Screening method for substances involved in collagen biosynthesis and / or post-biosynthetic processes

Abstract

To provide methods for screening agents participating in the collagen biosynthesis process and / or post biosynthesis process.SOLUTION: Disclosed is a method for screening an agent participating in the collagen biosynthesis process and / or post biosynthesis process comprising the steps of: (1) contacting a test substance to a cell to which a collagen promoter vector has been introduced, where the vector comprises a collagen promoter and a nucleic acid encoding a fusion protein between a collagen-related protein (preprocollagen protein, procollagen protein, collagen protein and a variant thereof) and a fluorescent or luminescent protein, where the fusion of the fluorescent or luminescent protein is one or more insertions thereof selected from the group consisting of: an insertion to a region of up to 30 amino acids from the C-terminus to the N-terminus of collagen, an insertion to a region from the 30 amino acids from the N-terminus to the C terminus of C-propeptide, and an insertion into N-propeptide; and (2) measuring the fluorescence or luminescence of the culture supernatant and / or the cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening for substances involved in collagen biosynthesis and / or post-biosynthesis processes, and the present invention makes it possible to efficiently screen for substances involved in collagen biosynthesis and / or post-biosynthesis processes in vivo. [Background technology]

[0002] Collagen accounts for 30% of the proteins in the body and is an important protein with functions such as skeletal support and cell adhesion. It is a major component of tissues in the human body, including bones, cartilage, ligaments, tendons, corneal stroma, skin, liver, and muscle. Collagen is classified into fibrillar collagen, which forms collagen fibers, and non-fibrillar collagen, which does not form fibers. Fibrillar collagen is composed of three collagen polypeptide chains that assemble into a triple helix structure (tropocollagen). This triple helix tropocollagen self-organizes to form collagen fibrils (collagen microfibrils), with a shift of 1 / 4 of the molecular length. These collagen fibrils then aggregate to form collagen fibers (collagen fiber bundles). Furthermore, in the corneal stroma and cortical bone, collagen fibers align in the same direction to form overlapping layers, and these layers are further stacked perpendicularly to each other, forming a three-dimensional plywood-like layered structure. Collagen with this three-dimensional plywood-like layered structure is transparent and highly durable.

[0003] Diseases caused by such collagen molecular abnormalities include collagen diseases (e.g., dermatomyositis, polymyositis, rheumatoid arthritis) and organ fibrosis, many of which have no established treatments. One reason for this is that the collagen biosynthesis process, the process from biosynthesis to secretion, and the process of formation of higher-order structures remain unknown. If it becomes possible to analyze the collagen biosynthesis process, the process from biosynthesis to secretion, and the process of formation of higher-order collagen structures, this could be used as an effective means for elucidating the causes of diseases caused by collagen molecular abnormalities and for developing treatments for these diseases. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2016 / 152882 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors attempted to develop a system capable of detecting the process of collagen secretion from cells, and found that by inserting a fluorescent protein into a preprocollagen protein, a procollagen protein, or a collagen protein, a system capable of detecting the secretion of procollagen and collagen proteins from animal cells can be constructed. The present inventors have used the above-mentioned system to screen for drugs that affect the collagen secretion process, but there is a need to further develop a screening system for drugs involved in collagen biosynthesis and / or post-biosynthetic processes. It is therefore an object of the present invention to provide a method for screening for agents involved in collagen biosynthesis and / or post-biosynthetic processes. [Means for solving the problem]

[0006] As a result of extensive research into methods for screening for drugs involved in collagen biosynthesis and / or post-biosynthesis processes, the present inventors have surprisingly found that drugs involved in collagen biosynthesis and / or post-biosynthesis processes can be screened efficiently by using a vector that uses a collagen promoter for the expression of a collagen-related protein. Furthermore, they have found that drugs involved in collagen biosynthesis and / or post-biosynthesis processes can be accurately screened by using a vector that uses a constitutive promoter for the expression of a collagen-related protein. The present invention is based on this finding. Therefore, the present invention provides [1] (1) A method for screening for a substance involved in collagen biosynthesis and / or post-biosynthesis processes, comprising: (1) a step of contacting a test substance with cells into which a collagen promoter vector containing a collagen promoter and a nucleic acid encoding a fusion protein of a collagen-related protein that is a preprocollagen protein, a procollagen protein, a collagen protein, or a variant thereof, and a fluorescent protein or a luminescent protein has been introduced, wherein the fusion of the fluorescent protein or the luminescent protein is one or more insertions selected from the group consisting of an insertion of 30 amino acids from the C-terminus of the collagen protein to the N-terminus, an insertion of 30 amino acids from the N-terminus of the C-propeptide to the C-terminus, and an insertion into the N-propeptide; and (2) a step of measuring fluorescence or luminescence in the culture supernatant of the cells and / or within the cells. [2] (3) A method for screening for a substance involved in collagen biosynthesis and / or post-biosynthesis processes according to [1], further comprising: (1) contacting a test substance with a cell into which a constitutive promoter vector containing a constitutive promoter and a nucleic acid encoding a fusion protein of a collagen-related protein that is a preprocollagen protein, a procollagen protein, a collagen protein, or a variant thereof, and a fluorescent protein or a luminescent protein has been introduced, wherein the fusion of the fluorescent protein or the luminescent protein is one or more insertions selected from the group consisting of an insertion of 30 amino acids from the C-terminus to the N-terminus of the collagen protein, an insertion of 30 amino acids from the N-terminus to the C-terminus of the C-propeptide, and an insertion into the N-propeptide; and (4) measuring the fluorescence or luminescence in the cell culture supernatant. [3] The method for screening for a substance involved in collagen biosynthesis and / or post-biosynthetic processes according to [1] or [2], wherein the collagen-related protein encoded by the collagen promoter vector and / or constitutive promoter vector is a preprocollagen protein or a procollagen protein, and the fluorescent protein or luminescent protein is (a) inserted 30 amino acids from the C-terminus of the collagen protein toward the N-terminus and inserted 30 amino acids from the N-terminus of the C-propeptide toward the C-terminus, (b) inserted 30 amino acids from the C-terminus of the collagen protein toward the N-terminus and inserted into the N-propeptide, or (c) inserted 30 amino acids from the N-terminus of the C-propeptide toward the C-terminus and inserted into the N-propeptide. [Effects of the Invention]

[0007] The screening method of the present invention allows for efficient screening of substances involved in collagen biosynthesis and / or post-biosynthesis processes, and also allows for accurate screening of substances involved in collagen biosynthesis and / or post-biosynthesis processes. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows an outline of a vector that uses a collagen promoter to encode a fusion protein in which GFP is inserted into a collagen protein and mCherry is inserted into the C-propeptide of a procollagen protein. [Figure 2] 1 shows photographs showing collagen biosynthesis in cells in the presence of TGF-β, using cells into which the collagen promoter vector obtained in Example 1 has been introduced. [Figure 3] 1 is a graph showing the results of measuring extracellular collagen secretion in the presence of TGF-β using cells into which the collagen promoter vector obtained in Example 1 has been introduced. [Figure 4]FIG. 1 shows an outline of a vector encoding a fusion protein using a CMV promoter, in which GFP is inserted into a collagen protein and mCherry is inserted into the C-propeptide of a procollagen protein. [Figure 5] 1 is a graph showing the measurement of the effect of test substances on promoting extracellular collagen secretion using cells into which a CMV promoter vector has been introduced. [Figure 6] FIG. 1 shows an outline of a vector encoding a fusion protein using a CMV promoter, in which GFP is inserted into the N-propeptide of procollagen protein and mCherry is inserted into the C-propeptide of procollagen protein. [Figure 7-1] This figure shows a schematic diagram of four types of constitutive promoter vectors (CMV promoter not shown) containing nucleic acids in which tilapia type I preprocollagen and CMV promoter are used to insert GFP into the collagen and mCherry into the C-propeptide, and also shows a photograph demonstrating extracellular collagen secretion. [Figure 7-2] This figure shows a schematic diagram of four types of constitutive promoter vectors (CMV promoter not shown) containing nucleic acids in which tilapia type I preprocollagen and CMV promoter are used to insert GFP into the collagen and mCherry into the C-propeptide, and also shows a photograph demonstrating extracellular collagen secretion. DETAILED DESCRIPTION OF THE INVENTION

[0009] [1] First embodiment of the screening method The screening method of the present invention is a method for screening for substances involved in collagen biosynthesis and / or post-biosynthesis processes, and comprises the steps of: (1) contacting a test substance with cells into which a collagen promoter vector containing a collagen promoter and a nucleic acid encoding a fusion protein of a collagen-related protein (preprocollagen protein, procollagen protein, collagen protein, or a variant thereof) with a fluorescent protein or a luminescent protein, and (2) measuring the fluorescence or luminescence in the culture supernatant and / or intracellularly of the cells. The fusion of the fluorescent protein or luminescent protein is one or more insertions selected from the group consisting of insertion of 30 amino acids from the C-terminus of the collagen protein to the N-terminus, insertion of 30 amino acids from the N-terminus of the C-propeptide to the C-terminus, and insertion into the N-propeptide.

[0010] Collagen promoter vector A first embodiment of the screening method of the present invention uses a collagen promoter vector containing a collagen promoter. That is, the first embodiment is screening using cells into which a collagen promoter vector containing a nucleic acid encoding a fusion protein and a collagen promoter have been introduced (hereinafter, sometimes referred to as first screening). The collagen promoter vector contains a nucleic acid encoding a collagen-related protein, such as a preprocollagen protein, a procollagen protein, a collagen protein, or a variant thereof, and these collagen-related proteins are naturally expressed by a collagen promoter. That is, a promoter is a nucleotide sequence involved in initiating transcription of RNA from DNA, and since the collagen promoter is the native promoter used for transcription of collagen, it is believed that natural expression of collagen-related proteins from the collagen promoter vector will be observed.

[0011] Collagen-related proteins The collagen-related protein is not particularly limited, as long as it has a repeating amino acid sequence of Gly-Xaa-Yaa (wherein Gly is glycine, and Xaa and Yaa are any amino acids), and examples thereof include preprocollagen proteins, procollagen proteins, collagen proteins, variants thereof, and fragments thereof. For example, human collagen proteins include types I to XXVIII collagen proteins, their preprocollagen proteins or procollagen proteins, variants thereof, and fragments thereof. A preprocollagen protein has a signal peptide and an N-propeptide at the N-terminus of the collagen protein, and a C-propeptide at the C-terminus. A procollagen protein has an N-propeptide at the N-terminus of the collagen protein, and a C-propeptide at the C-terminus. The Gly-Xaa-Yaa repeating amino acid sequence is called a collagen-like sequence, and the presence of this Gly-Xaa-Yaa collagen-like sequence is an important characteristic of collagen-related proteins. Furthermore, the biological species from which the collagen-related protein is derived is not limited, and examples thereof include mammals (e.g., humans, cows, pigs, sheep, goats, mice, rats, guinea pigs, or monkeys), birds (e.g., chickens, geese, ducks, or ostriches), reptiles (e.g., crocodiles), amphibians (e.g., frogs), fish (e.g., sturgeon, tilapia, sea bream, flounder, sharks, sardines, tuna, pufferfish, goldfish, cod, flounder, or carp), or invertebrates (e.g., jellyfish).

[0012] Collagen proteins (hereinafter sometimes simply referred to as "collagen") are mainly divided into fibrous collagens and non-fibrous collagens, and examples of fibrous collagens include type I collagen, type II collagen, type III collagen, type V collagen, and type XI collagen. For example, type I collagen has a molecular weight of approximately 300,000, consisting of three polypeptide chains with a molecular weight of approximately 100,000, forming a triple helix structure (tropocollagen). This triple helix structure resembles a rigid rod 300 nm long and 1.5 nm in diameter, and is called tropocollagen. This triple helix topocollagen self-organizes, aligning with a shift of 1 / 4 of the molecular length to form collagen fibrils (collagen microfibrils). These collagen fibrils then aggregate to form collagen fibers (collagen fiber bundles). Furthermore, in the corneal stroma and cortical bone, collagen fibers are aligned in the same direction to form overlapping layers, and these layers are further stacked perpendicularly to each other, forming a three-dimensional plywood-like layered structure. Collagen with this three-dimensional plywood-like layered structure is transparent and highly durable. In type I collagen, Xaa in Gly-Xaa-Yaa is often proline or 3-hydroxyproline, and Yaa is often 4-hydroxyproline or hydroxylysine. Hydroxyproline is an amino acid unique to collagen that is not found in normal proteins, and it is thought that the triple helix structure is stabilized by hydrogen bonds between the hydroxyl group of hydroxyproline and hydration water.

[0013] Collagen proteins are produced in cells as, but are not limited to, preprocollagen proteins (hereinafter sometimes simply referred to as procollagen) having a signal peptide and an N-propeptide at the N-terminus and a C-propeptide at the C-terminus. Examples of procollagens include, but are not limited to, types I to XXVIII collagen.

[0014] Variants of preprocollagen proteins, procollagen proteins, or collagen proteins are not particularly limited, as long as they have a Gly-Xaa-Yaa repeat amino acid sequence. Examples of variants include those in which amino acids have been deleted, substituted, inserted, and / or added to the amino acid sequence of a natural collagen protein (e.g., the aforementioned types I to XXVIII collagen proteins). The number of amino acids 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, even more preferably 10 or less, and most preferably 1 or several. For example, type I collagen consists of approximately 1,000 amino acids, but if it has more than 100 deletions, substitutions, insertions, and / or additions, it may not even function as collagen. Another example of a mutant collagen protein is one that has 80% or more identity to the amino acid sequence of a natural collagen protein (for example, the above-mentioned types I to XXVIII collagen proteins). The amino acid identity is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 97% or more. If the amino acid identity is less than 80%, the protein may not function as collagen.

[0015] The fragments of procollagen proteins, collagen proteins, or variants thereof are not particularly limited, as long as they are partial peptides of procollagen proteins, collagen proteins, or variants thereof, and examples thereof include types I to XXVIII collagens, partial peptides of these procollagens, or variants thereof. The amino acid chain length and cleavage site of the fragments are not particularly limited, and can be determined appropriately depending on the purpose of gene construction.

[0016] <Fluorescent protein or luminescent protein> The fusion protein includes a fluorescent protein that confers fluorescence or a luminescent protein that confers luminescence. Examples of fluorescent proteins or luminescent proteins include GFP, 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, and 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, and luciferase (e.g., Renilla luciferase, firefly luciferase, and bacterial luciferase).

[0017] Fusion Protein In one embodiment of the fusion protein, the fluorescent protein or luminescent protein is inserted into the collagen protein at a position 30 amino acids from the C-terminus toward the N-terminus. The insertion of the fluorescent protein or luminescent protein into the collagen protein is not limited, but is preferably inserted at a position 50 amino acids from the C-terminus toward the N-terminus, more preferably inserted at a position 100 amino acids from the C-terminus toward the N-terminus, even more preferably inserted at a position 200 amino acids from the C-terminus toward the N-terminus, even more preferably inserted at a position 300 amino acids from the C-terminus toward the N-terminus, even more preferably inserted at a position 400 amino acids from the C-terminus toward the N-terminus, even more preferably inserted at a position 500 amino acids from the C-terminus toward the N-terminus, and even more preferably inserted at a position 600 amino acids from the C-terminus toward the N-terminus.

[0018] In one embodiment of the fusion protein of the present invention, a fluorescent protein or luminescent protein is inserted into the C-propeptide at a position 30 amino acids from the N-terminus toward the C-terminus. The insertion of the fluorescent protein or luminescent protein into the C-propeptide is not limited, but is preferably inserted at a position 35 amino acids from the N-terminus toward the C-terminus, more preferably inserted at a position 40 amino acids from the N-terminus toward the C-terminus, and preferably inserted at a position 50 amino acids from the N-terminus toward the C-terminus.

[0019] When a fluorescent protein or a luminescent protein is inserted into the C-terminus of a collagen protein, or when a fluorescent protein or a luminescent protein is inserted into the N-terminus of the C-propeptide, the expressed collagen protein may not be able to form a triple helix structure and may not be secreted outside the cell.

[0020] Furthermore, in one embodiment of the fusion protein of the present invention, a fluorescent protein or a luminescent protein is inserted into the N-propeptide. The insertion position of the fluorescent protein or luminescent protein into the N-propeptide is not particularly limited, because the insertion of the fluorescent protein or luminescent protein into the N-propeptide does not affect the formation of the triple helix structure of collagen.

[0021] The insertion of a fluorescent protein or a luminescent protein into the collagen protein, C-propeptide, and N-propeptide may be a single insertion, a combination of two insertions (e.g., an insertion into the collagen protein and C-propeptide, an insertion into the collagen protein and N-propeptide, or an insertion into the C-propeptide and N-propeptide), or a combination of three insertions. Specifically, the combinations of the two insertions are (a) insertion of the fluorescent protein or luminescent protein into the collagen protein at a position 30 amino acids from the C-terminus toward the N-terminus and into the C-propeptide at a position 30 amino acids from the N-terminus, (b) insertion of the collagen protein at a position 30 amino acids from the C-terminus toward the N-terminus and into the N-propeptide, or (c) insertion of the C-propeptide at a position 30 amino acids from the N-terminus toward the C-terminus and into the N-propeptide. The insertion into the collagen protein or the C-propeptide may be at the preferred positions described above.

[0022] Collagen promoter The collagen promoter used in the present invention is not particularly limited as long as it is an original collagen promoter, and examples thereof include type I collagen α1 promoter (SEQ ID NO: 4), type I collagen α2 promoter, type II collagen α1 promoter, type III collagen α1 promoter, type IV collagen α1 promoter, type IV collagen α2 promoter, type IV collagen α3 promoter, type IV collagen α4 promoter, type IV collagen α5 promoter, type IV collagen α6 promoter, type V collagen α1 promoter, type V collagen α2 promoter, type V collagen α3 promoter, type V collagen α4 promoter, type VI collagen α1 promoter, type VI collagen α2 promoter, type VI collagen α3 promoter, type VII collagen α1 promoter, type VIII collagen α1 promoter, type VIII collagen α2 promoter, type IX collagen α1 promoter, type IX collagen α2 promoter, type IX collagen α3 promoter, type X collagen α1 promoter, type XI collagen α1 promoter, type XI collagen α2 promoter, type XI collagen α3 promoter, type XIII collagen α1 promoter, type XIV collagen α1 promoter, type XV collagen α1 promoter, type XVI collagen α1 promoter, type XVII collagen α1 promoter, type XVIII collagen α1 promoter, type XIX collagen α1 promoter, type XX collagen α1 promoter, type XXI collagen α1 promoter, type XXII collagen α1 promoter, type XXIII collagen α1 promoter, type XXIV collagen α1 promoter, type XXV collagen α1 promoter, type XXVI collagen α1 promoter, type XXVII collagen α1 promoter, or type XXVIII collagen α1 promoter. Since the collagen promoters are the natural promoters of collagen, the use of these promoters makes it possible to reproduce the natural expression of collagen-related proteins in cells.

[0023] (nucleic acid) The collagen promoter vector used in the present invention comprises a nucleic acid encoding the fusion protein and a collagen promoter. The nucleic acid encoding the fusion protein is not particularly limited, as long as it encodes a fusion protein of a collagen-related protein such as a preprocollagen protein, a procollagen protein, a collagen protein, or a variant or fragment thereof, with a fluorescent protein or the like. Nucleic acids encoding preprocollagen proteins, procollagen proteins, or collagen proteins can be extracted from, but are not limited to, tissues of collagen-containing organisms or isolated cells of those organisms. They can also be obtained from vectors containing already isolated cDNAs. They can also be prepared by DNA synthesis.

[0024] Furthermore, nucleic acids encoding the mutants can be obtained by introducing mutations into nucleic acids encoding, for example, preprocollagen proteins, procollagen proteins, or collagen proteins, so that amino acids are deleted, substituted, inserted, and / or added. Nucleic acid mutations (deletions, substitutions, insertions, and / or additions) can be introduced by methods known in the art. Furthermore, naturally occurring nucleic acids having mutations resulting in amino acid deletions, substitutions, insertions, and / or additions can also be used without genetic engineering.

[0025] Furthermore, the nucleic acid encoding the fragment can be a portion of a nucleic acid encoding a preprocollagen protein, a procollagen protein, a collagen protein, or a variant thereof. For example, a nucleic acid encoding a fragment of a preprocollagen protein, a procollagen protein, a collagen protein, or a variant thereof can be obtained by cleaving the nucleic acid with a restriction enzyme. The nucleic acid contained in the collagen promoter vector is not limited to, but may be, for example, DNA or RNA.

[0026] (vector) The backbone vector of the collagen promoter vector used in the present invention may be a cloning vector or an expression vector. Examples of RNA vectors include bacteriophages. Furthermore, viral vectors (e.g., adenoviruses or lentiviruses) can also be used. The backbone vector can be any vector commonly used in this field without limitation, including, for example, E. coli plasmids such as pBR322, pBR325, pUC118, pUC119, pKC30, pCFM536, pcDNA3.1, pcDNA3, pME, or pGEX. Commercially available vectors can also be used, including, for example, pQE70, pQE60, pQE-9 (Qiagen), pBluescript II, pTrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), and pET-11a (Novagen). The backbone vector contains a replication origin, a selection marker, and a promoter, and may also contain an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like, as necessary.

[0027] (Vector-containing host cells) The host cells containing the collagen promoter vector used in the present invention are not particularly limited, and examples thereof include Escherichia coli, actinomycetes, yeast, filamentous fungi, insect cells, and animal cells, with Escherichia coli or animal cells being preferred, and animal cells being more preferred. By using animal cells, collagen can be expressed in the native host cells. Furthermore, it is possible to analyze the formation process of the higher-order structure of collagen. The animal cells are not particularly limited as long as they are capable of expressing collagen, and examples of cells that can be used include cells derived from mammals (e.g., humans, cows, pigs, sheep, goats, mice, rats, guinea pigs, or monkeys), birds (e.g., chickens, geese, ducks, or ostriches), reptiles (e.g., crocodiles), amphibians (e.g., frogs), fish (e.g., sturgeon, tilapia, sea bream, flounder, sharks, sardines, tuna, pufferfish, goldfish, cod, flounder, or carp), or invertebrates (e.g., jellyfish). The method for introducing the vector into animal cells is not particularly limited, and any method known in the art can be used, such as lipofection, microinjection, electroporation, or infection with a viral vector.

[0028] (1) Contact process In the contacting step (1) of the screening method of the present invention, a test substance is contacted with cells into which a collagen promoter vector has been introduced.

[0029] Test Substance Test substances are not limited as long as they are substances that may be involved in collagen biosynthesis and / or post-biosynthetic processes. Examples include various known compounds (including peptides) registered in chemical files, compounds obtained by combinatorial chemistry techniques (Terrett, N.K. et al., Tetrahedron, 51, 8135-8137, 1995), random peptides generated using phage display (Felici, F. et al., J. Mol. Biol., 222, 301-310, 1991), high-molecular-weight compounds, medium-molecular-weight compounds, and low-molecular-weight compounds. Microbial culture supernatants, cell culture supernatants, biological fluids, natural components derived from plants or marine organisms, and animal tissue extracts can also be used as test substances for screening. The concentration of the test substance can be determined as appropriate, but since there is thought to be an optimum concentration at which the test substance affects the dynamics of collagen, it is preferable to test the substance after several serial dilutions.

[0030] (2) Measurement process In the measurement step of the screening method of the present invention, fluorescence or luminescence in the cell culture supernatant and / or intracellularly is measured. By measuring the fluorescence or luminescence in the cell culture supernatant, collagen-related proteins secreted into the culture supernatant can be measured. Therefore, it can be determined whether a test substance is a substance that is involved (increased or decreased) in a post-biosynthetic process of collagen-related proteins (e.g., secretion from cells). Here, the "post-biosynthetic process" of collagen or collagen-related proteins from cells involves dynamics such as triplex formation, vesicle transport (CGN), processing, secretory vesicles (TGN), and secretion after collagen-related protein biosynthesis within cells. Therefore, by measuring the fluorescence or luminescence in the cell culture supernatant, it is possible to screen for substances involved in these dynamics. The fluorescence or luminescence in the culture supernatant can be measured using, but is not particularly limited to, a plate reader or a spectrophotometer.

[0031] On the other hand, by measuring intracellular fluorescence or luminescence, collagen-related proteins biosynthesized in the cells can be measured, and therefore, it can be determined whether a test substance is a substance that is involved in (increases or decreases) the intracellular biosynthesis (induction of expression) of collagen-related proteins. Intracellular fluorescence or luminescence can be measured using a fluorescence microscope, flow cytometer, plate reader, spectrophotometer, or confocal fluorescence microscope. The fluorescence or luminescence of a cell lysate can also be measured using a plate reader or spectrophotometer. Furthermore, the fluorescence or luminescence of cells can be measured directly using a plate reader.

[0032] Analysis can be carried out essentially by comparing with cells not contacted with the test substance. That is, if the expression of the collagen fusion protein is increased or decreased compared to cells not contacted with the test substance, the test substance can be determined to be a substance involved in the biosynthesis and / or post-biosynthetic process of collagen-related proteins.

[0033] The screening method of the present invention can be used to screen for drugs (compounds (including proteins, amino acids, nucleic acids, lipids, extracts, etc.)) or mixtures for treating or preventing collagen-related diseases. Collagen-related diseases are not particularly limited, as long as collagen is thought to be related to the cause or symptoms of the disease, and examples include organ / tissue fibrosis such as liver fibrosis, pulmonary fibrosis, and renal fibrosis, collagen diseases such as dermatomyositis, polymyositis, and rheumatoid arthritis, and corneal opacity. The cells used in the screening methods of the present invention may be, for example, normal cells, cells isolated from patients with collagen-related diseases, or cells isolated and prepared from model animals. Examples of cells isolated from patients with collagen-related diseases include collagen-secreting cells, specifically hepatic stellate cells, fibroblasts, and chondrocytes isolated from patients with liver fibrosis. Examples of cells isolated and prepared from model animals include hepatic stellate cells and pulmonary fibroblasts. Furthermore, normal collagen-secreting cells can be activated, for example, with TGF-β and used as model cells for liver fibrosis, pulmonary fibrosis, renal fibrosis, or rheumatoid arthritis, and can be used as cells for screening drugs for the respective diseases.

[0034] Furthermore, if the collagen fusion protein is secreted extracellularly in greater or less amounts than in cells not contacted with the test substance, the screened test substance can be determined to be effective in the secretion of collagen or collagen-related proteins. Furthermore, if the intracellular transport of the collagen fusion protein is restored to normal compared to cells not contacted with the test substance, the screened test substance can be determined to be effective in the intracellular triplex formation and transport of collagen-related proteins. Furthermore, if the processing of the collagen fusion protein is restored to normal compared to cells not contacted with the test substance, the screened test substance can be determined to be effective in the processing of collagen-related proteins.

[0035] [2] Second embodiment of the screening method The screening method of the present invention preferably further comprises the steps of (3) contacting a test substance with cells into which a constitutive promoter vector containing a constitutive promoter and a nucleic acid encoding a fusion protein of a collagen-related protein, which is a preprocollagen protein, a procollagen protein, a collagen protein, or a variant thereof, with a fluorescent protein or a luminescent protein has been introduced, and (4) measuring the fluorescence or luminescence in the culture supernatant of the cells.

[0036] A second embodiment of the screening method of the present invention includes, in addition to the contacting step (1) and measuring step (2) using a collagen promoter vector, a contacting step (3) and measuring step (4) using a constitutive promoter vector. As described above, the contacting step (1) and measuring step (2) using a collagen promoter vector enable screening for substances involved in the biosynthesis and / or post-biosynthetic processes of collagen-related proteins. However, in the case of a substance that is not involved in the biosynthesis of collagen-related proteins but is involved only in the post-biosynthesis processes, collagen-related proteins may not be sufficiently biosynthesized in cells using the collagen promoter vector. If collagen-related proteins are not sufficiently biosynthesized in cells, it may be impossible to measure the effects of substances involved only in the post-biosynthesis processes. Alternatively, if the secretion amount of collagen fusion protein changes in measurements using cells using the collagen promoter vector, it is impossible to distinguish whether the cause is a change in the amount of protein produced or a change in a subsequent process. In cells containing a constitutive promoter vector, collagen-related proteins are sufficiently biosynthesized in the cells by the constitutive promoter. Therefore, substances involved only in the post-biosynthesis processes of collagen-related proteins can be accurately screened. In addition, in the screening method of the present invention, as a third embodiment, by performing only the contacting step (3) and the measuring step (4) using a constitutive promoter vector, it is possible to screen for a substance involved in a post-biosynthetic process of collagen-related proteins.

[0037] In a second embodiment of the screening method of the present invention, screening can be carried out in accordance with the contact step (1) and measurement step (2) described above, except that a constitutive promoter vector is used instead of a collagen promoter vector. That is, the "collagen-related protein," "fluorescent protein or luminescent protein," "fusion protein," "nucleic acid," "vector," "vector-containing host cell," "(1) contacting step," "test substance," and "measurement step (2)" described in "(1) First embodiment of screening method" above can be carried out in the same manner, except that a constitutive promoter vector is used. Constitutive promoter vectors will be described below.

[0038] Constitutive promoter vector In the contacting step (3) and the measuring step (4) of the second embodiment of the screening method of the present invention, a constitutive promoter vector containing a constitutive promoter is used. That is, the contacting step (3) and the measuring step (4) are screenings (hereinafter sometimes referred to as second screenings) using cells into which a nucleic acid encoding a fusion protein and a constitutive promoter vector containing a constitutive promoter have been introduced. A constitutive promoter vector contains a nucleic acid encoding a collagen-related protein, such as a preprocollagen protein, a procollagen protein, a collagen protein, or a variant thereof, and these collagen-related proteins are constitutively expressed by a constitutive promoter. That is, a promoter is a nucleotide sequence involved in initiating transcription of RNA from DNA, but the constitutive promoter is not the native promoter used for transcription of collagen, and can constitutively express the collagen-related protein in cells.

[0039] Constitutive promoter The constitutive promoter used in the present invention is not particularly limited as long as it is capable of constitutively expressing a collagen-related protein, but examples include the CMV promoter, EF1alpha promoter, CAG promoter, RSV promoter, PGK promoter, and LTR (GALV LTR, MSCV LTR) promoter. By using the constitutive promoter, it is possible to efficiently and accurately screen for the involvement of test substances in post-biosynthetic processes of collagen-related proteins in cells.

[0040] 《Action》 The mechanism by which the screening method of the present invention can accurately screen for substances involved in the biosynthesis and / or post-biosynthesis process of collagen-related proteins has not been elucidated in detail, but can be assumed as follows, although the present invention is not limited by the assumption below. In the first screening using cells introduced with a collagen promoter vector in the screening method of the present invention, the use of a collagen promoter allows for natural expression of collagen-related proteins. Therefore, it is possible to screen for substances involved in the natural biosynthesis of collagen-related proteins. Furthermore, it is also possible to screen for substances involved in the natural biosynthesis of collagen-related proteins. For example, in the case of a substance involved only in the biosynthesis of collagen-related proteins, it can be determined that the substance is involved in the biosynthesis of collagen-related proteins by measuring the increase or decrease in collagen-related proteins within the cells. Furthermore, in the case of a substance involved in the biosynthesis and post-biosynthetic processes of collagen-related proteins, it can be determined that the substance is involved in the biosynthesis and / or post-biosynthetic processes of collagen-related proteins by measuring the increase or decrease in collagen-related proteins within the cells and in the culture supernatant. On the other hand, in the case of a substance that is not involved in the biosynthesis of collagen-related proteins but is involved only in post-biosynthetic processes, the first screening may not result in sufficient collagen-related protein biosynthesis, making it impossible to measure post-biosynthetic processes. Furthermore, the first screening may reveal involvement in biosynthesis and / or secretion, but it is unable to distinguish between biosynthesis and post-biosynthesis processes. In this case, the second screening using cells into which the constitutive promoter vector has been introduced can be effectively used. In cells containing a constitutive promoter vector, the constitutive promoter drives sufficient intracellular biosynthesis of collagen-related proteins. Therefore, the second screening makes it possible to accurately screen for substances that are not dependent on collagen biosynthesis but are involved only in post-biosynthetic processes of collagen-related proteins. [Example]

[0041] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0042] Example 1 In this example, a collagen promoter vector was constructed using a collagen promoter, with GFP inserted two-thirds of the way from the N-terminus of type I collagen and mCherry inserted closer to the C-propeptide than the C-terminus. GFP was inserted into the BamHI site of the collagen protein, and mCherry was inserted into the EcoRI site of the C-propeptide. The colIα1 promoter was used as the collagen promoter. A schematic diagram of the vector is shown in Figure 1. The base sequence of the DNA containing the vector is shown in SEQ ID NO: 1. A DNA fragment with BamHI restriction enzyme sites added to both ends of GFP cDNA was created and inserted into the BamHI site within human preprocollagen Iα1 cDNA. During protein translation, adjustments were made to allow the collagen protein and GFP protein to link together. Next, a DNA fragment with EcoRI sites added to both ends of mCherry cDNA was created and inserted into the EcoRI site of human preprocollagen Iα1-GFP fusion cDNA. During protein translation, adjustments were made to allow the collagen protein and mCherry protein to link together, and the base sequence was confirmed at each step of the procedure. The colIα1 promoter vector was introduced into NIH3T3 cells by the polyethyleneimine (PEI) method.

[0043] Example 2 In this example, the effect of a test substance on collagen biosynthesis was examined using cells into which the collagen promoter vector obtained in Example 1 had been introduced. TGF-β, which is known to increase collagen biosynthesis, was used as the test substance. 1x10 5 Cells were seeded onto 35mm dishes in a lab and left to stand for 48 hours. TGF-β was then added at a concentration of 2 or 12.5ng / mL. Fluorescence in the cells was observed under a fluorescence microscope after 8, 24, 48, and 72 hours. As shown in Figure 2, the addition of TGF-β increased the fluorescence in the cells.

[0044] Example 3 In this example, the effect of a test substance on collagen secretion was examined using cells into which the collagen promoter vector obtained in Example 1 had been introduced. TGF-β, which is known to increase collagen biosynthesis, was used as the test substance. 5 Cells were seeded onto 35mm dishes in a PBS-MS / MS system and left to stand for 48 hours. TGF-β was then added at a concentration of 2 or 12.5ng / mL. After 8, 24, 48, and 72 hours, 200μL of the culture supernatant was collected and measured for fluorescence. As shown in Figure 3, the addition of TGF-β did not increase the fluorescence in the culture supernatant, suggesting that TGF-β is not involved in the post-synthesis process of collagen protein.

[0045] Example 4 In this example, a constitutive promoter vector was constructed using the CMV promoter, a constitutive promoter. GFP was inserted two-thirds of the way from the N-terminus of type I collagen, and mCherry was inserted closer to the C-propeptide than the C-terminus. The procedure of Example 1 was repeated to obtain a CMV promoter vector, except that a CMV promoter was used instead of the colIα1 promoter. The CMV promoter vector was introduced into NIH3T3 cells using the polyethyleneimine (PEI) method. A schematic diagram of the vector is shown in Figure 4. The base sequence of the DNA other than the vector is shown in SEQ ID NO: 2.

[0046] Example 5 In this example, the effects of test substances on collagen secretion were examined using the cells into which the CMV promoter vector obtained in Example 4 had been introduced. The test substances used were ascorbic acid, TGF-β1, and acene. The procedure of Example 3 was repeated, except that cells transfected with a CMV promoter vector were used instead of cells transfected with a collagen promoter vector, and that ascorbic acid (50 μg / mL), TGF-β1 (2 ng / mL), and aceca root were used as test substances. As shown in Figure 5, ascorbic acid (AA) and Acacia officinalis increased the extracellular secretion of collagen, whereas TGF-β1 did not.

[0047] Example 6 In this example, we attempted to construct a vector containing nucleic acids using the constitutive CMV promoter, with GFP inserted closer to the N-terminus of the N-propeptide of type I collagen and mCherry inserted closer to the C-terminus of the C-propeptide. GFP was inserted into the KpnI site of the N-propeptide, and mCherry was inserted into the EcoRI site of the C-propeptide. A schematic diagram of the vector is shown in Figure 6. The base sequence of the DNA other than the vector is shown in SEQ ID NO: 3. A DNA fragment with KpnI restriction enzyme sites added to both ends of GFP cDNA was created and inserted into the KpnI site within human preprocollagen Iα1 cDNA. Adjustments were made to ensure that the collagen and GFP proteins would link together during protein translation. Next, a DNA fragment with EcoRI sites added to both ends of mCherry cDNA was created and inserted into the EcoRI site of human preprocollagen Iα1-GFP fusion cDNA. Adjustments were also made to ensure that the collagen and mCherry proteins would link together during protein translation, and the base sequence was confirmed at each step of the procedure. The obtained constitutive promoter vector was transfected into NIH3T3 cells by the polyethyleneimine (PEI) method.

[0048] Example 7 In this example, four constitutive promoter vectors were constructed using tilapia type I collagen and the CMV promoter, carrying nucleic acids in which GFP was inserted into the type I collagen and mCherry was inserted into the C-propeptide. The procedure of Example 4 was repeated, except that tilapia type I collagen was used instead of human type I collagen and the GFP and mCherry insertion positions were different. The constructions are shown in Figures 7(A) to 7(D). In the first embodiment, GFP is inserted 305 amino acids from the N-terminus and 778 amino acids from the C-terminus of the collagen protein (Figure 7A). In the second embodiment, GFP is inserted 305 amino acids from the N-terminus and 778 amino acids from the C-terminus of the collagen protein, and mCherry is inserted 214 amino acids from the N-terminus and 25 amino acids from the C-terminus of the C-propeptide (Figure 7B). In the third embodiment, GFP is inserted 305 amino acids from the N-terminus and 778 amino acids from the C-terminus of the collagen protein, and mCherry is inserted 52 amino acids from the N-terminus and 188 amino acids from the C-propeptide (Figure 7C). In the fourth embodiment, GFP is inserted 32 amino acids from the N-terminus and 69 amino acids from the C-terminus of the N-propeptide, and mCherry is inserted 214 amino acids from the N-terminus and 25 amino acids from the C-terminus of the C-propeptide (D).

[0049] As shown in FIG. 7, extracellular fluorescence was observed in the four embodiments of tilapia collagen, confirming the extracellular secretion of collagen protein. [Industrial Applicability]

[0050] The screening method of the present invention can screen compounds (including drugs, extracts, proteins, amino acids, etc.) used in the treatment or prevention of collagen-related diseases.

Claims

1. (1) A step of contacting a test substance with a cell into which a collagen promoter vector containing a collagen promoter and a nucleic acid encoding a fusion protein of a collagen-related protein, which is a preprocollagen protein, a procollagen protein, a collagen protein, or a variant thereof, with a fluorescent protein or a luminescent protein has been introduced, the step in which the fusion of the fluorescent protein or the luminescent protein is one or more insertions selected from the group consisting of insertion into the collagen protein at 30 amino acids from the C-terminus toward the N-terminus, insertion into the C-propeptide at 30 amino acids from the N-terminus toward the C-terminus, and insertion into the N-propeptide; (2) measuring the fluorescence or luminescence in the culture supernatant and / or intracellularly of the cells; (3) A step of contacting the test substance in (1) with a cell into which a constitutive promoter vector containing a constitutive promoter and a nucleic acid encoding a fusion protein of a collagen-related protein, which is a preprocollagen protein, a procollagen protein, a collagen protein, or a variant thereof, with a fluorescent protein or a luminescent protein has been introduced, The step in which the fusion of a fluorescent protein or a luminescent protein is one or more insertions selected from the group consisting of insertion into the collagen protein at 30 amino acids from the C-terminus toward the N-terminus, insertion into the C-propeptide at 30 amino acids from the N-terminus toward the C-terminus, and insertion into the N-propeptide; and (4) measuring the fluorescence or luminescence in the cell culture supernatant; A method for screening for a substance involved in collagen biosynthesis and / or post-biosynthesis processes, comprising:

2. The collagen-related protein encoded by the collagen promoter vector and / or the constitutive promoter vector is a preprocollagen protein or a procollagen protein, and the fluorescent protein or luminescent protein is (a) inserted at the N-terminus of the collagen protein more than 30 amino acids from the C-terminus, and inserted at the C-terminus of the C-propeptide more than 30 amino acids from the N-terminus, (b) inserted at the N-terminus of the collagen protein more than 30 amino acids from the C-terminus and into the N-propeptide; (c) inserted 30 amino acids C-terminally from the N-terminus of the C-propeptide and inserted into the N-propeptide; A method for screening for substances involved in collagen biosynthesis and / or post-biosynthetic processes according to claim 1.

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