr His Glu Lys Phe Ile Arg Asp Ser Gln Glu 530 535 540 Lys Pro Lys Pro Val Pro Asp Lys Glu Asn Lys Lys Leu Leu Cys Arg 545 550 555 560 Lys Cys Lys Ala Leu Ala Cys Tyr Thr Ala Asp Val Arg Ile Glu 565 570 575 Glu Cys His Tyr Thr Val Leu Gly Asp Ala Phe Lys Glu Cys Phe Val 580 585 590 Ser Arg Pro His Pro Lys Pro Lys Gln Phe Ser Ser Phe Glu Lys Arg 595 600 605 Ala Lys Ile Phe Cys Ala Arg Gln Asn Cys Ser His Asp Trp Gly Ile 610 615 620 His Val Lys Tyr Lys Thr Phe Glu Ile Pro Val Ile Lys Ile Glu Ser 625 630 635 640 Phe Val Val Glu Asp Ile Ala Thr Gly Val Gln Thr Leu Tyr Ser Lys 645 650 655 Trp Lys Asp Phe His Phe Glu Lys Ile Pro Phe Asp Pro Ala Glu Met 660 665 670 Ser Lys <210> 11 <211> 708 <212> PRT <213> Artificial Sequence <220> <223> Linker 3 (H) + Helicase Domain + RD domain <400> 11 Glu Cys Gln Asn Leu Ser Glu Asn Ser Cys Pro Ser Ser Glu Val Ser 1 5 10 15 Asp Thr Asn Leu Tyr Ser Pro Phe Lys Pro Arg Asn Tyr Gln Leu Glu 20 25 30 Leu Ala Leu Pro Ala Met Lys Gly Lys Asn Thr Ile Ile Cys Ala Pro 35 40 45 Thr Gly Cys Gly Lys Thr Phe Val Ser Leu Leu Ile Cys Glu His His 50 55 60 Leu Lys Lys Phe Pro Gln Gly Gln Lys Gly Lys Val Val Phe Phe Ala 65 70 75 80 Asn Gln Ile Pro Val Tyr Glu Gln Gln Lys Ser Val Phe Ser Lys Tyr 85 90 95 Phe Glu Arg His Gly Tyr Arg Val Thr Gly Ile Ser Gly Ala Thr Ala 100 105 110 Glu Asn Val Pro Glu Gln Ile Val Glu Asn Asn Asp Ile Ile 115 120 125 Leu Thr Pro Gln Ile Leu Val Asn Asn Leu Lys Lys Gly Thr Ile Pro 130 135 140 Ser Leu Ser Ile Phe Thr Leu Met Ile Phe Asp Glu Cys His Asn Thr 145 150 155 160 Ser Lys Gln His Pro Tyr Asn Met Ile Met Phe Asn Tyr Leu Asp Gln 165 170 175 Lys Leu Gly Gly Ser Ser Gly Pro Leu Pro Gln Val Ile Gly Leu Thr 180 185 190 Ala Ser Val Gly Val Gly Asp Ala Lys Asn Thr Asp Glu Ala Leu Asp 195 200 205 Tyr Ile Cys Lys Leu Cys Ala Ser Leu Asp Ala Ser Val Ile Ala Thr 210 215 220 Val Lys His Asn Leu Glu Glu Leu Glu Gln Val Val Tyr Lys Pro Gln 225 230 235 240 Lys Phe Phe Arg Lys Val Glu Ser Arg Ile Ser Asp Lys Phe Lys Tyr 245 250 255 Ile Ile Ala Gln Leu Met Arg Asp Thr Glu Ser Leu Ala Lys Arg Ile 260 265 270 Cys Lys Asp Leu Glu Asn Leu Ser Gln Ile Gln Asn Arg Glu Phe Gly 275 280 285 Thr Gln Lys Tyr Glu Gln Trp Ile Val Thr Val Gln Lys Ala Cys Met 290 295 300 Val Phe Gln Met Pro Asp Lys Asp Glu Glu Ser Arg Ile Cys Lys Ala 305 310 315 320 Leu Phe Leu Tyr Thr Ser His Leu Arg Lys Tyr Asn Asp Ala Leu Ile 325 330 335 Ile Ser Glu His Ala Arg Met Lys Asp Ala Leu Asp Tyr Leu Lys Asp 340 345 350 Phe Phe Ser Asn Val Arg Ala Ala Gly Phe Asp Glu Ile Glu Gln Asp 355 360 365 Leu Thr Gln Arg Phe Glu Glu Lys Leu Gln Glu Leu Glu Ser Val Ser 370 375 380 Arg Asp Pro Ser Asn Glu Asn Pro Lys Leu Glu Asp Leu Cys Phe Ile 385 390 395 400 Leu Gln Glu Glu Tyr His Leu Asn Pro Glu Thr Ile Thr Ile Leu Phe 405 410 415 Val Lys Thr Arg Ala Leu Val Asp Ala Leu Lys Asn Trp Ile Glu Gly 420 425 430 Asn Pro Lys Leu Ser Phe Leu Lys Pro Gly Ile Leu Thr Gly Arg Gly 435 440 445 Lys Thr Asn Gln Asn Thr Gly Met Thr Leu Pro Ala Gln Lys Cys Ile 450 455 460 Leu Asp Ala Phe Lys Ala Ser Gly Asp His Asn Ile Leu Ile Ala Thr 465 470 475 480 Ser Val Ala Asp Glu Gly Ile Asp Ile Ala Gln Cys Asn Leu Val Ile 485 490 495 Leu Tyr Glu Tyr Val Gly Asn Val Ile Lys Met Ile Gln Thr Arg Gly 500 505 510 Arg Gly Arg Ala Arg Gly Ser Lys Cys Phe Leu Leu Thr Ser Asn Ala 515 520 525 Gly Val Ile Glu Lys Glu Gln Ile Asn Met Tyr Lys Glu Lys Met Met 530 535 540 Asn Asp Ser Ile Leu Arg Leu Gln Thr Trp Asp Glu Ala Val Phe Arg 545 550 555 560 Glu Lys Ile Leu His Ile Gln Thr His Glu Lys Phe Ile Arg Asp Ser 565 570 575 Gln Glu Lys Pro Lys Pro Val Pro Asp Lys Glu Asn Lys Lys Leu Leu 580 585 590 Cys Arg Lys Cys Lys Ala Leu Ala Cys Tyr Thr Ala Asp Val Arg Val 595 600 605 Ile Glu Glu Cys His Tyr Thr Val Leu Gly Asp Ala Phe Lys Glu Cys 610 615 620 Phe Val Ser Arg Pro His Pro Lys Pro Lys Gln Phe Ser Ser Phe Glu 625 630 635 640 Lys Arg Ala Lys Ile Phe Cys Ala Arg Gln Asn Cys Ser His Asp Trp 645 650 655 Gly Ile His Val Lys Tyr Lys Thr Phe Glu Ile Pro Val Ile Lys Ile 660 665 670 Glu Ser Phe Val Glu Asp Ile Ala Thr Gly Val Gln Thr Leu Tyr 675 680 685 Ser Lys Trp Lys Asp Phe His Phe Glu Lys Ile Pro Phe Asp Pro Ala 690 695 700 Glu Met Ser Lys 705 <210> 12 <211> 926 <212> PRT <213> Artificial Sequence <220> <223> RIG-I (M) <400> 12 Met Thr Ala Glu Gln Arg Gln Asn Leu Gln Ala Phe Arg Asp Tyr Ile 1 5 10 15 Lys Lys Ile Leu Asp Pro Thr Tyr Ile Leu Ser Tyr Met Ser Ser Trp 20 25 30 Leu Glu Asp Glu Glu Val Gln Tyr Ile Gln Ala Glu Lys Asn Asn Lys 35 40 45 Gly Pro Met Glu Ala Ala Ser Leu Phe Leu Gln Tyr Leu Leu Lys Leu 50 55 60 Gln Ser Glu Gly Trp Phe Gln Ala Phe Leu Asp Ala Leu Tyr His Ala 65 70 75 80 Gly Tyr Cys Gly Leu Cys Glu Ala Ile Glu Ser Trp Asp Phe Gln Lys 85 90 95 Ile Glu Lys Leu Glu Glu His Arg Leu Leu Leu Arg Arg Leu Glu Pro 100 105 110 Glu Phe Lys Ala Thr Val Asp Pro Asn Asp Ile Leu Ser Glu Leu Ser 115 120 125 Glu Cys Leu Ile Asn Gln Glu Cys Glu Glu Ile Arg Gln Ile Arg Asp 130 135 140 Thr Lys Gly Arg Met Ala Gly Ala Glu Lys Met Ala Glu Cys Leu Ile 145 150 155 160 Arg Ser Asp Lys Glu Asn Trp Pro Lys Val Leu Gln Leu Ala Leu Glu 165 170 175 Lys Asp Asn Ser Lys Phe Ser Glu Leu Trp Ile Val Asp Lys Gly Phe 180 185 190 Lys Arg Ala Glu Ser Lys Ala Asp Glu Asp Asp Gly Ala Glu Ala Ser 195 200 205 Ser Ile Gln Ile Phe Ile Gln Glu Glu Pro Glu Cys Gln Asn Leu Ser 210 215 220 Gln Asn Pro Gly Pro Pro Ser Glu Ala Ser Ser Asn Asn Leu His Ser 225 230 235 240 Pro Leu Lys Pro Arg Asn Tyr Gln Leu Glu Leu Ala Leu Pro Ala Lys 245 250 255 Lys Gly Lys Asn Thr Ile Ile Cys Ala Pro Thr Gly Cys Gly Lys Thr 260 265 270 Phe Val Ser Leu Leu Ile Cys Glu His His Leu Lys Lys Phe Pro Cys 275 280 285 Gly Gln Lys Gly Lys Val Val Phe Phe Ala Asn Gln Ile Pro Val Tyr 290 295 300 Glu Gln Gln Ala Thr Val Phe Ser Arg Tyr Phe Glu Arg Leu Gly Tyr 305 310 315 320 Asn Ile Ala Ser Ile Ser Gly Ala Thr Ser Asp Ser Val Ser Val Gln 325 330 335 His Ile Ile Glu Asp Asn Asp Ile Ile Leu Thr Pro Gln Ile Leu 340 345 350 Val Asn Asn Leu Asn Asn Gly Ala Ile Pro Ser Leu Ser Val Phe Thr 355 360 365 Leu Met Ile Phe Asp Glu Cys His Asn Thr Ser Lys Asn His Pro Tyr 370 375 380 Asn Gln Ile Met Phe Arg Tyr Leu Asp His Lys Leu Gly Glu Ser Arg 385 390 395 400 Asp Pro Leu Pro Gln Val Val Gly Leu Thr Ala Ser Val Gly Val Gly 405 410 415 Asp Ala Lys Thr Ala Glu Glu Ala Met Gln His Ile Cys Lys Leu Cys 420 425 430 Ala Ala Leu Asp Ala Ser Val Ile Ala Thr Val Asp Asn Val Ala 435 440 445 Glu Leu Glu Gln Val Val Tyr Lys Pro Gln Lys Ile Ser Arg Lys Val 450 455 460 Ala Ser Arg Thr Ser Asn Thr Phe Lys Cys Ile Ile Ser Gln Leu Met 465 470 475 480 Lys Glu Thr Glu Lys Leu Ala Lys Asp Val Ser Glu Glu Leu Gly Lys 485 490 495 Leu Phe Gln Ile Gln Asn Arg Glu Phe Gly Thr Gln Lys Tyr Glu Gln 500 505 510 Trp Ile Val Gly Val His Lys Ala Cys Ser Val Phe Gln Met Ala Asp 515 520 525 Lys Glu Glu Glu Ser Arg Val Cys Lys Ala Leu Phe Leu Tyr Thr Ser 530 535 540 His Leu Arg Lys Tyr Asn Asp Ala Leu Ile Ile Ser Glu Asp Ala Gln 545 550 555 560 Met Thr Asp Ala Leu Asn Tyr Leu Lys Ala Phe Phe His Asp Val Arg 565 570 575 Glu Ala Ala Phe Asp Glu Thr Glu Arg Glu Leu Thr Arg Arg Phe Glu 580 585 590 Glu Lys Leu Glu Glu Leu Glu Lys Val Ser Arg Asp Pro Ser Asn Glu 595 600 605 Asn Pro Lys Leu Arg Asp Leu Tyr Leu Val Leu Gln Glu Glu Tyr His 610 615 620 Leu Lys Pro Glu Thr Lys Thr Ile Leu Phe Val Lys Thr Arg Ala Leu 625 630 635 640 Val Asp Ala Leu Lys Lys Trp Ile Glu Glu Asn Pro Ala Leu Ser Phe 645 650 655 Leu Lys Pro Gly Ile Leu Thr Gly Arg Gly Arg Thr Asn Arg Ala Thr 660 665 670 Gly Met Thr Leu Pro Ala Gln Lys Cys Val Leu Glu Ala Phe Arg Ala 675 680 685 Ser Gly Asp Asn Asn Ile Leu Ile Ala Thr Ser Val Ala Asp Glu Gly 690 695 700 Ile Asp Ile Ala Glu Cys Asn Leu Val Ile Leu Tyr Glu Tyr Val Gly 705 710 715 720 Asn Val Ile Lys Met Ile Gln Thr Arg Gly Arg Gly Arg Ala Arg Asp 725 730 735 Ser Lys Cys Phe Leu Leu Thr Ser Ser Ala Asp Val Ile Glu Lys Glu 740 745 750 Lys Ala Asn Met Ile Lys Glu Lys Ile Met Asn Glu Ser Ile Leu Arg 755 760 765 Leu Gln Thr Trp Asp Glu Met Lys Phe Gly Lys Thr Val His Arg Ile 770 775 780 Gln Val Asn Glu Lys Leu Leu Arg Asp Ser Gln His Lys Pro Gln Pro 785 790 795 800 Val Pro Asp Lys Glu Asn Lys Lys Leu Leu Cys Gly Lys Cys Lys Asn 805 810 815 Phe Ala Cys Tyr Thr Ala Asp Ile Arg Val Val Glu Thr Ser His Tyr 820 825 830 Thr Val Leu Gly Asp Ala Phe Lys Glu Arg Phe Val Cys Lys Pro His 835 840 845 Pro Lys Pro Lys Ile Tyr Asp Asn Phe Glu Lys Lys Ala Lys Ile Phe 850 855 860 Cys Ala Lys Gln Asn Cys Ser His Asp Trp Gly Ile Phe Val Arg Tyr 865 870 875 880 Lys Thr Phe Glu Ile Pro Val Ile Lys Ile Glu Ser Phe Val Val Glu 885 890 895 Asp Ile Val Ser Gly Val Gln Asn Arg His Ser Lys Trp Lys Asp Phe 900 905 910 His Phe Glu Arg Ile Gln Phe Asp Pro Ala Glu Met Ser Val 915 920 925 <210> 13 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> CARD 1 (M) <400> 13 Met Thr Ala Glu Gln Arg Gln Asn Leu Gln Ala Phe Arg Asp Tyr Ile 1 5 10 15 Lys Lys Ile Leu Asp Pro Thr Tyr Ile Leu Ser Tyr Met Ser Ser Trp 20 25 30 Leu Glu Asp Glu Glu Val Gln Tyr Ile Gln Ala Glu Lys Asn Asn Lys 35 40 45 Gly Pro Met Glu Ala Ala Ser Leu Phe Leu Gln Tyr Leu Leu Lys Leu 50 55 60 Gln Ser Glu Gly Trp Phe Gln Ala Phe Leu Asp Ala Leu Tyr His Ala 65 70 75 80 Gly Tyr Cys Gly Leu Cys Glu 85 <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Linker 1 (M) <400> 14 Ala Ile Glu Ser One <210> 15 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> CARD 2 (M) <400> 15 Trp Asp Phe Gln Lys Ile Glu Lys Leu Glu Glu His Arg Leu Leu Leu 1 5 10 15 Arg Arg Leu Glu Pro Glu Phe Lys Ala Thr Val Asp Pro Asn Asp Ile 20 25 30 Leu Ser Glu Leu Ser Glu Cys Leu Ile Asn Gln Glu Cys Glu Glu Ile 35 40 45 Arg Gln Ile Arg Asp Thr Lys Gly Arg Met Ala Gly Ala Glu Lys Met 50 55 60 Ala Glu Cys Leu Ile Arg Ser Asp Lys Glu Asn Trp Pro Lys Val Leu 65 70 75 80 Gln <210> 16 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Linker 2 (M) <400> 16 Leu Ala Leu Glu Lys Asp Asn Ser Lys Phe Ser Glu Leu Trp Ile Val 1 5 10 15 Asp Lys Gly Phe Lys Arg Ala Glu Ser Lys Ala Asp Glu Asp Asp Gly 20 25 30 Ala Glu Ala Ser Ser Ile Gln Ile Phe Ile Gln Glu Glu Pro 35 40 45 <210> 17 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> Linker 3 (M) <400> 17 Glu Cys Gln Asn Leu Ser Gln Asn Pro Gly Pro Pro Ser Glu Ala Ser 1 5 10 15 Ser Asn Asn Leu His Ser Pro Leu Lys Pro Arg Asn Tyr Gln Leu Glu 20 25 30 Leu Ala <210> 18 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Helicase Domain (M) <400> 18 Leu Pro Ala Lys Lys Gly Lys Asn Thr Ile Ile Cys Ala Pro Thr Gly 1 5 10 15 Cys Gly Lys Thr Phe Val Ser Leu Leu Ile Cys Glu His His Leu Lys 20 25 30 Lys Phe Pro Cys Gly Gln Lys Gly Lys Val Val Phe Phe Ala Asn Gln 35 40 45 Ile Pro Val Tyr Glu Gln Gln Ala Thr Val Phe Ser Arg Tyr Phe Glu 50 55 60 Arg Leu Gly Tyr Asn Ile Ala Ser Ile Ser Gly Ala Thr Ser Asp Ser 65 70 75 80 Val Ser Val Gln His Ile Ile Glu Asp Asn Asp Ile Ile Ile Leu Thr 85 90 95 Pro Gln Ile Leu Val Asn Asn Leu Asn Asn Gly Ala Ile Pro Ser Leu 100 105 110 Ser Val Phe Thr Leu Met Ile Phe Asp Glu Cys His Asn Thr Ser Lys 115 120 125 Asn His Pro Tyr Asn Gln Ile Met Phe Arg Tyr Leu Asp His Lys Leu 130 135 140 Gly Glu Ser Arg Asp Pro Leu Pro Gln Val Val Gly Leu Thr Ala Ser 145 150 155 160 Val Gly Val Gly Asp Ala Lys Thr Ala Glu Glu Ala Met Gln His Ile 165 170 175 Cys Lys Leu Cys Ala Ala Leu Asp Ala Ser Val Ile Ala Thr Val Arg 180 185 190 Asp Asn Val Ala Glu Leu Glu Gln Val Val Tyr Lys Pro Gln Lys Ile 195 200 205 Ser Arg Lys Val Ala Ser Arg Thr Ser Asn Thr Phe Lys Cys Ile Ile 210 215 220 Ser Gln Leu Met Lys Glu Thr Glu Lys Leu Ala Lys Asp Val Ser Glu 225 230 235 240 Glu Leu Gly Lys Leu Phe Gln Ile Gln Asn Arg Glu Phe Gly Thr Gln 245 250 255 Lys Tyr Glu Gln Trp Ile Val Gly Val His Lys Ala Cys Ser Val Phe 260 265 270 Gln Met Ala Asp Lys Glu Glu Glu Ser Arg Val Cys Lys Ala Leu Phe 275 280 285 Leu Tyr Thr Ser Leu Arg Lys Tyr Asn Asp Ala Leu Ile Ile Ser 290 295 300 Glu Asp Ala Gln Met Thr Asp Ala Leu Asn Tyr Leu Lys Ala Phe Phe 305 310 315 320 His Asp Val Arg Glu Ala Ala Phe Asp Glu Thr Glu Arg Glu Leu Thr 325 330 335 Arg Arg Phe Glu Glu Lys Leu Glu Glu Leu Glu Lys Val Ser Arg Asp 340 345 350 Pro Ser Asn Glu Asn Pro Lys Leu Arg Asp Leu Tyr Leu Val Leu Gln 355 360 365 Glu Glu Tyr His Leu Lys Pro Glu Thr Lys Thr Ile Leu Phe Val Lys 370 375 380 Thr Arg Ala Leu Val Asp Ala Leu Lys Lys Trp Ile Glu Glu Asn Pro 385 390 395 400 Ala Leu Ser Phe Leu Lys Pro Gly Ile Leu Thr Gly Arg Gly Arg Thr 405 410 415 Asn Arg Ala Thr Gly Met Thr Leu Pro Ala Gln Lys Cys Val Leu Glu 420 425 430 Ala Phe Arg Ala Ser Gly Asp Asn Asn Ile Leu Ile Ala Thr Ser Val 435 440 445 Ala Asp Glu Gly Ile Asp Ile Ala Glu Cys Asn Leu Val Ile Leu Tyr 450 455 460 Glu Tyr Val Gly Asn Val Ile Lys Met Ile Gln Thr Arg Gly Arg Gly 465 470 475 480 Arg Ala Arg <210> 19 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> RD Domain (M) <400> 19 Asp Ser Lys Cys Phe Leu Leu Thr Ser Ser Ala Asp Val Ile Glu Lys 1 5 10 15 Glu Lys Ala Asn Met Ile Lys Glu Lys Ile Met Asn Glu Ser Ile Leu 20 25 30 Arg Leu Gln Thr Trp Asp Glu Met Lys Phe Gly Lys Thr Val His Arg 35 40 45 Ile Gln Val Asn Glu Lys Leu Leu Arg Asp Ser Gln His Lys Pro Gln 50 55 60 Pro Val Pro Asp Lys Glu Asn Lys Lys Leu Leu Cys Gly Lys Cys Lys 65 70 75 80 Asn Phe Ala Cys Tyr Thr Ala Asp Ile Arg Val Val Glu Thr Ser His 85 90 95 Tyr Thr Val Leu Gly Asp Ala Phe Lys Glu Arg Phe Val Cys Lys Pro 100 105 110 His Pro Lys Pro Lys Ile Tyr Asp Asn Phe Glu Lys Lys Ala Lys Ile 115 120 125 Phe Cys Ala Lys Gln Asn Cys Ser His Asp Trp Gly Ile Phe Val Arg 130 135 140 Tyr Lys Thr Phe Glu Ile Pro Val Ile Lys Ile Glu Ser Phe Val Val 145 150 155 160 Glu Asp Ile Val Ser Gly Val Gln Asn Arg His Ser Lys Trp Lys Asp 165 170 175 Phe His Phe Glu Arg Ile Gln Phe Asp Pro Ala Glu Met Ser Val 180 185 190 <210> 20 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> CARD Domain (M) <400> 20 Met Thr Ala Glu Gln Arg Gln Asn Leu Gln Ala Phe Arg Asp Tyr Ile 1 5 10 15 Lys Lys Ile Leu Asp Pro Thr Tyr Ile Leu Ser Tyr Met Ser Ser Trp 20 25 30 Leu Glu Asp Glu Glu Val Gln Tyr Ile Gln Ala Glu Lys Asn Asn Lys 35 40 45 Gly Pro Met Glu Ala Ala Ser Leu Phe Leu Gln Tyr Leu Leu Lys Leu 50 55 60 Gln Ser Glu Gly Trp Phe Gln Ala Phe Leu Asp Ala Leu Tyr His Ala 65 70 75 80 Gly Tyr Cys Gly Leu Cys Glu Ala Ile Glu Ser Trp Asp Phe Gln Lys 85 90 95 Ile Glu Lys Leu Glu Glu His Arg Leu Leu Leu Arg Arg Leu Glu Pro 100 105 110 Glu Phe Lys Ala Thr Val Asp Pro Asn Asp Ile Leu Ser Glu Leu Ser 115 120 125 Glu Cys Leu Ile Asn Gln Glu Cys Glu Glu Ile Arg Gln Ile Arg Asp 130 135 140 Thr Lys Gly Arg Met Ala Gly Ala Glu Lys Met Ala Glu Cys Leu Ile 145 150 155 160 Arg Ser Asp Lys Glu Asn Trp Pro Lys Val Leu Gln Leu Ala Leu Glu 165 170 175 Lys Asp Asn Ser Lys Phe Ser Glu Leu Trp Ile Val Asp Lys Gly Phe 180 185 190 Lys Arg Ala Glu Ser Lys Ala Asp Glu Asp Asp Gly Ala Glu Ala Ser 195 200 205 Ser Ile Gln Ile Phe Ile Gln Glu Glu Pro 210 215 <210> 21 <211> 674 <212> PRT <213> Artificial Sequence <220> <223> Helicase Domain + RD domain (M) <400> 21 Leu Pro Ala Lys Lys Gly Lys Asn Thr Ile Ile Cys Ala Pro Thr Gly 1 5 10 15 Cys Gly Lys Thr Phe Val Ser Leu Leu Ile Cys Glu His His Leu Lys 20 25 30 Lys Phe Pro Cys Gly Gln Lys Gly Lys Val Val Phe Phe Ala Asn Gln 35 40 45 Ile Pro Val Tyr Glu Gln Gln Ala Thr Val Phe Ser Arg Tyr Phe Glu 50 55 60 Arg Leu Gly Tyr Asn Ile Ala Ser Ile Ser Gly Ala Thr Ser Asp Ser 65 70 75 80 Val Ser Val Gln His Ile Ile Glu Asp Asn Asp Ile Ile Ile Leu Thr 85 90 95 Pro Gln Ile Leu Val Asn Asn Leu Asn Asn Gly Ala Ile Pro Ser Leu 100 105 110 Ser Val Phe Thr Leu Met Ile Phe Asp Glu Cys His Asn Thr Ser Lys 115 120 125 Asn His Pro Tyr Asn Gln Ile Met Phe Arg Tyr Leu Asp His Lys Leu 130 135 140 Gly Glu Ser Arg Asp Pro Leu Pro Gln Val Val Gly Leu Thr Ala Ser 145 150 155 160 Val Gly Val Gly Asp Ala Lys Thr Ala Glu Glu Ala Met Gln His Ile 165 170 175 Cys Lys Leu Cys Ala Ala Leu Asp Ala Ser Val Ile Ala Thr Val Arg 180 185 190 Asp Asn Val Ala Glu Leu Glu Gln Val Val Tyr Lys Pro Gln Lys Ile 195 200 205 Ser Arg Lys Val Ala Ser Arg Thr Ser Asn Thr Phe Lys Cys Ile Ile 210 215 220 Ser Gln Leu Met Lys Glu Thr Glu Lys Leu Ala Lys Asp Val Ser Glu 225 230 235 240 Glu Leu Gly Lys Leu Phe Gln Ile Gln Asn Arg Glu Phe Gly Thr Gln 245 250 255 Lys Tyr Glu Gln Trp Ile Val Gly Val His Lys Ala Cys Ser Val Phe 260 265 270 Gln Met Ala Asp Lys Glu Glu Glu Ser Arg Val Cys Lys Ala Leu Phe 275 280 285 Leu Tyr Thr Ser Leu Arg Lys Tyr Asn Asp Ala Leu Ile Ile Ser 290 295 300 Glu Asp Ala Gln Met Thr Asp Ala Leu Asn Tyr Leu Lys Ala Phe Phe 305 310 315 320 His Asp Val Arg Glu Ala Ala Phe Asp Glu Thr Glu Arg Glu Leu Thr 325 330 335 Arg Arg Phe Glu Glu Lys Leu Glu Glu Leu Glu Lys Val Ser Arg Asp 340 345 350 Pro Ser Asn Glu Asn Pro Lys Leu Arg Asp Leu Tyr Leu Val Leu Gln 355 360 365 Glu Glu Tyr His Leu Lys Pro Glu Thr Lys Thr Ile Leu Phe Val Lys 370 375 380 Thr Arg Ala Leu Val Asp Ala Leu Lys Lys Trp Ile Glu Glu Asn Pro 385 390 395 400 Ala Leu Ser Phe Leu Lys Pro Gly Ile Leu Thr Gly Arg Gly Arg Thr 405 410 415 Asn Arg Ala Thr Gly Met Thr Leu Pro Ala Gln Lys Cys Val Leu Glu 420 425 430 Ala Phe Arg Ala Ser Gly Asp Asn Asn Ile Leu Ile Ala Thr Ser Val 435 440 445 Ala Asp Glu Gly Ile Asp Ile Ala Glu Cys Asn Leu Val Ile Leu Tyr 450 455 460 Glu Tyr Val Gly Asn Val Ile Lys Met Ile Gln Thr Arg Gly Arg Gly 465 470 475 480 Arg Ala Arg Asp Ser Lys Cys Phe Leu Leu Thr Ser Ser Ala Asp Val 485 490 495 Ile Glu Lys Glu Lys Ala Asn Met Ile Lys Glu Lys Ile Met Asn Glu 500 505 510 Ser Ile Leu Arg Leu Gln Thr Trp Asp Glu Met Lys Phe Gly Lys Thr 515 520 525 Val His Arg Ile Gln Val Asn Glu Lys Leu Leu Arg Asp Ser Gln His 530 535 540 Lys Pro Gln Pro Val Pro Asp Lys Glu Asn Lys Lys Leu Leu Cys Gly 545 550 555 560 Lys Cys Lys Asn Phe Ala Cys Tyr Thr Ala Asp Ile Arg Val Val Glu 565 570 575 Thr Ser His Tyr Thr Val Leu Gly Asp Ala Phe Lys Glu Arg Phe Val 580 585 590 Cys Lys Pro His Pro Lys Pro Lys Ile Tyr Asp Asn Phe Glu Lys Lys 595 600 605 Ala Lys Ile Phe Cys Ala Lys Gln Asn Cys Ser His Asp Trp Gly Ile 610 615 620 Phe Val Arg Tyr Lys Thr Phe Glu Ile Pro Val Ile Lys Ile Glu Ser 625 630 635 640 Phe Val Val Glu Asp Ile Val Ser Gly Val Gln Asn Arg His Ser Lys 645 650 655 Trp Lys Asp Phe His Phe Glu Arg Ile Gln Phe Asp Pro Ala Glu Met 660 665 670 Ser Val <210> 22 <211> 708 <212> PRT <213> Artificial Sequence <220> <223> Linker 3 (M) + Helicase domain + RD domain (M) <400> 22 Glu Cys Gln Asn Leu Ser Gln Asn Pro Gly Pro Pro Ser Glu Ala Ser 1 5 10 15 Ser Asn Asn Leu His Ser Pro Leu Lys Pro Arg Asn Tyr Gln Leu Glu 20 25 30 Leu Ala Leu Pro Ala Lys Lys Gly Lys Asn Thr Ile Ile Cys Ala Pro 35 40 45 Thr Gly Cys Gly Lys Thr Phe Val Ser Leu Leu Ile Cys Glu His His 50 55 60 Leu Lys Lys Phe Pro Cys Gly Gln Lys Gly Lys Val Val Phe Phe Ala 65 70 75 80 Asn Gln Ile Pro Val Tyr Glu Gln Gln Ala Thr Val Phe Ser Arg Tyr 85 90 95 Phe Glu Arg Leu Gly Tyr Asn Ile Ala Ser Ile Ser Gly Ala Thr Ser 100 105 110 Asp Ser Val Ser Val Glu Ile Ile Glu Asp Asn Asp Ile Ile Ile 115 120 125 Leu Thr Pro Gln Ile Leu Val Asn Asn Leu Asn Asn Gly Ala Ile Pro 130 135 140 Ser Leu Ser Val Phe Thr Leu Met Ile Phe Asp Glu Cys His Asn Thr 145 150 155 160 Ser Lys Asn His Pro Tyr Asn Gln Ile Met Phe Arg Tyr Leu Asp His 165 170 175 Lys Leu Gly Glu Ser Arg Asp Pro Leu Pro Gln Val Val Gly Leu Thr 180 185 190 Ala Ser Val Gly Val Gly Asp Ala Lys Thr Ala Glu Glu Ala Met Gln 195 200 205 His Ile Cys Lys Leu Cys Ala Ala Leu Asp Ala Ser Val Ile Ala Thr 210 215 220 Val Arg Asp Asn Val Ala Glu Leu Glu Gln Val Val Tyr Lys Pro Gln 225 230 235 240 Lys Ile Ser Arg Lys Val Ala Ser Arg Thr Ser Asn Thr Phe Lys Cys 245 250 255 Ile Ile Ser Gln Leu Met Lys Glu Thr Glu Lys Leu Ala Lys Asp Val 260 265 270 Ser Glu Glu Leu Gly Lys Leu Phe Gln Ile Gln Asn Arg Glu Phe Gly 275 280 285 Thr Gln Lys Tyr Glu Gln Trp Ile Val Gly Val His Lys Ala Cys Ser 290 295 300 Val Phe Gln Met Ala Asp Lys Glu Glu Glu Ser Arg Val Cys Lys Ala 305 310 315 320 Leu Phe Leu Tyr Thr Ser His Leu Arg Lys Tyr Asn Asp Ala Leu Ile 325 330 335 Ile Ser Glu Asp Ala Gln Met Thr Asp Ala Leu Asn Tyr Leu Lys Ala 340 345 350 Phe Phe His Asp Val Arg Glu Ala Ala Phe Asp Glu Thr Glu Arg Glu 355 360 365 Leu Thr Arg Arg Phe Glu Glu Lys Leu Glu Glu Leu Glu Lys Val Ser 370 375 380 Arg Asp Pro Ser Asn Glu Asn Pro Lys Leu Arg Asp Leu Tyr Leu Val 385 390 395 400 Leu Gln Glu Glu Tyr His Leu Lys Pro Glu Thr Lys Thr Ile Leu Phe 405 410 415 Val Lys Thr Arg Ala Leu Val Asp Ala Leu Lys Lys Trp Ile Glu Glu 420 425 430 Asn Pro Ala Leu Ser Phe Leu Lys Pro Gly Ile Leu Thr Gly Arg Gly 435 440 445 Arg Thr Asn Arg Ala Thr Gly Met Thr Leu Pro Ala Gln Lys Cys Val 450 455 460 Leu Glu Ala Phe Arg Ala Ser Gly Asp Asn Asn Ile Leu Ile Ala Thr 465 470 475 480 Ser Val Ala Asp Glu Gly Ile Asp Ile Ala Glu Cys Asn Leu Val Ile 485 490 495 Leu Tyr Glu Tyr Val Gly Asn Val Ile Lys Met Ile Gln Thr Arg Gly 500 505 510 Arg Gly Arg Ala Arg Asp Ser Lys Cys Phe Leu Leu Thr Ser Ser Ala 515 520 525 Asp Val Ile Glu Lys Glu Lys Ale Asn Met Ile Lys Glu Lys Ile Met 530 535 540 Asn Glu Ser Ile Leu Arg Leu Gln Thr Trp Asp Glu Met Lys Phe Gly 545 550 555 560 Lys Thr Val His Arg Ile Gln Val Asn Glu Lys Leu Leu Arg Asp Ser 565 570 575 Gln His Lys Pro Gln Pro Val Pro Asp Lys Glu Asn Lys Lys Leu Leu 580 585 590 Cys Gly Lys Cys Lys Asn Phe Ala Cys Tyr Thr Ala Asp Ile Arg Val 595 600 605 Val Glu Thr Ser His Tyr Thr Val Leu Gly Asp Ala Phe Lys Glu Arg 610 615 620 Phe Val Cys Lys Pro His Pro Lys Pro Lys Ile Tyr Asp Asn Phe Glu 625 630 635 640 Lys Lys Ala Lys Ile Phe Cys Ala Lys Gln Asn Cys Ser His Asp Trp 645 650 655 Gly Ile Phe Val Arg Tyr Lys Thr Phe Glu Ile Pro Val Ile Lys Ile 660 665 670 Glu Ser Phe Val Val Glu Asp Ile Val Ser Gly Val Gln Asn Arg His 675 680 685 Ser Lys Trp Lys Asp Phe His Phe Glu Arg Ile Gln Phe Asp Pro Ala 690 695 700 Glu Met Ser Val 705 <210> 23 <211> 2775 <212> DNA <213> Artificial Sequence <220> ≪ 223 > RIG-I (H) <400> 23 atgaccaccg agcagcgacg cagcctgcaa gccttccagg attatatccg gaagaccctg 60 gaccctacct acatcctgag ctacatggcc ccctggttta gggaggaaga ggtgcagtat 120 attcaggctg agaaaaacaa caagggccca atggaggctg ccacactttt tctcaagttc 180 ctgttggagc tccaggagga aggctggttc cgtggctttt tggatgccct agaccatgca 240 ggttattctg gactttatga agccattgaa agttgggatt tcaaaaaaat tgaaaagttg 300 gaggagtata gattactttt aaaacgttta caaccagaat ttaaaaccag aattatccca 360 accgatatca tttctgatct gtctgaatgt ttaattaatc aggaatgtga agaaattcta 420 cagatttgct ctactaaggg gatgatggca ggtgcagaga aattggtgga atgccttctc 480 agatcagaca aggaaaactg gcccaaaact ttgaaacttg ctttggagaa agaaaggaac 540 aagttcagtg aactgtggat tgtagagaaa ggtataaaag atgttgaaac agaagatctt 600 gaggataaga tggaaacttc tgacatacag attttctacc aagaagatcc agaatgccag 660 aatcttagtg agaattcatg tccaccttca gaagtgtctg atacaaactt gtacagccca 720 tttaaaccaa gaaattacca attagagctt gctttgcctg ctatgaaagg aaaaaacaca 780 ataatatgtg ctcctacagg ttgtggaaaa acctttgttt cactgcttat atgtgaacat 840 catcttaaaa aattcccaca aggacaaaag gggaaagttg tcttttttgc gaatcagatc 900 ccagtgtatg aacagcagaa atctgtattc tcaaaatact ttgaaagaca tgggtataga 960 gttacaggca tttctggagc aacagctgag aatgtcccag tggaacagat tgttgagaac 1020 aatgacatca tcattttaac tccacagatt cttgtgaaca accttaaaaa gggaacgatt 1080 ccatcactat ccatctttac tttgatgata tttgatgaat gccacaacac tagtaaacaa 1140 cacccgtaca atatgatcat gtttaattat ctagatcaga aacttggagg atcttcaggc 1200 ccactgcccc aggtcattgg gctgactgcc tcggttggtg ttggggatgc caaaaacaca 1260 gatgaagcct tggattatat ctgcaagctg tgtgcttctc ttgatgcgtc agtgatagca 1320 acagtcaaac acaatctgga ggaactggag caagttgttt ataagcccca gaagtttttc 1380 aggaaagtgg aatcacggat tagcgacaaa tttaaataca tcatagctca gctgatgagg 1440 gacacagaga gtctggcaaa gagaatctgc aaagacctcg aaaacttatc tcaaattcaa 1500 aatagggaat ttggaacaca gaaatatgaa caatggattg ttacagttca gaaagcatgc 1560 atggtgttcc agatgccaga caaagatgaa gagagcagga tttgtaaagc cctgttttta 1620 tacacttcac atttgcggaa atataatgat gccctcatta tcagtgagca tgcacgaatg 1680 aaagatgctc tggattactt gaaagacttc ttcagcaatg tccgagcagc aggattcgat 1740 gagattgagc aagatcttac tcagagattt gaagaaaagc tgcaggaact agaaagtgtt 1800 tccagggatc ccagcaatga gaatcctaaa cttgaagacc tctgcttcat cttacaagaa 1860 gagtaccact taaacccaga gacaataaca attctctttg tgaaaaccag agcacttgtg 1920 gacgctttaa aaaattggat tgaaggaaat cctaaactca gttttctaaa acctggcata 1980 ttgactggac gtggcaaaac aaatcagaac acaggaatga ccctcccggc acagaagtgt 2040 atattggatg cattcaaagc cagtggagat cacaatattc tgattgccac ctcagttgct 2100 gatgaaggca ttgacattgc acagtgcaat cttgtcatcc tttatgagta tgtgggcaat 2160 gtcatcaaaa tgatccaaac cagaggcaga ggaagagcaa gaggtagcaa gtgcttcctt 2220 ctgactagta atgctggtgt aattgaaaaa gaacaaataa acatgtacaa agaaaaaatg 2280 atgaatgact ctattttacg ccttcagaca tgggacgaag cagtatttag ggaaaagatt 2340 ctgcatatac agactcatga aaaattcatc agagatagtc aagaaaaacc aaaacctgta 2400 cctgataagg aaaataaaaa actgctctgc agaaagtgca aagccttggc atgttacaca 2460 gctgacgtaa gagtgataga ggaatgccat tacactgtgc ttggagatgc ttttaaggaa 2520 tgctttgtga gtagaccaca tcccaagcca aagcagtttt caagttttga aaaaagagca 2580 aagatattct gtgcccgaca gaactgcagc catgactggg gaatccatgt gaagtacaag 2640 acatttgaga ttccagttat aaaaattgaa agttttgtgg tggaggatat tgcaactgga 2700 gttcagacac tgtactcgaa gtggaaggac tttcattttg agaagatacc atttgatcca 2760 gcagaaatgt ccaaa 2775 <210> 24 <211> 261 <212> DNA <213> Artificial Sequence <220> <223> CARD 1 (H) <400> 24 atgaccaccg agcagcgacg cagcctgcaa gccttccagg attatatccg gaagaccctg 60 gaccctacct acatcctgag ctacatggcc ccctggttta gggaggaaga ggtgcagtat 120 attcaggctg agaaaaacaa caagggccca atggaggctg ccacactttt tctcaagttc 180 ctgttggagc tccaggagga aggctggttc cgtggctttt tggatgccct agaccatgca 240 ggttattctg gactttatga a 261 <210> 25 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Linker 1 (H) <400> 25 gccattgaaa gt 12 <210> 26 <211> 243 <212> DNA <213> Artificial Sequence <220> <223> CARD 2 (H) <400> 26 tgggatttca aaaaaattga aaagttggag gagtatagat tacttttaaa acgtttacaa 60 ccagaattta aaaccagaat tatcccaacc gatatcattt ctgatctgtc tgaatgttta 120 attaatcagg aatgtgaaga aattctacag atttgctcta ctaaggggat gatggcaggt 180 gcagagaaat tggtggaatg ccttctcaga tcagacaagg aaaactggcc caaaactttg 240 aaa 243 <210> 27 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> Linker 2 (H) <400> 27 cttgctttgg agaaagaaag gaacaagttc agtgaactgt ggattgtaga gaaaggtata 60 aaagatgttg aaacagaaga tcttgaggat aagatggaaa cttctgacat acagattttc 120 taccaagaag atcca 135 <210> 28 <211> 102 <212> DNA <213> Artificial Sequence <220> <223> Linker 3 (H) <400> 28 gaatgccaga atcttagtga gaattcatgt ccaccttcag aagtgtctga tacaaacttg 60 tacagcccat ttaaaccaag aaattaccaa ttagagcttg ct 102 <210> 29 <211> 1449 <212> DNA <213> Artificial Sequence <220> <223> Helicase Domain (H) <400> 29 ttgcctgcta tgaaaggaaa aaacacaata atatgtgctc ctacaggttg tggaaaaacc 60 tttgtttcac tgcttatatg tgaacatcat cttaaaaaat tcccacaagg acaaaagggg 120 aaagttgtct tttttgcgaa tcagatccca gtgtatgaac agcagaaatc tgtattctca 180 aaatactttg aaagacatgg gtatagagtt acaggcattt ctggagcaac agctgagaat 240 gtcccagtgg aacagattgt tgagaacaat gacatcatca ttttaactcc acagattctt 300 gtaacaacc ttaaaaaggg aacgattcca tcactatcca tctttacttt gatgatattt 360 gatgaatgcc acaacactag taaacaacac ccgtacaata tgatcatgtt taattatcta 420 gatcagaaac ttggaggatc ttcaggccca ctgccccagg tcattgggct gactgcctcg 480 gttggtgttg gggatgccaa aaacacagat gaagccttgg attatatctg caagctgtgt 540 gcttctcttg atgcgtcagt gatagcaaca gtcaaacaca atctggagga actggagcaa 600 gttgtttata agccccagaa gtttttcagg aaagtggaat cacggattag cgacaaattt 660 aaatacatca tagctcagct gatgagggac acagagagtc tggcaaagag aatctgcaaa 720 gacctcgaaa acttatctca aattcaaaat agggaatttg gaacacagaa atatgaacaa 780 tggattgtta cagttcagaa agcatgcatg gtgttccaga tgccagacaa agatgaagag 840 agcaggattt gtaaagccct gtttttatac acttcacatt tgcggaaata taatgatgcc 900 ctcattatca gtgagcatgc acgaatgaaa gatgctctgg attacttgaa agacttcttc 960 agcaatgtcc gagcagcagg attcgatgag attgagcaag atcttactca gagatttgaa 1020 gaaaagctgc aggaactaga aagtgtttcc agggatccca gcaatgagaa tcctaaactt 1080 gaagacctct gcttcatctt acaagaagag taccacttaa acccagagac aataacaatt 1140 ctctttgtga aaaccagagc acttgtggac gctttaaaaa attggattga aggaaatcct 1200 aaactcagtt ttctaaaacc tggcatattg actggacgtg gcaaaacaaa tcagaacaca 1260 ggaatgaccc tcccggcaca gaagtgtata ttggatgcat tcaaagccag tggagatcac 1320 aatattctga ttgccacctc agttgctgat gaaggcattg acattgcaca gtgcaatctt 1380 gtcatccttt atgagtatgt gggcaatgtc atcaaaatga tccaaaccag aggcagagga 1440 agagcaaga 1449 <210> 30 <211> 573 <212> DNA <213> Artificial Sequence <220> <223> RD Domain (H) <400> 30 ggtagcaagt gcttccttct gactagtaat gctggtgtaa ttgaaaaaga acaaataaac 60 atgtacaaag aaaaaatgat gaatgactct attttacgcc ttcagacatg ggacgaagca 120 gtatttaggg aaaagattct gcatatacag actcatgaaa aattcatcag agatagtcaa 180 gaaaaaccaa aacctgtacc tgataaggaa aataaaaaac tgctctgcag aaagtgcaaa 240 gccttggcat gttacacagc tgacgtaaga gtgatagagg aatgccatta cactgtgctt 300 ggagatgctt ttaaggaatg ctttgtgagt agaccacatc ccaagccaaa gcagttttca 360 agttttgaaa aaagagcaaa gatattctgt gcccgacaga actgcagcca tgactgggga 420 atccatgtga agtacaagac atttgagatt ccagttataa aaattgaaag ttttgtggtg 480 gaggatattg caactggagt tcagacactg tactcgaagt ggaaggactt tcattttgag 540 aagataccat ttgatccagc agaaatgtcc aaa 573 <210> 31 <211> 651 <212> DNA <213> Artificial Sequence <220> <223> CARD Domain (H) <400> 31 atgaccaccg agcagcgacg cagcctgcaa gccttccagg attatatccg gaagaccctg 60 gaccctacct acatcctgag ctacatggcc ccctggttta gggaggaaga ggtgcagtat 120 attcaggctg agaaaaacaa caagggccca atggaggctg ccacactttt tctcaagttc 180 ctgttggagc tccaggagga aggctggttc cgtggctttt tggatgccct agaccatgca 240 ggttattctg gactttatga agccattgaa agttgggatt tcaaaaaaat tgaaaagttg 300 gaggagtata gattactttt aaaacgttta caaccagaat ttaaaaccag aattatccca 360 accgatatca tttctgatct gtctgaatgt ttaattaatc aggaatgtga agaaattcta 420 cagatttgct ctactaaggg gatgatggca ggtgcagaga aattggtgga atgccttctc 480 agatcagaca aggaaaactg gcccaaaact ttgaaacttg ctttggagaa agaaaggaac 540 aagttcagtg aactgtggat tgtagagaaa ggtataaaag atgttgaaac agaagatctt 600 gaggataaga tggaaacttc tgacatacag attttctacc aagaagatcc a 651 <210> 32 <211> 2022 <212> DNA <213> Artificial Sequence <220> <223> Helicase Domain + RD domain (H) <400> 32 ttgcctgcta tgaaaggaaa aaacacaata atatgtgctc ctacaggttg tggaaaaacc 60 tttgtttcac tgcttatatg tgaacatcat cttaaaaaat tcccacaagg acaaaagggg 120 aaagttgtct tttttgcgaa tcagatccca gtgtatgaac agcagaaatc tgtattctca 180 aaatactttg aaagacatgg gtatagagtt acaggcattt ctggagcaac agctgagaat 240 gtcccagtgg aacagattgt tgagaacaat gacatcatca ttttaactcc acagattctt 300 gtaacaacc ttaaaaaggg aacgattcca tcactatcca tctttacttt gatgatattt 360 gatgaatgcc acaacactag taaacaacac ccgtacaata tgatcatgtt taattatcta 420 gatcagaaac ttggaggatc ttcaggccca ctgccccagg tcattgggct gactgcctcg 480 gttggtgttg gggatgccaa aaacacagat gaagccttgg attatatctg caagctgtgt 540 gcttctcttg atgcgtcagt gatagcaaca gtcaaacaca atctggagga actggagcaa 600 gttgtttata agccccagaa gtttttcagg aaagtggaat cacggattag cgacaaattt 660 aaatacatca tagctcagct gatgagggac acagagagtc tggcaaagag aatctgcaaa 720 gacctcgaaa acttatctca aattcaaaat agggaatttg gaacacagaa atatgaacaa 780 tggattgtta cagttcagaa agcatgcatg gtgttccaga tgccagacaa agatgaagag 840 agcaggattt gtaaagccct gtttttatac acttcacatt tgcggaaata taatgatgcc 900 ctcattatca gtgagcatgc acgaatgaaa gatgctctgg attacttgaa agacttcttc 960 agcaatgtcc gagcagcagg attcgatgag attgagcaag atcttactca gagatttgaa 1020 gaaaagctgc aggaactaga aagtgtttcc agggatccca gcaatgagaa tcctaaactt 1080 gaagacctct gcttcatctt acaagaagag taccacttaa acccagagac aataacaatt 1140 ctctttgtga aaaccagagc acttgtggac gctttaaaaa attggattga aggaaatcct 1200 aaactcagtt ttctaaaacc tggcatattg actggacgtg gcaaaacaaa tcagaacaca 1260 ggaatgaccc tcccggcaca gaagtgtata ttggatgcat tcaaagccag tggagatcac 1320 aatattctga ttgccacctc agttgctgat gaaggcattg acattgcaca gtgcaatctt 1380 gtcatccttt atgagtatgt gggcaatgtc atcaaaatga tccaaaccag aggcagagga 1440 agagcaagag gtagcaagtg cttccttctg actagtaatg ctggtgtaat tgaaaaagaa 1500 caaataaaca tgtacaaaga aaaaatgatg aatgactcta ttttacgcct tcagacatgg 1560 gacgaagcag tatttaggga aaagattctg catatacaga ctcatgaaaa attcatcaga 1620 gatagtcaag aaaaaccaaa acctgtacct gataaggaaa ataaaaaact gctctgcaga 1680 aagtgcaaag ccttggcatg ttacacagct gacgtaagag tgatagagga atgccattac 1740 actgtgcttg gagatgcttt taaggaatgc tttgtgagta gaccacatcc caagccaaag 1800 cagttttcaa gttttgaaaa aagagcaaag atattctgtg cccgacagaa ctgcagccat 1860 gactggggaa tccatgtgaa gtacaagaca tttgagattc cagttataaa aattgaaagt 1920 tttgtggtgg aggatattgc aactggagtt cagacactgt actcgaagtg gaaggacttt 1980 cattttgaga agataccatt tgatccagca gaaatgtcca aa 2022 <210> 33 <211> 2124 <212> DNA <213> Artificial Sequence <220> Linker 3 (H) + Helicase domain + RD domain (H) <400> 33 gaatgccaga atcttagtga gaattcatgt ccaccttcag aagtgtctga tacaaacttg 60 tacagcccat ttaaaccaag aaattaccaa ttagagcttg ctttgcctgc tatgaaagga 120 aaaaacacaa taatatgtgc tcctacaggt tgtggaaaaa cctttgtttc actgcttata 180 tgtgaacatc atcttaaaaa attcccacaa ggacaaaagg ggaaagttgt cttttttgcg 240 aatcagatcc cagtgtatga acagcagaaa tctgtattct caaaatactt tgaaagacat 300 gggtatagag ttacaggcat ttctggagca acagctgaga atgtcccagt ggaacagatt 360 gttgagaaca atgacatcat cattttaact ccacagattc ttgtgaacaa ccttaaaaag 420 ggaacgattc catcactatc catctttact ttgatgatat ttgatgaatg ccacaacact 480 agtaaacaac acccgtacaa tatgatcatg tttaattatc tagatcagaa acttggagga 540 tcttcaggcc cactgcccca ggtcattggg ctgactgcct cggttggtgt tggggatgcc 600 aaaaacacag atgaagcctt ggattatatc tgcaagctgt gtgcttctct tgatgcgtca 660 gtgatagcaa cagtcaaaca caatctggag gaactggagc aagttgttta taagccccag 720 aagtttttca ggaaagtgga atcacggatt agcgacaaat ttaaatacat catagctcag 780 ctgatgaggg acacagagag tctggcaaag agaatctgca aagacctcga aaacttatct 840 caaattcaaa atagggaatt tggaacacag aaatatgaac aatggattgt tacagttcag 900 aaagcatgca tggtgttcca gatgccagac aaagatgaag agagcaggat ttgtaaagcc 960 ctgtttttat acacttcaca tttgcggaaa tataatgatg ccctcattat cagtgagcat 1020 gcacgaatga aagatgctct ggattacttg aaagacttct tcagcaatgt ccgagcagca 1080 ggattcgatg agattgagca agatcttact cagagatttg aagaaaagct gcaggaacta 1140 gaaagtgttt ccagggatcc cagcaatgag aatcctaaac ttgaagacct ctgcttcatc 1200 ttacaagaag agtaccactt aaacccagag acaataacaa ttctctttgt gaaaaccaga 1260 gcacttgtgg acgctttaaa aaattggatt gaaggaaatc ctaaactcag ttttctaaaa 1320 cctggcatat tgactggacg tggcaaaaca aatcagaaca caggaatgac cctcccggca 1380 cagaagtgta tattggatgc attcaaagcc agtggagatc acaatattct gattgccacc 1440 tcagttgctg atgaaggcat tgacattgca cagtgcaatc ttgtcatcct ttatgagtat 1500 gtgggcaatg tcatcaaaat gatccaaacc agaggagag gaagagcaag aggtagcaag 1560 tgcttccttc tgactagtaa tgctggtgta attgaaaaag aacaaataaa catgtacaaa 1620 gaaaaaatga tgaatgactc tattttacgc cttcagacat gggacgaagc agtatttagg 1680 gaaaagattc tgcatataca gactcatgaa aaattcatca gagatagtca agaaaaacca 1740 aaacctgtac ctgataagga aaataaaaaa ctgctctgca gaaagtgcaa agccttggca 1800 tgttacacag ctgacgtaag agtgatagag gaatgccatt acactgtgct tggagatgct 1860 tttaaggaat gctttgtgag tagaccacat cccaagccaa agcagttttc aagttttgaa 1920 aaaagagcaa agatattctg tgcccgacag aactgcagcc atgactgggg aatccatgtg 1980 aagtacaaga catttgagat tccagttata aaaattgaaa gttttgtggt ggaggatatt 2040 gcaactggag ttcagacact gtactcgaag tggaaggact ttcattttga gaagatacca 2100 tttgatccag cagaaatgtc caaa 2124 <210> 34 <211> 2778 <212> DNA <213> Artificial Sequence <220> <223> RIG-I (M) <400> 34 atgacagcgg agcagcggca gaatctgcaa gcattcagag actatatcaa gaagattctg 60 gaccccacct acatcctcag ctacatgagt tcctggctcg aggatgagga ggtgcagtac 120 attcaggctg agaagaacaa caagggccca atggaagctg cctcactctt cctccagtac 180 ctgttgaagc tgcagtcaga gggctggttc caggcctttt tggatgccct gtaccatgca 240 ggttactgtg gactttgtga agccatcgaa agttgggact ttcaaaaaat tgaaaagtta 300 gaggaacaca gattactttt aagacgttta gaaccagaat ttaaggccac agttgatcca 360 aatgatatcc tttctgaact atccgaatgt ttgattaatc aggaatgtga agaaatcaga 420 cagatccgag acactaaagg gagaatggca ggtgcggaga agatggccga atgtcttatc 480 agatccgaca aggaaaactg gccaaaggtc ttgcaacttg ctttggagaa agacaacagc 540 aagtttagtg aattgtggat tgttgataaa ggtttcaaaa gggctgaaag caaggctgat 600 gaggatgatg gagcggaggc gtccagcatc cagattttca ttcaggaaga gccagagtgt 660 cagaatctca gtcagaatcc cgggcctcct tcagaagcgt cttctaataa tttacacagc 720 ccattgaaac caagaaatta ccaactggag cttgccctgc ctgccaagaa agggaaaaat 780 acaataatat gtgcccctac tggttgtgga aaaacctttg tgtcgcttct tatatgtgaa 840 caccatctta aaaaattccc atgtggacaa aaagggaaag tggtcttctt cgctaaccaa 900 attcctgtct atgagcagca ggcaactgtg ttctcacgat attttgaaag acttgggtac 960 aacattgcga gcatttctgg ggcaacatct gatagcgtct cagtgcagca catcattgaa 1020 gacaatgata tcatcatcct gacaccccag attcttgtga acaatctcaa caacggagcc 1080 atcccctcgt tgtctgtctt cactctgatg atatttgatg agtgtcataa cactagcaaa 1140 aaccacccat acaatcagat catgttcaga tacctagacc acaaacttgg agagtcacgg 1200 gcccactgc ctcaggtcgt tgggctgact gcctccgtcg gcgttggaga tgctaagacc 1260 gcggaggaag ccatgcaaca tatctgtaaa ctctgtgccg ccctggatgc ctccgtgatt 1320 gccacagtca gagacaacgt tgcagaactg gaacaggtcg tttataagcc ccagaaaatt 1380 tccaggaaag tggcatcccg gacttcgaac acgtttaaat gcatcatctc tcagctgatg 1440 aaggagacag agaagctagc caaggatgtc tccgaggaac ttggaaagct ttttcaaatt 1500 caaaacagag aattcggcac ccagaaatat gaacagtgga ttgtcggcgt ccacaaagcg 1560 tgctcagtgt ttcagatggc agacaaagag gaggagagcc gggtctgcaa agcgctcttc 1620 ctgtacacat cacatttgcg gaaatacaac gatgcactca tcatcagtga ggatgcacag 1680 atgacagacg ctctaaatta cctcaaagcc ttcttccacg atgtccgaga agcagcattc 1740 gatgagaccg agcgagagct tactcggagg tttgaagaaa aactagagga attagaaaaa 1800 gtttccaggg atcccagcaa tgagaatcct aaactaagag acctctactt ggtcttacaa 1860 gaagagtacc acttaaagcc agagaccaag accattctct tcgtgaagac cagagcactc 1920 gtggatgctc tgaagaaatg gattgaagaa aatcctgcac taagctttct aaagcctggc 1980 atactgactg ggcgtggcag aacaaaccgg gcaacaggaa tgacgctccc ggcacagaag 2040 tgtgtgctgg aggcattcag agccagcgga gataacaata ttctgattgc tacctcggtc 2100 gctgatgaag gcattgacat tgctgagtgc aatctcgtca ttctctatga gtacgtgggc 2160 aacgtcatca agatgatcca aaccagaggc cgaggaagag cacgagatag caagtgcttc 2220 ctcctgacca gcagcgctga cgtgattgaa aaagaaaagg cgaacatgat caaggaaaaa 2280 ataatgaatg aatccatctt aagactgcag acatgggatg aaatgaaatt tggaaagacg 2340 gttcaccgca tacaggtgaa tgaaaaactc ctcagagaca gtcagcacaa accacaacct 2400 gttcctgaca aagaaaacaa gaaactgctg tgtggaaagt gcaagaattt tgcgtgctac 2460 acagctgaca ttcgagtggt tgagacgtcc cactacactg tccttggaga cgcttttaag 2520 gagcgctttg tgtgtaagcc acaccctaaa ccaaagatct atgacaattt tgagaagaaa 2580 gcaaagatat tctgcgccaa acagaactgt agccacgact ggggaatttt tgtgagatac 2640 aagacgttcg agattccagt cataaaaatt gaaagtttcg tcgtggaaga tattgtgagc 2700 ggagttcaga accggcactc aaagtggaag gactttcatt ttgaaaggat acagttcgat 2760 cctgcagaaa tgtccgta 2778 <210> 35 <211> 261 <212> DNA <213> Artificial Sequence <220> <223> CARD 1 (M) <400> 35 atgacagcgg agcagcggca gaatctgcaa gcattcagag actatatcaa gaagattctg 60 gaccccacct acatcctcag ctacatgagt tcctggctcg aggatgagga ggtgcagtac 120 attcaggctg agaagaacaa caagggccca atggaagctg cctcactctt cctccagtac 180 ctgttgaagc tgcagtcaga gggctggttc caggcctttt tggatgccct gtaccatgca 240 ggttactgtg gactttgtga a 261 <210> 36 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Linker 1 (M) <400> 36 gccatcgaaa gt 12 <210> 37 <211> 243 <212> DNA <213> Artificial Sequence <220> <223> CARD 2 (M) <400> 37 tgggactttc aaaaaattga aaagttagag gaacacagat tacttttaag acgtttagaa 60 ccagaattta aggccacagt tgatccaaat gatatccttt ctgaactatc cgaatgtttg 120 attaatcagg aatgtgaaga aatcagacag atccgagaca ctaaagggag aatggcaggt 180 gcggagaaga tggccgaatg tcttatcaga tccgacaagg aaaactggcc aaaggtcttg 240 caa 243 <210> 38 <211> 138 <212> DNA <213> Artificial Sequence <220> <223> Linker 2 (M) <400> 38 cttgctttgg agaaagacaa cagcaagttt agtgaattgt ggattgttga taaaggtttc 60 aaaagggctg aaagcaaggc tgatgaggat gatggagcgg aggcgtccag catccagatt 120 ttcattcagg aagagcca 138 <210> 39 <211> 102 <212> DNA <213> Artificial Sequence <220> <223> Linker 3 (M) <400> 39 gagtgtcaga atctcagtca gaatcccggg cctccttcag aagcgtcttc taataattta 60 cacagcccat tgaaaccaag aaattaccaa ctggagcttg cc 102 <210> 40 <211> 1449 <212> DNA <213> Artificial Sequence <220> <223> Helicase Domain (M) <400> 40 ctgcctgcca agaaagggaa aaatacaata atatgtgccc ctactggttg tggaaaaacc 60 tttgtgtcgc ttcttatatg tgaacaccat cttaaaaaat tcccatgtgg acaaaaaggg 120 aaagtggtct tcttcgctaa ccaaattcct gtctatgagc agcaggcaac tgtgttctca 180 cgatattttg aaagacttgg gtacaacatt gcgagcattt ctggggcaac atctgatagc 240 gtctcagtgc agcacatcat tgaagacaat gatatcatca tcctgacacc ccagattctt 300 gtgaacaatc tcaacaacgg agccatcccc tcgttgtctg tcttcactct gatgatattt 360 gatgagtgtc ataacactag caaaaaccac ccatacaatc agatcatgtt cagataccta 420 gaccacaaac ttggagagtc acgggaccca ctgcctcagg tcgttgggct gactgcctcc 480 gtcggcgttg gagatgctaa gaccgcggag gaagccatgc aacatatctg taaactctgt 540 gccgccctgg atgcctccgt gattgccaca gtcagagaca acgttgcaga actggaacag 600 gtcgtttata agccccagaa aatttccagg aaagtggcat cccggacttc gaacacgttt 660 aaatgcatca tctctcagct gatgaaggag acagagaagc tagccaagga tgtctccgag 720 gaacttggaa agctttttca aattcaaaac agagaattcg gcacccagaa atatgaacag 780 tggattgtcg gcgtccacaa agcgtgctca gtgtttcaga tggcagacaa agaggaggag 840 agccgggtct gcaaagcgct cttcctgtac acatcacatt tgcggaaata caacgatgca 900 ctcatcatca gtgaggatgc acagatgaca gacgctctaa attacctcaa agccttcttc 960 cacgatgtcc gagaagcagc attcgatgag accgagcgag agcttactcg gaggtttgaa 1020 gaaaaactag aggaattaga aaaagtttcc agggatccca gcaatgagaa tcctaaacta 1080 agagacctct acttggtctt acaagaagag taccacttaa agccagagac caagaccatt 1140 ctcttcgtga agaccagagc actcgtggat gctctgaaga aatggattga agaaaatcct 1200 gcactaagct ttctaaagcc tggcatactg actgggcgtg gcagaacaaa ccgggcaaca 1260 ggaatgacgc tcccggcaca gaagtgtgtg ctggaggcat tcagagccag cggagataac 1320 aatattctga ttgctacctc ggtcgctgat gaaggcattg acattgctga gtgcaatctc 1380 gtcattctct atgagtacgt gggcaacgtc atcaagatga tccaaaccag aggccgagga 1440 agagcacga 1449 <210> 41 <211> 573 <212> DNA <213> Artificial Sequence <220> <223> RD Domain (M) <400> 41 gatagcaagt gcttcctcct gaccagcagc gctgacgtga ttgaaaaaga aaaggcgaac 60 atgatcaagg aaaaaataat gaatgaatcc atcttaagac tgcagacatg ggatgaaatg 120 aaatttggaa agacggttca ccgcatacag gtgaatgaaa aactcctcag agacagtcag 180 cacaaaccac aacctgttcc tgacaaagaa aacaagaaac tgctgtgtgg aaagtgcaag 240 aattttgcgt gctacacagc tgacattcga gtggttgaga cgtcccacta cactgtcctt 300 ggagacgctt ttaaggagcg ctttgtgtgt aagccacacc ctaaaccaaa gatctatgac 360 aattttgaga agaaagcaaa gatattctgc gccaaacaga actgtagcca cgactgggga 420 atttttgtga gatacaagac gttcgagatt ccagtcataa aaattgaaag tttcgtcgtg 480 gaagatattg tgagcggagt tcagaaccgg cactcaaagt ggaaggactt tcattttgaa 540 aggatacagt tcgatcctgc agaaatgtcc gta 573 <210> 42 <211> 654 <212> DNA <213> Artificial Sequence <220> <223> CARD Domain (M) <400> 42 atgacagcgg agcagcggca gaatctgcaa gcattcagag actatatcaa gaagattctg 60 gaccccacct acatcctcag ctacatgagt tcctggctcg aggatgagga ggtgcagtac 120 attcaggctg agaagaacaa caagggccca atggaagctg cctcactctt cctccagtac 180 ctgttgaagc tgcagtcaga gggctggttc caggcctttt tggatgccct gtaccatgca 240 ggttactgtg gactttgtga agccatcgaa agttgggact ttcaaaaaat tgaaaagtta 300 gaggaacaca gattactttt aagacgttta gaaccagaat ttaaggccac agttgatcca 360 aatgatatcc tttctgaact atccgaatgt ttgattaatc aggaatgtga agaaatcaga 420 cagatccgag acactaaagg gagaatggca ggtgcggaga agatggccga atgtcttatc 480 agatccgaca aggaaaactg gccaaaggtc ttgcaacttg ctttggagaa agacaacagc 540 aagtttagtg aattgtggat tgttgataaa ggtttcaaaa gggctgaaag caaggctgat 600 gaggatgatg gagcggaggc gtccagcatc cagattttca ttcaggaaga gcca 654 <210> 43 <211> 2022 <212> DNA <213> Artificial Sequence <220> <223> Helicase Domain + RD domain (M) <400> 43 ctgcctgcca agaaagggaa aaatacaata atatgtgccc ctactggttg tggaaaaacc 60 tttgtgtcgc ttcttatatg tgaacaccat cttaaaaaat tcccatgtgg acaaaaaggg 120 aaagtggtct tcttcgctaa ccaaattcct gtctatgagc agcaggcaac tgtgttctca 180 cgatattttg aaagacttgg gtacaacatt gcgagcattt ctggggcaac atctgatagc 240 gtctcagtgc agcacatcat tgaagacaat gatatcatca tcctgacacc ccagattctt 300 gtgaacaatc tcaacaacgg agccatcccc tcgttgtctg tcttcactct gatgatattt 360 gatgagtgtc ataacactag caaaaaccac ccatacaatc agatcatgtt cagataccta 420 gaccacaaac ttggagagtc acgggaccca ctgcctcagg tcgttgggct gactgcctcc 480 gtcggcgttg gagatgctaa gaccgcggag gaagccatgc aacatatctg taaactctgt 540 gccgccctgg atgcctccgt gattgccaca gtcagagaca acgttgcaga actggaacag 600 gtcgtttata agccccagaa aatttccagg aaagtggcat cccggacttc gaacacgttt 660 aaatgcatca tctctcagct gatgaaggag acagagaagc tagccaagga tgtctccgag 720 gaacttggaa agctttttca aattcaaaac agagaattcg gcacccagaa atatgaacag 780 tggattgtcg gcgtccacaa agcgtgctca gtgtttcaga tggcagacaa agaggaggag 840 agccgggtct gcaaagcgct cttcctgtac acatcacatt tgcggaaata caacgatgca 900 ctcatcatca gtgaggatgc acagatgaca gacgctctaa attacctcaa agccttcttc 960 cacgatgtcc gagaagcagc attcgatgag accgagcgag agcttactcg gaggtttgaa 1020 gaaaaactag aggaattaga aaaagtttcc agggatccca gcaatgagaa tcctaaacta 1080 agagacctct acttggtctt acaagaagag taccacttaa agccagagac caagaccatt 1140 ctcttcgtga agaccagagc actcgtggat gctctgaaga aatggattga agaaaatcct 1200 gcactaagct ttctaaagcc tggcatactg actgggcgtg gcagaacaaa ccgggcaaca 1260 ggaatgacgc tcccggcaca gaagtgtgtg ctggaggcat tcagagccag cggagataac 1320 aatattctga ttgctacctc ggtcgctgat gaaggcattg acattgctga gtgcaatctc 1380 gtcattctct atgagtacgt gggcaacgtc atcaagatga tccaaaccag aggccgagga 1440 agagcacgag atagcaagtg cttcctcctg accagcagcg ctgacgtgat tgaaaaagaa 1500 aaggcgaaca tgatcaagga aaaaataatg aatgaatcca tcttaagact gcagacatgg 1560 gatgaaatga aatttggaaa gacggttcac cgcatacagg tgaatgaaaa actcctcaga 1620 gacagtcagc acaaaccaca acctgttcct gacaaagaaa acaagaaact gctgtgtgga 1680 aagtgcaaga attttgcgtg ctacacagct gacattcgag tggttgagac gtcccactac 1740 actgtccttg gagacgcttt taaggagcgc tttgtgtgta agccacaccc taaaccaaag 1800 atctatgaca attttgagaa gaaagcaaag atattctgcg ccaaacagaa ctgtagccac 1860 gactggggaa tttttgtgag atacaagacg ttcgagattc cagtcataaa aattgaaagt 1920 ttcgtcgtgg aagatattgt gagcggagtt cagaaccggc actcaaagtg gaaggacttt 1980 cattttgaaa ggatacagtt cgatcctgca gaaatgtccg ta 2022 <210> 44 <211> 2124 <212> DNA <213> Artificial Sequence <220> <223> Linker 3 (M) + Helicase domain + RD domain (M) <400> 44 gagtgtcaga atctcagtca gaatcccggg cctccttcag aagcgtcttc taataattta 60 cacagcccat tgaaaccaag aaattaccaa ctggagcttg ccctgcctgc caagaaaggg 120 aaaaatacaa taatatgtgc ccctactggt tgtggaaaaa cctttgtgtc gcttcttata 180 tgtgaacacc atcttaaaaa attcccatgt ggacaaaaag ggaaagtggt cttcttcgct 240 aaccaaattc ctgtctatga gcagcaggca actgtgttct cacgatattt tgaaagactt 300 gggtacaaca ttgcgagcat ttctggggca acatctgata gcgtctcagt gcagcacatc 360 attgaagaca atgatatcat catcctgaca ccccagattc ttgtgaacaa tctcaacaac 420 ggagccatcc cctcgttgtc tgtcttcact ctgatgatat ttgatgagtg tcataacact 480 agcaaaaacc acccatacaa tcagatcatg ttcagatacc tagaccacaa acttggagag 540 tcacgggacc cactgcctca ggtcgttggg ctgactgcct ccgtcggcgt tggagatgct 600 aagaccgcgg aggaagccat gcaacatatc tgtaaactct gtgccgccct ggatgcctcc 660 gtgattgcca cagtcagaga caacgttgca gaactggaac aggtcgttta taagccccag 720 aaaatttcca ggaaagtggc atcccggact tcgaacacgt ttaaatgcat catctctcag 780 ctgatgaagg agacagagaa gctagccaag gatgtctccg aggaacttgg aaagcttttt 840 caaattcaaa acagagaatt cggcacccag aaatatgaac agtggattgt cggcgtccac 900 aaagcgtgct cagtgtttca gatggcagac aaagaggagg agagccgggt ctgcaaagcg 960 ctcttcctgt acacatcaca tttgcggaaa tacaacgatg cactcatcat cagtgaggat 1020 gcacagatga cagacgctct aaattacctc aaagccttct tccacgatgt ccgagaagca 1080 gcattcgatg agaccgagcg agagcttact cggaggtttg aagaaaaact agaggaatta 1140 gaaaaagttt ccagggatcc cagcaatgag aatcctaaac taagagacct ctacttggtc 1200 ttacaagaag agtaccactt aaagccagag accaagacca ttctcttcgt gaagaccaga 1260 gcactcgtgg atgctctgaa gaaatggatt gaagaaaatc ctgcactaag ctttctaaag 1320 cctggcatac tgactgggcg tggcagaaca aaccgggcaa caggaatgac gctcccggca 1380 cagaagtgtg tgctggaggc attcagagcc agcggagata acaatattct gattgctacc 1440 tcggtcgctg atgaaggcat tgacattgct gagtgcaatc tcgtcattct ctatgagtac 1500 gtggcaacg tcatcaagat gatccaaacc agaggccgag gaagagcacg agatagcaag 1560 tgcttcctcc tgaccagcag cgctgacgtg attgaaaaag aaaaggcgaa catgatcaag 1620 gaaaaaataa tgaatgaatc catcttaaga ctgcagacat gggatgaaat gaaatttgga 1680 aagacggttc accgcataca ggtgaatgaa aaactcctca gagacagtca gcacaaacca 1740 caacctgttc ctgacaaaga aaacaagaaa ctgctgtgtg gaaagtgcaa gaattttgcg 1800 tgctacacag ctgacattcg agtggttgag acgtcccact acactgtcct tggagacgct 1860 tttaaggagc gctttgtgtg taagccacac cctaaaccaa agatctatga caattttgag 1920 aagaaagcaa agatattctg cgccaaacag aactgtagcc acgactgggg aatttttgtg 1980 agatacaaga cgttcgagat tccagtcata aaaattgaaa gtttcgtcgt ggaagatatt 2040 gtgagcggag ttcagaaccg gcactcaaag tggaaggact ttcattttga aaggatacag 2100 ttcgatcctg cagaaatgtc cgta 2124 <210> 45 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Control siRNA <400> 45 uucccgaac gugucacgut t 21 <210> 46 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> PGC1 siRNA <400> 46 gccaaaccaa caacuuuauu u 21 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MTCO-1_F <400> 47 ccccgatatg gcgtttcccc g 21 <210> 48 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MTCO-1_R <400> 48 gggagagata ggagaagtag g 21 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2_F <400> 49 ggccaccaat ggtactgaac 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2_R <400> 50 cgggaattgc atctgttttt 20 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ND-2_F <400> 51 tagccccctt tcacttctga 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ND-2_R <400> 52 gcgtagctgg gtttggttta 20 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> ≪ 223 > 18S rDNA_F <400> 53 cgccgcgctc taccttacct 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> ≪ 223 > 18S rDNA_R <400> 54 taggagagga gcgagcgacc 20 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Actin_F <400> 55 catgtacgtt gctatccagg c 21 <210> 56 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Actin_R <400> 56 ctccttaatg tcacgcacga t 21 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hTFAM_F <400> 57 ccgaggtggt tttcatctgt 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hTFAM_R <400> 58 agtcttcagc ttttcctgcg 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hNRF12_F <400> 59 agcaaaagca gagggtttca 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hNRF1_R <400> 60 ggtgactgcg ctgtctgata 20 <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPGC1a_F <400> 61 cactggttga ccctgttcct 20 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hPGC1a_R <400> 62 gtgtttcagg gcttctctgc 20
Claims (28)
Wherein the helicase domain comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 18, and the RD domain comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 19. 29. A composition for promoting mitochondrial proliferation and activity,
Wherein the metabolic syndrome related disease is type 2 diabetes, obesity, cardiovascular disease, hypertension, hyperglycemia, hyperlipidemia, obesity, atherosclerosis, aging or fatty liver.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020160019941A KR101793269B1 (en) | 2016-02-19 | 2016-02-19 | Pharmaceutical composition for preventing or treating metabolic syndrome |
Applications Claiming Priority (1)
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Non-Patent Citations (3)
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| Immunity, Vol. 34, No. 5, pp. 680-692 (2011.05.27.) |
| Nature, Vol. 479, No. 7373, pp. 423-427 (온라인 공개일 : 2011.09.25.)* |
| RNA, Vol. 18, pp. 2118-2127 (2012.) |
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