KR102501639B1 - Novel intracellular protein delivery method - Google Patents

Abstract

The present invention relates to novel intracellular protein delivery methods.
Eukaryotic cells are surrounded by a cell membrane of a phospholipid bilayer, and it is almost impossible for hydrophilic substances outside the cell to pass through the cell membrane and move into the cell due to the hydrophobicity of the inside of the bilayer. limited
Therefore, the present invention has been devised to solve the problems of the prior art as described above. Using the method of the present invention, it is possible to introduce the target material into the cytoplasm with high efficiency while maintaining the original function of the target material, and the target material Since it is possible to stay in the cytoplasm without moving to the nucleus, it is expected to be greatly used in the medical and bio fields.

Description

Novel intracellular protein delivery method {Novel intracellular protein delivery method}

The present invention relates to novel intracellular protein delivery methods.

Pathogen infection induces activation of the body's immune response. Among them, viral infection mediates the production of interferon in infected cells, and interferon binds to interferon receptors in other cells to activate the signal transduction system, to express several interferon-stimulated genes (ISGs) and to protect the cells. causes viral action.

Mx (Interferon-induced GTP-binding protein Mx) is a type of ISG whose production is induced by type I interferon in most vertebrates and belongs to GTPases (dynamin-like large guanosine triphosphatases). Mx protein has potential as an antiviral agent in that it is involved in the innate immune response to various viral infections and inhibits viral activation to help the host survive and recover from disease. Since this protein acts to inhibit the proliferation of viruses in the cytoplasm, the process of introducing the protein into the cell is essential to use the Mx protein as an antiviral agent.

Eukaryotic cells are surrounded by a cell membrane of a phospholipid bilayer, and it is almost impossible for hydrophilic substances outside the cell to pass through the cell membrane and move into the cell due to the hydrophobicity of the inside of the bilayer. To overcome these barriers and deliver drugs into cells, drug carriers such as liposomes have been developed. PTD (Protein transduction domain) is a kind of method used to overcome the material transport obstacle in the cell membrane and move materials into the cell, and is a peptide composed of about 10 basic amino acids. When PTD is bound, substances such as peptides and DNA can pass through cell membranes and enter cells without special receptors. Technologies used for gene delivery have been developed.

The present invention aims to develop a PTD-MxA fusion protein containing a novel PTD sequence. While studying myxovirus resistance A (MxA), the present inventors found that the peptide sequence (named REKKKFLKRR, cytoPTD) derived from the nuclear localization signal (NLS) of the mouse-derived Mx1 protein can function as a PTD. found something In particular, the cytoPTD has an excellent effect of transferring substances into the cytoplasm and then retaining them in the cytoplasm. In addition, when cytoPTD is connected to both sides of a substance to be delivered into the cytoplasm, the delivery efficiency is very high compared to the case where cytoPTD is connected to one side. Therefore, the present invention is expected to be widely used in the medical and bio fields.

The present invention has been made to solve the problems of the prior art as described above, and an object of the present invention is to provide a novel intracellular protein delivery method.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, numerous specific details are set forth, such as specific forms, compositions, and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, particular features, forms, compositions, or characteristics may be combined in one or more embodiments in any suitable way.

Unless otherwise defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention belongs.

In the present specification, a protein transduction domain is a peptide composed of 3 to 30 amino acids, which binds to various substances such as proteins, peptides, and DNA to move substances into cells without a special receptor. It refers to a peptide. Preferably, the sequence shown in Table 1 below, that is, a peptide derived from the nuclear localization signal (NLS) of the Mx1 protein, a sequence in which 5 to 10 arginines are bound, a trans-activator of transcription , TAT), ANTP, Vp22, MTS, Pep-1, Pep-2, etc., but it is not limited as long as it binds to the desired target protein to pass through the cell membrane and promote cell-to-cell movement. .

Justice order Peptides derived from the nuclear localization signal (NLS) of the Mx1 protein REKKKFLKRR 5 to 10 arginine bonds RRRRR to RRRRRRRRRR transcription transactivator (TAT) YGRKKRRORRR ANTP RQIKIWFQNRRMKWKK Vp22 DAATATRGRSAASRPTERPRAPARRSASRPRRPVE MTS AAVALLPAVLLALLAPAAADQNQLMP Pep-1 KETWWETWWTEWSQPKKKRKV Pep-2 KETWFETWFTEWSQPKKKRKV

In the present specification, antiviral refers to a preventive or therapeutic action against viral infection that is parasitic on living cells such as animals, plants, bacteria, and radioactive bacteria and can proliferate only within cells, and the virus is the denatured MxA protein of the present invention. It is not limited as long as the virus is inhibited from growing by, but preferably refers to an orthomyxoviridae influenza virus.

In the present specification, the target material means a target material to be delivered from the outside of the cell to the inside of the cell, more specifically, into the cytoplasm. The target material may be, but is not limited to, nucleic acid, protein, inorganic substance, organic substance, ion, nanoparticle, sugar, or lipid, and in the present invention, it is preferably a nucleic acid or protein, and more preferably a protein. The target material may be coupled to the protein transduction domain on one side or both ends to increase the transfer efficiency into the inside of the cell.

In the present specification, the myxovirus resistance A (MxA) protein is an antiviral protein belonging to the dynamin superfamily of large GTPases, including orthomyxoviruses and bunyaviruses. It exhibits intrinsic antiviral activity against many different viruses, and its expression is tightly controlled by type I (α/β) or type III (λ) interferons. The myxovirus resistant A protein consists of 662 amino acids (SEQ ID NO: 1), has a total size of 75.520 Da, and is capable of self-assembling highly ordered oligomers to form ring-like structures.

Myxovirus resistance A protein is divided into G domain (G domain, or GTPase binding domain), middle domain, and GTPase effect domain (GED) from the N-terminus, and the middle domain and GED are α1N, α1C, α2, It is divided into α3, α4, α6, L1 between α1N and α1C, L2 between α1C and α2, L3 between α3 and α4, and L4 between α3 and α4. In particular, the L4 and α4 regions corresponding to GED are parts that regulate the antiviral activity of the myxovirus resistance A protein and are very important for antiviral activity (Corinna Patzina et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, No. 9 , pp. 6020-6027). However, additional studies have shown that the MxA protein dissolves in an aqueous solution under high-concentration salt conditions containing 300 mM or more NaCl, but self-aggregates when the salt concentration is lowered to a physiological level (about 150 mM) to form a large oligomer of 3 MDa or more and insoluble. It was confirmed to form a precipitate (Georg Kochs et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 277, NO. 16, pp. 14172-14176, 2002). As described above, it is known that the MxA protein is difficult to transfer into cells due to its self-aggregating property, and thus is limited in exhibiting antiviral activity in cells. Therefore, it has not been commercialized as an antiviral agent.

In the present specification, a pharmaceutical composition means a composition administered for a specific purpose. For the purpose of the present invention, the pharmaceutical composition of the present invention is for preventing or treating influenza virus infection, and may include a compound involved therein and a pharmaceutically acceptable carrier, excipient or diluent. In addition, the pharmaceutical composition according to the present invention contains 0.1 to 50% by weight of the active ingredient of the present invention based on the total weight of the composition. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In the present specification, administration means introducing the composition of the present invention to a patient by any suitable method, and the administration route of the composition of the present invention may be administered through any general route as long as it can reach the target tissue. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, intrarectal administration, intracavity administration, intraperitoneal administration, intrathecal administration may be performed, but are not limited thereto. don't In the present invention, the effective amount is the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, weight, general health condition, sex and diet, administration time, administration route And it can be adjusted according to various factors including the secretion rate of the composition, the treatment period, and drugs used simultaneously. In the case of adults, the therapeutic pharmaceutical composition can be administered to the body in an amount of 50 ml to 500 ml once, and in the case of a compound, 0.1 ng/kg-10 mg/kg, and in the case of a monoclonal antibody, 0.1 ng/kg-10 mg /kg may be administered. The administration interval may be 1 to 12 times a day, and in the case of administration 12 times a day, it may be administered once every 2 hours. In addition, the pharmaceutical composition of the present invention may be administered alone or with other therapies known in the art, such as chemotherapy, radiation and surgery, for the treatment of desired cancer stem cells. In addition, the pharmaceutical composition of the present invention may be administered in combination with other therapies designed to enhance the immune response, such as adjuvants or cytokines (or nucleic acids encoding cytokines) well known in the art. Other standard delivery methods may also be used, such as biolistic delivery or ex vivo treatment. In ex vivo treatment, for example, antigen presenting cells (APCs), dendritic cells, peripheral blood mononuclear cells, or bone marrow cells may be obtained from a patient or a suitable donor, activated in vitro with the present pharmaceutical composition, and then administered to the patient. there is.

In the present specification, the food composition is intended to prevent or improve influenza virus infection for the purpose of the present invention, but is not limited thereto. Food compositions containing the composition of the present invention as an active ingredient are various foods, for example, beverages, milk, lactic acid bacteria-containing dairy products, gum, tea, vitamin complexes, powders, granules, pills, tablets, capsules, confectionery, rice cakes, and bread. It can be manufactured in the form of etc. Since the food composition of the present invention has little toxicity and side effects, it can be safely used even when taken for a long period of time for preventive purposes. When the composition of the present invention is included in the food composition, the amount may be added in an amount of 0.1 to 100% of the total weight. Here, when the food composition is prepared in the form of a beverage, there is no particular limitation except that the food composition is contained in the indicated ratio, and various flavors or natural carbohydrates may be included as additional ingredients as in conventional beverages. That is, natural carbohydrates may include monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as starch, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. there is. Examples of the flavoring agent include natural flavoring agents (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). Others of the present invention The food composition of is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, It may contain stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. These components may be used independently or in combination The ratio of these additives is not so important, but the composition of the present invention It is generally selected from the range of 0.1 to 100 parts by weight per part.

In one embodiment of the present invention, a recombinant protein characterized in that a protein transduction domain (PTD) is linked to the 5' end and the 3' end of the target protein is provided, and the recombinant protein is a cell nuclear membrane It provides a recombinant protein characterized in that it stays between and the cytoplasmic membrane, wherein the protein transduction domain is a peptide derived from a nuclear localization signal (NLS) of the Mx1 protein, a peptide to which 5 to 10 arginines are linked, transcription Provides a recombinant protein selected from the group consisting of trans-activator of transcription (TAT), ANTP, Vp22, MTS, Pep-1, and Pep-2, derived from the NLS of the Mx1 protein One peptide provides a recombinant protein that is a peptide represented by SEQ ID NO: 1, and the target protein provides a recombinant protein encoded by a nucleic acid represented by SEQ ID NO: 3, and the recombinant protein is SEQ ID NO: 6 Provided is a recombinant protein that is the indicated peptide.

In another embodiment of the present invention, a recombinant gene encoding the recombinant protein is provided.

In another embodiment of the present invention, a recombinant vector containing the recombinant gene is provided, wherein the vector is an E. coli overexpression vector.

In another embodiment of the present invention, a non-human transformant transformed with the recombinant vector is provided.

In another embodiment of the present invention, a pharmaceutical composition for preventing or treating influenza virus infection comprising the recombinant protein as an active ingredient is provided.

In another embodiment of the present invention, a food composition for preventing or improving influenza virus infection containing the recombinant protein as an active ingredient is provided.

In another embodiment of the present invention, linking a protein transduction domain (PTD) to the 5'end and 3'end of a gene encoding a target protein; do.

In another embodiment of the present invention, (a) preparing a recombinant gene by binding a protein transduction domain (PTD) to the 5' end and 3' end of a gene encoding a target protein; And, (b) expressing the recombinant gene in a non-human subject; provides a method for producing a recombinant protein comprising the.

In another embodiment of the present invention, (a) preparing a recombinant gene by binding a protein transduction domain (PTD) to the 5' end and 3' end of a gene encoding a target protein; (b) producing a recombinant protein by expressing the recombinant gene in a non-human organism; And, (c) purifying the recombinant protein; provides a method for producing a pharmaceutical composition for preventing or treating influenza virus infection, including.

Hereinafter, the present invention will be described in detail step by step.

The present invention relates to a novel intracellular protein delivery method, and more particularly, to a novel intracellular protein delivery method with increased efficiency of target material introduction into the cytoplasm. By using the method of the present invention, it is possible to introduce the target material into the cytoplasm with high efficiency while maintaining the original function of the target material, and it is possible to make the target material stay in the cytoplasm without moving to the nucleus, so it is expected to be widely used in the medical and bio fields. It is expected.

1 is a schematic diagram of a novel recombinant MxA protein cytoPTD-MxA-cytoPTD according to an embodiment of the present invention.
2 shows the results of introducing MxA protein, MxA-cytoPTD protein, or cytoPTD-MxA-cytoPTD protein into cells according to an embodiment of the present invention.
Figure 3 shows the results of verifying the antiviral effect of the cytoPTD-MxA-cytoPTD recombinant protein according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Construction of recombinant cytoPTD-MxA-cytoPTD expression vector

In order to confirm whether the novel cytoPTD sequence (SEQ ID NO: 1) can introduce the target protein into the cytoplasm, the present inventors combined a peptide having the amino acid sequence described in SEQ ID NO: 1 on both sides of the entire sequence of human-derived MxA, and its expression A vector was prepared.

Specifically, according to a preferred embodiment of the present invention, the gene sequence (SEQ ID NO: 2) encoding the peptide of SEQ ID NO: 1 is added to the 5' end and the 3' end of the human-derived MxA gene sequence (SEQ ID NO: 3), In order to add restriction enzyme (Nhe I and Not I) cleavage sites on both sides, PCR was performed using SEQ ID NOs: 4 and 5, which are novel primer sequences, as forward and reverse primers.

After performing PCR, restriction enzymes and DNA splicing enzymes were used to insert the amplified sequences into the vector. Specifically, the present inventors used pET28a, an E. coli overexpression vector. Since the pET28a vector itself contains a His tag at the multicloning site, the His tag, which facilitates protein extraction, can be made to bind to the recombinant protein. The PCR product and the vector were treated using Nhe I and Not I restriction enzymes, and the PCR product was inserted into the vector using T4 DNA ligase. The recombinant MxA protein (SEQ ID NO: 6) prepared through this process was named cytoPTD-MxA-cytoPTD, and a schematic thereof is shown in FIG. 1 . The vector expressing the cytoPTD-MxA-cytoPTD protein of the present invention was named pET28a-cytoPTD-MxA-cytoPTD expression vector.

Example 2: Expression of pET28a-cytoPTD-MxA-cytoPTD in E. coli

After transforming E. coli BL21 (DE3) with the pET28a-cytoPTD-MxA-cytoPTD vector prepared in Example 1, it was cultured in LB solid medium containing kanamycin at 37°C for 12 hours. At this time, the formed single colony was inoculated into LB liquid medium containing kanamycin, incubated at 37 ° C for 3 hours, the culture medium was cooled to 18 ° C, and IPTG (isopropyl-β-D-thiogalactopyranosid), an expression inducer, was added to 0.125 It was added to a concentration of mM and further cultured for 72 hours to express the cytoPTD-MxA-cytoPTD recombinant protein.

The cells precipitated by high-speed centrifugation of the culture solution were suspended in a cell lysis buffer and lysed using an ultrasonic generator. The lysis buffer used at this time included HEPES, MgCl ₂ , NaCl, imidazole, DNAse, and β-mercaptoethanol, and a protease inhibitor phenylmethanesulfonylfluoride (PMSF) was added to prevent protein degradation. The dissolved cells were again subjected to high-speed centrifugation, and the supernatant was applied to a Ni-NTA agarose resin column to adsorb proteins to the resin. Then, an elution buffer solution containing an excess of imidazole was added to separate and extract the recombinant protein adsorbed to the resin, and endotoxin in the recombinant protein solution was removed using Pierce's High-Capacity Endotoxin Removal Resin. Thereafter, imidazole contained in the protein solution was removed through DPBS dialysis, and the recombinant protein after dialysis was identified through a 2 - 20% SDS-PAGE gel.

Example 3: Confirmation of structural characteristics and introduction into cells of recombinant cytoPTD-MxA-cytoPTD

In order to confirm the structural characteristics and intracellular introduction of the recombinant cytoPTD-MxA-cytoPTD protein extracted in Example 2, MxA protein, MxA-cytoPTD protein, or cytoPTD-MxA were added to the dog-derived epidermal MDCK cell line at a concentration of 50 μg/ml. - After treatment with cytoPTD protein, it was incubated at 37°C for 6 hours. After washing the medium, the cells were fixed using 4% paraformaldehyde, and the MxA protein introduced into the cells was confirmed under a fluorescence microscope using an antibody that specifically binds to MxA. The results are shown in FIG. 2 . As a result of the experiment, the same amount of MxA protein was hardly introduced into cells. The recombinant MxA-cytoPTD protein, in which cytoPTD was bound to the C-terminus of MxA protein, was also better than MxA protein, but its introduction into cells was very insignificant. However, it was observed that the cytoPTD-MxA-cytoPTD protein, in which cytoPTD is bound to both the N and C termini of the MxA protein, introduces the target protein into cells sufficiently. confirmed that it can be done. In addition, when cytoPTD, previously known to function as a nuclear localization signal, is present as a recombinant protein combined with a target protein, it was confirmed that the target does not migrate to the nucleus and remains only in the cytoplasm. It was verified that it mediated only the introduction into the cytoplasm of .

Example 4: Confirmation of enhancing effect of recombinant cytoPTD-MxA-cytoPTD protein on defense against influenza virus

In Example 3, whether the recombinant cytoPTD-MxA-cytoPTD protein introduced into cells enhances the protective efficacy against influenza virus was verified in vitro and at the level. Specifically, MDCK cell lines were infected with influenza virus PR8 (subtype H1N1) at an MOI of 0.01, and 4 hours later, the cytoPTD-MxA-cytoPTD recombinant protein was treated at a concentration of 80 μg/ml. Then, 48 hours after infection, the expression of viral RNA in cells was measured by RT-qPCR method. As a result, it was confirmed that the expression of viral RNA was reduced by about 1/5 to 1/10 when the cytoPTD-MxA-cytoPTD recombinant protein was treated compared to the control group. was confirmed to inhibit. This is shown in Figure 3.

From the results of Examples 1 to 3, it can be seen that binding cytoPTD to both sides of the target protein is very effective in introducing the target substance into cells and can maintain the intrinsic function of the target protein. This means that the structure of cytoPTD-target substance-cytoPTD prepared in the present invention can be very useful for delivering a target protein into cells.

<110> Korea Advanced Institute of Science and Technology <120> Novel intracellular protein delivery method <130> PDPB204100 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT <213> artificial sequence <220> <223> truncated sequence from Mx1 <400> 1 Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg 1 5 10 <210> 2 <211> 30 <212> DNA <213> artificial sequence <220> <223> truncated sequence from Mx1 <400> 2 agagagaaga agaagttcct gaagaggcgg 30 <210> 3 <211> 1983 <212> DNA <213> Homo sapiens <400> 3 gttgtttccg aagtggacat cgcaaaagct gatccagctg ctgcatccca ccctctatta 60 ctgaatggag atgctactgt ggcccagaaa aatccaggct cggtggctga gaacaacctg 120 tgcagccagt atgaggagaa ggtgcgcccc tgcatcgacc tcattgactc cctgcgggct 180 ctaggtgtgg agcaggacct ggccctgcca gccatcgccg tcatcgggga ccagagctcg 240 ggcaagagct ccgtgttgga ggcactgtca ggagttgccc ttccccagagg cagcgggatc 300 gtgaccagat gcccgctggt gctgaaactg aagaaacttg tgaacgaaga taagtggaga 360 ggcaaggtca gttaccagga ctacgagatt gagatttcgg atgcttcaga ggtagaaaag 420 gaaattaata aagcccagaa tgccatcgcc ggggaaggaa tgggaatcag tcatgagcta 480 atcaccctgg agatcagctc ccgagatgtc ccggatctga ctctaataga ccttcctggc 540 ataaccagag tggctgtggg caatcagcct gctgacattg ggtataagat caagacactc 600 atcaagaagt acatccagag gcaggagaca atcagcctgg tggtggtccc cagtaatgtg 660 gacatcgcca ccacagaggc tctcagcatg gcccaggagg tggaccccga gggagacagg 720 accatcggaa tcttgacgaa gcctgatctg gtggacaaag gaactgaaga caaggttggg 780 gacgtggtgc ggaacctcgt gttccacctg aagaagggtt acatgattgt caagtgccgg 840 ggccagcagg agatccagga ccagctgagc ctgtccgaag ccctgcagag agagaagatc 900 ttctttgaga accacccata tttcagggat ctgctggagg aaggaaaggc cacggttccc 960 tgcctggcag aaaaacttac cagcgagctc atcacacata tctgtaaatc tctgcccctg 1020 ttagaaaatc aaatcaagga gactcaccag agaataacag aggagctaca aaagtatggt 1080 gtcgacatac cggaagacga aaatgaaaaa atgttcttcc tgatagataa aattaatgcc 1140 tttaatcagg acatcactgc tctcatgcaa ggagaggaaa ctgtagggga ggaagacatt 1200 cggctgttta ccagactccg acacgagttc cacaaatgga gtacaataat tgaaaacaat 1260 tttcaagaag gccataaaat tttgagtaga aaaatccaga aatttgaaaa tcagtatcgt 1320 ggtagagagc tgccaggctt tgtgaattac aggacatttg agacaatcgt gaaacagcaa 1380 atcaaggcac tggaagagcc ggctgtggat atgctacaca ccgtgacgga tatggtccgg 1440 cttgctttca cagatgtttc gataaaaaat tttgaagagt tttttaacct ccacagaacc 1500 gccaagtcca aaattgaaga cattagagca gaacaagaga gagaaggtga gaagctgatc 1560 cgcctccact tccagatgga acagattgtc tactgccagg accaggtata caggggtgca 1620 ttgcagaagg tcagagagaa ggagctggaa gaagaaaaga agaagaaatc ctgggatttt 1680 ggggctttcc agtccagctc ggcaacagac tcttccatgg aggagatctt tcagcacctg 1740 atggcctatc accaggaggc cagcaagcgc atctccagcc acatcccttt gatcatccag 1800 ttcttcatgc tccagacgta cggccagcag cttcagaagg ccatgctgca gctcctgcag 1860 gacaaggaca cctacagctg gctcctgaag gagcggagcg acaccagcga caagcggaag 1920 ttcctgaagg agcggcttgc acggctgacg caggctcggc gccggcttgc ccagttcccc 1980 ggt 1983 <210> 4 <211> 60 <212> DNA <213> artificial sequence <220> <223> forward primer <400> 4 ctagctagca gagagaagaa gaagttcctg aagaggcggg ttgtttccga agtggacatc 60 60 <210> 5 <211> 64 <212> DNA <213> artificial sequence <220> <223> reverse primer <400> 5 ataagaatgc ggccgcttac cgcctcttca ggaacttctt cttctctcta ccggggaact 60 gggc 64 <210> 6 <211> 704 <212> PRT <213> artificial sequence <220> <223> cytoPTD-MxA-cytoPTD <400> 6 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Arg Glu Lys Lys Lys Phe Leu Lys Arg 20 25 30 Arg Val Val Ser Glu Val Asp Ile Ala Lys Ala Asp Pro Ala Ala Ala 35 40 45 Ser His Pro Leu Leu Leu Asn Gly Asp Ala Thr Val Ala Gln Lys Asn 50 55 60 Pro Gly Ser Val Ala Glu Asn Asn Leu Cys Ser Gln Tyr Glu Glu Lys 65 70 75 80 Val Arg Pro Cys Ile Asp Leu Ile Asp Ser Leu Arg Ala Leu Gly Val 85 90 95 Glu Gln Asp Leu Ala Leu Pro Ala Ile Ala Val Ile Gly Asp Gln Ser 100 105 110 Ser Gly Lys Ser Ser Val Leu Glu Ala Leu Ser Gly Val Ala Leu Pro 115 120 125 Arg Gly Ser Gly Ile Val Thr Arg Cys Pro Leu Val Leu Lys Leu Lys 130 135 140 Lys Leu Val Asn Glu Asp Lys Trp Arg Gly Lys Val Ser Tyr Gln Asp 145 150 155 160 Tyr Glu Ile Glu Ile Ser Asp Ala Ser Glu Val Glu Lys Glu Ile Asn 165 170 175 Lys Ala Gln Asn Ala Ile Ala Gly Glu Gly Met Gly Ile Ser His Glu 180 185 190 Leu Ile Thr Leu Glu Ile Ser Ser Arg Asp Val Pro Asp Leu Thr Leu 195 200 205 Ile Asp Leu Pro Gly Ile Thr Arg Val Ala Val Gly Asn Gln Pro Ala 210 215 220 Asp Ile Gly Tyr Lys Ile Lys Thr Leu Ile Lys Lys Tyr Ile Gln Arg 225 230 235 240 Gln Glu Thr Ile Ser Leu Val Val Val Pro Ser Asn Val Asp Ile Ala 245 250 255 Thr Thr Glu Ala Leu Ser Met Ala Gln Glu Val Asp Pro Glu Gly Asp 260 265 270 Arg Thr Ile Gly Ile Leu Thr Lys Pro Asp Leu Val Asp Lys Gly Thr 275 280 285 Glu Asp Lys Val Val Asp Val Val Arg Asn Leu Val Phe His Leu Lys 290 295 300 Lys Gly Tyr Met Ile Val Lys Cys Arg Gly Gln Gln Glu Ile Gln Asp 305 310 315 320 Gln Leu Ser Leu Ser Glu Ala Leu Gln Arg Glu Lys Ile Phe Phe Glu 325 330 335 Asn His Pro Tyr Phe Arg Asp Leu Leu Glu Glu Gly Lys Ala Thr Val 340 345 350 Pro Cys Leu Ala Glu Lys Leu Thr Ser Glu Leu Ile Thr His Ile Cys 355 360 365 Lys Ser Leu Pro Leu Leu Glu Asn Gln Ile Lys Glu Thr His Gln Arg 370 375 380 Ile Thr Glu Glu Leu Gln Lys Tyr Gly Val Asp Ile Pro Glu Asp Glu 385 390 395 400 Asn Glu Lys Met Phe Phe Leu Ile Asp Lys Ile Asn Ala Phe Asn Gln 405 410 415 Asp Ile Thr Ala Leu Met Gln Gly Glu Glu Thr Val Gly Glu Glu Asp 420 425 430 Ile Arg Leu Phe Thr Arg Leu Arg His Glu Phe His Lys Trp Ser Thr 435 440 445 Ile Ile Glu Asn Asn Phe Gln Glu Gly His Lys Ile Leu Ser Arg Lys 450 455 460 Ile Gln Lys Phe Glu Asn Gln Tyr Arg Gly Arg Glu Leu Pro Gly Phe 465 470 475 480 Val Asn Tyr Arg Thr Phe Glu Thr Ile Val Lys Gln Gln Ile Lys Ala 485 490 495 Leu Glu Glu Pro Ala Val Asp Met Leu His Thr Val Thr Asp Met Val 500 505 510 Arg Leu Ala Phe Thr Asp Val Ser Ile Lys Asn Phe Glu Glu Phe Phe 515 520 525 Asn Leu His Arg Thr Ala Lys Ser Lys Ile Glu Asp Ile Arg Ala Glu 530 535 540 Gln Glu Arg Glu Gly Glu Lys Leu Ile Arg Leu His Phe Gln Met Glu 545 550 555 560 Gln Ile Val Tyr Cys Gln Asp Gln Val Tyr Arg Gly Ala Leu Gln Lys 565 570 575 Val Arg Glu Lys Glu Leu Glu Glu Glu Lys Lys Lys Lys Ser Trp Asp 580 585 590 Phe Gly Ala Phe Gln Ser Ser Ser Ala Thr Asp Ser Ser Met Glu Glu 595 600 605 Ile Phe Gln His Leu Met Ala Tyr His Gln Glu Ala Ser Lys Arg Ile 610 615 620 Ser Ser His Ile Pro Leu Ile Ile Gln Phe Phe Met Leu Gln Thr Tyr 625 630 635 640 Gly Gln Gln Leu Gln Lys Ala Met Leu Gln Leu Leu Gln Asp Lys Asp 645 650 655 Thr Tyr Ser Trp Leu Leu Lys Glu Arg Ser Asp Thr Ser Asp Lys Arg 660 665 670 Lys Phe Leu Lys Glu Arg Leu Ala Arg Leu Thr Gln Ala Arg Arg Arg 675 680 685 Leu Ala Gln Phe Pro Gly Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg 690 695 700

Claims (15)

A recombinant protein characterized in that a protein transduction domain (PTD) is bound to the N-terminus and C-terminus of the target protein,
The target protein is an Mx protein,
The protein transduction domain is a peptide represented by SEQ ID NO: 1,
The recombinant protein is a peptide represented by SEQ ID NO: 6, the recombinant protein.
According to claim 1,
The recombinant protein is characterized in that the recombinant protein stays between the nuclear membrane and the cytoplasmic membrane of the cell.
delete According to claim 1,
The peptide represented by SEQ ID NO: 1 is a peptide derived from a nuclear localization signal (NLS) of the Mx1 protein.
delete delete A recombinant gene encoding the recombinant protein of claim 1.
A recombinant vector comprising the recombinant gene of claim 7.
According to claim 8,
The vector is an E. coli overexpression vector, a recombinant vector.
Transformants other than humans transformed with the vector of claim 8.
A pharmaceutical composition for preventing or treating influenza virus infection, comprising the recombinant protein of claim 1 as an active ingredient.
A food composition for preventing or improving influenza virus infection, comprising the recombinant protein of claim 1 as an active ingredient.
As a method for producing a recombinant gene comprising the steps of combining a gene encoding a protein transduction domain (PTD) to the 5' end and 3' end of the gene encoding the target protein,
The gene encoding the protein transduction domain is the gene represented by SEQ ID NO: 2,
The recombinant gene is a gene encoding the peptide represented by SEQ ID NO: 6, the production method.
(a) preparing a recombinant gene by binding a gene encoding a protein transduction domain (PTD) to the 5'end and 3'end of a gene encoding a target protein; and
(b) expressing the recombinant gene in a non-human organism; a method for producing a recombinant protein comprising the steps of:
The gene encoding the protein transduction domain is the gene represented by SEQ ID NO: 2,
The recombinant gene is a gene encoding the peptide represented by SEQ ID NO: 6, the production method.
(a) preparing a recombinant gene by binding a gene encoding a protein transduction domain (PTD) to the 5'end and 3'end of a gene encoding a target protein;
(b) producing a recombinant protein by expressing the recombinant gene in a non-human organism; and
(c) purifying the recombinant protein; as a method for producing a pharmaceutical composition for preventing or treating influenza virus infection,
The gene encoding the protein transduction domain is the gene represented by SEQ ID NO: 2,
The recombinant gene is a gene encoding the peptide represented by SEQ ID NO: 6, the production method.

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