Vector peptide fragments and uses thereof

Abstract

According to the present invention, it is possible to provide a novel vector peptide fragment having cell membrane permeability, the vector peptide fragment according to the present invention comprising the following amino acid sequence: RKLAVYWSTYKRSR (SEQ ID NO: 1).

Description

Technical Field

[0001] The present invention relates to a method for introducing (transferring) an exogenous substance from the outside of a eukaryotic cell to the inside of the cell, and a carrier peptide fragment used in the method. It should be noted that this application claims priority based on Japanese Patent Application No. 2022-141808 filed on September 7, 2022, and the entire contents of the application are introduced as reference contents in this specification. Background Art

[0002] Conventionally, exogenous substances such as polypeptides, especially physiologically active substances, are introduced into cells (eukaryotic cells) of humans and non-human mammals to cause biotransformation of the cells (and tissues and organs composed of the cells, etc.) or to improve and enhance the functions of the cells.

[0003] For example, Patent Document 1 discloses a variety of polypeptides, including a nucleolar localization signal (NoLS) known as a signal sequence for localizing a protein to the nucleolus in the nucleus, and a cell penetrating peptide (CPP) capable of functioning as a cell penetrating peptide (CPP) that can introduce exogenous substances from the outside of the cell through the cell membrane into the cytoplasm.

[0004] Prior art literature Patent Literature Patent Document 1: International Publication No. 2011 / 013699 Summary of the invention

[0005] Problems to be solved by the invention However, in a construct comprising a CPP and an exogenous substance, the combination of the amino acid sequence of the CPP and the type of the exogenous substance may lead to fluctuations in cell membrane penetration efficiency. In recent years, drug delivery technology using CPPs has attracted much attention, and there is a need to diversify CPP development to select CPPs that are adapted to the exogenous substances used (e.g., nucleic acid drugs, etc.).

[0006] The technology disclosed in this specification is an invention created in view of the above situation. The main purpose of the present invention is to provide a novel carrier peptide fragment with cell membrane permeability. In addition, another purpose of the present invention is to provide a construct for introducing an exogenous substance having the carrier peptide fragment. In addition, another purpose of the present invention is to provide a method for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell.

[0007] The carrier peptide fragment of the present invention is a carrier peptide fragment introduced from the outside of a eukaryotic cell into at least the cytoplasm of the cell. The carrier peptide fragment is composed of the amino acid sequence: RKLAVYWSTYKRSR (SEQ ID NO. 1).

[0008] The carrier peptide fragment of this composition is an artificially synthesized peptide found by the inventor of the present invention after in-depth research. The carrier peptide fragment has cell membrane permeability and can introduce exogenous substances from the outside of the cell into the inside of the cell.

[0009] In addition, according to the present invention, a construct for introducing an exogenous substance (hereinafter also referred to as a "construct") prepared to achieve the above-mentioned purpose and to introduce a target exogenous substance from the outside of a eukaryotic cell at least into the cytoplasm of the cell can be provided. The construct of the present invention comprises the carrier peptide fragment described in this specification and an exogenous substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment. With such a construct, the target exogenous substance can be introduced from the outside of the cell into the inside of the cell.

[0010] In one embodiment of the construct of the present invention, the exogenous substance may be at least one organic compound selected from the group consisting of polypeptides, nucleic acids, pigments, and drugs.

[0011] Among them, "polypeptide" refers to a polymer having a structure in which multiple amino acids are bound by peptide bonds. Polypeptides are not limited by the number of peptide bonds (i.e., the number of amino acid residues). That is, polypeptides include compounds generally called peptides with an amino acid residue number of more than 10 and less than 300, and compounds generally called proteins (polymer compounds generally composed of more than 300 amino acid residues). In this technical field, there is no strict distinction between polypeptides and proteins. In this specification, polymers (including oligomers) formed by multiple amino acid residues are collectively referred to as polypeptides.

[0012] In addition, "nucleic acid" refers to a polymer of nucleotides, including DNA and RNA. "Nucleic acid" is not limited by the number of bases.

[0013] In one embodiment of the construct of the present invention, the exogenous substance may be disposed on the C-terminal side of the carrier peptide fragment.

[0014] In addition, according to the present invention, a method for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell in an in vitro or in vivo environment can be provided. The method of the present invention comprises (1) a step of preparing the construct of the present invention; and (2) a step of supplying the construct to a sample containing the target eukaryotic cell. In this way, the target exogenous substance can be efficiently introduced into the eukaryotic cell.

[0015] In one embodiment of the method of the present invention, the eukaryotic cell into which the construct is introduced may be a human or a non-human mammalian cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a histogram obtained by adding a construct related to one embodiment or FAM to the culture medium of HeLa cells and culturing the cells, and then analyzing the cells using a flow cytometer. DETAILED DESCRIPTION

[0017] The following will describe the implementation methods of the technology described in this specification. Matters required for the implementation of the present invention other than those specifically mentioned in this specification (such as general matters such as chemical synthesis methods of peptides, cell culture techniques, and preparation of constructs containing peptides and nucleic acids as components) can be understood as designable matters by those skilled in the art based on existing technologies in the fields of cell engineering, physiology, medicine, pharmacy, organic chemistry, biochemistry, genetic engineering, protein engineering, molecular biology, genetics, etc.

[0018] In addition, the technology described in this specification can be implemented based on the content recorded in this specification and the technical common sense in this field. It should be noted that in this specification, there is a situation where amino acids are represented by single-letter symbols that conform to the amino acid nomenclature shown in the IUPAC-IUB Guide. It should also be noted that in this specification, unless otherwise stated, "amino acid residue" is a term that includes the N-terminal amino acid and the C-terminal amino acid of the peptide chain.

[0019] In addition, in this specification, "synthetic peptide" refers to a peptide fragment that is not stable and independent in nature, but is manufactured by artificial chemical synthesis or biosynthesis (i.e., production based on genetic engineering) and can be stably present in a given composition. Among them, "peptide" refers to a term for amino acid polymers having multiple peptide bonds, and is not limited to the number of amino acid residues.

[0020] Furthermore, in the present specification, unless otherwise specified, the amino acid residues constituting peptides and proteins may be either L isomers or D isomers.

[0021] It should be noted that, in the amino acid sequence described in this specification, the left side always indicates the N-terminal side, and the right side indicates the C-terminal side.

[0022] The carrier peptide fragment described in this specification contains the amino acid sequence shown in sequence number 1: RKLAVYWSTYKRSR, and is usually composed of the amino acid sequence shown in sequence number 1. The amino acid sequence has cell membrane permeability and can be introduced from the outside of a eukaryotic cell into the inside of the cell.

[0023] The carrier peptide fragment described in this specification can also be based on a modified sequence of the amino acid sequence shown in SEQ ID NO. 1, as long as it does not impair cell membrane permeability. Wherein, "modified sequence" refers to an amino acid sequence (modified amino acid sequence) formed by replacing, deleting and / or adding (inserting) one or more (usually two or three) amino acid residues. Therefore, the carrier peptide fragment described in the present invention can be composed of, for example, 14 to 20, 14 to 17 or 14 to 15 amino acid residues.

[0024] As typical examples of modified sequences in the present specification, there can be cited sequences produced by so-called conservative amino acid replacements of, for example, 1, 2 or 3 amino acid residues conservatively replaced, and sequences in which 1, 2 or 3 amino acid residues are added (inserted) or deleted in the amino acid sequence, etc. As typical examples of similar replacements, there can be cited sequences in which, for example, basic amino acid residues are replaced by other basic amino acid residues (e.g., mutual replacement of lysine residues and arginine residues), sequences in which hydrophobic amino acid residues are replaced by other hydrophobic amino acid residues (e.g., mutual replacement of leucine residues, isoleucine residues and valine residues), etc.

[0025] The construct for introducing a foreign substance of the present invention comprises the carrier peptide fragment of the present invention and a foreign substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment.

[0026] The construct of the present invention can be designed and constructed by directly or indirectly combining (linking) with the N-terminal side and / or C-terminal side of the above-mentioned carrier peptide fragment via an appropriate linker.

[0027] The linker is not particularly limited and may be a peptide linker or a non-peptide linker. It is preferred, but not particularly limited, that the amino acid sequence constituting the peptide linker is an amino acid sequence that does not cause steric hindrance and is flexible. The peptide linker may be a linker comprising, for example, one or more of 10 or less amino acid residues (more preferably 1 or more and 5 or less, for example 1, 2, 3, 4 or 5 amino acid residues) selected from glycine, alanine, serine, etc. In addition, as such a linker, β-alanine may also be used. As a non-peptide linker, for example, an alkyl linker, a PEG (polyethylene glycol) linker, an aminocaproyl spacer, etc. may be used but is not particularly limited.

[0028] Exogenous substances can be organic compounds such as polypeptides, nucleic acids, pigments, drugs, etc.

[0029] In the case where the exogenous substance is a polypeptide, a peptide chain is designed in such a manner as to include the amino acid sequence constituting the polypeptide and the amino acid sequence constituting the carrier peptide fragment, and the peptide chain is biosynthesized or chemically synthesized, thereby preparing a construct for introducing the target exogenous substance. In addition, a construct can be prepared by directly or indirectly binding an organic compound that functions as a nucleic acid such as various DNA or RNA, a pigment (e.g., various fluorescent pigment compounds such as FAM and FITC), or a drug (e.g., an antitumor agent including a nucleic acid-based antitumor agent such as 5-fluorouracil (5FU), an antiviral agent such as azidothymidine (AZT), etc.) to the N-terminal side and / or C-terminal side of the above-mentioned carrier peptide fragment using various conventionally known scientific methods.

[0030] The functions of the exogenous substance may be, but are not particularly limited to, promotion of stem cell differentiation induction (stem cell differentiation inducing activity), inhibition of tumor cell proliferation (anti-tumor activity), inhibition of virus-infected cell proliferation (anti-viral activity), etc.

[0031] In the construct of the present invention, the number of exogenous substances bound to the carrier peptide fragment is not particularly limited. For example, one or more exogenous substances may be bound to one carrier peptide fragment. For example, it may be, but is not particularly limited to, binding a polypeptide, nucleic acid, drug, etc. to the C-terminal side of one carrier peptide fragment, and binding a pigment to the N-terminal side. By binding the pigment to the carrier peptide fragment, it is easy to evaluate the efficiency of the construct introduced into eukaryotic cells and its localization in the cell, and therefore it is preferred.

[0032] It should be noted that when the exogenous substance is a polypeptide, the polypeptide (amino acid sequence) used is not particularly limited. For example, a substance with a relatively large number of amino acid residues such as a polypeptide having about 100 to 1000 amino acid residues or a protein can be used as the exogenous substance.

[0033] Generally, the total number of amino acid residues constituting a synthetic peptide prepared as a construct for introduction of an exogenous substance is several to several tens (e.g., 10) or more, and a suitable value is 1000 or less, preferably 600 or less, more preferably 500 or less, and particularly preferably 300 or less (e.g., 10 to 300). Polypeptides of this length are easy to synthesize (biosynthesize, chemically synthesize) and easy to use.

[0034] As exogenous substances, mature polypeptides or polypeptide precursors (including pro- and pre-pro-) related to functions such as development, differentiation, proliferation, carcinogenesis, homeostasis (constancy), and metabolic regulation of various cells and tissues (organs), etc. are preferred. In addition, the exogenous substance introduction method of the present invention can also be implemented to introduce a polypeptide whose function is previously unknown into a cell and to clarify the function of the polypeptide in the cell (in a living tissue).

[0035] For example, when the eukaryotic cells to be introduced into the exogenous substance are stem cells of humans and other mammals, it is preferred to use mature polypeptides or polypeptide precursors having various physiological activities related to the differentiation induction of the stem cells. It should be noted that "stem cells" include somatic stem cells, embryonic stem cells, and artificially induced pluripotent stem cells (Induced pluri-potent stem cells: iPS cells). In addition, when the eukaryotic cells to be introduced into the exogenous substance are cancer cells (tumor cells), it is preferred to use various polypeptides related to the apoptosis induction of the cancer cells (tumor cells). Alternatively, in this case, it is preferred to use polypeptides that can hinder the function of cancer cells (tumor cells) to inhibit the immune surveillance mechanism. In addition, when the eukaryotic cells to be introduced into the object are bacterially infected cells or virally infected cells, it is preferred to use various polypeptides related to the apoptosis induction of the infected cells, polypeptides that can inhibit the proliferation of bacteria or viruses in the infected cells, or polypeptides that can inhibit the spread of bacterial or viral infections from the infected cells.

[0036] It should be noted that the polypeptide and carrier peptide fragment as exogenous substances may also contain modified amino acid sequences formed by replacement, deletion and / or addition (insertion) of one or more amino acid residues, as long as their functions are maintained.

[0037] In the construct in which the foreign substance is bound to the C-terminal side of the carrier peptide fragment, the α-amino group of the amino acid residue on the N-terminal side of the carrier peptide fragment is preferably acetylated. Although the detailed mechanism has not been ascertained, since the α-amino group of the amino acid on the N-terminal side of the protein in most eukaryotic cells is acetylated, such a configuration can improve the intracellular stability of the construct.

[0038] The amino acid residue on the C-terminal side of the construct is preferably amidated. If the carboxyl group of the amino acid residue (usually the C-terminal amino acid residue of the peptide chain) is amidated, the structural stability (e.g., protease resistance) of the construct in the cytoplasm and nucleolus can be improved. In addition, by amidating the carboxyl group, the hydrophilicity of the construct can be improved, thereby improving the solubility of the construct in an aqueous solvent. As such an aqueous solvent, for example, water, various buffer solutions, physiological saline (e.g., PBS), cell culture fluid, etc. can be cited.

[0039] For example, in the case of a construct in which an exogenous substance is bound to the N-terminal side of a carrier peptide fragment, the carboxyl group of the amino acid residue on the C-terminal side of the carrier peptide fragment is preferably amidated. In addition, for example, in the case in which the exogenous substance is a polypeptide and the polypeptide is bound to the C-terminal side of a carrier peptide fragment, the carboxyl group of the C-terminal amino acid residue of the polypeptide is preferably amidated.

[0040] Products with shorter peptide chains (including polypeptides, carrier peptide fragments and peptide linkers constituted as exogenous substances) in the construct can be easily manufactured according to conventional chemical synthesis methods. For example, any of the conventional known solid phase synthesis methods or liquid phase synthesis methods can be used. It is suitable to use a solid phase synthesis method using Boc (tert-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group for the amino group. That is, by using a solid phase synthesis method using commercially available peptide synthesis equipment, it is also possible to synthesize the above-mentioned peptide chain having the desired amino acid sequence and modified (N-terminal acetylation, C-terminal amidation, etc.) parts. It should be noted that it is also possible to synthesize only a part of the peptide chain by the above method, for example, only synthesize the carrier peptide fragment, or synthesize a peptide chain containing the carrier peptide fragment and the peptide linker part.

[0041] Alternatively, the peptide portion can also be biosynthesized based on genetic engineering methods. That is, a polynucleotide (usually DNA) encoding a nucleotide sequence (including an ATG start codon) encoding the desired amino acid sequence is synthesized. Then, a recombinant vector having an expression gene construct including the synthesized polynucleotide (DNA) and various acting factors (including promoters, ribosome binding sites, terminators, enhancers, and various cis-acting factors that control the expression level) for expressing the amino acid sequence in the host cell is constructed according to the host cell.

[0042] The recombinant vector is introduced into a predetermined host cell (e.g., yeast, insect cell, plant cell) by conventional techniques, and the host cell or a tissue or individual containing the cell is cultured under predetermined conditions. Thus, the target peptide can be produced in the cell. Then, the target peptide portion can be obtained by isolating the peptide portion from the host cell (in the culture medium if secretion has already occurred), and refolding, refining, etc. as needed.

[0043] It should be noted that the method for constructing a recombinant vector and the method for introducing the constructed recombinant vector into a host cell can directly adopt the methods that have been used in the art in the past. Since these methods themselves are not technical contents that bring specific characteristics to the technology described in this specification, detailed descriptions are omitted.

[0044] For example, in order to achieve efficient and large-scale production in host cells, a fusion protein expression system can be used. That is, a gene (DNA) encoding the amino acid sequence of the target polypeptide is chemically synthesized, and the synthetic gene is introduced into a suitable site of an appropriate fusion protein expression vector (for example, the pET series provided by Novogene and the pGEX series provided by Amersham Biosciences, etc. GST (glutathione S-transferase) fusion protein expression vector). Then, the host cell (usually Escherichia coli) is biotransformed using the vector. The resulting biotransformant is cultured to prepare the target fusion protein. The protein is then extracted and refined. The resulting refined fusion protein is then cleaved with a predetermined enzyme (protease), and the released target peptide fragment (i.e., the designed artificial polypeptide) is recovered by affinity chromatography and the like. By adopting this conventional known fusion protein expression system (for example, the GST / His system provided by Amersham Biosciences can be used), the target construct (artificial polypeptide) can be manufactured.

[0045] Alternatively, a cell-free protein synthesis system can be constructed using a template DNA (i.e., a synthetic gene fragment containing a nucleotide sequence encoding the amino acid sequence of the peptide portion of the construct), and various compounds required for synthesizing the peptide portion (ATP, RNA polymerase, amino acids, etc.) can be used to synthesize the target polypeptide in vitro using a so-called cell-free protein synthesis system. For cell-free protein synthesis systems, reference can be made to, for example, the paper by Shimizu et al. (Shimizu et al., Nature Biotechnology , 19, 751-755 (2001)) and Madin et al. Proc.Natl.Acad.Sci. USA, 97 (2), 559-564 (2000)). Based on the technologies described in these papers, many companies have been engaged in OEM production of polypeptides at the time of filing this application, and they have been marketed as kits for cell-free protein synthesis (for example, available from Cellfree Science Co., Ltd. in Japan).

[0046] A single-stranded or double-stranded polynucleotide including a nucleotide sequence encoding the peptide portion of the construct and / or a nucleotide sequence complementary to the sequence can be easily produced (synthesized) by conventionally known methods. That is, by selecting codons corresponding to each amino acid residue constituting the designed amino acid sequence, a nucleotide sequence corresponding to the amino acid sequence can be easily determined and provided. Moreover, once the nucleotide sequence is determined, a polynucleotide (single-stranded) corresponding to the desired nucleotide sequence can be easily obtained using a DNA synthesis device or the like. The obtained single-stranded DNA can then be used as a template to obtain a target double-stranded DNA using various enzymatic synthesis methods (usually PCR). In addition, the polynucleotide may be in the form of DNA or in the form of RNA (mRNA, etc.). DNA may be provided in a double-stranded or single-stranded form. When provided in a single-stranded form, it may be either a coding strand (sense strand) or a non-coding strand (antisense strand) having a complementary sequence thereto.

[0047] The polynucleotide thus obtained can be used as a material for constructing a recombinant gene (expression cassette) for peptide production in various host cells or cell-free protein synthesis systems as described above.

[0048] The construct of the present invention can be used as an active component of a composition for use based on the function of an exogenous substance. It should be noted that the construct can also be in the form of a salt, as long as the function of the exogenous substance is not lost. For example, an acid addition salt obtained by an addition reaction of an inorganic acid or an organic acid, which is usually used according to a conventional method, can be used. Therefore, the "construct" recorded in this specification and claims also includes the form of such a salt.

[0049] The construct can also be used as an active ingredient of a composition which may contain various pharmaceutically acceptable carriers depending on the form of use.

[0050] As the above-mentioned carrier, it is preferred that the carrier is commonly used in peptide drugs as a diluent, excipient, etc. As such a carrier, although it may be appropriately different depending on the purpose and form of the construct for the introduction of exogenous substances, water, physiological buffer and various organic solvents can be generally cited. In addition, the carrier can also be a non-drying oil such as an alcohol (ethanol, etc.) aqueous solution, glycerol, olive oil, etc. of an appropriate concentration, or it can also be a liposome. In addition, as a minor component that may be included in the pharmaceutical composition, various fillers, fillers, binders, wetting agents, surfactants, pigments, fragrances, etc. can be cited.

[0051] The form of the composition is not particularly limited. Examples thereof include liquids, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, ointments, and the like. In addition, for use in injections, a lyophilized product or granulated product that is dissolved in physiological saline or an appropriate buffer (e.g., PBS) or the like to prepare a drug solution immediately before use may also be used.

[0052] The process of preparing various forms of drugs (compositions) using the construct (main component) and various carriers (secondary components) as materials can be carried out according to the conventionally known methods. These manufacturing methods themselves are not methods that bring characteristics to the technology of the present invention, so detailed descriptions are omitted. As a source of information on the details of the formulation, for example, "Comprehensive Medicinal Chemistry" edited by Corwin Hansch and published by Pergamon Press can be cited. Comprehensive Medicinal Chemistry ) (1990).

[0053] According to the present invention, a method for introducing exogenous substances in vivo or in vitro using the construct of the present invention is provided. The method mainly comprises the following steps (1) to (2): (1) a step of preparing a construct for introducing an exogenous substance comprising the carrier peptide fragment of the present invention and a target exogenous substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment; and (2) A step of supplying the construct to a sample containing target eukaryotic cells. In addition, the method of the present invention may further include, after the step (2), a step (3) of incubating the sample to which the construct has been supplied, and introducing the construct into the eukaryotic cells in the sample.

[0054] In vivo, the above-mentioned "eukaryotic cells" include, for example, various tissues, viscera, organs, blood, lymph, etc. In vitro, the above-mentioned "eukaryotic cells" include, for example, various cell clusters, tissues, viscera, organs, blood, lymph, and cell lines removed from living organisms. Examples of eukaryotic cells include cells derived from the animal kingdom, such as mammals, birds, fish, amphibians, reptiles, and insects, cells derived from the fungal kingdom, and cells derived from the plant kingdom. Preferably, the cells may be human or mammals other than human.

[0055] The composition comprising the construct of the present invention can be used in vivo in a method and dosage corresponding to its form and purpose. For example, as a liquid preparation, it can be administered to the affected part (e.g., malignant tumor tissue, virus-infected tissue, inflammatory tissue, etc.) of the patient (i.e., living organism) by intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal injection in a suitable required amount. Alternatively, solid form preparations such as tablets, gel-like or aqueous gel-like preparations such as ointments can be directly applied to a set tissue (i.e., an affected part such as a tissue or organ including, for example, tumor cells, virus-infected cells, inflammatory cells, etc.). Alternatively, solid form preparations such as tablets can also be administered orally. In the case of oral administration, it is preferred to use a capsule or protective (coating) material that can inhibit the decomposition of digestive enzymes in the digestive tract.

[0056] Alternatively, for eukaryotic cells cultured in vitro, an appropriate amount of the construct may be supplied to the target eukaryotic cell culture medium at least once. The amount supplied each time and the number of times supplied vary depending on the type of eukaryotic cells cultured, cell density (cell density at the start of culture), number of passages, culture conditions, type of culture medium, and other conditions, and are therefore not particularly limited. For example, it is preferred to add once, twice, or more times so that the concentration of the carrier peptide fragment in the culture medium is generally in the range of 0.05 μM or more and 100 μM or less, for example, in the range of 0.5 μM or more and 50 μM or less, or, for example, in the range of 1 μM or more and 30 μM or less. In addition, the incubation time after adding the construct also varies depending on the type of eukaryotic cells and various conditions, and is therefore not particularly limited. For example, it may be more than 0.5 hours, more than 1 hour, more than 4 hours, more than 8 hours, or more than 20 hours. It should be noted that the incubation conditions also vary depending on the type of eukaryotic cells, and are therefore not particularly limited, and may be incubated at a temperature of 37° C. in an environment of 5% CO2.

[0057] It should be noted that in the test examples described below, an example of an in vitro introduction method will be given.

[0058] The method for evaluating the introduction efficiency of the construct is not particularly limited. For example, when a pigment (usually a fluorescent pigment compound) is bound to the construct, the introduction efficiency into eukaryotic cells can be evaluated using microscopic observation (e.g., fluorescence microscopic observation), flow cytometry, etc. In addition, the introduction efficiency of the construct can also be evaluated by immunochemical methods (e.g., Western Blotting, immunocytological staining, etc.) using antibodies that specifically recognize the peptide portion of the construct.

[0059] As described above, as specific embodiments of the technology described in this specification, the following items can be cited.

[0060] Item 1: A carrier peptide fragment, wherein it consists of the amino acid sequence: RKLAVYWSTYKRSR (SEQ ID NO: 1).

[0061] Item 2: A construct comprising the carrier peptide fragment described in Item 1 and a foreign substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment.

[0062] Item 3: The construct according to Item 2, wherein the exogenous substance is at least one organic compound selected from the group consisting of polypeptides, nucleic acids, pigments and drugs.

[0063] Item 4: The construct according to Item 2 or Item 3, wherein the exogenous substance is disposed on the C-terminal side of the carrier peptide fragment.

[0064] Item 5: A method for introducing a target exogenous substance from outside a eukaryotic cell into at least the cytoplasm of the cell in vitro, comprising: (1) a step of preparing the construct according to any one of items 2 to 4; and (2) A step of supplying the above construct to a sample containing target eukaryotic cells.

[0065] Item 6: The method according to Item 5, wherein the eukaryotic cell into which the construct is introduced is a cell of a human or a mammal other than a human.

[0066] Several relevant test examples of the technology described in this specification are described below, but it is not intended to limit the technology described in this specification to the contents shown in these test examples.

[0067] In Example 1, the constructs shown in Table 1 were prepared as sample 1. Specifically, a synthetic peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in which the α-amino group of the arginine residue at the N-terminal side is acetylated, and a fluorescent dye FAM (C 21 H 12 O7: 5(6)-carboxyfluorescein, molecular weight 376.3, excitation wavelength 495 nm, fluorescence wavelength 520 nm). The construct was diluted with dimethyl sulfoxide (DMSO) to prepare a sample solution 1 with a sample concentration of 2 mM.

[0068] HeLa cells (a mature cell line derived from human cervical cancer cells) were used as eukaryotic cells to evaluate the cell membrane permeability of sample 1. In Example 1, the sample solution 1 prepared above was used, and in Example 12, a FAM solution was used.

[0069] HeLa cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Catalog No. 044-29765) containing 10% FBS (fetal bovine serum).

[0070] After washing the HeLa cells adhered to the culture plate with PBS, a 0.25% trypsin / EDTA solution was added and incubated at 37°C for 3 minutes. After the incubation, the DMEM containing 10% FBS was added to inactivate the trypsin, and then the cells were centrifuged at 150×g for 5 minutes to precipitate the cells. After removing the supernatant generated by centrifugation, the DMEM containing 10% FBS was added to the precipitate (cell pellet) to prepare approximately 2×10 5 1 mL of the cell suspension was added to the wells of a commercially available 6-well plate (manufactured by AJEXA TECHNO GLASS CO., LTD.), and cells (approximately 2×10 5 cells / well). In addition, the 2 mM sample solution 1 was diluted with the DMEM containing 10% FBS to prepare a sample solution 1 with a sample 1 concentration of 20 μM. Then, 1 mL of the 20 μM sample solution 1 was added to the well (i.e., the sample 1 concentration in the medium in the well was 10 μM, and the DMSO concentration was 0.5%). The cells were then incubated for 20 hours at 37°C under 5% CO2.

[0071] After incubation for 20 hours, the culture supernatant was removed from the wells, and the cells in the wells were washed twice with 1 mL of PBS. Next, 200 μL of 0.25% trypsin / EDTA solution was added to the wells, and incubated at 37°C for 3 minutes. After the incubation, 400 μL of the above-mentioned DMEM containing 10% FBS was added to the wells to inactivate trypsin, and then the cell suspension in the wells was pipetted into a test tube to recover the cells. Thereafter, 600 μL of PBS was further added to the wells to wash the wells. Then, the PBS in the wells was pipetted into the above-mentioned test tubes to recover the cells remaining in the wells into the above-mentioned test tubes. The test tubes were centrifuged at 4°C and 210×g for 5 minutes. After centrifugation, the supernatant was removed, and the precipitate (cell pellet) was suspended (washed) with 1 mL of PBS and centrifuged under the same conditions as above. This operation was repeated twice, and then the supernatant was removed to obtain cells (cell pellets) cultured in the medium containing Sample 1.

[0072] The cell membrane permeability of the sample 1 of the cells (cell pellets) obtained above was analyzed using a flow cytometer. As the flow cytometer, an On-Chip flow cytometer (manufactured by On-Chip Biotechnologies Co., LTD.) was used.

[0073] For this analysis, the cell pellet obtained above was suspended in 100 μL of On-Chip T buffer to prepare a cell suspension for analysis.

[0074] The flow cytometer was used to perform selection based on forward scatter (FSC) and side scatter (SSC), set gates for the cell population to be analyzed, and measured the fluorescence intensity of the cell population within the gate. It should be noted that the analysis was performed in a state where the number of cells in the cell population was at least 10,000. The fluorescence intensity was measured using the fluorescence detector FL2 of the flow cytometer that can detect the FAM fluorescence wavelength (the optimal detection wavelength is around 543nm). The commercially available analysis software "FlowJo ® ” (manufactured by TreeStar) The measurement results were analyzed to obtain the median fluorescence intensity (MFI) of the measurement target cell group.

[0075] In Example 2, the same method as in Example 1 was used except that the sample solution 1 was changed to a FAM solution diluted with DMSO. It should be noted that the concentration of the FAM solution was the same as that of the sample solution 1 (i.e., the FAM concentration of the culture medium in the well was 10 μM and the DMSO concentration was 0.5%). The results of Examples 1 and 2 are shown in Tables 1 and Figure 1 . Figure 1 Schematic diagram of the histogram obtained by flow cytometry.

[0076] [Table 1] like Figure 1 As shown in the figure, the histogram of Example 1 is shifted to the right compared to the histogram of Example 2. It can be seen that the addition of FAM to the synthetic peptide formed by the amino acid sequence shown in SEQ ID NO: 1 has higher cell membrane permeability than using FAM alone. In other words, the synthetic peptide is a carrier peptide fragment that exhibits cell membrane permeability.

[0077] The above detailed description of the specific examples of the technology described in the present invention is only for illustration and does not limit the scope of protection claimed. The technology described in the scope of protection claimed includes various modifications and changes of the specific examples described above.

[0078] It should be noted that, although detailed data are not shown, the research of the inventors of the present invention has confirmed that the construct having the above-mentioned synthetic peptide can be efficiently introduced into the cytoplasm from the outside of the cell not only when the exogenous substance is a fluorescent pigment, but also when it is any one of polypeptides, nucleic acids and drugs.

[0079] Industrial Applicability According to the technology described in the present invention, there is provided an artificially manufactured carrier peptide fragment and a construct having the carrier peptide fragment for introducing a target exogenous substance from the outside of a eukaryotic cell (especially a human cell without a cell wall and various animal cells represented by non-human mammals) into the cytoplasm. By using the construct, the target exogenous substance can be more effectively introduced into the target cell, and living tissues such as cells into which the exogenous substance has been introduced and organs containing cells containing the exogenous substance can be obtained. In addition, by using the construct, a therapeutic drug targeted at a disease can be provided.

Claims

1. A carrier peptide fragment, characterized in that: It is composed of the following amino acid sequence: RKLAVYWSTYKRSR (serial number 1).

2. A construct, characterized in that Include: The carrier peptide fragment according to claim 1, and A foreign substance bound to the N-terminal side and / or the C-terminal side of the carrier peptide fragment.

3. The construct according to claim 2, characterized in that: The exogenous substance is at least one organic compound selected from polypeptides, nucleic acids, pigments and drugs.

4. The construct according to claim 2, characterized in that: The exogenous substance is disposed on the C-terminal side of the carrier peptide fragment.

5. A method for introducing a target exogenous substance from the outside of a eukaryotic cell into at least the cytoplasm of the cell in vitro, characterized in that: Include: (1) a step of preparing the construct according to any one of claims 2 to 4; and (2) A step of supplying the construct to a sample containing target eukaryotic cells.

6. The method according to claim 5, characterized in that: The eukaryotic cell to be introduced with the construct is a cell of a human or a mammal other than a human.

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