WO2016152882A1 - Protéine de fusion à base de collagène et procédé de criblage de médicaments l'utilisant - Google Patents

Protéine de fusion à base de collagène et procédé de criblage de médicaments l'utilisant Download PDF

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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
protein
vector
cells
fusion protein
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Japanese (ja)
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田中　利明
生駒　俊之
田中　順三
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国立大学法人東京工業大学
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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, 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

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  • 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

Abstract

L'objet de la présente invention est de construire un système permettant de détecter une sécrétion de procollagène par des cellules animales. Cet objet peut être obtenu par une protéine de fusion constituée d'une protéine fluorescente ou d'une protéine luminescente et d'une protéine de type collagène qui est une protéine de collagène, une protéine de procollagène, une protéine de préprocollagène ayant une séquence d'acides aminés Gly-Xaa-Yaa se répétant (où Gly représente la glycine et Xaa et Yaa représentent des acides aminés arbitraires), ou un variant ou un fragment de celle-ci.
PCT/JP2016/059066 2015-03-20 2016-03-22 Protéine de fusion à base de collagène et procédé de criblage de médicaments l'utilisant WO2016152882A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019098246A1 (fr) * 2017-11-14 2019-05-23 国立大学法人筑波大学 Protéine modifiée de collagène et son application
JPWO2019098246A1 (ja) * 2017-11-14 2021-01-07 国立大学法人 筑波大学 改変されたコラーゲンタンパク質およびその用途
JP7303551B2 (ja) 2017-11-14 2023-07-05 国立大学法人 筑波大学 改変されたコラーゲンタンパク質およびその用途
US11697679B2 (en) 2017-11-14 2023-07-11 University Of Tsukuba Modified collagen protein and application of same

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