WO2016152882A1 - Collagen fusion protein and drug screening method using same - Google Patents
Collagen fusion protein and drug screening method using same Download PDFInfo
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- WO2016152882A1 WO2016152882A1 PCT/JP2016/059066 JP2016059066W WO2016152882A1 WO 2016152882 A1 WO2016152882 A1 WO 2016152882A1 JP 2016059066 W JP2016059066 W JP 2016059066W WO 2016152882 A1 WO2016152882 A1 WO 2016152882A1
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- collagen
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- fusion protein
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
Definitions
- the present invention relates to a collagen fusion protein and a method for screening a drug using the same, a vector containing a nucleic acid encoding the collagen fusion protein, E. coli containing the vector, and methods for producing them. According to the present invention, candidate substances for drugs for treating or preventing collagen-related diseases can be screened. Further, according to the present invention, a vector having a collagen fusion protein and a normal nucleic acid encoding it can be obtained.
- Collagen accounts for 30% of proteins in the body and is an important protein having functions such as skeletal support and cell adhesion. For example, bone / cartilage, ligament / tendon, corneal stroma, skin, liver, It is a major component of tissues such as muscle.
- Collagen includes fibrous collagen that forms collagen fibers and non-fibrous collagen that does not form fibers. Fibrous collagen forms a triple helical structure (tropocollagen) by gathering three polypeptide chains of collagen. The topocollagen having a triple helical structure is self-assembled to be aligned with a quarter of the molecular length to form collagen fibrils (collagen fibrils). Furthermore, many collagen fibrils are aggregated to form collagen fibers (collagen fiber bundles).
- the collagen fibers form a two-dimensional layer aligned in the same direction, and this layer has a plywood-like three-dimensional layer structure in which each layer is orthogonally stacked.
- Collagen having this plywood-like three-dimensional layered plate structure is transparent and has high strength.
- diseases caused by such molecular abnormality of collagen include collagen disease (for example, dermatomyositis, polymyositis) or corneal cloudiness, but there are many treatment methods that have not been established. This is partly due to the fact that the secretory process of collagen and the formation process of higher-order structures are still unclear. If it is possible to analyze the secretion process of collagen and the formation process of higher-order structures, it will be used as an effective means for elucidating the causes of diseases caused by collagen molecular abnormalities and developing treatments for them. be able to.
- collagen disease for example, dermatomyositis, polymyositis
- corneal cloudiness corneal cloudiness
- an object of the present invention is to construct a system capable of detecting procollagen and collagen secretion from animal cells.
- the present inventors used genetic recombination for the construction of the system. For example, Patent Document 1 reports on genetic manipulation of collagen proteins.
- the present inventors attempted to construct an expression vector using Escherichia coli by incorporating a nucleic acid encoding collagen into a vector.
- the present inventors when transformed with Escherichia coli using a vector that is usually used for protein expression, have deletions, insertions, and inhibition of restriction enzyme cleavage in genes encoding collagen proteins. I noticed it happened. That is, the present inventors could not obtain an expression vector containing the target collagen gene.
- This gene deletion / inhibition of restriction enzyme cleavage did not occur at specific positions, but occurred at random positions in the collagen gene. For example, it is known that deletion occurs at a specific position of a gene by homologous recombination.
- Another object of the present invention is to provide a vector having a nucleic acid which does not inhibit deletion, insertion or restriction enzyme cleavage encoding a collagen protein.
- the present invention [1] Preprocollagen protein, procollagen protein, collagen protein having a repetitive amino acid sequence of Gly-Xaa-Yaa (where Gly is glycine, and Xaa and Yaa are arbitrary amino acids), or mutations thereof A fusion protein of a collagen-related protein and a fluorescent protein or photoprotein, which is a body or a fragment thereof, [2] The fusion protein according to [1], wherein the fluorescent protein or photoprotein is bound or inserted into the N-terminal N-proprotein, collagen protein, or C-terminal C-proprotein of the procollagen protein.
- the fluorescent protein or photoprotein is inserted from the C-terminal of the collagen protein to the N-terminal side from 30 amino acids, and / or the fluorescent protein or photoprotein is C-terminal from the N-terminal of the C-proprotein to 30 amino acids
- the fusion protein according to [1] or [2], which is inserted on the side, [4] A collagen-related disease comprising a step of bringing a collagen-related disease cell expressing the fusion protein according to any one of [1] to [3] into contact with a test substance, and a step of analyzing the dynamics of the fusion protein
- Gly-Xaa and Yaa are arbitrary amino acids
- the Escherichia coli is DH10B, TOP10F ′, MC1061, XL1-Blue MRF ′, AG1, BL21 (DE3), DB3.1, DH1, DH5 ⁇ , DH5 ⁇ Turbo, DH12S, DM1, E. coli.
- a method for producing collagen comprising: (1) introducing the vector according to claim 6 into an animal cell; and (2) secreting collagen from the animal cell into which the vector has been introduced, and [14] (1) Introducing the vector according to claim 6 into an animal cell, and (2) detecting fluorescence or luminescence in the animal cell into which the vector is introduced.
- Luminescence detection method About.
- the fusion protein of the present invention can detect the secretion of procollagen from animal cells. Since collagen-related proteins form a triple helical structure, it is believed that when a fluorescent protein is inserted into a procollagen protein, the structure of the protein changes and a triple helical structure cannot be formed. However, by inserting a fluorescent protein at a specific position of collagen protein and / or C-proprotein, it becomes possible to maintain a triple helical structure and to construct a system that can detect the secretion of procollagen from animal cells. It was. In addition, by inserting a fluorescent protein at a specific position of N-proprotein, a triple helical structure can be maintained, and a system capable of detecting procollagen secretion from animal cells could be constructed.
- a gene encoding a collagen protein that does not cause inhibition of deletion, insertion, or cleavage with a restriction enzyme can be obtained.
- the vector-containing Escherichia coli of the present invention it is possible to obtain a vector containing a gene that encodes a collagen protein and does not have a defect such as a deletion.
- the method for producing a vector-containing Escherichia coli and the method for producing a vector of the present invention it is possible to produce a vector containing a gene encoding a collagen protein that does not have a defect such as a deletion and E. coli containing the vector.
- Collagen can be produced from animal cells using the vector obtained according to the present invention. Further, by using the vector obtained by the present invention, it is possible to analyze the formation process of the higher-order structure of collagen.
- FIG. 2 is a photograph (A) of electrophoresis of a vector amplified with TOP10F ′ and a vector amplified with XL-1 blue, and a photograph (B) of electrophoresis of a vector amplified with DH10B and XL-1 blue. It is the figure which showed the outline of the vector which codes the fusion protein by which EGFP was inserted in N proprotein of N terminal of procollagen protein, and mCherry was inserted in C proprotein of C terminal of procollagen protein.
- FIG. 2 is a photograph of the fusion protein of Example 1 expressed in mouse NIH3T3 cells.
- 2 is a photograph of the fusion protein of Example 1 expressed in mouse NIH3T3 cells. It is the photograph which expressed the fusion protein of Example 2 in the NIH3T3 cell of a mouse
- a fluorescence micrograph showing the dynamics of the expressed collagen fibers by introducing the vector of the present invention into normal hepatic stellate cells (2G92 cells) (A) and hepatic fibrosis hepatic stellate cells (5H cells) (C), and normal liver It is a photograph (B) in which TGF- ⁇ is added to stellate cells (2G92 cells) to activate collagen secretion.
- Fusion protein is a protein in which a collagen-related protein and a fluorescent protein or photoprotein are fused.
- collagen-related proteins will be described.
- Collagen-related protein is preprocollagen protein, procollagen protein, collagen protein having a repetitive amino acid sequence of Gly-Xaa-Yaa (where Gly is glycine, and Xaa and Yaa are arbitrary amino acids), or Or a fragment thereof.
- the collagen-related protein is not particularly limited as long as it has the Gly-Xaa-Yaa repetitive amino acid sequence.
- human type I to XXVIII type collagen protein, its pre-procollagen protein or procollagen protein Mention may be made of their variants, or fragments thereof.
- the preprocollagen protein has a signal peptide and N-proprotein on the N-terminal side of the collagen protein, and C-proprotein on the C-terminal side.
- the procollagen protein has N-proprotein on the N-terminal side of the collagen protein and C-proprotein on the C-terminal side.
- the repetitive amino acid sequence of Gly-Xaa-Yaa is called a collagen-like sequence, and having a collagen-like sequence of Gly-Xaa-Yaa is an important feature of collagen-related proteins.
- the biological species from which the collagen-related protein is derived is not limited, for example, mammals (for example, cows, pigs, sheep, goats, mice, rats, guinea pigs, or monkeys), birds (for example, chickens, Goose, duck, or ostrich), reptile (eg, crocodile), amphibian (eg, frog), fish (eg, sturgeon, tilapia, thailand, flounder, shark, sardine, tuna, puffer, goldfish, cod, flounder, or carp ), Or invertebrates (eg, jellyfish).
- mammals for example, cows, pigs, sheep, goats, mice, rats, guinea pigs, or monkeys
- birds for example, chickens, Goose, duck, or ostrich
- reptile eg, crocodile
- amphibian eg, frog
- fish eg, sturgeon, tilapia, thail
- the number of Gly-Xaa-Yaa repeating amino acid sequences contained in the collagen-related protein is not particularly limited as long as it is 2 or more, preferably 10 or more, more preferably 20 or more, More preferably, it is 30 or more, and most preferably 50 or more.
- the collagen-related protein can include an amino acid sequence other than the collagen-like sequence, and can include, for example, a signal peptide, proprotein, and telopeptide at the N-terminus or C-terminus.
- Collagen proteins are classified into fibrous collagen and non-fibrous collagen.
- fibrous collagen type I collagen, type II collagen, type III collagen, type V collagen are used.
- XI type collagen type I collagen has a “triple helical structure (tropocollagen)” formed by gathering three polypeptide chains having a molecular weight of about 100,000, and has a molecular weight of about 300,000. This triple helical structure has a length of 300 nm and is shaped like one hard rod having a diameter of 1.5 nm, and is called tropocollagen.
- the topocollagen having a triple helical structure is self-assembled to be aligned with a quarter of the molecular length to form collagen fibrils (collagen fibrils). Furthermore, many collagen fibrils are aggregated to form collagen fibers (collagen fiber bundles). Furthermore, in the corneal stroma and cortical bone, the collagen fibers form a two-dimensional layer aligned in the same direction, and this layer has a plywood-like three-dimensional layer structure in which each layer is orthogonally stacked. . Collagen having this plywood-like three-dimensional layered plate structure is transparent and has high strength.
- Xly of Gly-Xaa-Yaa is often proline or 3-hydroxyproline
- Yaa is 4-hydroxyproline or hydroxylysine. Hydroxyproline is not contained in ordinary proteins and is an amino acid unique to collagen, but it is considered that the triple helical structure is stabilized by hydrogen bonding between the hydroxyl group of hydroxyproline and hydrated water.
- the collagen protein is not limited in the cell, but, for example, a procollagen protein having N-proprotein on the N-terminal side and C-proprotein on the C-terminal side (hereinafter sometimes simply referred to as procollagen). ).
- the procollagen is not particularly limited, and examples include procollagen of type I to XXVIII collagen.
- the preprocollagen protein, the procollagen protein or the variant of the collagen protein is not particularly limited as long as it has a repetitive amino acid sequence of Gly-Xaa-Yaa.
- Examples of the mutant include those in which an amino acid is deleted, substituted, inserted, and / or added in the amino acid sequence of a natural collagen protein (for example, the above-mentioned type I to XXVIII collagen proteins).
- the number of amino acids to be deleted, substituted, inserted and / or added is not particularly limited, but is, for example, 100 or less, preferably 50 or less, more preferably 30 or less, and still more preferably 10 or less, most preferably 1 or several.
- type I collagen consists of about 1000 amino acids, but if it has more than 100 deletions, substitutions, insertions, and / or additions, it may not function as collagen.
- a variant collagen protein having 80% or more identity to the amino acid sequence of a natural collagen protein (for example, the above-mentioned type I to XXVIII collagen proteins) can be mentioned.
- the amino acid identity is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 97% or more. This is because when the amino acid identity is less than 80%, it may not function as collagen.
- the fragment of procollagen protein, collagen protein, or a variant thereof is not particularly limited as long as it is a partial peptide of procollagen protein, collagen protein, or variant thereof.
- Type I to XXVIII type collagen Partial peptides of those procollagens, or mutants thereof.
- the amino acid chain length, the cleavage site, and the like of the fragment are not particularly limited, and can be appropriately determined according to the purpose of gene construction.
- Fluorescent protein or photoprotein examples include, but are not limited to, a fluorescent protein that imparts fluorescence and a photoprotein that imparts luminescence.
- Fluorescent proteins or photoproteins include GFP, EGFP, mCherry, Sirius, EBFP, SBP2, EBP2, Azure, mKalama1, TagBFP, mBlueberry, mTurquoise, ECFP, CelCul, TP TurboGFP, CFP, AcGFP, TagGFP, AG (Azami-Green), mAG1, ZsGreen, EmGFP (Emerald), GP2, T-Sapphire, HyPer, TagYFP, mAmetrine, EYFP, YPh, FPhYFP turboYFP, ZsYello w, mBanana, mKO1, KO (Kusterrorism Orange), mOrange, mOrange2, mKO2, Keima570, TurboRFP, DsRe
- Collagen-related protein and fusion protein can be prepared using an expression vector described later, but can also be prepared by peptide synthesis.
- the fluorescent protein or photoprotein is inserted from the C-terminal of the collagen protein to the N-terminal side from 30 amino acids.
- a fluorescent protein or a luminescent protein is inserted from the N-terminal of C-proprotein to the C-terminal side from 30 amino acids.
- a fluorescent protein or photoprotein is inserted from the C-terminus of the collagen protein to the N-terminal side from 30 amino acids, and the fluorescent protein or photoprotein is introduced from the N-terminus of the C-proprotein. It is inserted into the C-terminal side from 30 amino acids.
- the insertion site of the fluorescent protein or luminescent protein into the collagen protein is more preferably N-terminal from 45 amino acids to the C-terminal of the collagen protein, and still more preferably N-terminal from 60 amino acids.
- the insertion site of the fluorescent protein or luminescent protein into C-proprotein is more preferably 45 amino acids from the N-terminus of C-proprotein to the C-terminal side, and even more preferably 60 amino acids from the C-terminal side.
- the nucleic acid of the present invention is a nucleic acid encoding the collagen-related protein or the fusion protein (hereinafter sometimes collectively referred to as “collagen nucleic acid”).
- Collagen nucleic acid a nucleic acid encoding a collagen-related protein such as the procollagen protein, collagen protein, or a variant thereof, or a fragment thereof, or a fusion protein of a collagen-related protein and a fluorescent protein or the like.
- the collagen nucleic acid of the present invention is a nucleic acid encoding a collagen-related protein such as the procollagen protein, collagen protein, or a variant thereof, or a fragment thereof, or a fusion protein of a collagen-related protein and a fluorescent protein or the like.
- the collagen nucleic acid of the present invention is a nucleic acid encoding a collagen-related protein such as the procollagen protein, collagen protein, or a variant thereof, or a fragment thereof, or a fusion protein of a collagen-related protein and
- the procollagen protein or the nucleic acid encoding the collagen protein is not limited, and can be extracted from, for example, a tissue of an organism having collagen or a separated cell of the organism. It can also be obtained from a vector containing the already isolated cDNA. It can also be prepared by DNA synthesis.
- the nucleic acid encoding the mutant can be obtained by introducing a mutation of the nucleic acid so that an amino acid is deleted, substituted, inserted, and / or added to, for example, a procollagen protein or a nucleic acid encoding a collagen protein.
- Nucleic acid mutations can be introduced by methods known in the art.
- mutation by which the amino acid which exists in nature is deleted, substituted, inserted, and / or added without using genetic manipulation can also be used.
- nucleic acid encoding the fragment a part of the nucleic acid encoding procollagen protein, collagen protein, or a variant thereof can be used.
- a nucleic acid encoding a procollagen protein, a collagen protein, or a fragment of a variant thereof can be obtained by cleaving the nucleic acid with a restriction enzyme.
- the nucleic acid contained in the vector of the present invention is not limited, and examples thereof include DNA or RNA.
- the vector of the present invention is not limited as long as it contains the collagen nucleic acid.
- the skeleton vector into which the collagen nucleic acid is introduced may be a cloning vector or an expression vector.
- bacteriophage can be mentioned as an RNA vector.
- the backbone vector a vector usually used in this field can be used without limitation.
- pBR322, pBR325, pUC118, pUC119, pKC30, pCFM536, pcDNA3.1, pcDNA3, pME, or pGEX examples include E. coli plasmids.
- the backbone vector includes a replication origin, a selection marker, and a promoter, and may include an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like as necessary.
- the collagen nucleic acid-containing vector can contain a nucleic acid other than a collagen nucleic acid.
- a fusion protein can be obtained by binding other proteins to collagen protein or the like, but a collagen nucleic acid-containing vector is a nucleic acid that encodes a protein to be bound to this collagen (hereinafter sometimes referred to as binding protein).
- binding protein can be included.
- the binding protein can be bound to the N-terminus or C-terminus of collagen protein or the like, or can be inserted into collagen protein or the like. That is, the nucleic acid encoding the binding protein may be bound to the 5 ′ side or 3 ′ side of the collagen nucleic acid, or may be inserted into the collagen nucleic acid.
- the vector of the present invention is not limited, but is preferably obtained from Escherichia coli having a specific gene mutation described below. That is, those introduced into E. coli having a specific gene mutation and recovered from the E. coli are preferred. This is because it is possible to obtain a vector containing a collagen nucleic acid in which deletion, insertion, or inhibition of cleavage with a restriction enzyme has not occurred.
- the vector-containing host cell of the present invention comprises the vector of the present invention.
- the host is not particularly limited, and examples include E. coli, actinomycetes, yeast, filamentous fungi, or animal cells, preferably E. coli or animal cells, more preferably E. coli having a specific gene mutation. Or it is an animal cell. This is because by using Escherichia coli having a specific gene mutation, a vector containing a collagen nucleic acid in which deletion, insertion, or inhibition of cleavage with a restriction enzyme has not occurred can be obtained. Further, collagen can be produced by using animal cells. Furthermore, it is possible to analyze the formation process of the higher-order structure of collagen.
- the Escherichia coli having a gene mutation containing the vector of the present invention is not limited, but preferably mcrA, ⁇ (mrr-hsdRMS-mcrBC), ⁇ 80lacZ ⁇ M15, ⁇ lacX74, deoR, recA1, endA1, araD139, ⁇ (ara , leu) 7697, galU, galK , rpsL (str r), E. coli having nupG, galE, hsdS, and gene mutation selected from the group consisting of combinations of two or more thereof.
- the combination of gene mutations is not limited, but preferably mcrA, ⁇ (mrr-hsdRMS-mcrBC), ⁇ 80lacZ ⁇ M15, ⁇ lacX74, deoR, araD139, ⁇ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG, and a genetic mutation selected from the group consisting of two or more thereof, more preferably mcrA, ⁇ (mrr-hsdRMS-mcrBC), recA, hsdS and two or more thereof
- a gene mutation selected from the group consisting of a combination more preferably a gene mutation selected from the group consisting of mcrA, ⁇ (mrr-hsdRMS-mcrBC), recA and combinations of two or more thereof, most Preferably, it is a gene mutation selected from the group consisting of mcrA, ⁇ (mrr-hsdRMS-m
- Escherichia coli having a gene mutation includes DH10B, TOP10F ′, MC1061, XL1-Blue MRF ′, AG1, BL21 (DE3), DB3.1, DH1, DH5 ⁇ , DH5 ⁇ Turbo, DH12S, DM1, E. coli.
- a method usually used in this field can be used without limitation.
- use of competent cells treated with calcium chloride, a protoplast method, a calcium phosphate method, a lipofection method, an electroporation method, or the like can be used.
- the vector-containing Escherichia coli production method of the present invention comprises mcrA, ⁇ (mrr-hsdRMS-mcrBC), ⁇ 80lacZ ⁇ M15, ⁇ lacX74, deoR, recA1, introduced into E. coli having a genetic mutation selected from the group consisting of endA1, araD139, ⁇ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG, galE, hsdS, and combinations of two or more thereof It is characterized by doing.
- the method of producing the vector of the present invention includes mcrA, ⁇ (mrr-hsdRMS-mcrBC), ⁇ 80lacZ ⁇ M15, ⁇ lacX74, deoR, recA1, endA1, araD139, ⁇ (ara, leu) 7697, galU, galK. , RpsL (str r ), nupG, galE, hsdS, and a step of introducing the vector into the E. coli having a genetic mutation selected from the group consisting of two or more thereof, and recovering the vector, To do.
- nucleic acid encoding a procollagen protein, collagen protein, or a variant thereof, or a fragment thereof “vector”, and “mcrA, ⁇ ( mrr-hsdRMS-mcrBC), ⁇ 80lacZ ⁇ M15, ⁇ lacX74, deoR, recA1, endA1, araD139, ⁇ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG, galE, hsdS, and two or more thereof
- Escherichia coli having a gene mutation selected from the group consisting of combinations” and the like are described in the above-mentioned sections “[1] Fusion protein”, “[2] Nucleic acid and vector”, and “[3] Vector-containing host cell”.
- a method for recovering the vector can be a method known in the art.
- a collagen nucleic acid-containing vector into which the vector has been introduced is cultured in an LB medium or the like, Grow E. coli.
- the proliferated Escherichia coli is recovered, and the vector DNA can be recovered using, for example, alkaline SDS method, boiling method, ethidium / cesium chloride density gradient centrifugation method, glass bead purification method, or a modified method thereof.
- the method for producing collagen of the present invention comprises (1) a step of introducing the vector of the present invention into an animal cell, and (2) a step of secreting collagen from the animal cell into which the vector has been introduced. ,including.
- the “vector” used in the method for producing collagen of the present invention is the “vector” described in the sections “[1] fusion protein”, “[2] nucleic acid and vector”, and “[3] vector-containing host cell”. Can be used.
- Animal cells used in the method for producing collagen of the present invention are not particularly limited as long as they are cells capable of expressing collagen.
- mammals for example, cows, pigs, sheep, goats, mice, Rats, guinea pigs, or monkeys
- birds eg, chickens, geese, ducks, or ostriches
- reptiles eg, crocodiles
- amphibians eg, frogs
- fishes eg, sturgeon, tilapia, thailand, flounder, shark
- invertebrates eg, jellyfish
- the method for introducing the vector into animal cells is not particularly limited, and a method known in the art can be used. For example, an electroporation method can be used.
- a method for secreting collagen from animal cells into which a vector has been introduced is not particularly limited, but can be performed by culturing animal cells in a medium.
- the culture temperature is not particularly limited, but can be cultured at, for example, 30 to 42, preferably 35 to 39 ° C.
- secretion outside the cell can be optimized by adjusting the protein concentration in the animal cell. For secretion, it is preferable that cells form a multilayer.
- the collagen fluorescence or luminescence detection method of the present invention comprises (1) a step of introducing the vector of the present invention into an animal cell, and (2) an animal cell into which the vector has been introduced. Detecting fluorescence or luminescence.
- the “vector” used in the method for detecting fluorescence or luminescence of collagen of the present invention is described in the above “[1] fusion protein”, “[2] nucleic acid and vector”, and “[3] vector-containing host cell”. A “vector” can be used.
- the animal cell used in the method for producing collagen of the present invention is not particularly limited as long as it is a cell capable of expressing collagen, but the animal cell described in the above “[5] Method for producing collagen”. Can be mentioned.
- Fluorescence or luminescence from animal cells can also be detected by a known method in this field, but can be detected by using, for example, a fluorescence microscope, a spectrophotometer, a confocal fluorescence microscope, or the like.
- Method for screening a substance for treating or preventing a collagen-related disease comprises contacting a collagen-related disease cell expressing the fusion protein with a test substance, And analyzing the kinetics of the fusion protein.
- the collagen-related disease is not particularly limited as long as collagen is considered to be related to the cause or symptoms of the disease, but for example, organ / tissue fibers such as liver fibrosis, pulmonary fibrosis, renal fibrosis, etc. Symptom, collagen disease, or indirect rheumatism.
- the collagen-related disease cell used in the screening method of the present invention may be a cell isolated from the aforementioned collagen-related disease patient or a cell prepared from a model animal.
- cells isolated from patients with collagen-related diseases for example, cells that secrete collagen can be used. Specific examples include hepatic stellate cells, fibroblasts, and chondrocytes isolated from patients with liver fibrosis. be able to.
- hepatic stellate cells or lung fibroblasts can be mentioned as cells produced from model animals.
- normal collagen-secreting cells are activated by, for example, TGF- ⁇ , and used as model cells for liver fibrosis, pulmonary fibrosis, renal fibrosis, collagen disease, or indirect rheumatism, and used as screening cells for each drug. be able to.
- test substance is not limited as long as it contains substances that may be related to the kinetics of collagen, or includes such substances.
- various known compounds peptides registered in the chemical file
- a compound group obtained by combinatorial chemistry technology (Terrett, NK et al., Tetrahedron, 51, 8135-8137, 1995), or phage display method (Felici, F. et al., J. Mol. Biol., 222, 301-310, 1991) or the like can be used, or a random peptide group or a low molecular weight compound can be used.
- microorganism culture supernatant, cell culture supernatant, in-vivo body fluid, natural components derived from plants or marine organisms, or animal tissue extracts can also be used as test substances for screening.
- the contacting step in the screening method of the present invention is a step of bringing a collagen-related disease cell expressing the fusion protein into contact with a test substance.
- concentration of the test substance can also be determined as appropriate. However, since it is considered that the test substance has an optimum concentration that affects the dynamics of collagen, it is preferable to dilute the test substance in several stages.
- the dynamic analysis step in the present invention is a step of analyzing the dynamics of the collagen fusion protein.
- Examples of the analysis of collagen dynamics include analysis of the expression level of collagen fusion protein, analysis of extracellular secretion of collagen fusion protein, analysis of intracellular transport of collagen fusion protein, or analysis of processing of collagen fusion protein. Can do.
- the kinetic analysis can be basically performed by comparing with a cell not contacted with a test substance. That is, when the expression of the collagen fusion protein is increased or decreased as compared to cells not contacted with the test substance, the test substance can be determined as a candidate for a therapeutic drug for collagen-related diseases.
- the test substance when the collagen fusion protein is secreted extracellularly compared to cells not contacted with the test substance, it can be determined that the test substance is effective for collagen-related diseases. Furthermore, when the intracellular transport of the collagen fusion protein is restored as compared with cells not contacted with the test substance, it can be determined that the test substance is effective for collagen-related diseases. Further, when the processing of the collagen fusion protein is restored normally as compared with the cells not contacted with the test substance, it can be determined that the test substance is effective for collagen-related diseases.
- a candidate substance is used.
- yellow fluorescence consisting of fluorescence of EGFP and m-Cherry is observed in the cells.
- a candidate substance having a therapeutic effect is brought into contact, the C-proprotein with m-Cherry inserted is cleaved, and the collagen protein with EGFP inserted is normally secreted outside the cell, and green fluorescence is observed.
- the extracellular secretion of collagen protein is observed even in normal hepatic stellate cells, and it can be determined that a candidate substance exhibiting such kinetics is effective for liver fibrosis.
- the determination of kinetic analysis used for screening is not limited to these, and it may be determined that it has an effect on collagen-related diseases when it shows different kinetics compared to cells not contacted with the test substance. it can.
- Comparative Example 1 An attempt was made to construct a vector having a nucleic acid in which GFP was inserted in the center of type 1 collagen and mCherry was bound to the C-terminus. GFP was inserted into the BamH1 site of the collagen protein, and mCherry was inserted into the EcoR1 site of the C-proprotein. A schematic diagram of the vector is shown in FIG. The nucleotide sequence of DNA other than the vector is shown in SEQ ID NO: 1. A DNA fragment in which the restriction enzyme BamH1 site was added to both ends of the EGFP cDNA was prepared, and this was inserted into BamH1site inside the human preprocollagen1 ⁇ 1 cDNA.
- the protein translation was adjusted so that the collagen protein and the EGFP protein were connected.
- a DNA fragment in which EcoR1 site was added to both ends of the mCherry cDNA was prepared, and this was inserted into the EcoR1 site of the human preprocollagen 1 ⁇ 1-EGFP fusion cDNA.
- protein translation was adjusted so that the collagen protein and the mCherry protein were connected, and the base sequence was confirmed in each step of the operation.
- the XL-1 blue competent cell (transformation-receptive cell) was prepared in accordance with “Genetic Library Preparation Method” (edited by Hiroshi Nojima, Yodosha).
- the cryopreserved competent cells were thawed and immediately 100 ng of plasmid DNA was added and mixed. The mixture was allowed to stand on ice for 30 minutes, then warmed at 42 ° C. for 1 minute and 30 seconds, and immediately cooled on ice. 1 mL of medium was added thereto, and 100 ⁇ L was spread on an LB plate to which ampicillin had been added. Incubated overnight at 37 ° C., and the colonies that appeared were used.
- the DNA contained in the obtained vector was shorter than the target length, longer than the target length, or not cut with NotI or SmaI.
- FIG. 2A shows a longer one than the target length, one that could not be cut with NotI, and SmaI.
- ⁇ Comparative Example 2 An attempt was made to construct a vector having a nucleic acid having EGFP bound to the N-terminal of type 1 collagen and mCherry bound to the C-terminus. EGFP was inserted into the Kpn1 site of N-proprotein, and mCherry was inserted into the EcoR1 site of C-proprotein. A schematic diagram of the vector is shown in FIG. The nucleotide sequence of DNA other than the vector is shown in SEQ ID NO: 2. ⁇ Vector Construction Method> A DNA fragment in which the restriction enzyme KpnI site was added to both ends of the EGFP cDNA was prepared and inserted into the KpnI site inside the human preprocollagen1 ⁇ 1 cDNA.
- the protein translation was adjusted so that the collagen protein and the EGFP protein were connected.
- a DNA fragment in which EcoR1 sites were added to both ends of the mCherry cDNA was prepared, and this was inserted into the EcoR1 site of the human preprocollagen 1 ⁇ 1-EGFP fusion cDNA.
- protein translation was adjusted so that the collagen protein and the mCherry protein were connected, and the base sequence was confirmed in each step of the operation.
- the XL-1 blue competent cell (transformation-receptive cell) was prepared in accordance with “Genetic Library Preparation Method” (edited by Hiroshi Nojima, Yodosha).
- the cryopreserved competent cells were thawed and immediately 100 ng of plasmid DNA was added and mixed. The mixture was allowed to stand on ice for 30 minutes, then warmed at 42 ° C. for 1 minute and 30 seconds, and immediately cooled on ice. 1 mL of medium was added thereto, and 100 ⁇ L was spread on an LB plate to which ampicillin had been added. Incubated overnight at 37 ° C., and the colonies that appeared were used.
- the DNA contained in the obtained vector was shorter than the target length, longer than the target length, or not cut with NotI or SmaI.
- FIG. 2B shows a photograph cut with EcoRI, but all clones could not be cut with EcoRI.
- Example 1 A vector was obtained by repeating the operation of Comparative Example 1 except that TOP10F ′ was used instead of XL-1 blue. The obtained vector was subjected to nucleotide sequencing and confirmed to contain a nucleic acid of the desired length (FIG. 2A).
- Example 2 A vector was obtained by repeating the operation of Comparative Example 2 except that DH10B was used instead of XL-1 blue. The resulting vector contained the desired length of nucleic acid.
- FIG. 2B shows a photograph cut with EcoRI. All clones were cut with EcoRI. On the other hand, when XL-1 blue was used as a host, a normal clone that could be cleaved with EcoRI could not be obtained.
- Example 3 the fluorescence of collagen secreted extracellularly was observed.
- the vector obtained in Example 1 was introduced into NIH3T3 cells. Long-term culture for one month or longer was performed while changing the medium every two days to obtain a cell population consisting of multiple layers.
- the fluorescence signal of EGFP was observed with a fluorescence microscope. As shown in FIG. 4, when EGFP is inserted at the N-terminal side of the collagen protein, the collagen protein forms a triple helical structure and accumulates extracellularly (green fluorescence of EGFP). Was confirmed.
- Example 4 the fluorescence of collagen in the cells was observed.
- the vector obtained in Example 1 was introduced into NIH3T3 cells.
- the fluorescence signals of EGFP and mCherry were observed 48 hours later using a fluorescence microscope.
- EGFP is inserted into the N-terminal side of the collagen protein and mCherry is inserted into the C-terminal side of the C-proprotein, so that yellow that is procollagen and green that is collagen after processing are green. It was confirmed that a fluorescent signal was present in the cytoplasm.
- Collagen protein is also secreted extracellularly, but unlike Example 3, since the culture period was short, the amount of collagen protein accumulated was small, and EGFP green fluorescence was not observed.
- Example 5 the vector of FIG. 3 obtained by the same procedure as in Example 2 was introduced into mouse NIH3T3 cells, and fluorescence was detected with a confocal fluorescence microscope.
- the vector of FIG. 3 was introduced into NIH3T3 cells.
- the fluorescence signals of EGFP and mCherry were observed 48 hours later using a fluorescence microscope.
- EGFP is inserted into the N proprotein and mCherry is inserted into the C proprotein.
- the procollagen immediately after synthesis is indicated by a yellow signal, and the N proprotein cleaved by processing is A green fluorescent signal confirmed the presence in the cytoplasm.
- N-proprotein is also secreted extracellularly, but since it diffuses into the medium, EGFP green fluorescence was not observed.
- MCherry was inserted into the restriction enzyme AccIII site present in the C proprotein of the preprocollagen cDNA.
- the AccIII site is present at a site corresponding to 27 amino acids from the N-terminus of C-proprotein (FIG. 12A).
- the insertion of mCherry from the N-terminal of C-proprotein to the N-terminal side of 30 amino acids inhibits collagen fibrosis (triple helical structure construction). That is, collagen protein was accumulated in the ER-Golgi body and was not secreted extracellularly (FIGS. 12B and C).
- a fusion protein in which EGFP was bound to the C-terminus of the collagen protein was prepared (FIG. 12A). This fusion protein also inhibits collagen fibrosis (triple helical structure construction). That is, the collagen protein was localized in the cytoplasm and not secreted extracellularly (FIG. 12B).
- Example 6 a screening system for drugs for liver fibrosis was constructed.
- (1) Introduction of vector into hepatic stellate cells Steadily activated hepatic stellate cells (5H cells) that cause liver fibrosis isolated from the liver fibrosis rat obtained in Example 1 and activation Not transfected hepatic stellate cells (2G92 cells). Specifically, it was introduced into the cells using a transfection reagent (Fugne 6). As shown in FIG. 7, green fluorescence indicating normal processing and secretion of collagen protein was observed in non-activated hepatic stellate cells (FIG. 7A). However, only a yellow signal indicating abnormal collagen processing and secretion was observed in steady activated hepatic stellate cells (5H cells), and no green fluorescence indicating normal collagen secretion was observed (FIG. 7C). .
- Example 7 a screening system for candidate substances for therapeutic agents for liver fibrosis using the activated hepatic stellate cells (activated 2G92 cells) and stationary activated hepatic stellate cells (5H cells) described in Example 6 Built.
- activated 2G92 cells activated 2G92 cells
- stationary activated hepatic stellate cells 5H cells
- TGF- ⁇ was added at a final concentration of 1 ng / mL and cultured for 8 hours.
- 2G92 cells to which SB431542 was added were secreted with normal processing of collagen protein (FIG.
- Example 8 lung fibroblasts and renal tubular epithelial cells were used to produce cells used for screening pulmonary fibrosis therapeutic agents and cells used for screening renal fibrosis therapeutic agents.
- Example 6 (1) except that lung fibroblasts (TIG-3-20 cells and A549 cells) and renal tubular epithelial cells (MDCK cells) were used instead of hepatic stellate cells.
- TIG-3-20 cells, A549 cells, and MDCK cells in which collagen protein was expressed by the vector of the present invention were prepared.
- TIG-3-20 cells FIG. 10A
- A549 cells FIG. 10C
- MDCK cells FIG. 10D
- TGF- ⁇ was used to activate the cells. Except that pulmonary fibroblasts (TIG-3-20 cells) were used instead of hepatic stellate cells, the procedure of Example 6 (2) was repeated to activate TIG-3-20 cells. It was. As shown in FIG. 10B, only yellow signals indicating abnormal collagen processing and secretion are observed in TIG-3-20 cells, confirming that they can be used as model cells for pulmonary fibrosis. It was. These cells can be used to screen for pulmonary fibrosis drugs.
- the vector of the present invention can be effectively used for analysis of the collagen formation process.
- the analysis of the collagen formation process can be used as an effective means for elucidating the cause of a disease caused by collagen molecular abnormality and developing a treatment method thereof.
- the screening method of the present invention can be used for screening drugs for treating or preventing collagen-related diseases.
- this invention was demonstrated along the specific aspect, the deformation
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Abstract
The purpose of the present invention is to construct a system whereby secretion of procollagen from an animal cells can be detected. This purpose can be achieved by a fusion protein of a fluorescent protein or luminescent protein and a collagen-related protein which is a collagen protein, a procollagen protein, a preprocollagen protein having a repeating amino acid sequence of Gly-Xaa-Yaa (where Gly represents glycine and Xaa and Yaa represent arbitrary amino acids), or a variant or fragment thereof.
Description
本発明は、コラーゲン融合タンパク質及びそれを用いた薬剤のスクリーニング方法、並びにコラーゲン融合タンパク質をコードする核酸を含むベクター、ベクターを含む大腸菌、及びそれらの製造方法に関する。本発明によれば、コラーゲン関連疾患の治療用又は予防用薬剤の候補物質をスクリーニングすることができる。また、本発明によれば、コラーゲン融合タンパク質、及びそれをコードする正常な核酸を有するベクターを得ることができる。
The present invention relates to a collagen fusion protein and a method for screening a drug using the same, a vector containing a nucleic acid encoding the collagen fusion protein, E. coli containing the vector, and methods for producing them. According to the present invention, candidate substances for drugs for treating or preventing collagen-related diseases can be screened. Further, according to the present invention, a vector having a collagen fusion protein and a normal nucleic acid encoding it can be obtained.
コラーゲンは、生体内のタンパク質の３０％を占め、骨格支持及び細胞接着などの機能を有する重要なタンパク質であり、例えば、ヒトの身体の骨・軟骨、靭帯・腱、角膜実質、皮膚、肝臓、筋肉などの組織の主要構成成分である。コラーゲンには、コラーゲン線維を形成する線維性コラーゲンと、線維を形成しない非線維性コラーゲンが存在する。線維性コラーゲンは、コラーゲンのポリペプチド鎖が３本集まって３重らせん構造（トロポコラーゲン）を形成している。この３重らせん構造のトポコラーゲンは、自己組織化することによって、分子長の１／４ずつずれて整列し、コラーゲン原線維（コラーゲン細線維）を形成する。更に、このコラーゲン原線維が、多数集合して、コラーゲン線維（コラーゲン線維束）を形成している。更に、角膜実質や皮質骨においては、コラーゲン線維は同一方向に整列した二次元の層を形成し、更にこの層が１層ごとに直行して積層したベニヤ板様三次元層板構造を取っている。このベニヤ板様三次元層板構造を有するコラーゲンは、透明で且つ高い強度を有している。
Collagen accounts for 30% of proteins in the body and is an important protein having functions such as skeletal support and cell adhesion. For example, bone / cartilage, ligament / tendon, corneal stroma, skin, liver, It is a major component of tissues such as muscle. Collagen includes fibrous collagen that forms collagen fibers and non-fibrous collagen that does not form fibers. Fibrous collagen forms a triple helical structure (tropocollagen) by gathering three polypeptide chains of collagen. The topocollagen having a triple helical structure is self-assembled to be aligned with a quarter of the molecular length to form collagen fibrils (collagen fibrils). Furthermore, many collagen fibrils are aggregated to form collagen fibers (collagen fiber bundles). Furthermore, in the corneal stroma and cortical bone, the collagen fibers form a two-dimensional layer aligned in the same direction, and this layer has a plywood-like three-dimensional layer structure in which each layer is orthogonally stacked. . Collagen having this plywood-like three-dimensional layered plate structure is transparent and has high strength.
このようなコラーゲンの分子異常を起因とする疾患として膠原病（例えば、皮膚筋炎、多発性筋炎）又は角膜の白濁などが挙げられるが、治療法が確立されていないものが多い。これは、コラーゲンの分泌過程および高次構造の形成過程が未解明であることも、１つの原因である。コラーゲンの分泌過程および高次構造の形成過程の解析を行うことが可能であれば、コラーゲンの分子異常を起因とする疾患の原因の解明、及びそれらの治療法の開発にも有効な手段として用いることができる。
Examples of diseases caused by such molecular abnormality of collagen include collagen disease (for example, dermatomyositis, polymyositis) or corneal cloudiness, but there are many treatment methods that have not been established. This is partly due to the fact that the secretory process of collagen and the formation process of higher-order structures are still unclear. If it is possible to analyze the secretion process of collagen and the formation process of higher-order structures, it will be used as an effective means for elucidating the causes of diseases caused by collagen molecular abnormalities and developing treatments for them. be able to.
本発明者らは、動物細胞内でのコラーゲンの動態及び動物細胞から分泌されたコラーゲンの動態を解析するために、コラーゲンを動物細胞で発現させるシステムの構築を試みた。具体的には、プロコラーゲンのＣ末端に蛍光タンパク質を結合させた融合タンパク質を構築した。この融合タンパク質を動物細胞に導入して、動物細胞からのプロコラーゲンおよびコラーゲンの分泌の検出を試みた。しかしながら、動物細胞からのプロコラーゲンおよびコラーゲンの分泌は検出できなかった。
従って、本発明の目的は、動物細胞からのプロコラーゲンおよびコラーゲンの分泌を検出できるシステムの構築である。
一方、本発明者らは、前記のシステムの構築に、遺伝子組換えを用いた。コラーゲンタンパク質の遺伝子操作については、例えば特許文献１に報告されている。本発明者らは、コラーゲンをコードする核酸を、ベクターに組み込み、大腸菌を用いて発現ベクターなどの構築を試みた。しかしながら、本発明者らは、通常タンパク質の発現に用いられているベクターを用い、大腸菌で形質転換を行った場合、コラーゲンタンパク質をコードする遺伝子に欠失、挿入、制限酵素での切断の阻害が生じることに気がついた。すなわち、本発明者らは、目的のコラーゲン遺伝子を含む発現ベクターを得ることができなかった。この遺伝子の欠失挿入、制限酵素での切断の阻害は、特定の位置で発生するものではなく、コラーゲン遺伝子のランダムな位置で発生した。例えば、相同組換えにより、遺伝子の特定の位置に欠失が起きることは知られている。しかしながら、ベクターの構築において、不特定の位置で欠失、挿入、制限酵素での切断の阻害が生じることは、本発明者の知る限りにおいて、知られておらず、非常に珍しい現象であると思われる。
従って、本発明のもう１つの目的は、コラーゲンタンパク質をコードする欠失、挿入、制限酵素での切断の阻害のない核酸を有するベクターを提供することである。 In order to analyze the dynamics of collagen in animal cells and the secretion of collagen secreted from animal cells, the present inventors tried to construct a system for expressing collagen in animal cells. Specifically, a fusion protein in which a fluorescent protein was bound to the C-terminus of procollagen was constructed. This fusion protein was introduced into animal cells to attempt detection of procollagen and collagen secretion from animal cells. However, procollagen and collagen secretion from animal cells could not be detected.
Therefore, an object of the present invention is to construct a system capable of detecting procollagen and collagen secretion from animal cells.
On the other hand, the present inventors used genetic recombination for the construction of the system. For example,Patent Document 1 reports on genetic manipulation of collagen proteins. The present inventors attempted to construct an expression vector using Escherichia coli by incorporating a nucleic acid encoding collagen into a vector. However, the present inventors, when transformed with Escherichia coli using a vector that is usually used for protein expression, have deletions, insertions, and inhibition of restriction enzyme cleavage in genes encoding collagen proteins. I noticed it happened. That is, the present inventors could not obtain an expression vector containing the target collagen gene. This gene deletion / inhibition of restriction enzyme cleavage did not occur at specific positions, but occurred at random positions in the collagen gene. For example, it is known that deletion occurs at a specific position of a gene by homologous recombination. However, in the construction of a vector, deletion, insertion, and inhibition of cleavage with a restriction enzyme occur at unspecified positions, as far as the present inventors know, is a very rare phenomenon. Seem.
Accordingly, another object of the present invention is to provide a vector having a nucleic acid which does not inhibit deletion, insertion or restriction enzyme cleavage encoding a collagen protein.
従って、本発明の目的は、動物細胞からのプロコラーゲンおよびコラーゲンの分泌を検出できるシステムの構築である。
一方、本発明者らは、前記のシステムの構築に、遺伝子組換えを用いた。コラーゲンタンパク質の遺伝子操作については、例えば特許文献１に報告されている。本発明者らは、コラーゲンをコードする核酸を、ベクターに組み込み、大腸菌を用いて発現ベクターなどの構築を試みた。しかしながら、本発明者らは、通常タンパク質の発現に用いられているベクターを用い、大腸菌で形質転換を行った場合、コラーゲンタンパク質をコードする遺伝子に欠失、挿入、制限酵素での切断の阻害が生じることに気がついた。すなわち、本発明者らは、目的のコラーゲン遺伝子を含む発現ベクターを得ることができなかった。この遺伝子の欠失挿入、制限酵素での切断の阻害は、特定の位置で発生するものではなく、コラーゲン遺伝子のランダムな位置で発生した。例えば、相同組換えにより、遺伝子の特定の位置に欠失が起きることは知られている。しかしながら、ベクターの構築において、不特定の位置で欠失、挿入、制限酵素での切断の阻害が生じることは、本発明者の知る限りにおいて、知られておらず、非常に珍しい現象であると思われる。
従って、本発明のもう１つの目的は、コラーゲンタンパク質をコードする欠失、挿入、制限酵素での切断の阻害のない核酸を有するベクターを提供することである。 In order to analyze the dynamics of collagen in animal cells and the secretion of collagen secreted from animal cells, the present inventors tried to construct a system for expressing collagen in animal cells. Specifically, a fusion protein in which a fluorescent protein was bound to the C-terminus of procollagen was constructed. This fusion protein was introduced into animal cells to attempt detection of procollagen and collagen secretion from animal cells. However, procollagen and collagen secretion from animal cells could not be detected.
Therefore, an object of the present invention is to construct a system capable of detecting procollagen and collagen secretion from animal cells.
On the other hand, the present inventors used genetic recombination for the construction of the system. For example,
Accordingly, another object of the present invention is to provide a vector having a nucleic acid which does not inhibit deletion, insertion or restriction enzyme cleavage encoding a collagen protein.
本発明者らは、動物細胞からのプロコラーゲンの分泌を検出できるシステムについて、鋭意研究した結果、驚くべきことに、プロコラーゲンタンパク質のＮ末端のＮプロプロテイン、コラーゲンタンパク質、又はＣ末端のＣ－プロプロテインに蛍光タンパク質を結合又は挿入することにより、動物細胞からのプロコラーゲンおよびコラーゲンタンパク質の分泌を検出できるシステムを構築できることを見出した。そして、コラーゲンタンパク質に蛍光タンパク質又は発光タンパク質が挿入された融合タンパク質を発現したコラーゲン関連疾患の細胞を用いて、コラーゲン関連疾患の治療又は予防用薬剤のスクリーニングが可能であることを見出した。
また、本発明者らは、コラーゲンタンパク質をコードする欠失、挿入、制限酵素での切断の阻害のない核酸を有するベクターについて、鋭意研究した結果、驚くべきことに、特定の遺伝子変異を有する大腸菌を、コラーゲン遺伝子を含むベクターの宿主として用いることにより、コラーゲンタンパク質をコードする遺伝子に欠失、挿入、制限酵素での切断の阻害が生じないことを見出した。
本発明は、こうした知見に基づくものである。
従って、本発明は、
［１］Ｇｌｙ－Ｘａａ－Ｙａａ（式中、Ｇｌｙはグリシンであり、そしてＸａａ及びＹａａは任意のアミノ酸である）の繰り返しアミノ酸配列を有するプレプロコラーゲンタンパク質、プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体、又はそれらの断片である、コラーゲン関連タンパク質と、蛍光タンパク質又は発光タンパク質との融合タンパク質、
［２］前記蛍光タンパク質又は発光タンパク質が、プロコラーゲンタンパク質のＮ末端のＮ－プロプロテイン、コラーゲンタンパク質、又はＣ末端のＣ－プロプロテインに結合又は挿入されている、［１］に記載の融合タンパク質、
［３］前記蛍光タンパク質又は発光タンパク質がコラーゲンタンパク質のＣ末端から３０アミノ酸よりＮ末端側に挿入されており、及び／又は蛍光タンパク質又は発光タンパク質がＣ－プロプロテインのＮ末端から３０アミノ酸よりＣ末側に挿入されている、［１］又は［２］に記載の融合タンパク質、
［４］［１］～［３］のいずれかに記載の融合タンパク質を発現したコラーゲン関連疾患細胞と、試験物質とを接触させる工程、及び融合タンパク質の動態を解析する工程、を含むコラーゲン関連疾患の治療又は予防物質のスクリーニング方法、
［５］Ｇｌｙ－Ｘａａ－Ｙａａ（式中、Ｇｌｙはグリシンであり、そしてＸａａ及びＹａａは任意のアミノ酸である）の繰り返しアミノ酸配列を有するプレプロコラーゲンタンパク質、プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体、又はそれらの断片である、コラーゲン関連タンパク質、又は前記コラーゲン関連タンパク質と蛍光タンパク質又は発光タンパク質との融合タンパク質をコードする核酸、
［６］［５］に記載の核酸を含むベクター、
［７］［６］に記載のベクターを含む、ベクター含有宿主細胞、
［８］前記宿主細胞が、mcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌である、請求項７に記載のベクター含有宿主細胞、
［９］前記大腸菌が、ＤＨ１０Ｂ、ＴＯＰ１０Ｆ’、ＭＣ１０６１、ＸＬ１－Ｂｌｕｅ ＭＲＦ’、ＡＧ１、ＢＬ２１（ＤＥ３）、ＤＢ３．１、ＤＨ１、ＤＨ５α、ＤＨ５α Ｔｕｒｂｏ、ＤＨ１２Ｓ、ＤＭ１、Ｅ．Ｃｌｏｎｉ（ｒ）５ａｌｐｈａ、Ｅ．Ｃｌｏｎｉ（ｒ）１０Ｇ、Ｅ．Ｃｌｏｎｉ（ｒ）１０ＧＦ’、ＥＲ２２６７、ＨＢ１０１、ＨＭＳ１７４（ＤＥ３）、Ｈｉｇｈ－Ｃｏｎｔｒｏｌ（ｔｍ） ＢＬ２１（ＤＥ３）、Ｈｉｇｈ－Ｃｏｎｔｒｏｌ（ｔｍ）１０Ｇ、ＩＪ１１２６、ＪＭ１０８、ＪＭ１０９、Ｍａｃｈ１、ＭＣ１０６１、ＭＦＤｐｉｒ、ＯｍｎｉＭＡＸ２、ＯｖｅｒＥｘｐｒｅｓｓ（ｔｍ）Ｃ４１（ＤＥ３）、ＯｖｅｒＥｘｐｒｅｓｓ（ｔｍ）Ｃ４３（ＤＥ３）、Ｒｏｓｅｔｔａ（ＤＥ３）、ＳＯＬＲ、ＳＳ３２０、ＳＴＢＬ２、ＳＴＢＬ３、ＳＴＢＬ４、ＳＵＲＥ、ＳＵＲＥ２、ＴＧ１、ＴＯＰ１０、ＸＬ２－Ｂｌｕｅ、ＸＬ２－ＢｌｕｅＭＲＦ’、及びＸＬ１０－Ｇｏｌｄからなる群から選択される、［８］に記載のベクター含有宿主細胞、
［１０］［８］又は［９］に記載のベクター含有宿主細胞から得られるベクター、
［１１］［６］に記載のベクターをmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌に導入することを特徴とする、ベクター含有大腸菌の製造方法、
［１２］［６］に記載のベクターをmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌に導入する工程、及び前記ベクターを回収する工程を含むことを特徴とする、ベクターの製造方法、
［１３］（１）請求項６に記載のベクターを、動物細胞に導入する工程、及び（２）前記ベクターが導入された動物細胞からコラーゲンを分泌させる工程、を含む、コラーゲンの製造方法、及び
［１４］（１）請求項６に記載のベクターを、動物細胞に導入する工程、及び（２）前記ベクターが導入された動物細胞において蛍光又は発光を検出する工程、を含む、コラーゲンの蛍光又は発光検出方法、
に関する。 As a result of diligent research on a system capable of detecting secretion of procollagen from animal cells, the present inventors have surprisingly found that N proprotein, collagen protein at the N-terminal of procollagen protein, or C-terminal at C-terminal. It has been found that a system capable of detecting secretion of procollagen and collagen protein from animal cells can be constructed by binding or inserting a fluorescent protein into proprotein. And it discovered that the screening of the medicine for the treatment or prevention of a collagen related disease was possible using the cell of the collagen related disease which expressed the fusion protein by which fluorescent protein or photoprotein was inserted in collagen protein.
In addition, as a result of intensive research on a vector having a nucleic acid that does not inhibit the deletion, insertion, or restriction enzyme cleavage encoding a collagen protein, the present inventors have surprisingly found that E. coli having a specific gene mutation is present. Was used as a host of a vector containing a collagen gene, and it was found that deletion, insertion, and inhibition of restriction enzyme cleavage did not occur in the gene encoding the collagen protein.
The present invention is based on these findings.
Therefore, the present invention
[1] Preprocollagen protein, procollagen protein, collagen protein having a repetitive amino acid sequence of Gly-Xaa-Yaa (where Gly is glycine, and Xaa and Yaa are arbitrary amino acids), or mutations thereof A fusion protein of a collagen-related protein and a fluorescent protein or photoprotein, which is a body or a fragment thereof,
[2] The fusion protein according to [1], wherein the fluorescent protein or photoprotein is bound or inserted into the N-terminal N-proprotein, collagen protein, or C-terminal C-proprotein of the procollagen protein. ,
[3] The fluorescent protein or photoprotein is inserted from the C-terminal of the collagen protein to the N-terminal side from 30 amino acids, and / or the fluorescent protein or photoprotein is C-terminal from the N-terminal of the C-proprotein to 30 amino acids The fusion protein according to [1] or [2], which is inserted on the side,
[4] A collagen-related disease comprising a step of bringing a collagen-related disease cell expressing the fusion protein according to any one of [1] to [3] into contact with a test substance, and a step of analyzing the dynamics of the fusion protein A method of screening for a therapeutic or preventive substance for
[5] Preprocollagen protein, procollagen protein, collagen protein, or a mutation thereof having a repetitive amino acid sequence of Gly-Xaa-Yaa (where Gly is glycine, and Xaa and Yaa are arbitrary amino acids) A nucleic acid encoding a collagen-related protein, or a fusion protein of the collagen-related protein and a fluorescent protein or photoprotein, which is a body, or a fragment thereof,
[6] A vector comprising the nucleic acid according to [5],
[7] A vector-containing host cell comprising the vector according to [6],
[8] The host cell is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG A vector-containing host cell according to claim 7, which is E. coli having a genetic mutation selected from the group consisting of galE, hsdS, and combinations of two or more thereof,
[9] The Escherichia coli is DH10B, TOP10F ′, MC1061, XL1-Blue MRF ′, AG1, BL21 (DE3), DB3.1, DH1, DH5α, DH5α Turbo, DH12S, DM1, E. coli. Cloni (r) 5alpha, E.I. Cloni (r) 10G, E.I. Cloni (r) 10GF ', ER2267, HB101, HMS174 (DE3), High-Control (tm) BL21 (DE3), High-Control (tm) 10G, IJ1126, JM108, JM109, Mach1, MC1061, MFDpiMx, OFDpiMx, OFDpiMx (Tm) C41 (DE3), OverExpress (tm) C43 (DE3), Rosetta (DE3), SOLR, SS320, STBL2, STBL3, STBL4, SURE, SURE2, TG1, TOP10, XL2-Blue, XL2-BlueMRF ', and The vector-containing host cell according to [8], selected from the group consisting of XL10-Gold,
[10] A vector obtained from the vector-containing host cell according to [8] or [9],
[11] The vector described in [6] is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), NupG, galE, hsdS, and a method for producing a vector-containing Escherichia coli, which is introduced into Escherichia coli having a gene mutation selected from the group consisting of two or more combinations thereof,
[12] The vector described in [6] is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), NupG, galE, hsdS, and a step of introducing into E. coli having a genetic mutation selected from the group consisting of two or more combinations thereof, and a step of recovering the vector, Production method,
[13] A method for producing collagen, comprising: (1) introducing the vector according to claim 6 into an animal cell; and (2) secreting collagen from the animal cell into which the vector has been introduced, and [14] (1) Introducing the vector according to claim 6 into an animal cell, and (2) detecting fluorescence or luminescence in the animal cell into which the vector is introduced. Luminescence detection method,
About.
また、本発明者らは、コラーゲンタンパク質をコードする欠失、挿入、制限酵素での切断の阻害のない核酸を有するベクターについて、鋭意研究した結果、驚くべきことに、特定の遺伝子変異を有する大腸菌を、コラーゲン遺伝子を含むベクターの宿主として用いることにより、コラーゲンタンパク質をコードする遺伝子に欠失、挿入、制限酵素での切断の阻害が生じないことを見出した。
本発明は、こうした知見に基づくものである。
従って、本発明は、
［１］Ｇｌｙ－Ｘａａ－Ｙａａ（式中、Ｇｌｙはグリシンであり、そしてＸａａ及びＹａａは任意のアミノ酸である）の繰り返しアミノ酸配列を有するプレプロコラーゲンタンパク質、プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体、又はそれらの断片である、コラーゲン関連タンパク質と、蛍光タンパク質又は発光タンパク質との融合タンパク質、
［２］前記蛍光タンパク質又は発光タンパク質が、プロコラーゲンタンパク質のＮ末端のＮ－プロプロテイン、コラーゲンタンパク質、又はＣ末端のＣ－プロプロテインに結合又は挿入されている、［１］に記載の融合タンパク質、
［３］前記蛍光タンパク質又は発光タンパク質がコラーゲンタンパク質のＣ末端から３０アミノ酸よりＮ末端側に挿入されており、及び／又は蛍光タンパク質又は発光タンパク質がＣ－プロプロテインのＮ末端から３０アミノ酸よりＣ末側に挿入されている、［１］又は［２］に記載の融合タンパク質、
［４］［１］～［３］のいずれかに記載の融合タンパク質を発現したコラーゲン関連疾患細胞と、試験物質とを接触させる工程、及び融合タンパク質の動態を解析する工程、を含むコラーゲン関連疾患の治療又は予防物質のスクリーニング方法、
［５］Ｇｌｙ－Ｘａａ－Ｙａａ（式中、Ｇｌｙはグリシンであり、そしてＸａａ及びＹａａは任意のアミノ酸である）の繰り返しアミノ酸配列を有するプレプロコラーゲンタンパク質、プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体、又はそれらの断片である、コラーゲン関連タンパク質、又は前記コラーゲン関連タンパク質と蛍光タンパク質又は発光タンパク質との融合タンパク質をコードする核酸、
［６］［５］に記載の核酸を含むベクター、
［７］［６］に記載のベクターを含む、ベクター含有宿主細胞、
［８］前記宿主細胞が、mcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌である、請求項７に記載のベクター含有宿主細胞、
［９］前記大腸菌が、ＤＨ１０Ｂ、ＴＯＰ１０Ｆ’、ＭＣ１０６１、ＸＬ１－Ｂｌｕｅ ＭＲＦ’、ＡＧ１、ＢＬ２１（ＤＥ３）、ＤＢ３．１、ＤＨ１、ＤＨ５α、ＤＨ５α Ｔｕｒｂｏ、ＤＨ１２Ｓ、ＤＭ１、Ｅ．Ｃｌｏｎｉ（ｒ）５ａｌｐｈａ、Ｅ．Ｃｌｏｎｉ（ｒ）１０Ｇ、Ｅ．Ｃｌｏｎｉ（ｒ）１０ＧＦ’、ＥＲ２２６７、ＨＢ１０１、ＨＭＳ１７４（ＤＥ３）、Ｈｉｇｈ－Ｃｏｎｔｒｏｌ（ｔｍ） ＢＬ２１（ＤＥ３）、Ｈｉｇｈ－Ｃｏｎｔｒｏｌ（ｔｍ）１０Ｇ、ＩＪ１１２６、ＪＭ１０８、ＪＭ１０９、Ｍａｃｈ１、ＭＣ１０６１、ＭＦＤｐｉｒ、ＯｍｎｉＭＡＸ２、ＯｖｅｒＥｘｐｒｅｓｓ（ｔｍ）Ｃ４１（ＤＥ３）、ＯｖｅｒＥｘｐｒｅｓｓ（ｔｍ）Ｃ４３（ＤＥ３）、Ｒｏｓｅｔｔａ（ＤＥ３）、ＳＯＬＲ、ＳＳ３２０、ＳＴＢＬ２、ＳＴＢＬ３、ＳＴＢＬ４、ＳＵＲＥ、ＳＵＲＥ２、ＴＧ１、ＴＯＰ１０、ＸＬ２－Ｂｌｕｅ、ＸＬ２－ＢｌｕｅＭＲＦ’、及びＸＬ１０－Ｇｏｌｄからなる群から選択される、［８］に記載のベクター含有宿主細胞、
［１０］［８］又は［９］に記載のベクター含有宿主細胞から得られるベクター、
［１１］［６］に記載のベクターをmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌に導入することを特徴とする、ベクター含有大腸菌の製造方法、
［１２］［６］に記載のベクターをmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌に導入する工程、及び前記ベクターを回収する工程を含むことを特徴とする、ベクターの製造方法、
［１３］（１）請求項６に記載のベクターを、動物細胞に導入する工程、及び（２）前記ベクターが導入された動物細胞からコラーゲンを分泌させる工程、を含む、コラーゲンの製造方法、及び
［１４］（１）請求項６に記載のベクターを、動物細胞に導入する工程、及び（２）前記ベクターが導入された動物細胞において蛍光又は発光を検出する工程、を含む、コラーゲンの蛍光又は発光検出方法、
に関する。 As a result of diligent research on a system capable of detecting secretion of procollagen from animal cells, the present inventors have surprisingly found that N proprotein, collagen protein at the N-terminal of procollagen protein, or C-terminal at C-terminal. It has been found that a system capable of detecting secretion of procollagen and collagen protein from animal cells can be constructed by binding or inserting a fluorescent protein into proprotein. And it discovered that the screening of the medicine for the treatment or prevention of a collagen related disease was possible using the cell of the collagen related disease which expressed the fusion protein by which fluorescent protein or photoprotein was inserted in collagen protein.
In addition, as a result of intensive research on a vector having a nucleic acid that does not inhibit the deletion, insertion, or restriction enzyme cleavage encoding a collagen protein, the present inventors have surprisingly found that E. coli having a specific gene mutation is present. Was used as a host of a vector containing a collagen gene, and it was found that deletion, insertion, and inhibition of restriction enzyme cleavage did not occur in the gene encoding the collagen protein.
The present invention is based on these findings.
Therefore, the present invention
[1] Preprocollagen protein, procollagen protein, collagen protein having a repetitive amino acid sequence of Gly-Xaa-Yaa (where Gly is glycine, and Xaa and Yaa are arbitrary amino acids), or mutations thereof A fusion protein of a collagen-related protein and a fluorescent protein or photoprotein, which is a body or a fragment thereof,
[2] The fusion protein according to [1], wherein the fluorescent protein or photoprotein is bound or inserted into the N-terminal N-proprotein, collagen protein, or C-terminal C-proprotein of the procollagen protein. ,
[3] The fluorescent protein or photoprotein is inserted from the C-terminal of the collagen protein to the N-terminal side from 30 amino acids, and / or the fluorescent protein or photoprotein is C-terminal from the N-terminal of the C-proprotein to 30 amino acids The fusion protein according to [1] or [2], which is inserted on the side,
[4] A collagen-related disease comprising a step of bringing a collagen-related disease cell expressing the fusion protein according to any one of [1] to [3] into contact with a test substance, and a step of analyzing the dynamics of the fusion protein A method of screening for a therapeutic or preventive substance for
[5] Preprocollagen protein, procollagen protein, collagen protein, or a mutation thereof having a repetitive amino acid sequence of Gly-Xaa-Yaa (where Gly is glycine, and Xaa and Yaa are arbitrary amino acids) A nucleic acid encoding a collagen-related protein, or a fusion protein of the collagen-related protein and a fluorescent protein or photoprotein, which is a body, or a fragment thereof,
[6] A vector comprising the nucleic acid according to [5],
[7] A vector-containing host cell comprising the vector according to [6],
[8] The host cell is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG A vector-containing host cell according to claim 7, which is E. coli having a genetic mutation selected from the group consisting of galE, hsdS, and combinations of two or more thereof,
[9] The Escherichia coli is DH10B, TOP10F ′, MC1061, XL1-Blue MRF ′, AG1, BL21 (DE3), DB3.1, DH1, DH5α, DH5α Turbo, DH12S, DM1, E. coli. Cloni (r) 5alpha, E.I. Cloni (r) 10G, E.I. Cloni (r) 10GF ', ER2267, HB101, HMS174 (DE3), High-Control (tm) BL21 (DE3), High-Control (tm) 10G, IJ1126, JM108, JM109, Mach1, MC1061, MFDpiMx, OFDpiMx, OFDpiMx (Tm) C41 (DE3), OverExpress (tm) C43 (DE3), Rosetta (DE3), SOLR, SS320, STBL2, STBL3, STBL4, SURE, SURE2, TG1, TOP10, XL2-Blue, XL2-BlueMRF ', and The vector-containing host cell according to [8], selected from the group consisting of XL10-Gold,
[10] A vector obtained from the vector-containing host cell according to [8] or [9],
[11] The vector described in [6] is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), NupG, galE, hsdS, and a method for producing a vector-containing Escherichia coli, which is introduced into Escherichia coli having a gene mutation selected from the group consisting of two or more combinations thereof,
[12] The vector described in [6] is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), NupG, galE, hsdS, and a step of introducing into E. coli having a genetic mutation selected from the group consisting of two or more combinations thereof, and a step of recovering the vector, Production method,
[13] A method for producing collagen, comprising: (1) introducing the vector according to claim 6 into an animal cell; and (2) secreting collagen from the animal cell into which the vector has been introduced, and [14] (1) Introducing the vector according to claim 6 into an animal cell, and (2) detecting fluorescence or luminescence in the animal cell into which the vector is introduced. Luminescence detection method,
About.
本発明の融合タンパク質は、動物細胞からのプロコラーゲンの分泌を検出することができる。コラーゲン関連タンパク質は三重らせん構造を形成するため、プロコラーゲンタンパク質に蛍光タンパク質を挿入するとタンパク質の構造が変化し、三重らせん構造を形成できなくなると考えられている。しかしながら、コラーゲンタンパク質及び／又はＣ－プロプロテインの特定の位置に蛍光タンパク質を挿入することにより、三重らせん構造を維持することが可能となり、動物細胞からのプロコラーゲンの分泌を検出できるシステムを構築できた。また、Ｎ－プロプロテインの特定の位置に蛍光タンパク質を挿入することにより、三重らせん構造を維持することが可能となり、動物細胞からのプロコラーゲンの分泌を検出できるシステムを構築できた。
更に、本発明の特定の遺伝子変異を有する大腸菌から得られるベクターによれば、コラーゲンタンパク質をコードする、欠失、挿入、制限酵素での切断の阻害が生じていない遺伝子を得ることができる。また、本発明のベクター含有大腸菌によれば、コラーゲンタンパク質をコードする、欠失などの障害が生じていない遺伝子を含むベクターを得ることができる。本発明のベクター含有大腸菌の製造方法及びベクターの製造方法によれば、コラーゲンタンパク質をコードする、欠失などの障害が生じていない遺伝子を含むベクター及びそのベクターを含む大腸菌を製造することができる。
本発明によって得られたベクターを用いて、動物細胞でコラーゲンを製造することができる。また、本発明によって得られたベクターを用いることにより、コラーゲンの高次構造の形成過程を解析することが可能である。 The fusion protein of the present invention can detect the secretion of procollagen from animal cells. Since collagen-related proteins form a triple helical structure, it is believed that when a fluorescent protein is inserted into a procollagen protein, the structure of the protein changes and a triple helical structure cannot be formed. However, by inserting a fluorescent protein at a specific position of collagen protein and / or C-proprotein, it becomes possible to maintain a triple helical structure and to construct a system that can detect the secretion of procollagen from animal cells. It was. In addition, by inserting a fluorescent protein at a specific position of N-proprotein, a triple helical structure can be maintained, and a system capable of detecting procollagen secretion from animal cells could be constructed.
Furthermore, according to the vector obtained from Escherichia coli having the specific gene mutation of the present invention, a gene encoding a collagen protein that does not cause inhibition of deletion, insertion, or cleavage with a restriction enzyme can be obtained. Moreover, according to the vector-containing Escherichia coli of the present invention, it is possible to obtain a vector containing a gene that encodes a collagen protein and does not have a defect such as a deletion. According to the method for producing a vector-containing Escherichia coli and the method for producing a vector of the present invention, it is possible to produce a vector containing a gene encoding a collagen protein that does not have a defect such as a deletion and E. coli containing the vector.
Collagen can be produced from animal cells using the vector obtained according to the present invention. Further, by using the vector obtained by the present invention, it is possible to analyze the formation process of the higher-order structure of collagen.
更に、本発明の特定の遺伝子変異を有する大腸菌から得られるベクターによれば、コラーゲンタンパク質をコードする、欠失、挿入、制限酵素での切断の阻害が生じていない遺伝子を得ることができる。また、本発明のベクター含有大腸菌によれば、コラーゲンタンパク質をコードする、欠失などの障害が生じていない遺伝子を含むベクターを得ることができる。本発明のベクター含有大腸菌の製造方法及びベクターの製造方法によれば、コラーゲンタンパク質をコードする、欠失などの障害が生じていない遺伝子を含むベクター及びそのベクターを含む大腸菌を製造することができる。
本発明によって得られたベクターを用いて、動物細胞でコラーゲンを製造することができる。また、本発明によって得られたベクターを用いることにより、コラーゲンの高次構造の形成過程を解析することが可能である。 The fusion protein of the present invention can detect the secretion of procollagen from animal cells. Since collagen-related proteins form a triple helical structure, it is believed that when a fluorescent protein is inserted into a procollagen protein, the structure of the protein changes and a triple helical structure cannot be formed. However, by inserting a fluorescent protein at a specific position of collagen protein and / or C-proprotein, it becomes possible to maintain a triple helical structure and to construct a system that can detect the secretion of procollagen from animal cells. It was. In addition, by inserting a fluorescent protein at a specific position of N-proprotein, a triple helical structure can be maintained, and a system capable of detecting procollagen secretion from animal cells could be constructed.
Furthermore, according to the vector obtained from Escherichia coli having the specific gene mutation of the present invention, a gene encoding a collagen protein that does not cause inhibition of deletion, insertion, or cleavage with a restriction enzyme can be obtained. Moreover, according to the vector-containing Escherichia coli of the present invention, it is possible to obtain a vector containing a gene that encodes a collagen protein and does not have a defect such as a deletion. According to the method for producing a vector-containing Escherichia coli and the method for producing a vector of the present invention, it is possible to produce a vector containing a gene encoding a collagen protein that does not have a defect such as a deletion and E. coli containing the vector.
Collagen can be produced from animal cells using the vector obtained according to the present invention. Further, by using the vector obtained by the present invention, it is possible to analyze the formation process of the higher-order structure of collagen.
［１］融合タンパク質
本発明の融合タンパク質は、コラーゲン関連タンパク質と蛍光タンパク質又は発光タンパク質とが融合したタンパク質である。以下に、コラーゲン関連タンパク質について、説明する。 [1] Fusion protein The fusion protein of the present invention is a protein in which a collagen-related protein and a fluorescent protein or photoprotein are fused. Hereinafter, collagen-related proteins will be described.
本発明の融合タンパク質は、コラーゲン関連タンパク質と蛍光タンパク質又は発光タンパク質とが融合したタンパク質である。以下に、コラーゲン関連タンパク質について、説明する。 [1] Fusion protein The fusion protein of the present invention is a protein in which a collagen-related protein and a fluorescent protein or photoprotein are fused. Hereinafter, collagen-related proteins will be described.
《コラーゲン関連タンパク質》
コラーゲン関連タンパク質は、Ｇｌｙ－Ｘａａ－Ｙａａ（式中、Ｇｌｙはグリシンであり、そしてＸａａ及びＹａａは任意のアミノ酸である）の繰り返しアミノ酸配列を有するプレプロコラーゲンタンパク質、プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体、又はそれらの断片である。
前記コラーゲン関連タンパク質は、前記Ｇｌｙ－Ｘａａ－Ｙａａの繰り返しアミノ酸配列を有する限りにおいて、特に限定されるものではないが、例えばヒトのＩ型～XXVIII型コラーゲンタンパク質、そのプレプロコラーゲンタンパク質又はプロコラーゲンタンパク質、それらの変異体、又はそれらの断片を挙げることができる。プレプロコラーゲンタンパク質はコラーゲンタンパク質のＮ末側に、シグナルペプチド及びＮ－プロプロテインを有し、そしてＣ末側にＣ－プロプロテインを有している。またプロコラーゲンタンパク質は、コラーゲンタンパク質のＮ末側に、Ｎ－プロプロテインを有し、そしてＣ末側にＣ－プロプロテインを有している。前記Ｇｌｙ－Ｘａａ－Ｙａａの繰り返しアミノ酸配列はコラーゲン様配列と呼ばれ、このＧｌｙ－Ｘａａ－Ｙａａのコラーゲン様配列を有することがコラーゲン関連タンパク質の重要な特徴である。
また、前記コラーゲン関連タンパク質の由来する生物種も限定されるものではないが、例えば、哺乳類（例えば、ウシ、ブタ、ヒツジ、ヤギ、マウス、ラット、モルモット、又はサル）、鳥類（例えば、ニワトリ、ガチョウ、アヒル、又はダチョウ）、爬虫類（例えば、ワニ）、両生類（例えば、カエル）、魚類（例えば、チョウザメ、テラピア、タイ、ヒラメ、サメ、イワシ、マグロ、フグ、キンギョ、タラ、カレイ、又はコイ）、又は無脊椎動物（例えば、クラゲ）を挙げることができる。
前記コラーゲン関連タンパク質に含まれるＧｌｙ－Ｘａａ－Ｙａａの繰り返しアミノ酸配列の数は、２つ以上であれば特に限定されるものではないが、好ましくは１０以上であり、より好ましくは２０以上であり、更に好ましくは３０以上であり、最も好ましくは５０以上である。また、コラーゲン関連タンパク質は、前記コラーゲン様配列以外のアミノ酸配列を含むことができ、例えば、Ｎ末端又はＣ末端にシグナルペプチド、プロプロテイン、テロペプチドを含むことができる。 《Collagen-related protein》
Collagen-related protein is preprocollagen protein, procollagen protein, collagen protein having a repetitive amino acid sequence of Gly-Xaa-Yaa (where Gly is glycine, and Xaa and Yaa are arbitrary amino acids), or Or a fragment thereof.
The collagen-related protein is not particularly limited as long as it has the Gly-Xaa-Yaa repetitive amino acid sequence. For example, human type I to XXVIII type collagen protein, its pre-procollagen protein or procollagen protein, Mention may be made of their variants, or fragments thereof. The preprocollagen protein has a signal peptide and N-proprotein on the N-terminal side of the collagen protein, and C-proprotein on the C-terminal side. The procollagen protein has N-proprotein on the N-terminal side of the collagen protein and C-proprotein on the C-terminal side. The repetitive amino acid sequence of Gly-Xaa-Yaa is called a collagen-like sequence, and having a collagen-like sequence of Gly-Xaa-Yaa is an important feature of collagen-related proteins.
In addition, the biological species from which the collagen-related protein is derived is not limited, for example, mammals (for example, cows, pigs, sheep, goats, mice, rats, guinea pigs, or monkeys), birds (for example, chickens, Goose, duck, or ostrich), reptile (eg, crocodile), amphibian (eg, frog), fish (eg, sturgeon, tilapia, thailand, flounder, shark, sardine, tuna, puffer, goldfish, cod, flounder, or carp ), Or invertebrates (eg, jellyfish).
The number of Gly-Xaa-Yaa repeating amino acid sequences contained in the collagen-related protein is not particularly limited as long as it is 2 or more, preferably 10 or more, more preferably 20 or more, More preferably, it is 30 or more, and most preferably 50 or more. The collagen-related protein can include an amino acid sequence other than the collagen-like sequence, and can include, for example, a signal peptide, proprotein, and telopeptide at the N-terminus or C-terminus.
コラーゲン関連タンパク質は、Ｇｌｙ－Ｘａａ－Ｙａａ（式中、Ｇｌｙはグリシンであり、そしてＸａａ及びＹａａは任意のアミノ酸である）の繰り返しアミノ酸配列を有するプレプロコラーゲンタンパク質、プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体、又はそれらの断片である。
前記コラーゲン関連タンパク質は、前記Ｇｌｙ－Ｘａａ－Ｙａａの繰り返しアミノ酸配列を有する限りにおいて、特に限定されるものではないが、例えばヒトのＩ型～XXVIII型コラーゲンタンパク質、そのプレプロコラーゲンタンパク質又はプロコラーゲンタンパク質、それらの変異体、又はそれらの断片を挙げることができる。プレプロコラーゲンタンパク質はコラーゲンタンパク質のＮ末側に、シグナルペプチド及びＮ－プロプロテインを有し、そしてＣ末側にＣ－プロプロテインを有している。またプロコラーゲンタンパク質は、コラーゲンタンパク質のＮ末側に、Ｎ－プロプロテインを有し、そしてＣ末側にＣ－プロプロテインを有している。前記Ｇｌｙ－Ｘａａ－Ｙａａの繰り返しアミノ酸配列はコラーゲン様配列と呼ばれ、このＧｌｙ－Ｘａａ－Ｙａａのコラーゲン様配列を有することがコラーゲン関連タンパク質の重要な特徴である。
また、前記コラーゲン関連タンパク質の由来する生物種も限定されるものではないが、例えば、哺乳類（例えば、ウシ、ブタ、ヒツジ、ヤギ、マウス、ラット、モルモット、又はサル）、鳥類（例えば、ニワトリ、ガチョウ、アヒル、又はダチョウ）、爬虫類（例えば、ワニ）、両生類（例えば、カエル）、魚類（例えば、チョウザメ、テラピア、タイ、ヒラメ、サメ、イワシ、マグロ、フグ、キンギョ、タラ、カレイ、又はコイ）、又は無脊椎動物（例えば、クラゲ）を挙げることができる。
前記コラーゲン関連タンパク質に含まれるＧｌｙ－Ｘａａ－Ｙａａの繰り返しアミノ酸配列の数は、２つ以上であれば特に限定されるものではないが、好ましくは１０以上であり、より好ましくは２０以上であり、更に好ましくは３０以上であり、最も好ましくは５０以上である。また、コラーゲン関連タンパク質は、前記コラーゲン様配列以外のアミノ酸配列を含むことができ、例えば、Ｎ末端又はＣ末端にシグナルペプチド、プロプロテイン、テロペプチドを含むことができる。 《Collagen-related protein》
Collagen-related protein is preprocollagen protein, procollagen protein, collagen protein having a repetitive amino acid sequence of Gly-Xaa-Yaa (where Gly is glycine, and Xaa and Yaa are arbitrary amino acids), or Or a fragment thereof.
The collagen-related protein is not particularly limited as long as it has the Gly-Xaa-Yaa repetitive amino acid sequence. For example, human type I to XXVIII type collagen protein, its pre-procollagen protein or procollagen protein, Mention may be made of their variants, or fragments thereof. The preprocollagen protein has a signal peptide and N-proprotein on the N-terminal side of the collagen protein, and C-proprotein on the C-terminal side. The procollagen protein has N-proprotein on the N-terminal side of the collagen protein and C-proprotein on the C-terminal side. The repetitive amino acid sequence of Gly-Xaa-Yaa is called a collagen-like sequence, and having a collagen-like sequence of Gly-Xaa-Yaa is an important feature of collagen-related proteins.
In addition, the biological species from which the collagen-related protein is derived is not limited, for example, mammals (for example, cows, pigs, sheep, goats, mice, rats, guinea pigs, or monkeys), birds (for example, chickens, Goose, duck, or ostrich), reptile (eg, crocodile), amphibian (eg, frog), fish (eg, sturgeon, tilapia, thailand, flounder, shark, sardine, tuna, puffer, goldfish, cod, flounder, or carp ), Or invertebrates (eg, jellyfish).
The number of Gly-Xaa-Yaa repeating amino acid sequences contained in the collagen-related protein is not particularly limited as long as it is 2 or more, preferably 10 or more, more preferably 20 or more, More preferably, it is 30 or more, and most preferably 50 or more. The collagen-related protein can include an amino acid sequence other than the collagen-like sequence, and can include, for example, a signal peptide, proprotein, and telopeptide at the N-terminus or C-terminus.
コラーゲンタンパク質（以下、単に「コラーゲン」と称することがある）は、線維性コラーゲン及び非線維性コラーゲンに分けられ、線維性コラーゲンとしては、Ｉ型コラーゲン、II型コラーゲン、III型コラーゲン、Ｖ型コラーゲン、又はXI型コラーゲン等を挙げることができる。
例えば、Ｉ型コラーゲンは、分子量約１０万のポリペプチド鎖が３本集まって「３重らせん構造（トロポコラーゲン）」を作っており、分子量は約３０万である。この３重らせん構造は、長さ３００ｎｍで、直径１．５ｎｍの１本の硬い棒のような形態をしており、トロポコラーゲンと称される。この３重らせん構造のトポコラーゲンは、自己組織化することによって、分子長の１／４ずつずれて整列し、コラーゲン原線維（コラーゲン細線維）を形成する。更に、このコラーゲン原線維が、多数集合して、コラーゲン線維（コラーゲン線維束）を形成している。更に、角膜実質や皮質骨においては、コラーゲン線維は同一方向に整列した二次元の層を形成し、更にこの層が１層ごとに直行して積層したベニヤ板様三次元層板構造を取っている。このベニヤ板様三次元層板構造を有するコラーゲンは、透明で且つ高い強度を有している。また、Ｉ型コラーゲンにおいては、Ｇｌｙ－Ｘａａ－ＹａａのＸａａはプロリン又は３－ヒドロキシプロリン、そしてＹａａは４－ヒドロキシプロリン、又はヒドロキシリジンであることが多い。ヒドロキシプロリンは、通常のタンパク質に含まれておらず、コラーゲンに特有のアミノ酸であるが、ヒドロキシプロリンの水酸基と水和水との水素結合によって３重らせん構造が安定すると考えられる。 Collagen proteins (hereinafter sometimes simply referred to as “collagen”) are classified into fibrous collagen and non-fibrous collagen. As the fibrous collagen, type I collagen, type II collagen, type III collagen, type V collagen are used. Or XI type collagen.
For example, type I collagen has a “triple helical structure (tropocollagen)” formed by gathering three polypeptide chains having a molecular weight of about 100,000, and has a molecular weight of about 300,000. This triple helical structure has a length of 300 nm and is shaped like one hard rod having a diameter of 1.5 nm, and is called tropocollagen. The topocollagen having a triple helical structure is self-assembled to be aligned with a quarter of the molecular length to form collagen fibrils (collagen fibrils). Furthermore, many collagen fibrils are aggregated to form collagen fibers (collagen fiber bundles). Furthermore, in the corneal stroma and cortical bone, the collagen fibers form a two-dimensional layer aligned in the same direction, and this layer has a plywood-like three-dimensional layer structure in which each layer is orthogonally stacked. . Collagen having this plywood-like three-dimensional layered plate structure is transparent and has high strength. In type I collagen, Xly of Gly-Xaa-Yaa is often proline or 3-hydroxyproline, and Yaa is 4-hydroxyproline or hydroxylysine. Hydroxyproline is not contained in ordinary proteins and is an amino acid unique to collagen, but it is considered that the triple helical structure is stabilized by hydrogen bonding between the hydroxyl group of hydroxyproline and hydrated water.
例えば、Ｉ型コラーゲンは、分子量約１０万のポリペプチド鎖が３本集まって「３重らせん構造（トロポコラーゲン）」を作っており、分子量は約３０万である。この３重らせん構造は、長さ３００ｎｍで、直径１．５ｎｍの１本の硬い棒のような形態をしており、トロポコラーゲンと称される。この３重らせん構造のトポコラーゲンは、自己組織化することによって、分子長の１／４ずつずれて整列し、コラーゲン原線維（コラーゲン細線維）を形成する。更に、このコラーゲン原線維が、多数集合して、コラーゲン線維（コラーゲン線維束）を形成している。更に、角膜実質や皮質骨においては、コラーゲン線維は同一方向に整列した二次元の層を形成し、更にこの層が１層ごとに直行して積層したベニヤ板様三次元層板構造を取っている。このベニヤ板様三次元層板構造を有するコラーゲンは、透明で且つ高い強度を有している。また、Ｉ型コラーゲンにおいては、Ｇｌｙ－Ｘａａ－ＹａａのＸａａはプロリン又は３－ヒドロキシプロリン、そしてＹａａは４－ヒドロキシプロリン、又はヒドロキシリジンであることが多い。ヒドロキシプロリンは、通常のタンパク質に含まれておらず、コラーゲンに特有のアミノ酸であるが、ヒドロキシプロリンの水酸基と水和水との水素結合によって３重らせん構造が安定すると考えられる。 Collagen proteins (hereinafter sometimes simply referred to as “collagen”) are classified into fibrous collagen and non-fibrous collagen. As the fibrous collagen, type I collagen, type II collagen, type III collagen, type V collagen are used. Or XI type collagen.
For example, type I collagen has a “triple helical structure (tropocollagen)” formed by gathering three polypeptide chains having a molecular weight of about 100,000, and has a molecular weight of about 300,000. This triple helical structure has a length of 300 nm and is shaped like one hard rod having a diameter of 1.5 nm, and is called tropocollagen. The topocollagen having a triple helical structure is self-assembled to be aligned with a quarter of the molecular length to form collagen fibrils (collagen fibrils). Furthermore, many collagen fibrils are aggregated to form collagen fibers (collagen fiber bundles). Furthermore, in the corneal stroma and cortical bone, the collagen fibers form a two-dimensional layer aligned in the same direction, and this layer has a plywood-like three-dimensional layer structure in which each layer is orthogonally stacked. . Collagen having this plywood-like three-dimensional layered plate structure is transparent and has high strength. In type I collagen, Xly of Gly-Xaa-Yaa is often proline or 3-hydroxyproline, and Yaa is 4-hydroxyproline or hydroxylysine. Hydroxyproline is not contained in ordinary proteins and is an amino acid unique to collagen, but it is considered that the triple helical structure is stabilized by hydrogen bonding between the hydroxyl group of hydroxyproline and hydrated water.
コラーゲンタンパク質は、細胞内において、限定されるものではないが、例えばＮ末端側にＮ－プロプロテイン及びＣ末端側にＣ－プロプロテインを有するプロコラーゲンタンパク質（以下、単にプロコラーゲンと称することがある）として作られる。プロコラーゲンは、特に限定されるものではないが、Ｉ型～XXVIII型コラーゲンのプロコラーゲンを挙げることができる。
The collagen protein is not limited in the cell, but, for example, a procollagen protein having N-proprotein on the N-terminal side and C-proprotein on the C-terminal side (hereinafter sometimes simply referred to as procollagen). ). The procollagen is not particularly limited, and examples include procollagen of type I to XXVIII collagen.
プレプロコラーゲンタンパク質、プロコラーゲンタンパク質又はコラーゲンタンパク質の変異体は、Ｇｌｙ－Ｘａａ－Ｙａａの繰り返しアミノ酸配列を有する限りにおいて、特に限定されるものではない。変異体としては、例えば天然のコラーゲンタンパク質（例えば、前記Ｉ型～XXVIII型コラーゲンタンパク質）のアミノ酸配列において、アミノ酸が欠失、置換、挿入、及び／又は付加されたものを挙げることができる。前記欠失、置換、挿入、及び／又は付加されるアミノ数は、特に限定されないが、例えば１００個以下であり、好ましくは５０個以下であり、より好ましくは３０個以下であり、更に好ましくは１０個以下であり、最も好ましくは１又は数個である。例えば、Ｉ型コラーゲンは、約１０００個のアミノ酸からなるが、１００個を超える欠失、置換、挿入、及び／又は付加を有する場合、コラーゲンとしても機能を示さないことがあるからである。
また、別の変異体としては、天然のコラーゲンタンパク質（例えば、前記Ｉ型～XXVIII型コラーゲンタンパク質）のアミノ酸配列に対して、８０％以上の同一性を有する変異コラーゲンタンパク質を挙げることができる。アミノ酸の同一性は、好ましくは８５％以上であり、より好ましくは９０％以上であり、更に好ましくは９５％以上であり、最も好ましくは９７％以上である。アミノ酸の同一性が８０％未満である場合、コラーゲンとしても機能を示さないことがあるからである。 The preprocollagen protein, the procollagen protein or the variant of the collagen protein is not particularly limited as long as it has a repetitive amino acid sequence of Gly-Xaa-Yaa. Examples of the mutant include those in which an amino acid is deleted, substituted, inserted, and / or added in the amino acid sequence of a natural collagen protein (for example, the above-mentioned type I to XXVIII collagen proteins). The number of amino acids to be deleted, substituted, inserted and / or added is not particularly limited, but is, for example, 100 or less, preferably 50 or less, more preferably 30 or less, and still more preferably 10 or less, most preferably 1 or several. For example, type I collagen consists of about 1000 amino acids, but if it has more than 100 deletions, substitutions, insertions, and / or additions, it may not function as collagen.
As another variant, a variant collagen protein having 80% or more identity to the amino acid sequence of a natural collagen protein (for example, the above-mentioned type I to XXVIII collagen proteins) can be mentioned. The amino acid identity is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 97% or more. This is because when the amino acid identity is less than 80%, it may not function as collagen.
また、別の変異体としては、天然のコラーゲンタンパク質（例えば、前記Ｉ型～XXVIII型コラーゲンタンパク質）のアミノ酸配列に対して、８０％以上の同一性を有する変異コラーゲンタンパク質を挙げることができる。アミノ酸の同一性は、好ましくは８５％以上であり、より好ましくは９０％以上であり、更に好ましくは９５％以上であり、最も好ましくは９７％以上である。アミノ酸の同一性が８０％未満である場合、コラーゲンとしても機能を示さないことがあるからである。 The preprocollagen protein, the procollagen protein or the variant of the collagen protein is not particularly limited as long as it has a repetitive amino acid sequence of Gly-Xaa-Yaa. Examples of the mutant include those in which an amino acid is deleted, substituted, inserted, and / or added in the amino acid sequence of a natural collagen protein (for example, the above-mentioned type I to XXVIII collagen proteins). The number of amino acids to be deleted, substituted, inserted and / or added is not particularly limited, but is, for example, 100 or less, preferably 50 or less, more preferably 30 or less, and still more preferably 10 or less, most preferably 1 or several. For example, type I collagen consists of about 1000 amino acids, but if it has more than 100 deletions, substitutions, insertions, and / or additions, it may not function as collagen.
As another variant, a variant collagen protein having 80% or more identity to the amino acid sequence of a natural collagen protein (for example, the above-mentioned type I to XXVIII collagen proteins) can be mentioned. The amino acid identity is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 97% or more. This is because when the amino acid identity is less than 80%, it may not function as collagen.
プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体の断片は、プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体の部分ペプチドである限りにおいて、特に限定されるものではなく、Ｉ型～XXVIII型コラーゲン、それらのプロコラーゲン、又はそれらの変異体の部分ペプチドを挙げることができる。前記断片のアミノ酸鎖長、及び切断部位なども特に限定されるものではなく、遺伝子構築の目的に応じて、適宜決定することができる。
The fragment of procollagen protein, collagen protein, or a variant thereof is not particularly limited as long as it is a partial peptide of procollagen protein, collagen protein, or variant thereof. Type I to XXVIII type collagen , Partial peptides of those procollagens, or mutants thereof. The amino acid chain length, the cleavage site, and the like of the fragment are not particularly limited, and can be appropriately determined according to the purpose of gene construction.
《蛍光タンパク質又は発光タンパク質》
前記融合タンパク質に用いるタンパク質としては、限定されるものではないが、蛍光を付与する蛍光タンパク質、発光を付与する発光タンパク質を挙げることができる。
蛍光タンパク質又は発光タンパク質としては、ＧＦＰ、ＥＧＦＰ、ｍＣｈｅｒｒｙ、Ｓｉｒｉｕｓ、ＥＢＦＰ、ＳＢＰ２、ＥＢＰ２、Ａｚｕｒｉｔｅ、ｍＫａｌａｍａ１、ＴａｇＢＦＰ、ｍＢｌｕｅｂｅｒｒｙ、ｍＴｕｒｑｕｏｉｓｅ、ＥＣＦＰ、Ｃｅｒｕｌｅａｎ、ｍＣｅｒｕｌｅａｎ、ＴａｇＣＦＰ、ＡｍＣｙａｎ、ｍＴＰ１、ＭｉＣｙ（Ｍｉｄｏｒｉｉｓｈｉ Ｃｙａｎ）、ＴｕｒｂｏＧＦＰ、ＣＦＰ、ＡｃＧＦＰ、ＴａｇＧＦＰ、ＡＧ（Ａｚａｍｉ－Ｇｒｅｅｎ）、ｍＡＧ１、ＺｓＧｒｅｅｎ、ＥｍＧＦＰ（Ｅｍｅｒａｌｄ）、ＧＰ２、Ｔ－Ｓａｐｐｈｉｒｅ、ＨｙＰｅｒ、ＴａｇＹＦＰ、ｍＡｍｅｔｒｉｎｅ、ＥＹＦＰ、ＹＦＰ、Ｖｅｎｕｓ、Ｃｉｔｒｉｎｅ、ＰｈｉＹＦＰ、ＰｈｉＹＦＰ－ｍ、ｔｕｒｂｏＹＦＰ、ＺｓＹｅｌｌｏｗ、ｍＢａｎａｎａ、ｍＫＯ１、ＫＯ（Ｋｕｓａｂｉｒａ Ｏｒａｎｇｅ）、ｍＯｒａｎｇｅ、ｍＯｒａｎｇｅ２、ｍＫＯ２、Ｋｅｉｍａ５７０、ＴｕｒｂｏＲＦＰ、ＤｓＲｅｄ－Ｅｘｐｒｅｓｓ、ＤｓＲｅｄ、ＤｓＲｅｄ２、ＴａｇＲＦＰ、ＴａｇＲＦＰ－Ｔ、ＤｓＲｅｄ－Ｍｏｎｏｍｅｒ、ｍＡｐｐｌｅ、ＡｓＲｅｄ２、ｍＳｔｒａｗｂｅｒｒｙ、ＴｕｒｂｏＦＰ６０２、ｍＲＰ１、ＪＲｅｄ、ＫｉｌｌｅｒＲｅｄ、ＫｅｉｍａＲｅｄ、ＨｃＲｅｄ、ｍＲａｓｂｅｒｒｙ、ｍＫａｔｅ２、ＴａｇＦＰ６３５、ｍＰｌｕｍ、ｅｇＦＰ６５０、Ｎｅｐｔｕｎｅ、ｍＮｅｐｔｕｎｅ、ｅｇＦＰ６７０及びルシフェラーゼからなる群から選択されるすくなくとも１つの蛍光又は発光タンパク質を挙げることができる。 << Fluorescent protein or photoprotein >>
Examples of the protein used for the fusion protein include, but are not limited to, a fluorescent protein that imparts fluorescence and a photoprotein that imparts luminescence.
Fluorescent proteins or photoproteins include GFP, EGFP, mCherry, Sirius, EBFP, SBP2, EBP2, Azure, mKalama1, TagBFP, mBlueberry, mTurquoise, ECFP, CelCul, TP TurboGFP, CFP, AcGFP, TagGFP, AG (Azami-Green), mAG1, ZsGreen, EmGFP (Emerald), GP2, T-Sapphire, HyPer, TagYFP, mAmetrine, EYFP, YPh, FPhYFP turboYFP, ZsYello w, mBanana, mKO1, KO (Kusabila Orange), mOrange, mOrange2, mKO2, Keima570, TurboRFP, DsRed-Express, DsRed, DsRed2, TagRFP, TagRFP, TagRFP, TRFRF JRed, KillerRed, KeimaRed, HcRed, mRasberry, mKate2, TagFP635, mPlum, eggFP650, Neptune, mNeptune, eggFP670 and at least one fluorescent or luminescent protein selected from the group consisting of luciferase.
前記融合タンパク質に用いるタンパク質としては、限定されるものではないが、蛍光を付与する蛍光タンパク質、発光を付与する発光タンパク質を挙げることができる。
蛍光タンパク質又は発光タンパク質としては、ＧＦＰ、ＥＧＦＰ、ｍＣｈｅｒｒｙ、Ｓｉｒｉｕｓ、ＥＢＦＰ、ＳＢＰ２、ＥＢＰ２、Ａｚｕｒｉｔｅ、ｍＫａｌａｍａ１、ＴａｇＢＦＰ、ｍＢｌｕｅｂｅｒｒｙ、ｍＴｕｒｑｕｏｉｓｅ、ＥＣＦＰ、Ｃｅｒｕｌｅａｎ、ｍＣｅｒｕｌｅａｎ、ＴａｇＣＦＰ、ＡｍＣｙａｎ、ｍＴＰ１、ＭｉＣｙ（Ｍｉｄｏｒｉｉｓｈｉ Ｃｙａｎ）、ＴｕｒｂｏＧＦＰ、ＣＦＰ、ＡｃＧＦＰ、ＴａｇＧＦＰ、ＡＧ（Ａｚａｍｉ－Ｇｒｅｅｎ）、ｍＡＧ１、ＺｓＧｒｅｅｎ、ＥｍＧＦＰ（Ｅｍｅｒａｌｄ）、ＧＰ２、Ｔ－Ｓａｐｐｈｉｒｅ、ＨｙＰｅｒ、ＴａｇＹＦＰ、ｍＡｍｅｔｒｉｎｅ、ＥＹＦＰ、ＹＦＰ、Ｖｅｎｕｓ、Ｃｉｔｒｉｎｅ、ＰｈｉＹＦＰ、ＰｈｉＹＦＰ－ｍ、ｔｕｒｂｏＹＦＰ、ＺｓＹｅｌｌｏｗ、ｍＢａｎａｎａ、ｍＫＯ１、ＫＯ（Ｋｕｓａｂｉｒａ Ｏｒａｎｇｅ）、ｍＯｒａｎｇｅ、ｍＯｒａｎｇｅ２、ｍＫＯ２、Ｋｅｉｍａ５７０、ＴｕｒｂｏＲＦＰ、ＤｓＲｅｄ－Ｅｘｐｒｅｓｓ、ＤｓＲｅｄ、ＤｓＲｅｄ２、ＴａｇＲＦＰ、ＴａｇＲＦＰ－Ｔ、ＤｓＲｅｄ－Ｍｏｎｏｍｅｒ、ｍＡｐｐｌｅ、ＡｓＲｅｄ２、ｍＳｔｒａｗｂｅｒｒｙ、ＴｕｒｂｏＦＰ６０２、ｍＲＰ１、ＪＲｅｄ、ＫｉｌｌｅｒＲｅｄ、ＫｅｉｍａＲｅｄ、ＨｃＲｅｄ、ｍＲａｓｂｅｒｒｙ、ｍＫａｔｅ２、ＴａｇＦＰ６３５、ｍＰｌｕｍ、ｅｇＦＰ６５０、Ｎｅｐｔｕｎｅ、ｍＮｅｐｔｕｎｅ、ｅｇＦＰ６７０及びルシフェラーゼからなる群から選択されるすくなくとも１つの蛍光又は発光タンパク質を挙げることができる。 << Fluorescent protein or photoprotein >>
Examples of the protein used for the fusion protein include, but are not limited to, a fluorescent protein that imparts fluorescence and a photoprotein that imparts luminescence.
Fluorescent proteins or photoproteins include GFP, EGFP, mCherry, Sirius, EBFP, SBP2, EBP2, Azure, mKalama1, TagBFP, mBlueberry, mTurquoise, ECFP, CelCul, TP TurboGFP, CFP, AcGFP, TagGFP, AG (Azami-Green), mAG1, ZsGreen, EmGFP (Emerald), GP2, T-Sapphire, HyPer, TagYFP, mAmetrine, EYFP, YPh, FPhYFP turboYFP, ZsYello w, mBanana, mKO1, KO (Kusabila Orange), mOrange, mOrange2, mKO2, Keima570, TurboRFP, DsRed-Express, DsRed, DsRed2, TagRFP, TagRFP, TagRFP, TRFRF JRed, KillerRed, KeimaRed, HcRed, mRasberry, mKate2, TagFP635, mPlum, eggFP650, Neptune, mNeptune, eggFP670 and at least one fluorescent or luminescent protein selected from the group consisting of luciferase.
コラーゲン関連タンパク質及び融合タンパク質は、後述の発現ベクターを用いて調製することも可能であるが、ペプチド合成によって調製することも可能である。
Collagen-related protein and fusion protein can be prepared using an expression vector described later, but can also be prepared by peptide synthesis.
本発明の融合タンパク質の１つの態様として、前記蛍光タンパク質又は発光タンパク質がコラーゲンタンパク質のＣ末端から３０アミノ酸よりＮ末端側に挿入されている。また、本発明の融合タンパク質の１つの態様として、蛍光タンパク質又は発光タンパク質がＣ－プロプロテインのＮ末端から３０アミノ酸よりＣ末側に挿入されている。更に、本発明の融合タンパク質の１つの態様として、蛍光タンパク質又は発光タンパク質がコラーゲンタンパク質のＣ末端から３０アミノ酸よりＮ末端側に挿入され、そして蛍光タンパク質又は発光タンパク質がＣ－プロプロテインのＮ末端から３０アミノ酸よりＣ末側に挿入されている。
前記蛍光タンパク質又は発光タンパク質のコラーゲンタンパク質への挿入部位は、より好ましくはコラーゲンタンパク質のＣ末端から４５アミノ酸よりＮ末端側であり、更に好ましくは６０アミノ酸よりＮ末端側である。前記蛍光タンパク質又は発光タンパク質のＣ－プロプロテインへの挿入部位は、より好ましくはＣ－プロプロテインのＮ末端から４５アミノ酸よりＣ末端側であり、更に好ましくは６０アミノ酸よりＣ末端側である。
蛍光タンパク質又は発光タンパク質がコラーゲンタンパク質のＣ末側に挿入された場合、又は蛍光タンパク質又は発光タンパク質がＣ－プロプロテインのＮ末側に挿入された場合、発現されるコラーゲンタンパク質が、３重らせん構造を形成することができず、細胞外へ分泌されないことがある。 As one aspect of the fusion protein of the present invention, the fluorescent protein or photoprotein is inserted from the C-terminal of the collagen protein to the N-terminal side from 30 amino acids. In one embodiment of the fusion protein of the present invention, a fluorescent protein or a luminescent protein is inserted from the N-terminal of C-proprotein to the C-terminal side from 30 amino acids. Furthermore, as one embodiment of the fusion protein of the present invention, a fluorescent protein or photoprotein is inserted from the C-terminus of the collagen protein to the N-terminal side from 30 amino acids, and the fluorescent protein or photoprotein is introduced from the N-terminus of the C-proprotein. It is inserted into the C-terminal side from 30 amino acids.
The insertion site of the fluorescent protein or luminescent protein into the collagen protein is more preferably N-terminal from 45 amino acids to the C-terminal of the collagen protein, and still more preferably N-terminal from 60 amino acids. The insertion site of the fluorescent protein or luminescent protein into C-proprotein is more preferably 45 amino acids from the N-terminus of C-proprotein to the C-terminal side, and even more preferably 60 amino acids from the C-terminal side.
When fluorescent protein or photoprotein is inserted at the C-terminal side of collagen protein, or when fluorescent protein or photoprotein is inserted at the N-terminal side of C-proprotein, the expressed collagen protein has a triple helical structure May not be formed and may not be secreted outside the cell.
前記蛍光タンパク質又は発光タンパク質のコラーゲンタンパク質への挿入部位は、より好ましくはコラーゲンタンパク質のＣ末端から４５アミノ酸よりＮ末端側であり、更に好ましくは６０アミノ酸よりＮ末端側である。前記蛍光タンパク質又は発光タンパク質のＣ－プロプロテインへの挿入部位は、より好ましくはＣ－プロプロテインのＮ末端から４５アミノ酸よりＣ末端側であり、更に好ましくは６０アミノ酸よりＣ末端側である。
蛍光タンパク質又は発光タンパク質がコラーゲンタンパク質のＣ末側に挿入された場合、又は蛍光タンパク質又は発光タンパク質がＣ－プロプロテインのＮ末側に挿入された場合、発現されるコラーゲンタンパク質が、３重らせん構造を形成することができず、細胞外へ分泌されないことがある。 As one aspect of the fusion protein of the present invention, the fluorescent protein or photoprotein is inserted from the C-terminal of the collagen protein to the N-terminal side from 30 amino acids. In one embodiment of the fusion protein of the present invention, a fluorescent protein or a luminescent protein is inserted from the N-terminal of C-proprotein to the C-terminal side from 30 amino acids. Furthermore, as one embodiment of the fusion protein of the present invention, a fluorescent protein or photoprotein is inserted from the C-terminus of the collagen protein to the N-terminal side from 30 amino acids, and the fluorescent protein or photoprotein is introduced from the N-terminus of the C-proprotein. It is inserted into the C-terminal side from 30 amino acids.
The insertion site of the fluorescent protein or luminescent protein into the collagen protein is more preferably N-terminal from 45 amino acids to the C-terminal of the collagen protein, and still more preferably N-terminal from 60 amino acids. The insertion site of the fluorescent protein or luminescent protein into C-proprotein is more preferably 45 amino acids from the N-terminus of C-proprotein to the C-terminal side, and even more preferably 60 amino acids from the C-terminal side.
When fluorescent protein or photoprotein is inserted at the C-terminal side of collagen protein, or when fluorescent protein or photoprotein is inserted at the N-terminal side of C-proprotein, the expressed collagen protein has a triple helical structure May not be formed and may not be secreted outside the cell.
［２］核酸及びベクター
本発明の核酸は、前記コラーゲン関連タンパク質又は前記融合タンパク質をコードする核酸（以下、まとめて「コラーゲン核酸」と称することがある）である。
《コラーゲン核酸》
本発明のコラーゲン核酸は、前記プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体、又はそれらの断片などのコラーゲン関連タンパク質、或いはコラーゲン関連タンパク質と蛍光タンパク質等との融合タンパク質をコードする核酸である限りにおいて、特に限定されるものでない。
プロコラーゲンタンパク質、又はコラーゲンタンパク質をコードする核酸は、限定されるものではないが、例えばコラーゲンを有する生物の組織、又はその生物の分離された細胞から抽出して用いることができる。また、既に分離されたｃＤＮＡを含むベクターから得ることもできる。更に、ＤＮＡ合成によって、調製することも可能である。 [2] Nucleic acid and vector The nucleic acid of the present invention is a nucleic acid encoding the collagen-related protein or the fusion protein (hereinafter sometimes collectively referred to as “collagen nucleic acid”).
<Collagen nucleic acid>
As long as the collagen nucleic acid of the present invention is a nucleic acid encoding a collagen-related protein such as the procollagen protein, collagen protein, or a variant thereof, or a fragment thereof, or a fusion protein of a collagen-related protein and a fluorescent protein or the like. However, there is no particular limitation.
The procollagen protein or the nucleic acid encoding the collagen protein is not limited, and can be extracted from, for example, a tissue of an organism having collagen or a separated cell of the organism. It can also be obtained from a vector containing the already isolated cDNA. It can also be prepared by DNA synthesis.
本発明の核酸は、前記コラーゲン関連タンパク質又は前記融合タンパク質をコードする核酸（以下、まとめて「コラーゲン核酸」と称することがある）である。
《コラーゲン核酸》
本発明のコラーゲン核酸は、前記プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体、又はそれらの断片などのコラーゲン関連タンパク質、或いはコラーゲン関連タンパク質と蛍光タンパク質等との融合タンパク質をコードする核酸である限りにおいて、特に限定されるものでない。
プロコラーゲンタンパク質、又はコラーゲンタンパク質をコードする核酸は、限定されるものではないが、例えばコラーゲンを有する生物の組織、又はその生物の分離された細胞から抽出して用いることができる。また、既に分離されたｃＤＮＡを含むベクターから得ることもできる。更に、ＤＮＡ合成によって、調製することも可能である。 [2] Nucleic acid and vector The nucleic acid of the present invention is a nucleic acid encoding the collagen-related protein or the fusion protein (hereinafter sometimes collectively referred to as “collagen nucleic acid”).
<Collagen nucleic acid>
As long as the collagen nucleic acid of the present invention is a nucleic acid encoding a collagen-related protein such as the procollagen protein, collagen protein, or a variant thereof, or a fragment thereof, or a fusion protein of a collagen-related protein and a fluorescent protein or the like. However, there is no particular limitation.
The procollagen protein or the nucleic acid encoding the collagen protein is not limited, and can be extracted from, for example, a tissue of an organism having collagen or a separated cell of the organism. It can also be obtained from a vector containing the already isolated cDNA. It can also be prepared by DNA synthesis.
また、前記変異体をコードする核酸は、例えばプロコラーゲンタンパク質又はコラーゲンタンパク質をコードする核酸に、アミノ酸が欠失、置換、挿入、及び／又は付加されるように、核酸の変異を導入することによって得ることができる。核酸の変異（欠失、置換、挿入、及び／又は付加）は、本分野において公知の方法によって、導入することが可能である。また、遺伝子操作を用いずに、自然界に存在する、アミノ酸が欠失、置換、挿入、及び／又は付加される変異を有する核酸を用いることもできる。
In addition, the nucleic acid encoding the mutant can be obtained by introducing a mutation of the nucleic acid so that an amino acid is deleted, substituted, inserted, and / or added to, for example, a procollagen protein or a nucleic acid encoding a collagen protein. Obtainable. Nucleic acid mutations (deletions, substitutions, insertions, and / or additions) can be introduced by methods known in the art. Moreover, the nucleic acid which has the variation | mutation by which the amino acid which exists in nature is deleted, substituted, inserted, and / or added without using genetic manipulation can also be used.
更に、前記断片をコードする核酸は、プロコラーゲンタンパク質、コラーゲンタンパク質、又はそれらの変異体をコードする核酸の一部を用いることができる。例えば、前記核酸を制限酵素によって切断することによって、プロコラーゲンタンパク質、コラーゲンタンパク質、又はそれらの変異体の断片をコードする核酸を得ることができる。
Furthermore, as the nucleic acid encoding the fragment, a part of the nucleic acid encoding procollagen protein, collagen protein, or a variant thereof can be used. For example, a nucleic acid encoding a procollagen protein, a collagen protein, or a fragment of a variant thereof can be obtained by cleaving the nucleic acid with a restriction enzyme.
本発明のベクターに含まれる核酸としては、限定されるものではないが、例えばＤＮＡ又はＲＮＡを挙げることができる。
The nucleic acid contained in the vector of the present invention is not limited, and examples thereof include DNA or RNA.
《ベクター》
本発明のベクターは、前記コラーゲン核酸を含む限りにおいて、限定されるものではない。また、前記コラーゲン核酸が導入される骨格のベクターは、クローニングベクターでもよく、発現ベクターでもよい。また、ＲＮＡベクターとしては、バクテリオファージを挙げることができる。
骨格となるベクターとしては、本分野で通常用いられているベクターを制限なく用いることができるが、例えばｐＢＲ３２２、ｐＢＲ３２５、ｐＵＣ１１８、ｐＵＣ１１９、ｐＫＣ３０、ｐＣＦＭ５３６、ｐｃＤＮＡ３．１、ｐｃＤＮＡ３，ｐＭＥ、又はｐＧＥＸ等の大腸菌プラスミド、を挙げることができる。また、ベクターとしては市販されているものを使用することができ、例えばｐＱＥ７０、ｐＱＥ６０、ｐＱＥ－９（Ｑｉａｇｅｎ）、ｐＢｌｕｅｓｃｒｉｐｔＩＩ ＫＳ、ｐｔｒｃ９９ａ、ｐＫＫ２２３－３、ｐＤＲ５４０、ｐＲＩＴ２Ｔ（Ｐｈａｒｍａｃｉａ）、ｐＥＴ－１１ａ（Ｎｏｖａｇｅｎ）などを挙げることができる。骨格ベクターは、複製開始点、選択マーカー、プロモーターを含み、必要に応じてエンハンサー、転写終結配列（ターミネーター）、リボソーム結合部位、ポリアデニル化シグナル等を含んでいてもよい。 "vector"
The vector of the present invention is not limited as long as it contains the collagen nucleic acid. The skeleton vector into which the collagen nucleic acid is introduced may be a cloning vector or an expression vector. Moreover, bacteriophage can be mentioned as an RNA vector.
As the backbone vector, a vector usually used in this field can be used without limitation. For example, pBR322, pBR325, pUC118, pUC119, pKC30, pCFM536, pcDNA3.1, pcDNA3, pME, or pGEX. Examples include E. coli plasmids. Commercially available vectors can be used, such as pQE70, pQE60, pQE-9 (Qiagen), pBluescript II KS, ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), pET-11a (Novagen) ) And the like. The backbone vector includes a replication origin, a selection marker, and a promoter, and may include an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like as necessary.
本発明のベクターは、前記コラーゲン核酸を含む限りにおいて、限定されるものではない。また、前記コラーゲン核酸が導入される骨格のベクターは、クローニングベクターでもよく、発現ベクターでもよい。また、ＲＮＡベクターとしては、バクテリオファージを挙げることができる。
骨格となるベクターとしては、本分野で通常用いられているベクターを制限なく用いることができるが、例えばｐＢＲ３２２、ｐＢＲ３２５、ｐＵＣ１１８、ｐＵＣ１１９、ｐＫＣ３０、ｐＣＦＭ５３６、ｐｃＤＮＡ３．１、ｐｃＤＮＡ３，ｐＭＥ、又はｐＧＥＸ等の大腸菌プラスミド、を挙げることができる。また、ベクターとしては市販されているものを使用することができ、例えばｐＱＥ７０、ｐＱＥ６０、ｐＱＥ－９（Ｑｉａｇｅｎ）、ｐＢｌｕｅｓｃｒｉｐｔＩＩ ＫＳ、ｐｔｒｃ９９ａ、ｐＫＫ２２３－３、ｐＤＲ５４０、ｐＲＩＴ２Ｔ（Ｐｈａｒｍａｃｉａ）、ｐＥＴ－１１ａ（Ｎｏｖａｇｅｎ）などを挙げることができる。骨格ベクターは、複製開始点、選択マーカー、プロモーターを含み、必要に応じてエンハンサー、転写終結配列（ターミネーター）、リボソーム結合部位、ポリアデニル化シグナル等を含んでいてもよい。 "vector"
The vector of the present invention is not limited as long as it contains the collagen nucleic acid. The skeleton vector into which the collagen nucleic acid is introduced may be a cloning vector or an expression vector. Moreover, bacteriophage can be mentioned as an RNA vector.
As the backbone vector, a vector usually used in this field can be used without limitation. For example, pBR322, pBR325, pUC118, pUC119, pKC30, pCFM536, pcDNA3.1, pcDNA3, pME, or pGEX. Examples include E. coli plasmids. Commercially available vectors can be used, such as pQE70, pQE60, pQE-9 (Qiagen), pBluescript II KS, ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), pET-11a (Novagen) ) And the like. The backbone vector includes a replication origin, a selection marker, and a promoter, and may include an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like as necessary.
前記コラーゲン核酸含有ベクターは、コラーゲン核酸以外の核酸を含むことができる。例えば、コラーゲンタンパク質等に、他のタンパク質を結合させて融合タンパク質を得ることができるが、コラーゲン核酸含有ベクターは、このコラーゲンに結合させるタンパク質（以下、結合タンパク質と称することがある）をコードする核酸を含むことができる。
前記結合タンパク質は、コラーゲンタンパク質等のＮ末端、又はＣ末端に結合させることもでき、またコラーゲンタンパク質等の中に挿入することもできる。すなわち、結合タンパク質をコードする核酸は、コラーゲン核酸の５‘側、又は３’側に結合させてもよく、またコラーゲン核酸の中に挿入してもよい。 The collagen nucleic acid-containing vector can contain a nucleic acid other than a collagen nucleic acid. For example, a fusion protein can be obtained by binding other proteins to collagen protein or the like, but a collagen nucleic acid-containing vector is a nucleic acid that encodes a protein to be bound to this collagen (hereinafter sometimes referred to as binding protein). Can be included.
The binding protein can be bound to the N-terminus or C-terminus of collagen protein or the like, or can be inserted into collagen protein or the like. That is, the nucleic acid encoding the binding protein may be bound to the 5 ′ side or 3 ′ side of the collagen nucleic acid, or may be inserted into the collagen nucleic acid.
前記結合タンパク質は、コラーゲンタンパク質等のＮ末端、又はＣ末端に結合させることもでき、またコラーゲンタンパク質等の中に挿入することもできる。すなわち、結合タンパク質をコードする核酸は、コラーゲン核酸の５‘側、又は３’側に結合させてもよく、またコラーゲン核酸の中に挿入してもよい。 The collagen nucleic acid-containing vector can contain a nucleic acid other than a collagen nucleic acid. For example, a fusion protein can be obtained by binding other proteins to collagen protein or the like, but a collagen nucleic acid-containing vector is a nucleic acid that encodes a protein to be bound to this collagen (hereinafter sometimes referred to as binding protein). Can be included.
The binding protein can be bound to the N-terminus or C-terminus of collagen protein or the like, or can be inserted into collagen protein or the like. That is, the nucleic acid encoding the binding protein may be bound to the 5 ′ side or 3 ′ side of the collagen nucleic acid, or may be inserted into the collagen nucleic acid.
本発明のベクターは、限定されるものではないが、好ましくは後述の特定の遺伝子変異を有する大腸菌から得られるものである。すなわち、特定の遺伝子変異を有する大腸菌に導入され、その大腸菌から回収されたものが好ましい。欠失、挿入、制限酵素での切断の阻害が生じていないコラーゲン核酸を含むベクターを得ることができるからである。
The vector of the present invention is not limited, but is preferably obtained from Escherichia coli having a specific gene mutation described below. That is, those introduced into E. coli having a specific gene mutation and recovered from the E. coli are preferred. This is because it is possible to obtain a vector containing a collagen nucleic acid in which deletion, insertion, or inhibition of cleavage with a restriction enzyme has not occurred.
［３］ベクター含有宿主細胞
本発明のベクター含有宿主細胞は、本発明のベクターを含むものである。宿主は、特に限定されるものではなく、大腸菌、放線菌、酵母、糸状菌、又は動物細胞を挙げることができるが、好ましくは大腸菌又は動物細胞であり、更に好ましくは特定の遺伝子変異を有する大腸菌又は動物細胞である。特定の遺伝子変異を有する大腸菌を用いることにより、欠失、挿入、制限酵素での切断の阻害が生じていないコラーゲン核酸を含むベクターを得ることができるからである。また、動物細胞を用いることにより、コラーゲンを製造することができる。更に、コラーゲンの高次構造の形成過程を解析することが可能である。 [3] Vector-containing host cell The vector-containing host cell of the present invention comprises the vector of the present invention. The host is not particularly limited, and examples include E. coli, actinomycetes, yeast, filamentous fungi, or animal cells, preferably E. coli or animal cells, more preferably E. coli having a specific gene mutation. Or it is an animal cell. This is because by using Escherichia coli having a specific gene mutation, a vector containing a collagen nucleic acid in which deletion, insertion, or inhibition of cleavage with a restriction enzyme has not occurred can be obtained. Further, collagen can be produced by using animal cells. Furthermore, it is possible to analyze the formation process of the higher-order structure of collagen.
本発明のベクター含有宿主細胞は、本発明のベクターを含むものである。宿主は、特に限定されるものではなく、大腸菌、放線菌、酵母、糸状菌、又は動物細胞を挙げることができるが、好ましくは大腸菌又は動物細胞であり、更に好ましくは特定の遺伝子変異を有する大腸菌又は動物細胞である。特定の遺伝子変異を有する大腸菌を用いることにより、欠失、挿入、制限酵素での切断の阻害が生じていないコラーゲン核酸を含むベクターを得ることができるからである。また、動物細胞を用いることにより、コラーゲンを製造することができる。更に、コラーゲンの高次構造の形成過程を解析することが可能である。 [3] Vector-containing host cell The vector-containing host cell of the present invention comprises the vector of the present invention. The host is not particularly limited, and examples include E. coli, actinomycetes, yeast, filamentous fungi, or animal cells, preferably E. coli or animal cells, more preferably E. coli having a specific gene mutation. Or it is an animal cell. This is because by using Escherichia coli having a specific gene mutation, a vector containing a collagen nucleic acid in which deletion, insertion, or inhibition of cleavage with a restriction enzyme has not occurred can be obtained. Further, collagen can be produced by using animal cells. Furthermore, it is possible to analyze the formation process of the higher-order structure of collagen.
《遺伝子変異を有する大腸菌》
本発明のベクターを含む遺伝子変異を有する大腸菌としては、限定されるものではないが、好ましくはmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌である。遺伝子変異の組み合わせは、限定されるものではないが、好ましくはmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、及びそれらの2つ以上の組み合わせからなる群から選択される遺伝子変異であり、より好ましくは、mcrA、Δ(mrr-hsdRMS-mcrBC)、recA 、hsdS及びそれらの2つ以上の組み合わせからなる群から選択される遺伝子変異であり、更に好ましくは、mcrA、Δ(mrr-hsdRMS-mcrBC)、recA及びそれらの2つ以上の組み合わせからなる群から選択される遺伝子変異であり、最も好ましくはmcrA、Δ(mrr-hsdRMS-mcrBC)、及びそれらの組み合わせからなる群から選択される遺伝子変異である。遺伝子変異を有する大腸菌としては、より具体的にはＤＨ１０Ｂ、ＴＯＰ１０Ｆ’、ＭＣ１０６１、ＸＬ１－Ｂｌｕｅ ＭＲＦ’、ＡＧ１、ＢＬ２１（ＤＥ３）、ＤＢ３．１、ＤＨ１、ＤＨ５α、ＤＨ５α Ｔｕｒｂｏ、ＤＨ１２Ｓ、ＤＭ１、Ｅ．Ｃｌｏｎｉ（ｒ）５ａｌｐｈａ、Ｅ．Ｃｌｏｎｉ（ｒ）１０Ｇ、Ｅ．Ｃｌｏｎｉ（ｒ）１０ＧＦ’、ＥＲ２２６７、ＨＢ１０１、ＨＭＳ１７４（ＤＥ３）、Ｈｉｇｈ－Ｃｏｎｔｒｏｌ（ｔｍ） ＢＬ２１（ＤＥ３）、Ｈｉｇｈ－Ｃｏｎｔｒｏｌ（ｔｍ）１０Ｇ、ＩＪ１１２６、ＪＭ１０８、ＪＭ１０９、Ｍａｃｈ１、ＭＣ１０６１、ＭＦＤｐｉｒ、ＯｍｎｉＭＡＸ２、ＯｖｅｒＥｘｐｒｅｓｓ（ｔｍ）Ｃ４１（ＤＥ３）、ＯｖｅｒＥｘｐｒｅｓｓ（ｔｍ）Ｃ４３（ＤＥ３）、Ｒｏｓｅｔｔａ（ＤＥ３）、ＳＯＬＲ、ＳＳ３２０、ＳＴＢＬ２、ＳＴＢＬ３、ＳＴＢＬ４、ＳＵＲＥ、ＳＵＲＥ２、ＴＧ１、ＴＯＰ１０、ＸＬ２－Ｂｌｕｅ、ＸＬ２－ＢｌｕｅＭＲＦ’、及びＸＬ１０－Ｇｏｌｄを挙げることができるが、特にはＤＨ１０Ｂ、ＴＯＰ１０Ｆ’、ＴＯＰ１０、ＳＴＢＬ２、及びＳＴＢＬ４が好ましい。 <Escherichia coli with gene mutation>
The Escherichia coli having a gene mutation containing the vector of the present invention is not limited, but preferably mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara , leu) 7697, galU, galK , rpsL (str r), E. coli having nupG, galE, hsdS, and gene mutation selected from the group consisting of combinations of two or more thereof. The combination of gene mutations is not limited, but preferably mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG, and a genetic mutation selected from the group consisting of two or more thereof, more preferably mcrA, Δ (mrr-hsdRMS-mcrBC), recA, hsdS and two or more thereof A gene mutation selected from the group consisting of a combination, more preferably a gene mutation selected from the group consisting of mcrA, Δ (mrr-hsdRMS-mcrBC), recA and combinations of two or more thereof, most Preferably, it is a gene mutation selected from the group consisting of mcrA, Δ (mrr-hsdRMS-mcrBC), and combinations thereof. More specifically, Escherichia coli having a gene mutation includes DH10B, TOP10F ′, MC1061, XL1-Blue MRF ′, AG1, BL21 (DE3), DB3.1, DH1, DH5α, DH5α Turbo, DH12S, DM1, E. coli. Cloni (r) 5alpha, E.I. Cloni (r) 10G, E.I. Cloni (r) 10GF ', ER2267, HB101, HMS174 (DE3), High-Control (tm) BL21 (DE3), High-Control (tm) 10G, IJ1126, JM108, JM109, Mach1, MC1061, MFDpiMx, OFDpiMx, OFDpiMx (Tm) C41 (DE3), OverExpress (tm) C43 (DE3), Rosetta (DE3), SOLR, SS320, STBL2, STBL3, STBL4, SURE, SURE2, TG1, TOP10, XL2-Blue, XL2-BlueMRF ', and XL10-Gold can be mentioned, and DH10B, TOP10F ′, TOP10, STBL2 and STBL4 are particularly preferable.
本発明のベクターを含む遺伝子変異を有する大腸菌としては、限定されるものではないが、好ましくはmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌である。遺伝子変異の組み合わせは、限定されるものではないが、好ましくはmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、及びそれらの2つ以上の組み合わせからなる群から選択される遺伝子変異であり、より好ましくは、mcrA、Δ(mrr-hsdRMS-mcrBC)、recA 、hsdS及びそれらの2つ以上の組み合わせからなる群から選択される遺伝子変異であり、更に好ましくは、mcrA、Δ(mrr-hsdRMS-mcrBC)、recA及びそれらの2つ以上の組み合わせからなる群から選択される遺伝子変異であり、最も好ましくはmcrA、Δ(mrr-hsdRMS-mcrBC)、及びそれらの組み合わせからなる群から選択される遺伝子変異である。遺伝子変異を有する大腸菌としては、より具体的にはＤＨ１０Ｂ、ＴＯＰ１０Ｆ’、ＭＣ１０６１、ＸＬ１－Ｂｌｕｅ ＭＲＦ’、ＡＧ１、ＢＬ２１（ＤＥ３）、ＤＢ３．１、ＤＨ１、ＤＨ５α、ＤＨ５α Ｔｕｒｂｏ、ＤＨ１２Ｓ、ＤＭ１、Ｅ．Ｃｌｏｎｉ（ｒ）５ａｌｐｈａ、Ｅ．Ｃｌｏｎｉ（ｒ）１０Ｇ、Ｅ．Ｃｌｏｎｉ（ｒ）１０ＧＦ’、ＥＲ２２６７、ＨＢ１０１、ＨＭＳ１７４（ＤＥ３）、Ｈｉｇｈ－Ｃｏｎｔｒｏｌ（ｔｍ） ＢＬ２１（ＤＥ３）、Ｈｉｇｈ－Ｃｏｎｔｒｏｌ（ｔｍ）１０Ｇ、ＩＪ１１２６、ＪＭ１０８、ＪＭ１０９、Ｍａｃｈ１、ＭＣ１０６１、ＭＦＤｐｉｒ、ＯｍｎｉＭＡＸ２、ＯｖｅｒＥｘｐｒｅｓｓ（ｔｍ）Ｃ４１（ＤＥ３）、ＯｖｅｒＥｘｐｒｅｓｓ（ｔｍ）Ｃ４３（ＤＥ３）、Ｒｏｓｅｔｔａ（ＤＥ３）、ＳＯＬＲ、ＳＳ３２０、ＳＴＢＬ２、ＳＴＢＬ３、ＳＴＢＬ４、ＳＵＲＥ、ＳＵＲＥ２、ＴＧ１、ＴＯＰ１０、ＸＬ２－Ｂｌｕｅ、ＸＬ２－ＢｌｕｅＭＲＦ’、及びＸＬ１０－Ｇｏｌｄを挙げることができるが、特にはＤＨ１０Ｂ、ＴＯＰ１０Ｆ’、ＴＯＰ１０、ＳＴＢＬ２、及びＳＴＢＬ４が好ましい。 <Escherichia coli with gene mutation>
The Escherichia coli having a gene mutation containing the vector of the present invention is not limited, but preferably mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara , leu) 7697, galU, galK , rpsL (str r), E. coli having nupG, galE, hsdS, and gene mutation selected from the group consisting of combinations of two or more thereof. The combination of gene mutations is not limited, but preferably mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG, and a genetic mutation selected from the group consisting of two or more thereof, more preferably mcrA, Δ (mrr-hsdRMS-mcrBC), recA, hsdS and two or more thereof A gene mutation selected from the group consisting of a combination, more preferably a gene mutation selected from the group consisting of mcrA, Δ (mrr-hsdRMS-mcrBC), recA and combinations of two or more thereof, most Preferably, it is a gene mutation selected from the group consisting of mcrA, Δ (mrr-hsdRMS-mcrBC), and combinations thereof. More specifically, Escherichia coli having a gene mutation includes DH10B, TOP10F ′, MC1061, XL1-Blue MRF ′, AG1, BL21 (DE3), DB3.1, DH1, DH5α, DH5α Turbo, DH12S, DM1, E. coli. Cloni (r) 5alpha, E.I. Cloni (r) 10G, E.I. Cloni (r) 10GF ', ER2267, HB101, HMS174 (DE3), High-Control (tm) BL21 (DE3), High-Control (tm) 10G, IJ1126, JM108, JM109, Mach1, MC1061, MFDpiMx, OFDpiMx, OFDpiMx (Tm) C41 (DE3), OverExpress (tm) C43 (DE3), Rosetta (DE3), SOLR, SS320, STBL2, STBL3, STBL4, SURE, SURE2, TG1, TOP10, XL2-Blue, XL2-BlueMRF ', and XL10-Gold can be mentioned, and DH10B, TOP10F ′, TOP10, STBL2 and STBL4 are particularly preferable.
大腸菌へのベクターの導入は、本分野で通常用いられている方法を、制限なく用いることができる。例えば、塩化カルシウム処理したコンピテント細胞の利用、プロトプラスト法、リン酸カルシウム法、リポフェクション法、又はエレクトロポレーション法などを用いることができる。
For introduction of a vector into E. coli, a method usually used in this field can be used without limitation. For example, use of competent cells treated with calcium chloride, a protoplast method, a calcium phosphate method, a lipofection method, an electroporation method, or the like can be used.
［４］ベクター含有大腸菌の製造方法及びベクターの製造方法
本発明のベクター含有大腸菌の製造方法は、本発明のベクターを、mcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、 galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌に導入することを特徴とする。
また、本発明のベクターの製造方法は、本発明のベクターをmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌に導入する工程、及び前記ベクターを回収する工程を含むことを特徴とする。 [4] Vector-containing Escherichia coli production method and vector production method The vector-containing Escherichia coli production method of the present invention comprises mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, introduced into E. coli having a genetic mutation selected from the group consisting of endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG, galE, hsdS, and combinations of two or more thereof It is characterized by doing.
In addition, the method of producing the vector of the present invention includes mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK. , RpsL (str r ), nupG, galE, hsdS, and a step of introducing the vector into the E. coli having a genetic mutation selected from the group consisting of two or more thereof, and recovering the vector, To do.
本発明のベクター含有大腸菌の製造方法は、本発明のベクターを、mcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、 galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌に導入することを特徴とする。
また、本発明のベクターの製造方法は、本発明のベクターをmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌に導入する工程、及び前記ベクターを回収する工程を含むことを特徴とする。 [4] Vector-containing Escherichia coli production method and vector production method The vector-containing Escherichia coli production method of the present invention comprises mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, introduced into E. coli having a genetic mutation selected from the group consisting of endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG, galE, hsdS, and combinations of two or more thereof It is characterized by doing.
In addition, the method of producing the vector of the present invention includes mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK. , RpsL (str r ), nupG, galE, hsdS, and a step of introducing the vector into the E. coli having a genetic mutation selected from the group consisting of two or more thereof, and recovering the vector, To do.
本発明のベクター含有大腸菌の製造方法、又はベクターの製造方法における「プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体、又はそれらの断片をコードする核酸」、「ベクター」、及び「mcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌」等は、前記の「［１］融合タンパク質」、「［２］核酸及びベクター」、及び「［３］ベクター含有宿主細胞」の項に記載のものを用いることができる。
また、本発明のベクターの製造方法における、ベクターを回収する方法は、本分野において公知の方法を用いることができるが、例えば前記ベクターが導入されたコラーゲン核酸含有ベクターをＬＢ培地などで培養し、大腸菌を増殖させる。増殖した大腸菌を回収し、例えばアルカリＳＤＳ法、煮沸法、エチジウム／塩化セシウム密度勾配遠心法、ガラスビーズ精製法、又はそれらの変法を用いて、ベクターＤＮＡを回収することができる。 In the method for producing a vector-containing Escherichia coli of the present invention, or the method for producing a vector, “nucleic acid encoding a procollagen protein, collagen protein, or a variant thereof, or a fragment thereof”, “vector”, and “mcrA, Δ ( mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG, galE, hsdS, and two or more thereof “Escherichia coli having a gene mutation selected from the group consisting of combinations” and the like are described in the above-mentioned sections “[1] Fusion protein”, “[2] Nucleic acid and vector”, and “[3] Vector-containing host cell”. Can be used.
Further, in the method for producing a vector of the present invention, a method for recovering the vector can be a method known in the art. For example, a collagen nucleic acid-containing vector into which the vector has been introduced is cultured in an LB medium or the like, Grow E. coli. The proliferated Escherichia coli is recovered, and the vector DNA can be recovered using, for example, alkaline SDS method, boiling method, ethidium / cesium chloride density gradient centrifugation method, glass bead purification method, or a modified method thereof.
また、本発明のベクターの製造方法における、ベクターを回収する方法は、本分野において公知の方法を用いることができるが、例えば前記ベクターが導入されたコラーゲン核酸含有ベクターをＬＢ培地などで培養し、大腸菌を増殖させる。増殖した大腸菌を回収し、例えばアルカリＳＤＳ法、煮沸法、エチジウム／塩化セシウム密度勾配遠心法、ガラスビーズ精製法、又はそれらの変法を用いて、ベクターＤＮＡを回収することができる。 In the method for producing a vector-containing Escherichia coli of the present invention, or the method for producing a vector, “nucleic acid encoding a procollagen protein, collagen protein, or a variant thereof, or a fragment thereof”, “vector”, and “mcrA, Δ ( mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG, galE, hsdS, and two or more thereof “Escherichia coli having a gene mutation selected from the group consisting of combinations” and the like are described in the above-mentioned sections “[1] Fusion protein”, “[2] Nucleic acid and vector”, and “[3] Vector-containing host cell”. Can be used.
Further, in the method for producing a vector of the present invention, a method for recovering the vector can be a method known in the art. For example, a collagen nucleic acid-containing vector into which the vector has been introduced is cultured in an LB medium or the like, Grow E. coli. The proliferated Escherichia coli is recovered, and the vector DNA can be recovered using, for example, alkaline SDS method, boiling method, ethidium / cesium chloride density gradient centrifugation method, glass bead purification method, or a modified method thereof.
［５］コラーゲンの製造方法
本発明のコラーゲンの製造方法は、（１）本発明のベクターを、動物細胞に導入する工程、及び（２）前記ベクターが導入された動物細胞からコラーゲンを分泌させる工程、を含む。
本発明のコラーゲンの製造方法に用いる「ベクター」は、前記「［１］融合タンパク質」、「［２］核酸及びベクター」、及び「［３］ベクター含有宿主細胞」の項に記載の「ベクター」を用いることができる。 [5] Method for Producing Collagen The method for producing collagen of the present invention comprises (1) a step of introducing the vector of the present invention into an animal cell, and (2) a step of secreting collagen from the animal cell into which the vector has been introduced. ,including.
The “vector” used in the method for producing collagen of the present invention is the “vector” described in the sections “[1] fusion protein”, “[2] nucleic acid and vector”, and “[3] vector-containing host cell”. Can be used.
本発明のコラーゲンの製造方法は、（１）本発明のベクターを、動物細胞に導入する工程、及び（２）前記ベクターが導入された動物細胞からコラーゲンを分泌させる工程、を含む。
本発明のコラーゲンの製造方法に用いる「ベクター」は、前記「［１］融合タンパク質」、「［２］核酸及びベクター」、及び「［３］ベクター含有宿主細胞」の項に記載の「ベクター」を用いることができる。 [5] Method for Producing Collagen The method for producing collagen of the present invention comprises (1) a step of introducing the vector of the present invention into an animal cell, and (2) a step of secreting collagen from the animal cell into which the vector has been introduced. ,including.
The “vector” used in the method for producing collagen of the present invention is the “vector” described in the sections “[1] fusion protein”, “[2] nucleic acid and vector”, and “[3] vector-containing host cell”. Can be used.
《動物細胞》
本発明のコラーゲンの製造方法に用いる動物細胞は、コラーゲンを発現することのできる細胞である限りにおいて、特に限定されるものではないが、例えば哺乳類（例えば、ウシ、ブタ、ヒツジ、ヤギ、マウス、ラット、モルモット、又はサル）、鳥類（例えば、ニワトリ、ガチョウ、アヒル、又はダチョウ）、爬虫類（例えば、ワニ）、両生類（例えば、カエル）、魚類（例えば、チョウザメ、テラピア、タイ、ヒラメ、サメ、イワシ、マグロ、フグ、キンギョ、タラ、カレイ、又はコイ）、又は無脊椎動物（例えば、クラゲ）由来の細胞を用いることができる。
動物細胞への前記ベクターの導入方法は、特に限定されるものではなく、本分野で公知の方法を用いることができるが、例えばエレクトロポレーション法を用いることができる。 <Animal cells>
Animal cells used in the method for producing collagen of the present invention are not particularly limited as long as they are cells capable of expressing collagen. For example, mammals (for example, cows, pigs, sheep, goats, mice, Rats, guinea pigs, or monkeys), birds (eg, chickens, geese, ducks, or ostriches), reptiles (eg, crocodiles), amphibians (eg, frogs), fishes (eg, sturgeon, tilapia, thailand, flounder, shark, Cells derived from sardines, tuna, puffer fish, goldfish, cod, flounder, or carp), or invertebrates (eg, jellyfish) can be used.
The method for introducing the vector into animal cells is not particularly limited, and a method known in the art can be used. For example, an electroporation method can be used.
本発明のコラーゲンの製造方法に用いる動物細胞は、コラーゲンを発現することのできる細胞である限りにおいて、特に限定されるものではないが、例えば哺乳類（例えば、ウシ、ブタ、ヒツジ、ヤギ、マウス、ラット、モルモット、又はサル）、鳥類（例えば、ニワトリ、ガチョウ、アヒル、又はダチョウ）、爬虫類（例えば、ワニ）、両生類（例えば、カエル）、魚類（例えば、チョウザメ、テラピア、タイ、ヒラメ、サメ、イワシ、マグロ、フグ、キンギョ、タラ、カレイ、又はコイ）、又は無脊椎動物（例えば、クラゲ）由来の細胞を用いることができる。
動物細胞への前記ベクターの導入方法は、特に限定されるものではなく、本分野で公知の方法を用いることができるが、例えばエレクトロポレーション法を用いることができる。 <Animal cells>
Animal cells used in the method for producing collagen of the present invention are not particularly limited as long as they are cells capable of expressing collagen. For example, mammals (for example, cows, pigs, sheep, goats, mice, Rats, guinea pigs, or monkeys), birds (eg, chickens, geese, ducks, or ostriches), reptiles (eg, crocodiles), amphibians (eg, frogs), fishes (eg, sturgeon, tilapia, thailand, flounder, shark, Cells derived from sardines, tuna, puffer fish, goldfish, cod, flounder, or carp), or invertebrates (eg, jellyfish) can be used.
The method for introducing the vector into animal cells is not particularly limited, and a method known in the art can be used. For example, an electroporation method can be used.
《分泌工程》
ベクターが導入された動物細胞から、コラーゲンを分泌させる方法は、特に限定されるものではないが、動物細胞を培地で培養することによってできる。
培養温度も特に限定されるものではないが、例えば３０～４２で培養することができ、好ましくは３５～３９℃である。また、動物細胞中のタンパク質濃度を調整することによって、細胞外への分泌を最適にすることができる。また、分泌には、細胞が多層を形成することが、好ましい。 <Secret process>
A method for secreting collagen from animal cells into which a vector has been introduced is not particularly limited, but can be performed by culturing animal cells in a medium.
The culture temperature is not particularly limited, but can be cultured at, for example, 30 to 42, preferably 35 to 39 ° C. In addition, secretion outside the cell can be optimized by adjusting the protein concentration in the animal cell. For secretion, it is preferable that cells form a multilayer.
ベクターが導入された動物細胞から、コラーゲンを分泌させる方法は、特に限定されるものではないが、動物細胞を培地で培養することによってできる。
培養温度も特に限定されるものではないが、例えば３０～４２で培養することができ、好ましくは３５～３９℃である。また、動物細胞中のタンパク質濃度を調整することによって、細胞外への分泌を最適にすることができる。また、分泌には、細胞が多層を形成することが、好ましい。 <Secret process>
A method for secreting collagen from animal cells into which a vector has been introduced is not particularly limited, but can be performed by culturing animal cells in a medium.
The culture temperature is not particularly limited, but can be cultured at, for example, 30 to 42, preferably 35 to 39 ° C. In addition, secretion outside the cell can be optimized by adjusting the protein concentration in the animal cell. For secretion, it is preferable that cells form a multilayer.
［６］コラーゲンの蛍光又は発光検出方法
本発明のコラーゲンの蛍光又は発光検出方法は、（１）本発明のベクターを、動物細胞に導入する工程、及び（２）前記ベクターが導入された動物細胞において蛍光又は発光を検出する工程、を含む。
本発明のコラーゲンの蛍光又は発光検出方法に用いる「ベクター」は、前記「［１］融合タンパク質」、「［２］核酸及びベクター」、及び「［３］ベクター含有宿主細胞」の項に記載の「ベクター」を用いることができる。 [6] Collagen fluorescence or luminescence detection method The collagen fluorescence or luminescence detection method of the present invention comprises (1) a step of introducing the vector of the present invention into an animal cell, and (2) an animal cell into which the vector has been introduced. Detecting fluorescence or luminescence.
The “vector” used in the method for detecting fluorescence or luminescence of collagen of the present invention is described in the above “[1] fusion protein”, “[2] nucleic acid and vector”, and “[3] vector-containing host cell”. A “vector” can be used.
本発明のコラーゲンの蛍光又は発光検出方法は、（１）本発明のベクターを、動物細胞に導入する工程、及び（２）前記ベクターが導入された動物細胞において蛍光又は発光を検出する工程、を含む。
本発明のコラーゲンの蛍光又は発光検出方法に用いる「ベクター」は、前記「［１］融合タンパク質」、「［２］核酸及びベクター」、及び「［３］ベクター含有宿主細胞」の項に記載の「ベクター」を用いることができる。 [6] Collagen fluorescence or luminescence detection method The collagen fluorescence or luminescence detection method of the present invention comprises (1) a step of introducing the vector of the present invention into an animal cell, and (2) an animal cell into which the vector has been introduced. Detecting fluorescence or luminescence.
The “vector” used in the method for detecting fluorescence or luminescence of collagen of the present invention is described in the above “[1] fusion protein”, “[2] nucleic acid and vector”, and “[3] vector-containing host cell”. A “vector” can be used.
《動物細胞》
本発明のコラーゲンの製造方法に用いる動物細胞は、コラーゲンを発現することのできる細胞である限りにおいて、特に限定されるものではないが、前記「［５］コラーゲンの製造方法」に記載の動物細胞を挙げることができる。 <Animal cells>
The animal cell used in the method for producing collagen of the present invention is not particularly limited as long as it is a cell capable of expressing collagen, but the animal cell described in the above “[5] Method for producing collagen”. Can be mentioned.
本発明のコラーゲンの製造方法に用いる動物細胞は、コラーゲンを発現することのできる細胞である限りにおいて、特に限定されるものではないが、前記「［５］コラーゲンの製造方法」に記載の動物細胞を挙げることができる。 <Animal cells>
The animal cell used in the method for producing collagen of the present invention is not particularly limited as long as it is a cell capable of expressing collagen, but the animal cell described in the above “[5] Method for producing collagen”. Can be mentioned.
《検出工程》
動物細胞からの蛍光又は発光の検出も、本分野における公知の方法を用いることができるが、例えば蛍光顕微鏡、分光光度計、又は共焦点蛍光顕微鏡などを用いることによって、検出することができる。 << Detection process >>
Fluorescence or luminescence from animal cells can also be detected by a known method in this field, but can be detected by using, for example, a fluorescence microscope, a spectrophotometer, a confocal fluorescence microscope, or the like.
動物細胞からの蛍光又は発光の検出も、本分野における公知の方法を用いることができるが、例えば蛍光顕微鏡、分光光度計、又は共焦点蛍光顕微鏡などを用いることによって、検出することができる。 << Detection process >>
Fluorescence or luminescence from animal cells can also be detected by a known method in this field, but can be detected by using, for example, a fluorescence microscope, a spectrophotometer, a confocal fluorescence microscope, or the like.
［７］コラーゲン関連疾患の治療又は予防物質のスクリーニング方法
本発明のコラーゲン関連疾患の治療又は予防物質のスクリーニング方法は、前記融合タンパク質を発現したコラーゲン関連疾患細胞と、試験物質とを接触させる工程、及び融合タンパク質の動態を解析する工程、を含む。 [7] Method for screening a substance for treating or preventing a collagen-related disease The method for screening a substance for treating or preventing a collagen-related disease of the present invention comprises contacting a collagen-related disease cell expressing the fusion protein with a test substance, And analyzing the kinetics of the fusion protein.
本発明のコラーゲン関連疾患の治療又は予防物質のスクリーニング方法は、前記融合タンパク質を発現したコラーゲン関連疾患細胞と、試験物質とを接触させる工程、及び融合タンパク質の動態を解析する工程、を含む。 [7] Method for screening a substance for treating or preventing a collagen-related disease The method for screening a substance for treating or preventing a collagen-related disease of the present invention comprises contacting a collagen-related disease cell expressing the fusion protein with a test substance, And analyzing the kinetics of the fusion protein.
《コラーゲン関連疾患》
コラーゲン関連疾患は、コラーゲンがその疾患の原因、又は症状等に関連すると考えられる限りにおいて、特に限定されるものではないが、例えば肝線維症、肺線維症、腎線維症などの臓器・組織線維症、膠原病、又は間接リウマチを挙げることができる。
本発明のスクリーニング方法に用いるコラーゲン関連疾患細胞は、前記のコラーゲン関連疾患患者から分離された細胞でもよく、モデル動物から作製された細胞でもよい。コラーゲン関連疾患患者から分離された細胞としては、例えば、コラーゲン分泌を行う細胞を用いることができ、具体的には肝線維症患者から分離された肝星細胞、線維芽細胞、軟骨細胞、を挙げることができる。また、モデル動物から作製された細胞としては、肝星細胞、又は肺線維芽細胞、を挙げることができる。
更に、正常なコラーゲン分泌細胞を、例えばＴＧＦ－βにより活性化し、肝線維症、肺線維症、腎線維症、膠原病、又は間接リウマチのモデル細胞として用い、それぞれの薬剤のスクリーニング用細胞として用いることができる。 《Collagen-related diseases》
The collagen-related disease is not particularly limited as long as collagen is considered to be related to the cause or symptoms of the disease, but for example, organ / tissue fibers such as liver fibrosis, pulmonary fibrosis, renal fibrosis, etc. Symptom, collagen disease, or indirect rheumatism.
The collagen-related disease cell used in the screening method of the present invention may be a cell isolated from the aforementioned collagen-related disease patient or a cell prepared from a model animal. As cells isolated from patients with collagen-related diseases, for example, cells that secrete collagen can be used. Specific examples include hepatic stellate cells, fibroblasts, and chondrocytes isolated from patients with liver fibrosis. be able to. Moreover, hepatic stellate cells or lung fibroblasts can be mentioned as cells produced from model animals.
Further, normal collagen-secreting cells are activated by, for example, TGF-β, and used as model cells for liver fibrosis, pulmonary fibrosis, renal fibrosis, collagen disease, or indirect rheumatism, and used as screening cells for each drug. be able to.
コラーゲン関連疾患は、コラーゲンがその疾患の原因、又は症状等に関連すると考えられる限りにおいて、特に限定されるものではないが、例えば肝線維症、肺線維症、腎線維症などの臓器・組織線維症、膠原病、又は間接リウマチを挙げることができる。
本発明のスクリーニング方法に用いるコラーゲン関連疾患細胞は、前記のコラーゲン関連疾患患者から分離された細胞でもよく、モデル動物から作製された細胞でもよい。コラーゲン関連疾患患者から分離された細胞としては、例えば、コラーゲン分泌を行う細胞を用いることができ、具体的には肝線維症患者から分離された肝星細胞、線維芽細胞、軟骨細胞、を挙げることができる。また、モデル動物から作製された細胞としては、肝星細胞、又は肺線維芽細胞、を挙げることができる。
更に、正常なコラーゲン分泌細胞を、例えばＴＧＦ－βにより活性化し、肝線維症、肺線維症、腎線維症、膠原病、又は間接リウマチのモデル細胞として用い、それぞれの薬剤のスクリーニング用細胞として用いることができる。 《Collagen-related diseases》
The collagen-related disease is not particularly limited as long as collagen is considered to be related to the cause or symptoms of the disease, but for example, organ / tissue fibers such as liver fibrosis, pulmonary fibrosis, renal fibrosis, etc. Symptom, collagen disease, or indirect rheumatism.
The collagen-related disease cell used in the screening method of the present invention may be a cell isolated from the aforementioned collagen-related disease patient or a cell prepared from a model animal. As cells isolated from patients with collagen-related diseases, for example, cells that secrete collagen can be used. Specific examples include hepatic stellate cells, fibroblasts, and chondrocytes isolated from patients with liver fibrosis. be able to. Moreover, hepatic stellate cells or lung fibroblasts can be mentioned as cells produced from model animals.
Further, normal collagen-secreting cells are activated by, for example, TGF-β, and used as model cells for liver fibrosis, pulmonary fibrosis, renal fibrosis, collagen disease, or indirect rheumatism, and used as screening cells for each drug. be able to.
《試験物質》
試験物質は、コラーゲンの動態に関連する可能性のある物質、又はそのような物質を含むものである限りにおいて限定されるものではないが、例えば、ケミカルファイルに登録されている種々の公知化合物（ペプチドを含む）、コンビナトリアル・ケミストリー技術（Ｔｅｒｒｅｔｔ，Ｎ．Ｋ．ら，Ｔｅｔｒａｈｅｄｒｏｎ，５１，８１３５－８１３７，１９９５）によって得られた化合物群、あるいは、ファージ・ディスプレイ法（Ｆｅｌｉｃｉ，Ｆ．ら，Ｊ．Ｍｏｌ．Ｂｉｏｌ．，２２２，３０１－３１０，１９９１）などを応用して作成されたランダム・ペプチド群、又は低分子化合物を用いることができる。また、微生物の培養上清、細胞の培養上清、生体内の体液、植物若しくは海洋生物由来の天然成分、又は動物組織抽出物などもスクリーニングの試験物質として用いることができる。 <Test substance>
The test substance is not limited as long as it contains substances that may be related to the kinetics of collagen, or includes such substances. For example, various known compounds (peptides registered in the chemical file) A compound group obtained by combinatorial chemistry technology (Terrett, NK et al., Tetrahedron, 51, 8135-8137, 1995), or phage display method (Felici, F. et al., J. Mol. Biol., 222, 301-310, 1991) or the like can be used, or a random peptide group or a low molecular weight compound can be used. In addition, microorganism culture supernatant, cell culture supernatant, in-vivo body fluid, natural components derived from plants or marine organisms, or animal tissue extracts can also be used as test substances for screening.
試験物質は、コラーゲンの動態に関連する可能性のある物質、又はそのような物質を含むものである限りにおいて限定されるものではないが、例えば、ケミカルファイルに登録されている種々の公知化合物（ペプチドを含む）、コンビナトリアル・ケミストリー技術（Ｔｅｒｒｅｔｔ，Ｎ．Ｋ．ら，Ｔｅｔｒａｈｅｄｒｏｎ，５１，８１３５－８１３７，１９９５）によって得られた化合物群、あるいは、ファージ・ディスプレイ法（Ｆｅｌｉｃｉ，Ｆ．ら，Ｊ．Ｍｏｌ．Ｂｉｏｌ．，２２２，３０１－３１０，１９９１）などを応用して作成されたランダム・ペプチド群、又は低分子化合物を用いることができる。また、微生物の培養上清、細胞の培養上清、生体内の体液、植物若しくは海洋生物由来の天然成分、又は動物組織抽出物などもスクリーニングの試験物質として用いることができる。 <Test substance>
The test substance is not limited as long as it contains substances that may be related to the kinetics of collagen, or includes such substances. For example, various known compounds (peptides registered in the chemical file) A compound group obtained by combinatorial chemistry technology (Terrett, NK et al., Tetrahedron, 51, 8135-8137, 1995), or phage display method (Felici, F. et al., J. Mol. Biol., 222, 301-310, 1991) or the like can be used, or a random peptide group or a low molecular weight compound can be used. In addition, microorganism culture supernatant, cell culture supernatant, in-vivo body fluid, natural components derived from plants or marine organisms, or animal tissue extracts can also be used as test substances for screening.
（接触工程）
本発明のスクリーニング方法における接触工程は、前記融合タンパク質を発現したコラーゲン関連疾患細胞と、試験物質とを接触させる工程である。
試験物質の濃度も、適宜決定することができるが、試験物質がコラーゲンの動態に影響を与える最適濃度があると考えられるため、何段階かに希釈して試験することが好ましい。 (Contact process)
The contacting step in the screening method of the present invention is a step of bringing a collagen-related disease cell expressing the fusion protein into contact with a test substance.
The concentration of the test substance can also be determined as appropriate. However, since it is considered that the test substance has an optimum concentration that affects the dynamics of collagen, it is preferable to dilute the test substance in several stages.
本発明のスクリーニング方法における接触工程は、前記融合タンパク質を発現したコラーゲン関連疾患細胞と、試験物質とを接触させる工程である。
試験物質の濃度も、適宜決定することができるが、試験物質がコラーゲンの動態に影響を与える最適濃度があると考えられるため、何段階かに希釈して試験することが好ましい。 (Contact process)
The contacting step in the screening method of the present invention is a step of bringing a collagen-related disease cell expressing the fusion protein into contact with a test substance.
The concentration of the test substance can also be determined as appropriate. However, since it is considered that the test substance has an optimum concentration that affects the dynamics of collagen, it is preferable to dilute the test substance in several stages.
（融合タンパク質の動態解析工程）
本発明における動態解析工程は、コラーゲンの融合タンパク質の動態を解析する工程である。コラーゲンの動態の解析としては、例えばコラーゲン融合タンパク質の発現量の解析、コラーゲン融合タンパク質の細胞外の分泌の解析、コラーゲン融合タンパク質の細胞内輸送の解析、又はコラーゲン融合タンパク質のプロセシングの解析を挙げることができる。
動態解析は、基本的に試験物質を接触させない細胞と比較することによって行うことができる。すなわち、試験物質を接触させない細胞と比較して、コラーゲン融合タンパク質の発現が増加又は減少している場合、その試験物質をコラーゲン関連疾患の治療薬物の候補と判断することができる。また、試験物質を接触させない細胞と比較して、コラーゲン融合タンパク質が、細胞外に分泌されている場合、その試験物質をコラーゲン関連疾患に効果があるものと判断することができる。更に、試験物質を接触させない細胞と比較して、コラーゲン融合タンパク質の細胞内輸送が正常に回復した場合、その試験物質をコラーゲン関連疾患に効果があるものと判断することができる。また、試験物質を接触させない細胞と比較して、コラーゲン融合タンパク質のプロセシングが正常に回復した場合、その試験物質をコラーゲン関連疾患に効果があるものと判断することができる。 (Fusion protein dynamics analysis process)
The dynamic analysis step in the present invention is a step of analyzing the dynamics of the collagen fusion protein. Examples of the analysis of collagen dynamics include analysis of the expression level of collagen fusion protein, analysis of extracellular secretion of collagen fusion protein, analysis of intracellular transport of collagen fusion protein, or analysis of processing of collagen fusion protein. Can do.
The kinetic analysis can be basically performed by comparing with a cell not contacted with a test substance. That is, when the expression of the collagen fusion protein is increased or decreased as compared to cells not contacted with the test substance, the test substance can be determined as a candidate for a therapeutic drug for collagen-related diseases. In addition, when the collagen fusion protein is secreted extracellularly compared to cells not contacted with the test substance, it can be determined that the test substance is effective for collagen-related diseases. Furthermore, when the intracellular transport of the collagen fusion protein is restored as compared with cells not contacted with the test substance, it can be determined that the test substance is effective for collagen-related diseases. Further, when the processing of the collagen fusion protein is restored normally as compared with the cells not contacted with the test substance, it can be determined that the test substance is effective for collagen-related diseases.
本発明における動態解析工程は、コラーゲンの融合タンパク質の動態を解析する工程である。コラーゲンの動態の解析としては、例えばコラーゲン融合タンパク質の発現量の解析、コラーゲン融合タンパク質の細胞外の分泌の解析、コラーゲン融合タンパク質の細胞内輸送の解析、又はコラーゲン融合タンパク質のプロセシングの解析を挙げることができる。
動態解析は、基本的に試験物質を接触させない細胞と比較することによって行うことができる。すなわち、試験物質を接触させない細胞と比較して、コラーゲン融合タンパク質の発現が増加又は減少している場合、その試験物質をコラーゲン関連疾患の治療薬物の候補と判断することができる。また、試験物質を接触させない細胞と比較して、コラーゲン融合タンパク質が、細胞外に分泌されている場合、その試験物質をコラーゲン関連疾患に効果があるものと判断することができる。更に、試験物質を接触させない細胞と比較して、コラーゲン融合タンパク質の細胞内輸送が正常に回復した場合、その試験物質をコラーゲン関連疾患に効果があるものと判断することができる。また、試験物質を接触させない細胞と比較して、コラーゲン融合タンパク質のプロセシングが正常に回復した場合、その試験物質をコラーゲン関連疾患に効果があるものと判断することができる。 (Fusion protein dynamics analysis process)
The dynamic analysis step in the present invention is a step of analyzing the dynamics of the collagen fusion protein. Examples of the analysis of collagen dynamics include analysis of the expression level of collagen fusion protein, analysis of extracellular secretion of collagen fusion protein, analysis of intracellular transport of collagen fusion protein, or analysis of processing of collagen fusion protein. Can do.
The kinetic analysis can be basically performed by comparing with a cell not contacted with a test substance. That is, when the expression of the collagen fusion protein is increased or decreased as compared to cells not contacted with the test substance, the test substance can be determined as a candidate for a therapeutic drug for collagen-related diseases. In addition, when the collagen fusion protein is secreted extracellularly compared to cells not contacted with the test substance, it can be determined that the test substance is effective for collagen-related diseases. Furthermore, when the intracellular transport of the collagen fusion protein is restored as compared with cells not contacted with the test substance, it can be determined that the test substance is effective for collagen-related diseases. Further, when the processing of the collagen fusion protein is restored normally as compared with the cells not contacted with the test substance, it can be determined that the test substance is effective for collagen-related diseases.
例えば、実施例に示すように、コラーゲンタンパク質にＥＧＦＰが挿入され、Ｃ－プロプロテインにｍ－Ｃｈｅｒｒｙが挿入されたコラーゲン融合タンパク質が導入された肝線維症肝星細胞を用いた場合、候補物質を接触させない場合、プロコラーゲンタンパク質のプロセシング又は分泌が異常なため、細胞内にＥＧＦＰ及びｍ－Ｃｈｅｒｒｙの蛍光からなる黄色の蛍光が観察される。これに対して治療効果のある候補物質を接触させるとｍ－Ｃｈｅｒｒｙの挿入されたＣ－プロプロテインが切断され、ＥＧＦＰの挿入されたコラーゲンタンパク質が正常に細胞外に分泌され、緑の蛍光が観察される。コラーゲンタンパク質の細胞外への分泌は、正常な肝星細胞においても観察されるものであり、このような動態を示す候補物質は、肝線維症に効果があるものと判断することができる。
しかしながら、スクリーニングに用いられる動態解析の判断は、これらに限られるものではなく、試験物質を接触させない細胞と比較して、異なる動態を示す場合、コラーゲン関連疾患に効果があるものと判断することができる。 For example, as shown in the examples, when using hepatic fibrosis hepatic stellate cells into which a collagen fusion protein in which EGFP is inserted into a collagen protein and m-Cherry is inserted into a C-proprotein is used, a candidate substance is used. When not contacted, since the processing or secretion of procollagen protein is abnormal, yellow fluorescence consisting of fluorescence of EGFP and m-Cherry is observed in the cells. In contrast, when a candidate substance having a therapeutic effect is brought into contact, the C-proprotein with m-Cherry inserted is cleaved, and the collagen protein with EGFP inserted is normally secreted outside the cell, and green fluorescence is observed. Is done. The extracellular secretion of collagen protein is observed even in normal hepatic stellate cells, and it can be determined that a candidate substance exhibiting such kinetics is effective for liver fibrosis.
However, the determination of kinetic analysis used for screening is not limited to these, and it may be determined that it has an effect on collagen-related diseases when it shows different kinetics compared to cells not contacted with the test substance. it can.
しかしながら、スクリーニングに用いられる動態解析の判断は、これらに限られるものではなく、試験物質を接触させない細胞と比較して、異なる動態を示す場合、コラーゲン関連疾患に効果があるものと判断することができる。 For example, as shown in the examples, when using hepatic fibrosis hepatic stellate cells into which a collagen fusion protein in which EGFP is inserted into a collagen protein and m-Cherry is inserted into a C-proprotein is used, a candidate substance is used. When not contacted, since the processing or secretion of procollagen protein is abnormal, yellow fluorescence consisting of fluorescence of EGFP and m-Cherry is observed in the cells. In contrast, when a candidate substance having a therapeutic effect is brought into contact, the C-proprotein with m-Cherry inserted is cleaved, and the collagen protein with EGFP inserted is normally secreted outside the cell, and green fluorescence is observed. Is done. The extracellular secretion of collagen protein is observed even in normal hepatic stellate cells, and it can be determined that a candidate substance exhibiting such kinetics is effective for liver fibrosis.
However, the determination of kinetic analysis used for screening is not limited to these, and it may be determined that it has an effect on collagen-related diseases when it shows different kinetics compared to cells not contacted with the test substance. it can.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。
Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
《比較例１》
本比較例では、１型コラーゲンの中央にＧＦＰを挿入し、そしてＣ末端にｍＣｈｅｒｒｙを結合した核酸を有するベクターの構築を試みた。ＧＦＰはコラーゲンタンパク質のＢａｍＨ１の部位に挿入され、ｍＣｈｅｒｒｙはＣ－プロプロテインのＥｃｏＲ１の部位に挿入された。
ベクターの概略図を図１に示す。ベクター以外のＤＮＡの塩基配列を配列番号１に示す。
ＥＧＦＰ ｃＤＮＡの両端に制限酵素ＢａｍＨ１ ｓｉｔｅを付加したＤＮＡ断片を作成し、これをヒトｐｒｅｐｒｏｃｏｌｌａｇｅｎ１α１ｃＤＮＡ内部のＢａｍＨ１ｓｉｔｅに挿入した。この際、タンパク質翻訳において、コラーゲンタンパク質とＥＧＦＰタンパク質がつながるように調節を行った。次に、ｍＣｈｅｒｒｙ ｃＤＮＡの両端にＥｃｏＲ１ ｓｉｔｅを付加したＤＮＡ断片を作成し、これをヒトｐｒｅｐｒｏｃｏｌｌａｇｅｎ１α１－ＥＧＦＰ融合ｃＤＮＡ のＥｃｏＲ１ ｓｉｔｅに挿入した。この際にも、タンパク質翻訳において、コラーゲンタンパク質とｍＣｈｅｒｒｙタンパク質がつながるように調節を行い、操作の各ステップにおいて塩基配列の確認を行った。 << Comparative Example 1 >>
In this comparative example, an attempt was made to construct a vector having a nucleic acid in which GFP was inserted in the center oftype 1 collagen and mCherry was bound to the C-terminus. GFP was inserted into the BamH1 site of the collagen protein, and mCherry was inserted into the EcoR1 site of the C-proprotein.
A schematic diagram of the vector is shown in FIG. The nucleotide sequence of DNA other than the vector is shown in SEQ ID NO: 1.
A DNA fragment in which the restriction enzyme BamH1 site was added to both ends of the EGFP cDNA was prepared, and this was inserted into BamH1site inside the human preprocollagen1α1 cDNA. At this time, the protein translation was adjusted so that the collagen protein and the EGFP protein were connected. Next, a DNA fragment in which EcoR1 site was added to both ends of the mCherry cDNA was prepared, and this was inserted into the EcoR1 site of the human preprocollagen 1α1-EGFP fusion cDNA. Also at this time, protein translation was adjusted so that the collagen protein and the mCherry protein were connected, and the base sequence was confirmed in each step of the operation.
本比較例では、１型コラーゲンの中央にＧＦＰを挿入し、そしてＣ末端にｍＣｈｅｒｒｙを結合した核酸を有するベクターの構築を試みた。ＧＦＰはコラーゲンタンパク質のＢａｍＨ１の部位に挿入され、ｍＣｈｅｒｒｙはＣ－プロプロテインのＥｃｏＲ１の部位に挿入された。
ベクターの概略図を図１に示す。ベクター以外のＤＮＡの塩基配列を配列番号１に示す。
ＥＧＦＰ ｃＤＮＡの両端に制限酵素ＢａｍＨ１ ｓｉｔｅを付加したＤＮＡ断片を作成し、これをヒトｐｒｅｐｒｏｃｏｌｌａｇｅｎ１α１ｃＤＮＡ内部のＢａｍＨ１ｓｉｔｅに挿入した。この際、タンパク質翻訳において、コラーゲンタンパク質とＥＧＦＰタンパク質がつながるように調節を行った。次に、ｍＣｈｅｒｒｙ ｃＤＮＡの両端にＥｃｏＲ１ ｓｉｔｅを付加したＤＮＡ断片を作成し、これをヒトｐｒｅｐｒｏｃｏｌｌａｇｅｎ１α１－ＥＧＦＰ融合ｃＤＮＡ のＥｃｏＲ１ ｓｉｔｅに挿入した。この際にも、タンパク質翻訳において、コラーゲンタンパク質とｍＣｈｅｒｒｙタンパク質がつながるように調節を行い、操作の各ステップにおいて塩基配列の確認を行った。 << Comparative Example 1 >>
In this comparative example, an attempt was made to construct a vector having a nucleic acid in which GFP was inserted in the center of
A schematic diagram of the vector is shown in FIG. The nucleotide sequence of DNA other than the vector is shown in SEQ ID NO: 1.
A DNA fragment in which the restriction enzyme BamH1 site was added to both ends of the EGFP cDNA was prepared, and this was inserted into BamH1site inside the human preprocollagen1α1 cDNA. At this time, the protein translation was adjusted so that the collagen protein and the EGFP protein were connected. Next, a DNA fragment in which EcoR1 site was added to both ends of the mCherry cDNA was prepared, and this was inserted into the EcoR1 site of the human preprocollagen 1α1-EGFP fusion cDNA. Also at this time, protein translation was adjusted so that the collagen protein and the mCherry protein were connected, and the base sequence was confirmed in each step of the operation.
ＸＬ－１ ｂｌｕｅのコンピテントセル（形質転換受容性細胞）作成法は、「遺伝子ライブラリーの作成法」（編集・野島博、羊土社）に従った。凍結保存されたコンピテントセルを解凍し、すぐに１００ｎｇのプラスミドＤＮＡを入れ混合した。氷上で３０分間静置した後、４２℃で１分３０秒温め、すぐに氷冷した。ここに１ｍＬの培地を加え、１００μＬをアンピシリンを添加したＬＢプレートに塗り広げた。３７℃にて一晩保温し、現れたコロニーを使用した。
得られたベクターに含まれるＤＮＡは、目的の長さより短いもの、目的の長さより長いもの、ＮｏｔＩ、又はＳｍａＩで切断できないものであった。図２（Ａ）に、目的の長さより長いもの、ＮｏｔＩ、及びＳｍａＩで切断できなかったものを示す The XL-1 blue competent cell (transformation-receptive cell) was prepared in accordance with “Genetic Library Preparation Method” (edited by Hiroshi Nojima, Yodosha). The cryopreserved competent cells were thawed and immediately 100 ng of plasmid DNA was added and mixed. The mixture was allowed to stand on ice for 30 minutes, then warmed at 42 ° C. for 1 minute and 30 seconds, and immediately cooled on ice. 1 mL of medium was added thereto, and 100 μL was spread on an LB plate to which ampicillin had been added. Incubated overnight at 37 ° C., and the colonies that appeared were used.
The DNA contained in the obtained vector was shorter than the target length, longer than the target length, or not cut with NotI or SmaI. FIG. 2A shows a longer one than the target length, one that could not be cut with NotI, and SmaI.
得られたベクターに含まれるＤＮＡは、目的の長さより短いもの、目的の長さより長いもの、ＮｏｔＩ、又はＳｍａＩで切断できないものであった。図２（Ａ）に、目的の長さより長いもの、ＮｏｔＩ、及びＳｍａＩで切断できなかったものを示す The XL-1 blue competent cell (transformation-receptive cell) was prepared in accordance with “Genetic Library Preparation Method” (edited by Hiroshi Nojima, Yodosha). The cryopreserved competent cells were thawed and immediately 100 ng of plasmid DNA was added and mixed. The mixture was allowed to stand on ice for 30 minutes, then warmed at 42 ° C. for 1 minute and 30 seconds, and immediately cooled on ice. 1 mL of medium was added thereto, and 100 μL was spread on an LB plate to which ampicillin had been added. Incubated overnight at 37 ° C., and the colonies that appeared were used.
The DNA contained in the obtained vector was shorter than the target length, longer than the target length, or not cut with NotI or SmaI. FIG. 2A shows a longer one than the target length, one that could not be cut with NotI, and SmaI.
《比較例２》
本比較例では、１型コラーゲンのＮ末にＥＧＦＰを結合し、そしてＣ末端にｍＣｈｅｒｒｙを結合した核酸を有するベクターの構築を試みた。ＥＧＦＰはＮ－プロプロテインのＫｐｎ１の部位に挿入され、ｍＣｈｅｒｒｙはＣ－プロプロテインのＥｃｏＲ１の部位に挿入された。
ベクターの概略図を図３に示す。ベクター以外のＤＮＡの塩基配列を配列番号２に示す。
＜ベクター構築法＞ＥＧＦＰ ｃＤＮＡの両端に制限酵素ＫｐｎＩ ｓｉｔｅを付加したＤＮＡ断片を作成し、これをヒトｐｒｅｐｒｏｃｏｌｌａｇｅｎ１α１ ｃＤＮＡ内部のＫｐｎＩ ｓｉｔｅに挿入した。この際、タンパク質翻訳において、コラーゲンタンパク質とＥＧＦＰタンパク質がつながるように調節を行った。次に、ｍＣｈｅｒｒｙ ｃＤＮＡ の両端にＥｃｏＲ１ ｓｉｔｅを付加したＤＮＡ断片を作成し、これをヒトｐｒｅｐｒｏｃｏｌｌａｇｅｎ１α１－ＥＧＦＰ融合ｃＤＮＡのＥｃｏＲ１ ｓｉｔｅに挿入した。この際にも、タンパク質翻訳において、コラーゲンタンパク質とｍＣｈｅｒｒｙタンパク質がつながるように調節を行い、操作の各ステップにおいて塩基配列の確認を行った。 << Comparative Example 2 >>
In this comparative example, an attempt was made to construct a vector having a nucleic acid having EGFP bound to the N-terminal oftype 1 collagen and mCherry bound to the C-terminus. EGFP was inserted into the Kpn1 site of N-proprotein, and mCherry was inserted into the EcoR1 site of C-proprotein.
A schematic diagram of the vector is shown in FIG. The nucleotide sequence of DNA other than the vector is shown in SEQ ID NO: 2.
<Vector Construction Method> A DNA fragment in which the restriction enzyme KpnI site was added to both ends of the EGFP cDNA was prepared and inserted into the KpnI site inside the human preprocollagen1α1 cDNA. At this time, the protein translation was adjusted so that the collagen protein and the EGFP protein were connected. Next, a DNA fragment in which EcoR1 sites were added to both ends of the mCherry cDNA was prepared, and this was inserted into the EcoR1 site of the human preprocollagen 1α1-EGFP fusion cDNA. Also at this time, protein translation was adjusted so that the collagen protein and the mCherry protein were connected, and the base sequence was confirmed in each step of the operation.
本比較例では、１型コラーゲンのＮ末にＥＧＦＰを結合し、そしてＣ末端にｍＣｈｅｒｒｙを結合した核酸を有するベクターの構築を試みた。ＥＧＦＰはＮ－プロプロテインのＫｐｎ１の部位に挿入され、ｍＣｈｅｒｒｙはＣ－プロプロテインのＥｃｏＲ１の部位に挿入された。
ベクターの概略図を図３に示す。ベクター以外のＤＮＡの塩基配列を配列番号２に示す。
＜ベクター構築法＞ＥＧＦＰ ｃＤＮＡの両端に制限酵素ＫｐｎＩ ｓｉｔｅを付加したＤＮＡ断片を作成し、これをヒトｐｒｅｐｒｏｃｏｌｌａｇｅｎ１α１ ｃＤＮＡ内部のＫｐｎＩ ｓｉｔｅに挿入した。この際、タンパク質翻訳において、コラーゲンタンパク質とＥＧＦＰタンパク質がつながるように調節を行った。次に、ｍＣｈｅｒｒｙ ｃＤＮＡ の両端にＥｃｏＲ１ ｓｉｔｅを付加したＤＮＡ断片を作成し、これをヒトｐｒｅｐｒｏｃｏｌｌａｇｅｎ１α１－ＥＧＦＰ融合ｃＤＮＡのＥｃｏＲ１ ｓｉｔｅに挿入した。この際にも、タンパク質翻訳において、コラーゲンタンパク質とｍＣｈｅｒｒｙタンパク質がつながるように調節を行い、操作の各ステップにおいて塩基配列の確認を行った。 << Comparative Example 2 >>
In this comparative example, an attempt was made to construct a vector having a nucleic acid having EGFP bound to the N-terminal of
A schematic diagram of the vector is shown in FIG. The nucleotide sequence of DNA other than the vector is shown in SEQ ID NO: 2.
<Vector Construction Method> A DNA fragment in which the restriction enzyme KpnI site was added to both ends of the EGFP cDNA was prepared and inserted into the KpnI site inside the human preprocollagen1α1 cDNA. At this time, the protein translation was adjusted so that the collagen protein and the EGFP protein were connected. Next, a DNA fragment in which EcoR1 sites were added to both ends of the mCherry cDNA was prepared, and this was inserted into the EcoR1 site of the human preprocollagen 1α1-EGFP fusion cDNA. Also at this time, protein translation was adjusted so that the collagen protein and the mCherry protein were connected, and the base sequence was confirmed in each step of the operation.
ＸＬ－１ ｂｌｕｅのコンピテントセル（形質転換受容性細胞）作成法は、「遺伝子ライブラリーの作成法」（編集・野島博、羊土社）に従った。凍結保存されたコンピテントセルを解凍し、すぐに１００ｎｇのプラスミドＤＮＡを入れ混合した。氷上で３０分間静置した後、４２℃で１分３０秒温め、すぐに氷冷した。ここに１ｍＬの培地を加え、１００μＬをアンピシリンを添加したＬＢプレートに塗り広げた。３７℃にて一晩保温し、現れたコロニーを使用した。
得られたベクターに含まれるＤＮＡは、目的の長さより短いもの、目的の長さより長いもの、ＮｏｔＩ、又はＳｍａＩで切断できないものであった。図２ＢにＥｃｏＲＩでの切断した写真を示すが、全てのクローンがＥｃｏＲＩで切断できなかった。 The XL-1 blue competent cell (transformation-receptive cell) was prepared in accordance with “Genetic Library Preparation Method” (edited by Hiroshi Nojima, Yodosha). The cryopreserved competent cells were thawed and immediately 100 ng of plasmid DNA was added and mixed. The mixture was allowed to stand on ice for 30 minutes, then warmed at 42 ° C. for 1 minute and 30 seconds, and immediately cooled on ice. 1 mL of medium was added thereto, and 100 μL was spread on an LB plate to which ampicillin had been added. Incubated overnight at 37 ° C., and the colonies that appeared were used.
The DNA contained in the obtained vector was shorter than the target length, longer than the target length, or not cut with NotI or SmaI. FIG. 2B shows a photograph cut with EcoRI, but all clones could not be cut with EcoRI.
得られたベクターに含まれるＤＮＡは、目的の長さより短いもの、目的の長さより長いもの、ＮｏｔＩ、又はＳｍａＩで切断できないものであった。図２ＢにＥｃｏＲＩでの切断した写真を示すが、全てのクローンがＥｃｏＲＩで切断できなかった。 The XL-1 blue competent cell (transformation-receptive cell) was prepared in accordance with “Genetic Library Preparation Method” (edited by Hiroshi Nojima, Yodosha). The cryopreserved competent cells were thawed and immediately 100 ng of plasmid DNA was added and mixed. The mixture was allowed to stand on ice for 30 minutes, then warmed at 42 ° C. for 1 minute and 30 seconds, and immediately cooled on ice. 1 mL of medium was added thereto, and 100 μL was spread on an LB plate to which ampicillin had been added. Incubated overnight at 37 ° C., and the colonies that appeared were used.
The DNA contained in the obtained vector was shorter than the target length, longer than the target length, or not cut with NotI or SmaI. FIG. 2B shows a photograph cut with EcoRI, but all clones could not be cut with EcoRI.
《実施例１》
ＸＬ－１ ｂｌｕｅに代えて、ＴＯＰ１０Ｆ’を用いたことを除いては、比較例１の操作を繰り返し、ベクターを得た。得られたベクターは、塩基配列決定を行い目的の長さの核酸が含まれていることを確認した（図２Ａ）。 Example 1
A vector was obtained by repeating the operation of Comparative Example 1 except that TOP10F ′ was used instead of XL-1 blue. The obtained vector was subjected to nucleotide sequencing and confirmed to contain a nucleic acid of the desired length (FIG. 2A).
ＸＬ－１ ｂｌｕｅに代えて、ＴＯＰ１０Ｆ’を用いたことを除いては、比較例１の操作を繰り返し、ベクターを得た。得られたベクターは、塩基配列決定を行い目的の長さの核酸が含まれていることを確認した（図２Ａ）。 Example 1
A vector was obtained by repeating the operation of Comparative Example 1 except that TOP10F ′ was used instead of XL-1 blue. The obtained vector was subjected to nucleotide sequencing and confirmed to contain a nucleic acid of the desired length (FIG. 2A).
《実施例２》
ＸＬ－１ ｂｌｕｅに代えて、ＤＨ１０Ｂを用いたことを除いては、比較例２の操作を繰り返し、ベクターを得た。得られたベクターは、目的の長さの核酸を含んでいた。図２Ｂに、ＥｃｏＲＩでの切断した写真を示すが、全てのクローンがＥｃｏＲＩで切断することができた。一方、ＸＬ－１ ｂｌｕｅを宿主として用いた場合は、ＥｃｏＲＩで切断できる正常なクローンを取得することはできなかった。 Example 2
A vector was obtained by repeating the operation of Comparative Example 2 except that DH10B was used instead of XL-1 blue. The resulting vector contained the desired length of nucleic acid. FIG. 2B shows a photograph cut with EcoRI. All clones were cut with EcoRI. On the other hand, when XL-1 blue was used as a host, a normal clone that could be cleaved with EcoRI could not be obtained.
ＸＬ－１ ｂｌｕｅに代えて、ＤＨ１０Ｂを用いたことを除いては、比較例２の操作を繰り返し、ベクターを得た。得られたベクターは、目的の長さの核酸を含んでいた。図２Ｂに、ＥｃｏＲＩでの切断した写真を示すが、全てのクローンがＥｃｏＲＩで切断することができた。一方、ＸＬ－１ ｂｌｕｅを宿主として用いた場合は、ＥｃｏＲＩで切断できる正常なクローンを取得することはできなかった。 Example 2
A vector was obtained by repeating the operation of Comparative Example 2 except that DH10B was used instead of XL-1 blue. The resulting vector contained the desired length of nucleic acid. FIG. 2B shows a photograph cut with EcoRI. All clones were cut with EcoRI. On the other hand, when XL-1 blue was used as a host, a normal clone that could be cleaved with EcoRI could not be obtained.
《実施例３》
本実施例では、細胞外に分泌されたコラーゲンの蛍光を観察した。ＮＩＨ３Ｔ３細胞に実施例１で得られたベクターを導入した。２日おきに培地を交換しながら１ヶ月間以上の長期培養を実施し、多層からなる細胞集団を得た。蛍光顕微鏡によりＥＧＦＰの蛍光シグナルを観察した。
図４に示すようにＥＧＦＰが、コラーゲンタンパク質のＮ末側に挿入されていることにより、コラーゲンタンパク質が３重らせん構造を形成し、そして細胞外に蓄積していること（ＥＧＦＰの緑色の蛍光）を確認できた。 Example 3
In this example, the fluorescence of collagen secreted extracellularly was observed. The vector obtained in Example 1 was introduced into NIH3T3 cells. Long-term culture for one month or longer was performed while changing the medium every two days to obtain a cell population consisting of multiple layers. The fluorescence signal of EGFP was observed with a fluorescence microscope.
As shown in FIG. 4, when EGFP is inserted at the N-terminal side of the collagen protein, the collagen protein forms a triple helical structure and accumulates extracellularly (green fluorescence of EGFP). Was confirmed.
本実施例では、細胞外に分泌されたコラーゲンの蛍光を観察した。ＮＩＨ３Ｔ３細胞に実施例１で得られたベクターを導入した。２日おきに培地を交換しながら１ヶ月間以上の長期培養を実施し、多層からなる細胞集団を得た。蛍光顕微鏡によりＥＧＦＰの蛍光シグナルを観察した。
図４に示すようにＥＧＦＰが、コラーゲンタンパク質のＮ末側に挿入されていることにより、コラーゲンタンパク質が３重らせん構造を形成し、そして細胞外に蓄積していること（ＥＧＦＰの緑色の蛍光）を確認できた。 Example 3
In this example, the fluorescence of collagen secreted extracellularly was observed. The vector obtained in Example 1 was introduced into NIH3T3 cells. Long-term culture for one month or longer was performed while changing the medium every two days to obtain a cell population consisting of multiple layers. The fluorescence signal of EGFP was observed with a fluorescence microscope.
As shown in FIG. 4, when EGFP is inserted at the N-terminal side of the collagen protein, the collagen protein forms a triple helical structure and accumulates extracellularly (green fluorescence of EGFP). Was confirmed.
《実施例４》
本実施例では、細胞内でのコラーゲンの蛍光を観察した。ＮＩＨ３Ｔ３細胞に実施例１で得られたベクターを導入した。蛍光顕微鏡により４８時間後にＥＧＦＰおよびｍＣｈｅｒｒｙの蛍光シグナルを観察した。
図５に示すようにＥＧＦＰがコラーゲンタンパク質のＮ末側に挿入され、ｍＣｈｅｒｒｙがＣ－プロプロテインのＣ末側に挿入されていることにより、プロコラーゲンである黄色およびプロセシング後のコラーゲンである緑色の蛍光シグナルが細胞質に存在することを確認できた。なお、コラーゲンタンパク質は、細胞外にも分泌されているが、実施例３と異なり、培養期間が短いためにコラーゲンタンパク質の蓄積量が少なく、ＥＧＦＰ緑の蛍光が観察されなかった。 Example 4
In this example, the fluorescence of collagen in the cells was observed. The vector obtained in Example 1 was introduced into NIH3T3 cells. The fluorescence signals of EGFP and mCherry were observed 48 hours later using a fluorescence microscope.
As shown in FIG. 5, EGFP is inserted into the N-terminal side of the collagen protein and mCherry is inserted into the C-terminal side of the C-proprotein, so that yellow that is procollagen and green that is collagen after processing are green. It was confirmed that a fluorescent signal was present in the cytoplasm. Collagen protein is also secreted extracellularly, but unlike Example 3, since the culture period was short, the amount of collagen protein accumulated was small, and EGFP green fluorescence was not observed.
本実施例では、細胞内でのコラーゲンの蛍光を観察した。ＮＩＨ３Ｔ３細胞に実施例１で得られたベクターを導入した。蛍光顕微鏡により４８時間後にＥＧＦＰおよびｍＣｈｅｒｒｙの蛍光シグナルを観察した。
図５に示すようにＥＧＦＰがコラーゲンタンパク質のＮ末側に挿入され、ｍＣｈｅｒｒｙがＣ－プロプロテインのＣ末側に挿入されていることにより、プロコラーゲンである黄色およびプロセシング後のコラーゲンである緑色の蛍光シグナルが細胞質に存在することを確認できた。なお、コラーゲンタンパク質は、細胞外にも分泌されているが、実施例３と異なり、培養期間が短いためにコラーゲンタンパク質の蓄積量が少なく、ＥＧＦＰ緑の蛍光が観察されなかった。 Example 4
In this example, the fluorescence of collagen in the cells was observed. The vector obtained in Example 1 was introduced into NIH3T3 cells. The fluorescence signals of EGFP and mCherry were observed 48 hours later using a fluorescence microscope.
As shown in FIG. 5, EGFP is inserted into the N-terminal side of the collagen protein and mCherry is inserted into the C-terminal side of the C-proprotein, so that yellow that is procollagen and green that is collagen after processing are green. It was confirmed that a fluorescent signal was present in the cytoplasm. Collagen protein is also secreted extracellularly, but unlike Example 3, since the culture period was short, the amount of collagen protein accumulated was small, and EGFP green fluorescence was not observed.
《実施例５》
本実施例では、実施例２と同様の手順で得られた図３のベクターをマウスのＮＩＨ３Ｔ３細胞に導入し、共焦点蛍光顕微鏡で、蛍光を検出した。ＮＩＨ３Ｔ３細胞に図３のベクターを導入した。蛍光顕微鏡により４８時間後にＥＧＦＰおよびｍＣｈｅｒｒｙの蛍光シグナルを観察した。
図６に示すようにＥＧＦＰがＮプロプロテインに挿入され、ｍＣｈｅｒｒｙがＣプロプロテインに挿入していることにより、合成直後のプロコラーゲンは黄色のシグナルによって、また、プロセシングにより切断されたＮプロプロテインは緑色の蛍光シグナルによって、細胞質に存在することを確認できた。なお、Ｎ－プロプロテインは、細胞外にも分泌されているが、培地に拡散しているため、ＥＧＦＰ緑の蛍光は観察されなかった。 Example 5
In this example, the vector of FIG. 3 obtained by the same procedure as in Example 2 was introduced into mouse NIH3T3 cells, and fluorescence was detected with a confocal fluorescence microscope. The vector of FIG. 3 was introduced into NIH3T3 cells. The fluorescence signals of EGFP and mCherry were observed 48 hours later using a fluorescence microscope.
As shown in FIG. 6, EGFP is inserted into the N proprotein and mCherry is inserted into the C proprotein. As a result, the procollagen immediately after synthesis is indicated by a yellow signal, and the N proprotein cleaved by processing is A green fluorescent signal confirmed the presence in the cytoplasm. N-proprotein is also secreted extracellularly, but since it diffuses into the medium, EGFP green fluorescence was not observed.
本実施例では、実施例２と同様の手順で得られた図３のベクターをマウスのＮＩＨ３Ｔ３細胞に導入し、共焦点蛍光顕微鏡で、蛍光を検出した。ＮＩＨ３Ｔ３細胞に図３のベクターを導入した。蛍光顕微鏡により４８時間後にＥＧＦＰおよびｍＣｈｅｒｒｙの蛍光シグナルを観察した。
図６に示すようにＥＧＦＰがＮプロプロテインに挿入され、ｍＣｈｅｒｒｙがＣプロプロテインに挿入していることにより、合成直後のプロコラーゲンは黄色のシグナルによって、また、プロセシングにより切断されたＮプロプロテインは緑色の蛍光シグナルによって、細胞質に存在することを確認できた。なお、Ｎ－プロプロテインは、細胞外にも分泌されているが、培地に拡散しているため、ＥＧＦＰ緑の蛍光は観察されなかった。 Example 5
In this example, the vector of FIG. 3 obtained by the same procedure as in Example 2 was introduced into mouse NIH3T3 cells, and fluorescence was detected with a confocal fluorescence microscope. The vector of FIG. 3 was introduced into NIH3T3 cells. The fluorescence signals of EGFP and mCherry were observed 48 hours later using a fluorescence microscope.
As shown in FIG. 6, EGFP is inserted into the N proprotein and mCherry is inserted into the C proprotein. As a result, the procollagen immediately after synthesis is indicated by a yellow signal, and the N proprotein cleaved by processing is A green fluorescent signal confirmed the presence in the cytoplasm. N-proprotein is also secreted extracellularly, but since it diffuses into the medium, EGFP green fluorescence was not observed.
《比較例３》
プレプロコラーゲンｃＤＮＡのＣプロプロテイン内に存在する制限酵素ＡｃｃＩＩＩ部位にｍＣｈｅｒｒｙを挿入した。ＡｃｃＩＩＩ部位はＣ－プロプロテインのＮ末端から２７アミノ酸に相当する部位に存在する（図１２Ａ）。ｍＣｈｅｒｒｙがＣ－プロプロテインのＮ末端から３０アミノ酸よりＮ末端側に挿入されていることによりコラーゲンの線維化（３重らせん構造構築）が阻害される。すなわち、コラーゲンタンパク質は、ＥＲ－ゴルジ体に蓄積され、細胞外には分泌されなかった（図１２Ｂ、Ｃ）。
また、コラーゲンタンパク質のＣ末端に、ＥＧＦＰを結合した融合タンパク質を作成した（図１２Ａ）。この融合タンパク質もコラーゲンの線維化（３重らせん構造構築）が阻害される。すなわち、コラーゲンタンパク質は、細胞質に局在し細胞外には分泌されなかった（図１２Ｂ）。 << Comparative Example 3 >>
MCherry was inserted into the restriction enzyme AccIII site present in the C proprotein of the preprocollagen cDNA. The AccIII site is present at a site corresponding to 27 amino acids from the N-terminus of C-proprotein (FIG. 12A). The insertion of mCherry from the N-terminal of C-proprotein to the N-terminal side of 30 amino acids inhibits collagen fibrosis (triple helical structure construction). That is, collagen protein was accumulated in the ER-Golgi body and was not secreted extracellularly (FIGS. 12B and C).
Further, a fusion protein in which EGFP was bound to the C-terminus of the collagen protein was prepared (FIG. 12A). This fusion protein also inhibits collagen fibrosis (triple helical structure construction). That is, the collagen protein was localized in the cytoplasm and not secreted extracellularly (FIG. 12B).
プレプロコラーゲンｃＤＮＡのＣプロプロテイン内に存在する制限酵素ＡｃｃＩＩＩ部位にｍＣｈｅｒｒｙを挿入した。ＡｃｃＩＩＩ部位はＣ－プロプロテインのＮ末端から２７アミノ酸に相当する部位に存在する（図１２Ａ）。ｍＣｈｅｒｒｙがＣ－プロプロテインのＮ末端から３０アミノ酸よりＮ末端側に挿入されていることによりコラーゲンの線維化（３重らせん構造構築）が阻害される。すなわち、コラーゲンタンパク質は、ＥＲ－ゴルジ体に蓄積され、細胞外には分泌されなかった（図１２Ｂ、Ｃ）。
また、コラーゲンタンパク質のＣ末端に、ＥＧＦＰを結合した融合タンパク質を作成した（図１２Ａ）。この融合タンパク質もコラーゲンの線維化（３重らせん構造構築）が阻害される。すなわち、コラーゲンタンパク質は、細胞質に局在し細胞外には分泌されなかった（図１２Ｂ）。 << Comparative Example 3 >>
MCherry was inserted into the restriction enzyme AccIII site present in the C proprotein of the preprocollagen cDNA. The AccIII site is present at a site corresponding to 27 amino acids from the N-terminus of C-proprotein (FIG. 12A). The insertion of mCherry from the N-terminal of C-proprotein to the N-terminal side of 30 amino acids inhibits collagen fibrosis (triple helical structure construction). That is, collagen protein was accumulated in the ER-Golgi body and was not secreted extracellularly (FIGS. 12B and C).
Further, a fusion protein in which EGFP was bound to the C-terminus of the collagen protein was prepared (FIG. 12A). This fusion protein also inhibits collagen fibrosis (triple helical structure construction). That is, the collagen protein was localized in the cytoplasm and not secreted extracellularly (FIG. 12B).
《実施例６》
本実施例では、肝線維症の薬剤のスクリーニング系の構築を行った。
（１）肝星細胞へのベクターの導入
実施例１で得られたベクターを肝線維症のラットから分離した肝線維症の原因となる定常的活性化した肝星細胞（５Ｈ細胞）及び活性化していない肝星細胞（２Ｇ９２細胞）にトランスフェクトした。具体的には、トランスフェクション試薬（Fugne6）を用いて細胞に導入した。図７に示すように、活性化していない肝星細胞では、コラーゲンタンパク質の正常なプロセシングおよび分泌を示す緑色の蛍光が観察された（図７Ａ）。しかし、定常的活性化肝星細胞（５Ｈ細胞）ではコラーゲンのプロセシングおよび分泌の異常を示す黄色のシグナルのみが観察され、コラーゲンの正常な分泌を示す緑色の蛍光は見られなかった（図７Ｃ）。 Example 6
In this example, a screening system for drugs for liver fibrosis was constructed.
(1) Introduction of vector into hepatic stellate cells Steadily activated hepatic stellate cells (5H cells) that cause liver fibrosis isolated from the liver fibrosis rat obtained in Example 1 and activation Not transfected hepatic stellate cells (2G92 cells). Specifically, it was introduced into the cells using a transfection reagent (Fugne 6). As shown in FIG. 7, green fluorescence indicating normal processing and secretion of collagen protein was observed in non-activated hepatic stellate cells (FIG. 7A). However, only a yellow signal indicating abnormal collagen processing and secretion was observed in steady activated hepatic stellate cells (5H cells), and no green fluorescence indicating normal collagen secretion was observed (FIG. 7C). .
本実施例では、肝線維症の薬剤のスクリーニング系の構築を行った。
（１）肝星細胞へのベクターの導入
実施例１で得られたベクターを肝線維症のラットから分離した肝線維症の原因となる定常的活性化した肝星細胞（５Ｈ細胞）及び活性化していない肝星細胞（２Ｇ９２細胞）にトランスフェクトした。具体的には、トランスフェクション試薬（Fugne6）を用いて細胞に導入した。図７に示すように、活性化していない肝星細胞では、コラーゲンタンパク質の正常なプロセシングおよび分泌を示す緑色の蛍光が観察された（図７Ａ）。しかし、定常的活性化肝星細胞（５Ｈ細胞）ではコラーゲンのプロセシングおよび分泌の異常を示す黄色のシグナルのみが観察され、コラーゲンの正常な分泌を示す緑色の蛍光は見られなかった（図７Ｃ）。 Example 6
In this example, a screening system for drugs for liver fibrosis was constructed.
(1) Introduction of vector into hepatic stellate cells Steadily activated hepatic stellate cells (5H cells) that cause liver fibrosis isolated from the liver fibrosis rat obtained in Example 1 and activation Not transfected hepatic stellate cells (2G92 cells). Specifically, it was introduced into the cells using a transfection reagent (Fugne 6). As shown in FIG. 7, green fluorescence indicating normal processing and secretion of collagen protein was observed in non-activated hepatic stellate cells (FIG. 7A). However, only a yellow signal indicating abnormal collagen processing and secretion was observed in steady activated hepatic stellate cells (5H cells), and no green fluorescence indicating normal collagen secretion was observed (FIG. 7C). .
（２）ＴＧＦ－βによる正常肝星細胞の活性化
更に、２Ｇ９２細胞を用いて肝線維症の薬剤のスクリーニングに用いる細胞を以下のように作製した。活性化していない肝星細胞２Ｇ９２を２枚のディッシュ上でセミコンフルエントまで増殖させた。一方のディッシュには、１ｎｇ／ｍＬの最終濃度にて肝星細胞を活性化するＴＧＦ－βを添加した。添加後、８時間以降においてコラーゲンタンパク質のプロセシングおよび分泌を共焦点顕微鏡により検出した結果、ＴＧＦ－βを添加したディッシュではコラーゲンのプロセシングおよび分泌の異常を示す黄色のシグナルのみが観察されるようになり、肝線維症の原因となる活性化肝星細胞（活性化２Ｇ９２細胞）に変化したことが確認された（図７Ｂ）。 (2) Activation of normal hepatic stellate cells by TGF-β Further, 2G92 cells were used to prepare cells for screening for hepatic fibrosis drugs as follows. Non-activated hepatic stellate cells 2G92 were grown to semi-confluent on two dishes. One dish was added with TGF-β that activates hepatic stellate cells at a final concentration of 1 ng / mL. As a result of detecting the processing and secretion of collagen protein with a confocal microscope after 8 hours from the addition, only a yellow signal indicating abnormality in the processing and secretion of collagen is observed in the dish to which TGF-β is added. It was confirmed that the cells changed to activated hepatic stellate cells (activated 2G92 cells) that cause liver fibrosis (FIG. 7B).
更に、２Ｇ９２細胞を用いて肝線維症の薬剤のスクリーニングに用いる細胞を以下のように作製した。活性化していない肝星細胞２Ｇ９２を２枚のディッシュ上でセミコンフルエントまで増殖させた。一方のディッシュには、１ｎｇ／ｍＬの最終濃度にて肝星細胞を活性化するＴＧＦ－βを添加した。添加後、８時間以降においてコラーゲンタンパク質のプロセシングおよび分泌を共焦点顕微鏡により検出した結果、ＴＧＦ－βを添加したディッシュではコラーゲンのプロセシングおよび分泌の異常を示す黄色のシグナルのみが観察されるようになり、肝線維症の原因となる活性化肝星細胞（活性化２Ｇ９２細胞）に変化したことが確認された（図７Ｂ）。 (2) Activation of normal hepatic stellate cells by TGF-β Further, 2G92 cells were used to prepare cells for screening for hepatic fibrosis drugs as follows. Non-activated hepatic stellate cells 2G92 were grown to semi-confluent on two dishes. One dish was added with TGF-β that activates hepatic stellate cells at a final concentration of 1 ng / mL. As a result of detecting the processing and secretion of collagen protein with a confocal microscope after 8 hours from the addition, only a yellow signal indicating abnormality in the processing and secretion of collagen is observed in the dish to which TGF-β is added. It was confirmed that the cells changed to activated hepatic stellate cells (activated 2G92 cells) that cause liver fibrosis (FIG. 7B).
《実施例７》
本実施例では、実施例６に記載の活性化肝星細胞（活性化２Ｇ９２細胞）及び定常的活性化肝星細胞（５Ｈ細胞）を用いて、肝線維症の治療薬の候補物質のスクリーニング系を構築した。
セミコンフルエントまで増殖させた２Ｇ９２細胞に、肝線維症の治療効果を有するＳＢ４３１５４２（０．５μＭ）又は溶媒ＤＭＳＯ添加し、１時間培養した。その後１ｎｇ／ｍＬの最終濃度でＴＧＦ－βを添加し、８時間培養した。その結果、ＳＢ４３１５４２を添加した２Ｇ９２細胞は、コラーゲンタンパク質が正常にプロセンシングされて分泌された（図８）が、ＤＭＳＯを添加した２Ｇ９２細胞は、コラーゲンタンパク質のプロセシングおよび分泌の異常を示す黄色のシグナルのみが見られた（図なし）。ＳＢ４３１５４２を添加した活性化肝星細胞はコラーゲンの分泌が正常に変化しており、従って、本発明のベクターによってコラーゲンタンパク質を発現させた２Ｇ９２細胞を用いて、肝線維症の治療薬の候補物質をスクリーニングできることが分かった。
また、セミコンフルエントまで増殖させた５Ｈ細胞に、肝線維症の治療効果を有するＳＢ４３１５４２（０．５μＭ）又は溶媒ＤＭＳＯを添加し、１時間以上培養した。その結果、ＳＢ４３１５４２を添加した５Ｈ細胞は、コラーゲンタンパク質が正常にプロセンシングされて分泌された（図９）が、ＤＭＳＯを添加した５Ｈ細胞は、コラーゲンタンパク質のプロセシングおよび分泌の変化（正常化）は見られなかった（図なし）。ＳＢ４３１５４２を添加した定常的活性化肝星細胞はコラーゲンの分泌が正常に変化しており、従って、本発明のベクターによってコラーゲンタンパク質を発現させた５Ｈ細胞を用いて、肝線維症の治療薬の候補物質をスクリーニングできることが分かった。 Example 7
In this example, a screening system for candidate substances for therapeutic agents for liver fibrosis using the activated hepatic stellate cells (activated 2G92 cells) and stationary activated hepatic stellate cells (5H cells) described in Example 6 Built.
To 2G92 cells grown to semi-confluent, SB431542 (0.5 μM) having a therapeutic effect on liver fibrosis or solvent DMSO was added and cultured for 1 hour. Thereafter, TGF-β was added at a final concentration of 1 ng / mL and cultured for 8 hours. As a result, 2G92 cells to which SB431542 was added were secreted with normal processing of collagen protein (FIG. 8), whereas 2G92 cells to which DMSO was added showed yellow signal indicating abnormal processing and secretion of collagen protein. Only was seen (not shown). Activated hepatic stellate cells to which SB431542 is added have normal changes in collagen secretion. Therefore, using 2G92 cells in which collagen protein is expressed by the vector of the present invention, a candidate for a therapeutic agent for liver fibrosis can be obtained. It turns out that it can be screened.
Further, SB431542 (0.5 μM) having a therapeutic effect on liver fibrosis or the solvent DMSO was added to 5H cells grown to semi-confluent and cultured for 1 hour or longer. As a result, 5H cells to which SB431542 was added were secreted with collagen protein being normally processed (FIG. 9), whereas 5H cells to which DMSO was added had no changes in the processing and secretion of collagen protein (normalization). Not seen (not shown). The steadily activated hepatic stellate cells to which SB431542 has been added have normal changes in collagen secretion. Therefore, using 5H cells in which collagen protein is expressed by the vector of the present invention, candidates for therapeutic agents for liver fibrosis are obtained. It turns out that the substance can be screened.
本実施例では、実施例６に記載の活性化肝星細胞（活性化２Ｇ９２細胞）及び定常的活性化肝星細胞（５Ｈ細胞）を用いて、肝線維症の治療薬の候補物質のスクリーニング系を構築した。
セミコンフルエントまで増殖させた２Ｇ９２細胞に、肝線維症の治療効果を有するＳＢ４３１５４２（０．５μＭ）又は溶媒ＤＭＳＯ添加し、１時間培養した。その後１ｎｇ／ｍＬの最終濃度でＴＧＦ－βを添加し、８時間培養した。その結果、ＳＢ４３１５４２を添加した２Ｇ９２細胞は、コラーゲンタンパク質が正常にプロセンシングされて分泌された（図８）が、ＤＭＳＯを添加した２Ｇ９２細胞は、コラーゲンタンパク質のプロセシングおよび分泌の異常を示す黄色のシグナルのみが見られた（図なし）。ＳＢ４３１５４２を添加した活性化肝星細胞はコラーゲンの分泌が正常に変化しており、従って、本発明のベクターによってコラーゲンタンパク質を発現させた２Ｇ９２細胞を用いて、肝線維症の治療薬の候補物質をスクリーニングできることが分かった。
また、セミコンフルエントまで増殖させた５Ｈ細胞に、肝線維症の治療効果を有するＳＢ４３１５４２（０．５μＭ）又は溶媒ＤＭＳＯを添加し、１時間以上培養した。その結果、ＳＢ４３１５４２を添加した５Ｈ細胞は、コラーゲンタンパク質が正常にプロセンシングされて分泌された（図９）が、ＤＭＳＯを添加した５Ｈ細胞は、コラーゲンタンパク質のプロセシングおよび分泌の変化（正常化）は見られなかった（図なし）。ＳＢ４３１５４２を添加した定常的活性化肝星細胞はコラーゲンの分泌が正常に変化しており、従って、本発明のベクターによってコラーゲンタンパク質を発現させた５Ｈ細胞を用いて、肝線維症の治療薬の候補物質をスクリーニングできることが分かった。 Example 7
In this example, a screening system for candidate substances for therapeutic agents for liver fibrosis using the activated hepatic stellate cells (activated 2G92 cells) and stationary activated hepatic stellate cells (5H cells) described in Example 6 Built.
To 2G92 cells grown to semi-confluent, SB431542 (0.5 μM) having a therapeutic effect on liver fibrosis or solvent DMSO was added and cultured for 1 hour. Thereafter, TGF-β was added at a final concentration of 1 ng / mL and cultured for 8 hours. As a result, 2G92 cells to which SB431542 was added were secreted with normal processing of collagen protein (FIG. 8), whereas 2G92 cells to which DMSO was added showed yellow signal indicating abnormal processing and secretion of collagen protein. Only was seen (not shown). Activated hepatic stellate cells to which SB431542 is added have normal changes in collagen secretion. Therefore, using 2G92 cells in which collagen protein is expressed by the vector of the present invention, a candidate for a therapeutic agent for liver fibrosis can be obtained. It turns out that it can be screened.
Further, SB431542 (0.5 μM) having a therapeutic effect on liver fibrosis or the solvent DMSO was added to 5H cells grown to semi-confluent and cultured for 1 hour or longer. As a result, 5H cells to which SB431542 was added were secreted with collagen protein being normally processed (FIG. 9), whereas 5H cells to which DMSO was added had no changes in the processing and secretion of collagen protein (normalization). Not seen (not shown). The steadily activated hepatic stellate cells to which SB431542 has been added have normal changes in collagen secretion. Therefore, using 5H cells in which collagen protein is expressed by the vector of the present invention, candidates for therapeutic agents for liver fibrosis are obtained. It turns out that the substance can be screened.
《実施例８》
本実施例では、肺線維芽細胞、及び腎臓尿細管上皮細胞を用いて、肺線維症の治療薬のスクリーニングに用いる細胞、及び腎線維症の治療薬のスクリーニングに用いる細胞を作製した。
肝星細胞に代えて、肺線維芽細胞（ＴＩＧ－３－２０細胞、及びＡ５４９細胞）、及び腎臓尿細管上皮細胞（ＭＤＣＫ細胞）を用いたことを除いては、実施例６（１）の操作を繰り返して、本発明のベクターによってコラーゲンタンパク質を発現させたＴＩＧ－３－２０細胞、Ａ５４９細胞、及びＭＤＣＫ細胞を作製した。図１０に示すように、ＴＩＧ－３－２０細胞（図１０Ａ）、Ａ５４９細胞（図１０Ｃ）、及びＭＤＣＫ細胞（図１０Ｄ）は、コラーゲンを可視化することができた。
また、ＴＩＧ－３－２０細胞については、ＴＧＦ－βを用いて、細胞の活性化を行った。肝星細胞に代えて、肺線維芽細胞（ＴＩＧ－３－２０細胞）を用いたことを除いては、実施例６（２）の操作を繰り返して、ＴＩＧ－３－２０細胞を活性化させた。図１０Ｂに示すように、ＴＩＧ－３－２０細胞は、コラーゲンのプロセシングおよび分泌の異常を示す黄色のシグナルのみが観察されるようになり、肺線維症のモデル細胞として用いることができることが確認できた。この細胞を用いて、肺線維症の薬剤のスクリーングができる。 Example 8
In this example, lung fibroblasts and renal tubular epithelial cells were used to produce cells used for screening pulmonary fibrosis therapeutic agents and cells used for screening renal fibrosis therapeutic agents.
Example 6 (1) except that lung fibroblasts (TIG-3-20 cells and A549 cells) and renal tubular epithelial cells (MDCK cells) were used instead of hepatic stellate cells. By repeating the operation, TIG-3-20 cells, A549 cells, and MDCK cells in which collagen protein was expressed by the vector of the present invention were prepared. As shown in FIG. 10, TIG-3-20 cells (FIG. 10A), A549 cells (FIG. 10C), and MDCK cells (FIG. 10D) were able to visualize collagen.
For TIG-3-20 cells, TGF-β was used to activate the cells. Except that pulmonary fibroblasts (TIG-3-20 cells) were used instead of hepatic stellate cells, the procedure of Example 6 (2) was repeated to activate TIG-3-20 cells. It was. As shown in FIG. 10B, only yellow signals indicating abnormal collagen processing and secretion are observed in TIG-3-20 cells, confirming that they can be used as model cells for pulmonary fibrosis. It was. These cells can be used to screen for pulmonary fibrosis drugs.
本実施例では、肺線維芽細胞、及び腎臓尿細管上皮細胞を用いて、肺線維症の治療薬のスクリーニングに用いる細胞、及び腎線維症の治療薬のスクリーニングに用いる細胞を作製した。
肝星細胞に代えて、肺線維芽細胞（ＴＩＧ－３－２０細胞、及びＡ５４９細胞）、及び腎臓尿細管上皮細胞（ＭＤＣＫ細胞）を用いたことを除いては、実施例６（１）の操作を繰り返して、本発明のベクターによってコラーゲンタンパク質を発現させたＴＩＧ－３－２０細胞、Ａ５４９細胞、及びＭＤＣＫ細胞を作製した。図１０に示すように、ＴＩＧ－３－２０細胞（図１０Ａ）、Ａ５４９細胞（図１０Ｃ）、及びＭＤＣＫ細胞（図１０Ｄ）は、コラーゲンを可視化することができた。
また、ＴＩＧ－３－２０細胞については、ＴＧＦ－βを用いて、細胞の活性化を行った。肝星細胞に代えて、肺線維芽細胞（ＴＩＧ－３－２０細胞）を用いたことを除いては、実施例６（２）の操作を繰り返して、ＴＩＧ－３－２０細胞を活性化させた。図１０Ｂに示すように、ＴＩＧ－３－２０細胞は、コラーゲンのプロセシングおよび分泌の異常を示す黄色のシグナルのみが観察されるようになり、肺線維症のモデル細胞として用いることができることが確認できた。この細胞を用いて、肺線維症の薬剤のスクリーングができる。 Example 8
In this example, lung fibroblasts and renal tubular epithelial cells were used to produce cells used for screening pulmonary fibrosis therapeutic agents and cells used for screening renal fibrosis therapeutic agents.
Example 6 (1) except that lung fibroblasts (TIG-3-20 cells and A549 cells) and renal tubular epithelial cells (MDCK cells) were used instead of hepatic stellate cells. By repeating the operation, TIG-3-20 cells, A549 cells, and MDCK cells in which collagen protein was expressed by the vector of the present invention were prepared. As shown in FIG. 10, TIG-3-20 cells (FIG. 10A), A549 cells (FIG. 10C), and MDCK cells (FIG. 10D) were able to visualize collagen.
For TIG-3-20 cells, TGF-β was used to activate the cells. Except that pulmonary fibroblasts (TIG-3-20 cells) were used instead of hepatic stellate cells, the procedure of Example 6 (2) was repeated to activate TIG-3-20 cells. It was. As shown in FIG. 10B, only yellow signals indicating abnormal collagen processing and secretion are observed in TIG-3-20 cells, confirming that they can be used as model cells for pulmonary fibrosis. It was. These cells can be used to screen for pulmonary fibrosis drugs.
本発明のベクターは、コラーゲンの形成過程の解析に有効に用いることができる。このコラーゲンの形成過程の解析は、コラーゲンの分子異常を起因とする疾患の原因の解明、及びそれらの治療法の開発にも有効な手段として用いることができる。また、本発明のスクリーニング方法は、コラーゲン関連疾患の治療又は予防用薬剤のスクリーニングに用いることができる。
以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。 The vector of the present invention can be effectively used for analysis of the collagen formation process. The analysis of the collagen formation process can be used as an effective means for elucidating the cause of a disease caused by collagen molecular abnormality and developing a treatment method thereof. In addition, the screening method of the present invention can be used for screening drugs for treating or preventing collagen-related diseases.
As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.
以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。 The vector of the present invention can be effectively used for analysis of the collagen formation process. The analysis of the collagen formation process can be used as an effective means for elucidating the cause of a disease caused by collagen molecular abnormality and developing a treatment method thereof. In addition, the screening method of the present invention can be used for screening drugs for treating or preventing collagen-related diseases.
As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.
Claims (14)
- Ｇｌｙ－Ｘａａ－Ｙａａ（式中、Ｇｌｙはグリシンであり、そしてＸａａ及びＹａａは任意のアミノ酸である）の繰り返しアミノ酸配列を有するプレプロコラーゲンタンパク質、プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体、又はそれらの断片である、コラーゲン関連タンパク質と、蛍光タンパク質又は発光タンパク質との融合タンパク質。 A preprocollagen protein, procollagen protein, collagen protein, or a variant thereof having a repetitive amino acid sequence of Gly-Xaa-Yaa (where Gly is glycine and Xaa and Yaa are any amino acids), or A fusion protein of a collagen-related protein, which is a fragment thereof, and a fluorescent protein or photoprotein.
- 前記蛍光タンパク質又は発光タンパク質が、プロコラーゲンタンパク質のＮ末端のＮ－プロプロテイン、コラーゲンタンパク質、又はＣ末端のＣ－プロプロテインに結合又は挿入されている、請求項１に記載の融合タンパク質。 The fusion protein according to claim 1, wherein the fluorescent protein or photoprotein is bound to or inserted into N-proprotein, collagen protein, or C-proprotein at the N-terminus of procollagen protein.
- 前記蛍光タンパク質又は発光タンパク質がコラーゲンタンパク質のＣ末端から３０アミノ酸よりＮ末端側に挿入されており、及び／又は蛍光タンパク質又は発光タンパク質がＣ－プロプロテインのＮ末端から３０アミノ酸よりＣ末側に挿入されている、請求項１又は２に記載の融合タンパク質。 The fluorescent protein or photoprotein is inserted from the C-terminal of the collagen protein to the N-terminal side from 30 amino acids, and / or the fluorescent protein or photoprotein is inserted from the N-terminal of the C-proprotein to the C-terminal side from 30 amino acids. The fusion protein according to claim 1 or 2, wherein
- 請求項１～３のいずれか一項に記載の融合タンパク質を発現したコラーゲン関連疾患細胞と、試験物質とを接触させる工程、及び
融合タンパク質の動態を解析する工程、
を含むコラーゲン関連疾患の治療又は予防物質のスクリーニング方法。 A step of bringing a collagen-related disease cell expressing the fusion protein according to any one of claims 1 to 3 into contact with a test substance, and a step of analyzing the dynamics of the fusion protein;
A method for screening a substance for treating or preventing a collagen-related disease. - Ｇｌｙ－Ｘａａ－Ｙａａ（式中、Ｇｌｙはグリシンであり、そしてＸａａ及びＹａａは任意のアミノ酸である）の繰り返しアミノ酸配列を有するプレプロコラーゲンタンパク質、プロコラーゲンタンパク質、コラーゲンタンパク質、若しくはそれらの変異体、又はそれらの断片である、コラーゲン関連タンパク質、又は前記コラーゲン関連タンパク質と蛍光タンパク質又は発光タンパク質との融合タンパク質をコードする核酸。 A preprocollagen protein, procollagen protein, collagen protein, or a variant thereof having a repetitive amino acid sequence of Gly-Xaa-Yaa (where Gly is glycine and Xaa and Yaa are any amino acids), or A nucleic acid encoding a collagen-related protein or a fusion protein of the collagen-related protein and a fluorescent protein or photoprotein, which is a fragment thereof.
- 請求項５に記載の核酸を含むベクター。 A vector comprising the nucleic acid according to claim 5.
- 請求項６に記載のベクターを含む、ベクター含有宿主細胞。 A vector-containing host cell comprising the vector according to claim 6.
- 前記宿主細胞が、mcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌である、請求項７に記載のベクター含有宿主細胞。 The host cell is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG, galE, The vector-containing host cell according to claim 7, which is Escherichia coli having a gene mutation selected from the group consisting of hsdS and a combination of two or more thereof.
- 前記大腸菌が、ＤＨ１０Ｂ、ＴＯＰ１０Ｆ’、ＭＣ１０６１、ＸＬ１－Ｂｌｕｅ ＭＲＦ’、ＡＧ１、ＢＬ２１（ＤＥ３）、ＤＢ３．１、ＤＨ１、ＤＨ５α、ＤＨ５α Ｔｕｒｂｏ、ＤＨ１２Ｓ、ＤＭ１、Ｅ．Ｃｌｏｎｉ（ｒ）５ａｌｐｈａ、Ｅ．Ｃｌｏｎｉ（ｒ）１０Ｇ、Ｅ．Ｃｌｏｎｉ（ｒ）１０ＧＦ’、ＥＲ２２６７、ＨＢ１０１、ＨＭＳ１７４（ＤＥ３）、Ｈｉｇｈ－Ｃｏｎｔｒｏｌ（ｔｍ） ＢＬ２１（ＤＥ３）、Ｈｉｇｈ－Ｃｏｎｔｒｏｌ（ｔｍ）１０Ｇ、ＩＪ１１２６、ＪＭ１０８、ＪＭ１０９、Ｍａｃｈ１、ＭＣ１０６１、ＭＦＤｐｉｒ、ＯｍｎｉＭＡＸ２、ＯｖｅｒＥｘｐｒｅｓｓ（ｔｍ）Ｃ４１（ＤＥ３）、ＯｖｅｒＥｘｐｒｅｓｓ（ｔｍ）Ｃ４３（ＤＥ３）、Ｒｏｓｅｔｔａ（ＤＥ３）、ＳＯＬＲ、ＳＳ３２０、ＳＴＢＬ２、ＳＴＢＬ３、ＳＴＢＬ４、ＳＵＲＥ、ＳＵＲＥ２、ＴＧ１、ＴＯＰ１０、ＸＬ２－Ｂｌｕｅ、ＸＬ２－ＢｌｕｅＭＲＦ’、及びＸＬ１０－Ｇｏｌｄからなる群から選択される、請求項８に記載のベクター含有宿主細胞。 The E. coli is DH10B, TOP10F ', MC1061, XL1-Blue MRF', AG1, BL21 (DE3), DB3.1, DH1, DH5α, DH5α Turbo, DH12S, DM1, E. Cloni (r) 5alpha, E.I. Cloni (r) 10G, E.I. Cloni (r) 10GF ′, ER2267, HB101, HMS174 (DE3), High-Control (tm) BL21 (DE3), High-Control (tm) 10G, IJ1126, JM108, JM109, Mach1, MC1061, MFDpiMx, OFDpiMx, OFDpiMx (Tm) C41 (DE3), OverExpress (tm) C43 (DE3), Rosetta (DE3), SOLR, SS320, STBL2, STBL3, STBL4, SURE, SURE2, TG1, TOP10, XL2-Blue, XL2-BlueMRF ', and 9. The vector-containing host cell according to claim 8, which is selected from the group consisting of XL10-Gold.
- 請求項８又は９に記載のベクター含有宿主細胞から得られるベクター。 A vector obtained from the vector-containing host cell according to claim 8 or 9.
- 請求項６に記載のベクターをmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌に導入することを特徴とする、ベクター含有大腸菌の製造方法。 The vector of claim 6 is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG , GalE, hsdS, and a method for producing a vector-containing E. coli, which is introduced into E. coli having a genetic mutation selected from the group consisting of two or more combinations thereof.
- 請求項６に記載のベクターをmcrA、Δ(mrr-hsdRMS-mcrBC)、φ80lacZΔM15、ΔlacX74、deoR、recA1、endA1、araD139、Δ(ara, leu)7697、galU、galK、rpsL(strr)、nupG、galE、hsdS、及びそれらの２つ以上の組み合わせからなる群から選択される遺伝子変異を有する大腸菌に導入する工程、及び前記ベクターを回収する工程を含むことを特徴とする、ベクターの製造方法。 The vector of claim 6 is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK, rpsL (str r ), nupG , GalE, hsdS, and a method for producing a vector, comprising the steps of introducing into E. coli having a genetic mutation selected from the group consisting of two or more combinations thereof, and recovering the vector.
- （１）請求項６に記載のベクターを、動物細胞に導入する工程、及び
（２）前記ベクターが導入された動物細胞からコラーゲンを分泌させる工程、
を含む、コラーゲンの製造方法。 (1) a step of introducing the vector according to claim 6 into an animal cell, and (2) a step of secreting collagen from the animal cell into which the vector has been introduced,
A method for producing collagen, comprising: - （１）請求項６に記載のベクターを、動物細胞に導入する工程、及び
（２）前記ベクターが導入された動物細胞において蛍光又は発光を検出する工程、
を含む、コラーゲンの蛍光又は発光検出方法。 (1) a step of introducing the vector according to claim 6 into an animal cell, and (2) a step of detecting fluorescence or luminescence in the animal cell into which the vector has been introduced,
A method for detecting fluorescence or luminescence of collagen, comprising:
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| JP7303551B2 (en) | 2017-11-14 | 2023-07-05 | 国立大学法人 筑波大学 | Modified collagen protein and uses thereof |
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