JP6868232B2 - Screening method for collagen fusion protein and drugs using it - Google Patents

Description

The present invention relates to a screening method for a collagen fusion protein and a drug using the collagen fusion protein, a vector containing a nucleic acid encoding the collagen fusion protein, Escherichia coli containing the vector, and a method for producing the same. According to the present invention, candidate substances for therapeutic or prophylactic agents for 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 the same can be obtained.

Collagen accounts for 30% of proteins in the living body and is an important protein having functions such as skeletal support and cell adhesion. For example, bone / cartilage, ligaments / tendons, stroma, skin, liver, etc. of the human body. It is a major constituent of tissues such as muscle. Collagen includes fibrous collagen that forms collagen fibers and non-fibrous collagen that does not form fibers. In fibrous collagen, three polypeptide chains of collagen are gathered to form a triple helix structure (tropocollagen). By self-assembling, this triple-helical topocollagen is aligned with a deviation of 1/4 of the molecular length to form collagen fibrils (collagen fibrils). Furthermore, a large number of these collagen fibrils are assembled to form collagen fibers (collagen fiber bundles). Furthermore, in the stroma and cortical bone, collagen fibers form two-dimensional layers aligned in the same direction, and these layers form a veneer-like three-dimensional layered plate structure in which each layer is vertically laminated. .. Collagen having this plywood-like three-dimensional layer plate structure is transparent and has high strength.

Examples of diseases caused by such molecular abnormalities of collagen include collagen disease (for example, dermatomyositis and polymyositis) or cloudiness of the cornea, but many of them have no established treatment method. One reason for this is that the secretory process of collagen and the formation process of higher-order structures are unclear. If it is possible to analyze the collagen secretion process and the formation process of higher-order structures, it will be used as an effective means for elucidating the causes of diseases caused by molecular abnormalities of collagen and developing treatment methods for them. be able to.

Special Table 2000-508544

In order to analyze the dynamics of collagen in animal cells and the dynamics of collagen secreted from animal cells, the present inventors attempted to construct a system for expressing collagen in animal cells. Specifically, a fusion protein in which a fluorescent protein was bound to the C-terminal of procollagen was constructed. This fusion protein was introduced into animal cells to attempt to detect procollagen and collagen secretion from animal cells. However, procollagen and collagen secretion from animal cells could not be detected.
Therefore, an object of the present invention is to construct a system capable of detecting procollagen and collagen secretion from animal cells.
On the other hand, the present inventors used genetic recombination to construct the above system. Genetic engineering of collagen proteins is reported in, for example, Patent Document 1. The present inventors have incorporated a nucleic acid encoding collagen into a vector and attempted to construct an expression vector or the like using Escherichia coli. However, when transformation was performed in Escherichia coli using a vector normally used for protein expression, the present inventors could delete, insert, or inhibit cleavage with a restriction enzyme in the gene encoding the collagen protein. I noticed that it happened. That is, the present inventors could not obtain an expression vector containing the target collagen gene. Deletion insertion of this gene and inhibition of cleavage with restriction enzymes did not occur at specific positions, but occurred at random positions of the collagen gene. For example, it is known that homologous recombination causes a deletion at a specific position in a gene. However, it is not known to the present inventor that inhibition of deletion, insertion, or restriction enzyme cleavage occurs at an unspecified position in the construction of a vector, and it is a very rare phenomenon. Seem.
Therefore, another object of the present invention is to provide a vector having a nucleic acid that does not inhibit the deletion, insertion, or restriction enzyme cleavage encoding a collagen protein.

As a result of diligent research on a system capable of detecting the secretion of procollagen from animal cells, the present inventors surprisingly found that the N-terminal N-proprotein, collagen protein, or C-terminal C-protein of the procollagen protein was used. It has been found that by binding or inserting a fluorescent protein into a proprotein, a system capable of detecting the secretion of procollagen and collagen protein from animal cells can be constructed. Then, they have found that it is possible to screen a drug for treating or preventing a collagen-related disease by using cells of a collagen-related disease expressing a fusion protein in which a fluorescent protein or a photoprotein is inserted into the collagen protein.
In addition, as a result of diligent research on a vector having a nucleic acid that does not inhibit deletion, insertion, or restriction with a deletion, insertion, or restriction enzyme that encodes a collagen protein, the present inventors surprisingly found that Escherichia coli having a specific gene mutation. Was used as a host for a vector containing an Escherichia coli gene, and it was found that the gene encoding the collagen protein was not deleted, inserted, or inhibited by a restriction enzyme.
The present invention is based on these findings.
Therefore, the present invention
[1] Preprocollagen protein, procollagen protein, collagen protein, or a mutation thereof having a repeating amino acid sequence of Gly-Xaa-Yaa (in the formula, Gly is glycine, and Xaa and Yaa are arbitrary amino acids). A fusion protein of a collagen-related protein and a fluorescent protein or a luminescent protein, which is a body or a fragment thereof,
[2] The fusion protein according to [1], wherein the fluorescent protein or luminescent protein is bound or inserted into the N-terminal N-proprotein, collagen protein, or C-terminal C-proprotein of the procollagen protein. ,
[3] The fluorescent protein or luminescent protein 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 luminescent protein is C-terminal from the N-terminal to 30 amino acids of the C-proprotein. The fusion protein according to [1] or [2], which is inserted on the side.
[4] Collagen-related disease including a step of contacting a collagen-related disease cell expressing the fusion protein according to any one of [1] to [3] with a test substance, and a step of analyzing the dynamics of the fusion protein. Method of screening for therapeutic or prophylactic substances,
[5] Preprocollagen protein, procollagen protein, collagen protein, or a mutation thereof having a repeating amino acid sequence of Gly-Xaa-Yaa (in the formula, Gly is glycine, and Xaa and Yaa are arbitrary amino acids). A nucleic acid encoding a collagen-related protein, which is a body or a fragment thereof, or a fusion protein of the collagen-related protein and a fluorescent protein or a luminescent protein.
[6] A vector containing the nucleic acid according to [5].
[7] A vector-containing host cell containing the vector according to [6].
[8] The host cells are mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7679, galU, galK, rpsL (str ^r ), nupG. , GalE, hsdS, and the vector-containing host cell according to claim 7, which is an Escherichia coli having a gene mutation selected from the group consisting of two or more combinations thereof.
[9] The E. coli is DH10B, TOP10F', MC1061, XL1-Blue MRF', AG1, BL21 (DE3), DB3.1, DH1, DH5α, DH5α Turbo, DH12S, DM1, E. coli. Clone (r) 5 alpha, E.I. Clone (r) 10G, E.I. Clone (r) 10GF', ER2267, HB101, HMS174 (DE3), High-Control (tm) BL21 (DE3), High-Control (tm) 10G, IJ1126, JM108, JM109, Mach1, MC1061, MFDpir, OmpressMA (Tm) C41 (DE3), OverExpress (tm) C43 (DE3), Rosetta (DE3), SOLR, SS320, STBL2, STBL3, STBL4, SURE, SURE2, TG1, TOP10, XL2-Blue, XL2-BlueMRF', and The vector-containing host cell according to [8], selected from the group consisting of XL10-Gold.
[10] A vector obtained from the vector-containing host cell according to [8] or [9].
[11] The vector according to [6] is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7679, galU, galK, rpsL (str ^r). ), NupG, galE, hsdS, and a method for producing vector-containing Escherichia coli, which comprises introducing into Escherichia coli having a gene mutation selected from the group consisting of two or more combinations thereof.
[12] The vector according to [6] is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7679, galU, galK, rpsL (str ^r). ), NupG, galE, hsdS, and a step of introducing into Escherichia coli having a gene mutation selected from the group consisting of two or more combinations thereof, and a step of recovering the vector. Production method,
[13] A method for producing collagen, which comprises (1) a step of introducing the vector according to claim 6 into an animal cell, and (2) a step of secreting collagen from the animal cell into which the vector has been introduced. [14] Fluorescence of collagen or including (1) a step of introducing the vector according to claim 6 into an animal cell and (2) a step of detecting fluorescence or luminescence in the animal cell into which the vector has been introduced. Luminescence detection method,
Regarding.

The fusion protein of the present invention can detect the secretion of procollagen from animal cells. Since collagen-related proteins form a triple helix structure, it is thought that when a fluorescent protein is inserted into a procollagen protein, the protein structure changes and the triple helix structure cannot be formed. However, by inserting the fluorescent protein at a specific position of collagen protein and / or C-proprotein, it is possible to maintain the triple helix structure and construct a system that can detect the secretion of procollagen from animal cells. It was. In addition, by inserting the fluorescent protein at a specific position of the N-proprotein, it became possible to maintain the triple helix structure, and it was possible to construct a system capable of detecting the secretion of procollagen from animal cells.
Furthermore, according to a vector obtained from Escherichia coli having a specific gene mutation of the present invention, it is possible to obtain a gene encoding a collagen protein that is not inhibited by deletion, insertion, or restriction enzyme. Further, according to the vector-containing Escherichia coli of the present invention, it is possible to obtain a vector containing a gene encoding a collagen protein and having no disorder such as deletion. According to the method for producing 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 and not causing a disorder such as deletion, and an Escherichia coli containing the vector.
Collagen can be produced in animal cells using the vector obtained by the present invention. Moreover, by using the vector obtained by the present invention, it is possible to analyze the formation process of the higher-order structure of collagen.

It is a figure which showed the outline of the vector which encodes the fusion protein which EGFP was inserted into collagen protein, and mCherry was inserted into C proprotein of C terminal of procollagen protein. It is a photograph (A) of the electrophoresis of the vector amplified by TOP10F'and the vector amplified by XL-1 blue, and a photograph (B) of the electrophoresis of the vector amplified by DH10B and XL-1 blue. It is a figure which showed the outline of the vector which encodes the fusion protein which EGFP was inserted into the N proprotein of the N terminal of a procollagen protein, and mCherry was inserted into the C proprotein of the C terminal of a procollagen protein. It is a photograph which expressed the fusion protein of Example 1 in mouse NIH3T3 cells. It is a photograph which expressed the fusion protein of Example 1 in mouse NIH3T3 cells. It is a photograph which expressed the fusion protein of Example 2 in mouse NIH3T3 cells. A fluorescent micrograph showing the dynamics of collagen fibers expressed 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-β was added to an astral cell (2G92 cell) to activate collagen secretion. Drug screening system by contacting activated hepatic stellate cells (activated 2G92 cells) and constantly activated hepatic stellate cells (5H cells) into which the vector of the present invention has been introduced with SB431542, which has a therapeutic effect on hepatic fibrosis. It is a fluorescence micrograph which carried out the construction of. It is a fluorescence micrograph in which a screening system was constructed by contacting SB431542, which has a therapeutic effect on liver fibrosis, with constantly activated hepatic stellate cells (5H cells) into which the vector of the present invention was introduced. Collagen expression was confirmed by introducing the vector of the present invention into lung fibroblasts (TIG-3-20 cells (A), A549 cells (C)) and renal tubular epithelial cells (MDCK cells (D)). It is a photograph that was taken. It is a photograph which added TGF-β to TIG-3-20 cells and activated the cells (B). Outline of the vector in which mCherry was inserted at the site of 27 amino acids from the N-terminal of C-proprotein (A), and a fluorescence microscope showing that the fibrosis of collagen expressed from the vector (triple helix structure construction) was inhibited. It is a photograph (B, C). It is the outline (A) of the vector which bonded EGFP to the C-terminal of the collagen protein, and the fluorescence micrograph (B) which shows that the fibrosis (triple helix structure construction) of the collagen expressed from the vector was inhibited. ..

[1] Fusion protein The fusion protein of the present invention is a protein in which a collagen-related protein is fused with a fluorescent protein or a photoprotein. The collagen-related proteins will be described below.

《Collagen-related protein》
Collagen-related proteins are preprocollagen proteins, procollagen proteins, collagen proteins, or them, which have a repeating amino acid sequence of Gly-Xaa-Yaa (in the formula, Gly is glycine, and Xaa and Yaa are arbitrary amino acids). A variant of, or a fragment thereof.
The collagen-related protein is not particularly limited as long as it has the repeating amino acid sequence of Gly-Xaa-Yaa, but for example, human type I to XXVIII collagen proteins, preprocollagen proteins or procollagen proteins thereof, and the like. Variants thereof, or fragments thereof, can be mentioned. The preprocollagen protein has a signal peptide and N-proprotein on the N-terminal side of the collagen protein, and has a C-proprotein on the C-terminal side. Further, the procollagen protein has N-proprotein on the N-terminal side of the collagen protein and C-proprotein on the C-terminal side. The repeating amino acid sequence of Gly-Xaa-Yaa is called a collagen-like sequence, and having this Gly-Xaa-Yaa collagen-like sequence is an important feature of collagen-related proteins.
The species from which the collagen-related protein is derived is also not limited, but for example, mammals (eg, cows, pigs, ducks, goats, mice, rats, guinea pigs, or monkeys), birds (eg, chickens, etc.). Gutters, ducks, or ostriches), reptiles (eg, crocodile), amphibians (eg, frogs), fish (eg, butterfly sharks, terapias, ties, flatfish, sharks, sardines, tuna, pufferfish, goldfish, cod, curry, or carp ), Or invertebrates (eg, ostriches).
The number of repeating amino acid sequences of Gly-Xaa-Yaa contained in the collagen-related protein is not particularly limited as long as it is 2 or more, but is preferably 10 or more, more preferably 20 or more. It is more preferably 30 or more, and most preferably 50 or more. In addition, the collagen-related protein can contain an amino acid sequence other than the collagen-like sequence, and can include, for example, a signal peptide, a proprotein, and a telopeptide at the N-terminal or C-terminal.

Collagen protein (hereinafter, may be simply referred to as "collagen") is divided into fibrous collagen and non-fibrous collagen, and the fibrous collagen includes type I collagen, type II collagen, type III collagen, and type V collagen. , Or XI type collagen and the like.
For example, type I collagen has a "triple helix structure (tropocollagen)" in which three polypeptide chains having a molecular weight of about 100,000 are gathered to form a "triple helix structure (tropocollagen)", and the molecular weight is about 300,000. This triple helix structure has a length of 300 nm and a shape like a single hard rod having a diameter of 1.5 nm, and is called tropocollagen. By self-assembling, this triple-helical topocollagen is aligned with a deviation of 1/4 of the molecular length to form collagen fibrils (collagen fibrils). Furthermore, a large number of these collagen fibrils are assembled to form collagen fibers (collagen fiber bundles). Furthermore, in the stroma and cortical bone, collagen fibers form two-dimensional layers aligned in the same direction, and these layers form a veneer-like three-dimensional layered plate structure in which each layer is vertically laminated. .. Collagen having this plywood-like three-dimensional layer plate structure is transparent and has high strength. Further, in type I collagen, Xaa of Gly-Xaa-Yaa is often proline or 3-hydroxyproline, and Yaa is 4-hydroxyproline or hydroxylysine in many cases. Hydroxyproline is not contained in ordinary proteins and is an amino acid peculiar to collagen, but it is considered that the triple helix structure is stabilized by the hydrogen bond between the hydroxyl group of hydroxyproline and the hydrated water.

The collagen protein is not limited in cells, but is, for example, a procollagen protein having N-proprotein on the N-terminal side and C-proprotein on the C-terminal side (hereinafter, may be simply referred to as procollagen). ). The pro-collagen is not particularly limited, and examples thereof include pro-collagen of type I to type XXVIII collagen.

The pre-procollagen protein, pro-collagen protein or variant of collagen protein is not particularly limited as long as it has a repeating amino acid sequence of Gly-Xaa-Yaa. Examples of the mutant include those in which amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of a natural collagen protein (for example, the type I to XXVIII collagen proteins). The number of aminos 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, still more preferably. The number is 10 or less, and most preferably 1 or several. For example, type I collagen consists of about 1000 amino acids, but may not function as collagen if it has more than 100 deletions, substitutions, insertions, and / or additions.
Further, as another mutant, a mutant collagen protein having 80% or more identity with respect to the amino acid sequence of the natural collagen protein (for example, the 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 if the amino acid identity is less than 80%, it may not function as collagen.

The procollagen protein, collagen protein, or a fragment thereof is not particularly limited as long as it is a partial peptide of the procollagen protein, collagen protein, or a variant thereof, and type I to XXVIII collagen. , Their procollagen, or partial peptides of their variants. The amino acid chain length of the fragment, the cleavage site, and the like are not particularly limited, and can be appropriately determined depending on the purpose of gene construction.

<< Fluorescent protein or photoprotein >>
Examples of the protein used for the fusion protein include, but are not limited to, a fluorescent protein that imparts fluorescence and a luminescent protein that imparts luminescence.
Examples of the fluorescent protein or photoprotein include GFP, EGFP, mCherry, Siris, EBFP, SBP2, EBP2, Azurite, mKalama1, TagBFP, mBluebury, mTurquioise, ECFP, Cerulean, mCeryMian TurboGFP, CFP, AcGFP, TagGFP, AG (Azami-Green), mAG1, ZsGreen, EmGFP (Emerald), GP2, T-Snapfile, HyPer, TagYFP, mAmetrine, EYFP, YFPline, EYFP, YFP turboYFP, ZsYellow, mBanana, mKO1, KO (Kusabira Orange), mOrange, mOrange2, mKO2, Keima570, TurboRFP, DsRed-Express, DsRed, DsRed2, TagRFP, TagRFP-T, DsRed-Monomer, mApple, AsRed2, mStrawberry, TurboFP602, Fluorescence selected from the group consisting of mRP1, JRed, KillerRed, KeimaRed, HcRed, mRasbury, mKate2, TagFP635, mPlum, egFP650, Neptune, mNeptune, egFP670 and luciferase.

Collagen-related proteins and fusion proteins can be prepared using the expression vectors described below, but can also be prepared by peptide synthesis.

As one aspect of the fusion protein of the present invention, the fluorescent protein or photoprotein is inserted from the C-terminal of the collagen protein to the N-terminal side from 30 amino acids. Further, as one aspect of the fusion protein of the present invention, a fluorescent protein or a photoprotein is inserted from the N-terminal of the C-proprotein to the C-terminal side from 30 amino acids. Further, as one embodiment of the fusion protein of the present invention, a fluorescent protein or luminescent protein is inserted from the C-terminal of the collagen protein to the N-terminal side from 30 amino acids, and the fluorescent protein or luminescent protein is inserted from the N-terminal of the C-proprotein. It is inserted on the C-terminal side from 30 amino acids.
The insertion site of the fluorescent protein or photoprotein into the collagen protein is more preferably the N-terminal side from the C-terminal of the collagen protein to 45 amino acids, and further preferably the N-terminal side from 60 amino acids. The insertion site of the fluorescent protein or photoprotein into the C-proprotein is more preferably the C-terminal side from the N-terminal of the C-proprotein to the C-terminal side from 45 amino acids, and further preferably the C-terminal side from the 60 amino acids.
When the fluorescent protein or photoprotein is inserted on the C-terminal side of the collagen protein, or when the fluorescent protein or photoprotein is inserted on the N-terminal side of the C-proprotein, the expressed collagen protein has a triple helix structure. May not be formed and may not be secreted extracellularly.

[2] Nucleic Acid and Vector The nucleic acid of the present invention is a nucleic acid encoding the collagen-related protein or the fusion protein (hereinafter, may be collectively referred to as “collagen nucleic acid”).
《Collagen nucleic acid》
The collagen nucleic acid of the present invention is as long as it is a nucleic acid encoding a collagen-related protein such as the pro-collagen protein, a collagen protein, a variant thereof, or a fragment thereof, or a fusion protein of a collagen-related protein and a fluorescent protein or the like. Is not particularly limited.
The procollagen protein or the nucleic acid encoding the collagen protein can be used by extracting from, for example, the tissue of an organism having collagen or the isolated cells of the organism. It can also be obtained from a vector containing the cDNA that has already been separated. Furthermore, it can be prepared by DNA synthesis.

Further, the nucleic acid encoding the mutant is introduced by introducing a mutation in the nucleic acid so that the amino acid is deleted, substituted, inserted, and / or added to the procollagen protein or the nucleic acid encoding the collagen protein, for example. Obtainable. Nucleic acid mutations (deletion, substitution, insertion, and / or addition) can be introduced by methods known in the art. It is also possible to use naturally occurring nucleic acids having mutations in which amino acids are deleted, substituted, inserted, and / or added without using genetic engineering.

Further, as the nucleic acid encoding the fragment, a procollagen protein, a collagen protein, or a part of the nucleic acid encoding a mutant thereof can be used. For example, by cleaving the nucleic acid with a restriction enzyme, a nucleic acid encoding a procollagen protein, a collagen protein, or a fragment thereof can be obtained.

Examples of the nucleic acid contained in the vector of the present invention include, but are not limited to, DNA or RNA.

"vector"
The vector of the present invention is not limited as long as it contains the collagen nucleic acid. Further, the skeleton vector into which the collagen nucleic acid is introduced may be a cloning vector or an expression vector. Moreover, as an RNA vector, a bacteriophage can be mentioned.
As the skeleton vector, a vector usually used in this field can be used without limitation, and for example, pBR322, pBR325, pUC118, pUC119, pKC30, pCFM536, pcDNA3.1, pcDNA3, pME, pGEX and the like can be used. Escherichia coli plasmid can be mentioned. Commercially available vectors can be used, for example, pQE70, pQE60, pQE-9 (Qiagen), pBluescriptII KS, ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), pET-11a (Novagen). ) And so on. The skeletal vector contains a replication origin, a selectable marker, a promoter, and may optionally include an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like.

The collagen nucleic acid-containing vector can contain nucleic acids other than collagen nucleic acid. For example, a fusion protein can be obtained by binding another protein to a collagen protein or the like, and the collagen nucleic acid-containing vector is a nucleic acid encoding a protein to be bound to this collagen (hereinafter, may be referred to as a binding protein). Can be included.
The binding protein can be bound to the N-terminal or C-terminal 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, but is not limited to, preferably obtained from Escherichia coli having a specific gene mutation described later. That is, those introduced into Escherichia coli having a specific gene mutation and recovered from the Escherichia coli are preferable. This is because it is possible to obtain a vector containing a collagen nucleic acid that has not been deleted, inserted, or inhibited by a restriction enzyme.

[3] Vector-containing host cell The vector-containing host cell of the present invention contains the vector of the present invention. The host is not particularly limited and may include Escherichia coli, actinomycetes, yeast, filamentous fungi, or animal cells, but is preferably Escherichia coli or animal cells, and more preferably Escherichia coli having a specific gene mutation. Or it is an animal cell. This is because by using Escherichia coli having a specific gene mutation, it is possible to obtain a vector containing collagen nucleic acid that is not inhibited by deletion, insertion, or restriction enzyme. In addition, collagen can be produced by using animal cells. Furthermore, it is possible to analyze the formation process of higher-order structures of collagen.

<< Escherichia coli with gene mutation >>
Escherichia coli having a gene mutation containing the vector of the present invention is preferably, but not limited to, mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara). , leu) 7697, galU, galK, rpsL (str ^r ), nupG, galE, hsdS, and E. coli with a genetic mutation selected from the group consisting of two or more combinations thereof. The combination of gene mutations is not limited, but is preferably mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, araD139, Δ (ara, leu) 7679, galU, galK, rpsL (str). ^{A gene mutation selected from the group consisting of r} ), nupG, and two or more combinations thereof, more preferably mcrA, Δ (mrr-hsdRMS-mcrBC), recA, hsdS and two or more of them. It is a gene mutation selected from the group consisting of combinations, and more preferably a gene mutation selected from the group consisting of mcrA, Δ (mrr-hsdRMS-mcrBC), recA and two or more combinations thereof. It is preferably a gene mutation selected from the group consisting of mcrA, Δ (mrr-hsdRMS-mcrBC), and combinations thereof. More specifically, Escherichia coli having a gene mutation includes DH10B, TOP10F', MC1061, XL1-Blue MRF', AG1, BL21 (DE3), DB3.1, DH1, DH5α, DH5α Turbo, DH12S, DM1, E. coli. Clone (r) 5 alpha, E.I. Clone (r) 10G, E.I. Clone (r) 10GF', ER2267, HB101, HMS174 (DE3), High-Control (tm) BL21 (DE3), High-Control (tm) 10G, IJ1126, JM108, JM109, Mach1, MC1061, MFDpir, (Tm) C41 (DE3), OverExpress (tm) C43 (DE3), Rosetta (DE3), SOLR, SS320, STBL2, STBL3, STBL4, SURE, SURE2, TG1, TOP10, XL2-Blue, XL2-BlueMRF', and XL10-Gold can be mentioned, but DH10B, TOP10F', TOP10, STBL2, and STBL4 are particularly preferable.

For the introduction of the vector into Escherichia coli, the methods usually used in this field can be used without limitation. For example, utilization of competent cells treated with calcium chloride, a protoplast method, a calcium phosphate method, a lipofection method, an electroporation method, or the like can be used.

[4] Method for producing vector-containing Escherichia coli and method for producing vector In the method for producing vector-containing Escherichia coli of the present invention, the vector of the present invention is used as mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, Introduced into E. coli having a gene mutation selected from the group consisting of endA1, araD139, Δ (ara, leu) 7679, galU, galK, rpsL (str ^r ), nupG, galE, hsdS, and two or more combinations thereof. It is characterized by doing.
Further, in the method for producing the vector of the present invention, the vector of the present invention is used as mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7697, galU, galK. , RpsL (str ^r ), nupG, galE, hsdS and a step of introducing into Escherichia coli having a gene mutation selected from the group consisting of two or more combinations thereof, and a step of recovering the vector. To do.

The method for producing vector-containing Escherichia coli of the present invention, or "nucleic acid encoding a procollagen protein, collagen protein, or a mutant thereof, or a fragment thereof", "vector", and "mcrA, Δ ( mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7679, galU, galK, rpsL (str ^r ), nupG, galE, hsdS, and two or more of them. "Escherichia coli having a gene mutation selected from the group consisting of combinations" and the like are described in the above-mentioned "[1] fusion protein", "[2] nucleic acid and vector", and "[3] vector-containing host cell". Can be used.
Further, as a method for recovering a vector in the method for producing a vector of the present invention, a method known in the art can be used. For example, a collagen nucleic acid-containing vector into which the vector has been introduced is cultured in an LB medium or the like. Grow E. coli. The grown Escherichia coli can be recovered, and the vector DNA can be recovered by using, for example, an alkaline SDS method, a boiling method, an ethidium / cesium chloride density gradient centrifugation method, a glass bead purification method, or a modification thereof.

[5] Method for producing collagen The method for producing collagen of the present invention is as follows: (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》
The 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, but for example, mammals (for example, cows, pigs, ostriches, goats, mice, etc.). Rats, guinea pigs, or monkeys), birds (eg chickens, geese, ducks, or ostriches), reptiles (eg crocodile), amphibians (eg frogs), fish (eg butterfly sharks, terapias, ties, sharks, sharks, Cells derived from sardines, tuna, ducks, goldfish, cod, frogs, or carp), or invertebrates (eg, jellyfish) can be used.
The method for introducing the vector into animal cells is not particularly limited, and a method known in the art can be used, and for example, an electroporation method can be used.

<< Secretion process >>
The method for secreting collagen from the animal cells into which the vector has been introduced is not particularly limited, but can be achieved by culturing the animal cells in a medium.
The culture temperature is also not particularly limited, but can be cultured at, for example, 30 to 42, preferably 35 to 39 ° C. In addition, extracellular secretion can be optimized by adjusting the protein concentration in animal cells. In addition, it is preferable that cells form multiple layers for secretion.

[6] Fluorescence or luminescence detection method for collagen The fluorescence or luminescence detection method for collagen 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. Includes a step of detecting fluorescence or luminescence in.
The "vector" used in the method for detecting fluorescence or luminescence of collagen of the present invention is described in the sections of "[1] Fusion protein", "[2] Nucleic acid and vector", and "[3] Vector-containing host cell". A "vector" can be used.

《Animal cells》
The animal cell used in the method for producing collagen of the present invention is not particularly limited as long as it is a cell capable of expressing collagen, but the animal cell according to the above-mentioned "[5] Method for producing collagen". Can be mentioned.

<< Detection process >>
Fluorescence or luminescence from animal cells can also be detected by a method known in the art, but can be detected by using, for example, a fluorescence microscope, a spectrophotometer, a confocal fluorescence microscope, or the like.

[7] Method for Screening for Treatment or Preventive Substances for Collagen-Related Diseases The method for screening for treatment or preventive substances for collagen-related diseases of the present invention is a step of contacting a collagen-related disease cell expressing the fusion protein with a test substance. And the step of analyzing the dynamics of the fusion protein.

《Collagen-related diseases》
Collagen-related diseases are not particularly limited as long as collagen is considered to be related to the cause or symptom of the disease, but are not particularly limited, but are organ / tissue fibers such as liver fibrosis, pulmonary fibrosis, and renal fibrosis. Disease, collagen disease, or indirect rheumatism can be mentioned.
The collagen-related disease cells used in the screening method of the present invention may be cells isolated from the above-mentioned collagen-related disease patients, or cells prepared from model animals. Examples of cells isolated from collagen-related disease patients include cells that secrete collagen, and specific examples thereof include hepatic stellate cells, fibroblasts, and chondrocytes isolated from patients with hepatic fibrosis. be able to. In addition, examples of cells produced from model animals include hepatic stellate cells and lung fibroblasts.
Furthermore, normal collagen-secreting cells are activated by, for example, TGF-β and used as model cells for hepatic fibrosis, pulmonary fibrosis, renal fibrosis, collagen disease, or indirect rheumatism, and used as screening cells for each drug. be able to.

《Test substance》
The test substance is not limited to substances that may be related to the dynamics of collagen, or as long as it contains such substances, but for example, various known compounds (peptides) registered in the chemical file. Included), compounds obtained by combinatorial chemistry techniques (Terrett, NK et al., Tetrahedron, 51, 8135-8137, 1995), or phage display methods (Felici, F. et al., J. Mol. A random peptide group or a low molecular weight compound prepared by applying Biol., 222, 301-310, 1991) or the like can be used. Further, a culture supernatant of a microorganism, a culture supernatant of a cell, a body fluid in a living body, a natural component derived from a plant or a marine organism, an animal tissue extract, or the like can also be used as a test substance for screening.

(Contact process)
The contact step in the screening method of the present invention is a step of contacting the collagen-related disease cells expressing the fusion protein with the test substance.
The concentration of the test substance can also be determined as appropriate, but since it is considered that the test substance has an optimum concentration that affects the dynamics of collagen, it is preferable to dilute it in several steps for the test.

(Fusion protein dynamic analysis process)
The dynamic analysis step in the present invention is a step of analyzing the dynamics of the collagen fusion protein. Analysis of collagen dynamics includes, for example, analysis of collagen fusion protein expression level, analysis of collagen fusion protein extracellular secretion, analysis of collagen fusion protein intracellular transport, or analysis of collagen fusion protein processing. Can be done.
Dynamic analysis can basically be performed by comparing the test material with cells that are not in contact with the test substance. That is, when the expression of the collagen fusion protein is increased or decreased as compared with the cells not contacted with the test substance, the test substance can be judged as a candidate for a therapeutic drug for collagen-related diseases. In addition, when the collagen fusion protein is secreted extracellularly as compared with cells that are not in contact 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 to normal as compared with the cells not contacted with the test substance, it can be judged that the test substance is effective for collagen-related diseases. In addition, when the processing of the collagen fusion protein is restored to normal as compared with the cells not contacted with the test substance, it can be judged that the test substance is effective for collagen-related diseases.

For example, as shown in Examples, when EGFP is inserted into a collagen protein and a collagen fusion protein in which m-Cherry is inserted into a C-proprotein is introduced into a hepatic fibrosis hepatic stellate cell, a candidate substance is used. In the absence of contact, yellow fluorescence consisting of EGFP and m-Cherry fluorescence is observed intracellularly due to abnormal processing or secretion of procollagen protein. On the other hand, when a candidate substance having a therapeutic effect is brought into contact, the C-proprotein in which m-Cherry is inserted is cleaved, the collagen protein in which EGFP is inserted is normally secreted extracellularly, and green fluorescence is observed. Will be done. The extracellular secretion of collagen protein is also observed in normal hepatic stellate cells, and a candidate substance exhibiting such dynamics can be judged to be effective for hepatic fibrosis.
However, the judgment of the dynamic analysis used for screening is not limited to these, and it can be judged that it is effective for collagen-related diseases when it shows different dynamics as compared with cells not contacted with the test substance. it can.

Hereinafter, the present invention will be specifically described with reference to Examples, but these do not limit the scope of the present invention.

<< Comparative Example 1 >>
In this comparative example, we tried 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-terminal. GFP was inserted at the BamH1 site of the collagen protein and mCherry was inserted at the EcoR1 site of the C-proprotein.
A schematic diagram of the vector is shown in FIG. The base 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 the BamH1 site inside the human preprocollagen1α1 cDNA. At this time, in protein translation, adjustment was made so that the collagen protein and the EGFP protein were connected. Next, a DNA fragment in which EcoR1 site was added to both ends of the mCherry cDNA was prepared, and this was inserted into the EcoR1 site of the human preprocollagen1α1-EGFP fusion cDNA. Also at this time, in protein translation, adjustment was made so that the collagen protein and the mCherry protein were connected, and the base sequence was confirmed at each step of the operation.

The method for producing competent cells (transformation-accepting cells) of XL-1 blue was in accordance with "Method for producing gene library" (edited by Hiroshi Nojima, Yodosha). The cryopreserved competent cells were thawed, and immediately 100 ng of plasmid DNA was added and mixed. After allowing to stand on ice for 30 minutes, it was warmed at 42 ° C. for 1 minute and 30 seconds, and immediately ice-cooled. 1 mL of medium was added thereto, and 100 μL was spread on an LB plate supplemented with ampicillin. The colonies that appeared were used after being kept warm at 37 ° C. overnight.
The DNA contained in the obtained vector was shorter than the target length, longer than the target length, and could not be cleaved by NotI or SmaI. FIG. 2A shows those longer than the target length, those that could not be cut by NotI, and SmaI.

<< Comparative Example 2 >>
In this comparative example, an attempt was made to construct a vector having a nucleic acid in which EGFP was bound to the N-terminal of type 1 collagen and mCherry was bound to the C-terminal. EGFP was inserted at the Kpn1 site of the N-proprotein and mCherry was inserted at the EcoR1 site of the C-proprotein.
A schematic diagram of the vector is shown in FIG. The base 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 this was inserted into the KpnI site inside the human preprocollagen1α1 cDNA. At this time, in protein translation, adjustment was made so that the collagen protein and the EGFP protein were connected. Next, a DNA fragment in which EcoR1 site was added to both ends of the mCherry cDNA was prepared, and this was inserted into the EcoR1 site of the human preprocollagen1α1-EGFP fusion cDNA. Also at this time, in protein translation, adjustment was made so that the collagen protein and the mCherry protein were connected, and the base sequence was confirmed at each step of the operation.

The method for producing competent cells (transformation-accepting cells) of XL-1 blue was in accordance with "Method for producing gene library" (edited by Hiroshi Nojima, Yodosha). The cryopreserved competent cells were thawed, and immediately 100 ng of plasmid DNA was added and mixed. After allowing to stand on ice for 30 minutes, it was warmed at 42 ° C. for 1 minute and 30 seconds, and immediately ice-cooled. 1 mL of medium was added thereto, and 100 μL was spread on an LB plate supplemented with ampicillin. The colonies that appeared were used after being kept warm at 37 ° C. overnight.
The DNA contained in the obtained vector was shorter than the target length, longer than the target length, and could not be cleaved by NotI or SmaI. FIG. 2B shows a photograph cut by EcoRI, but all clones could not be cut by 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 sequenced and confirmed to contain 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 nucleic acid of the desired length. FIG. 2B shows a photograph cut by EcoRI, and all clones were able to be cut by EcoRI. On the other hand, when XL-1 blue was used as a host, a normal clone that could be cleaved by EcoRI could not be obtained.

<< Example 3 >>
In this example, the fluorescence of collagen secreted extracellularly was observed. The vector obtained in Example 1 was introduced into NIH3T3 cells. Long-term culture for 1 month or longer was carried out while changing the medium every 2 days to obtain a cell population composed of multiple layers. The fluorescence signal of EGFP was observed with a fluorescence microscope.
As shown in FIG. 4, when EGFP is inserted on the N-terminal side of collagen protein, the collagen protein forms a triple helix structure and accumulates extracellularly (green fluorescence of EGFP). I was able to confirm.

<< Example 4 >>
In this example, the fluorescence of collagen in the cells was observed. The vector obtained in Example 1 was introduced into NIH3T3 cells. Fluorescence signals of EGFP and mCherry were observed after 48 hours with a fluorescence microscope.
As shown in FIG. 5, EGFP is inserted into the N-terminal side of the collagen protein, and mCherry is inserted into the C-terminal side of the C-proprotein, so that the procollagen is yellow and the processed collagen is green. It was confirmed that the fluorescent signal was present in the cytoplasm. The collagen protein was also secreted extracellularly, but unlike Example 3, the amount of collagen protein accumulated was small due to the short culture period, and fluorescence of EGFP green was not observed.

<< Example 5 >>
In this example, the vector of FIG. 3 obtained by the same procedure as in Example 2 was introduced into mouse NIH3T3 cells, and fluorescence was detected with a confocal fluorescence microscope. The vector of FIG. 3 was introduced into NIH3T3 cells. Fluorescence signals of EGFP and mCherry were observed after 48 hours with a fluorescence microscope.
As shown in FIG. 6, EGFP is inserted into N-protein and mCherry is inserted into C-protein, so that pro-collagen immediately after synthesis is cleaved by a yellow signal, and N-proprotein cleaved by processing is The green fluorescent signal confirmed that it was present in the cytoplasm. Although N-proprotein was also secreted extracellularly, fluorescence of EGFP green was not observed because it was diffused in the medium.

<< Comparative Example 3 >>
MCherry was inserted into the restriction enzyme AccIII site present in the C proprotein of the preprocollagen cDNA. The AccIII site is located at the site corresponding to 27 amino acids from the N-terminal of C-proprotein (Fig. 12A). Collagen fibrosis (triple helix structure construction) is inhibited by inserting mCherry from the N-terminal of C-proprotein to the N-terminal side from 30 amino acids. That is, the collagen protein was accumulated in the ER-Golgi apparatus and was not secreted extracellularly (FIGS. 12B and C).
In addition, a fusion protein in which EGFP was bound to the C-terminal of the collagen protein was prepared (FIG. 12A). This fusion protein also inhibits collagen fibrosis (triplex helix structure construction). That is, the collagen protein was localized in the cytoplasm and was not secreted extracellularly (Fig. 12B).

<< Example 6 >>
In this example, a screening system for a drug for liver fibrosis was constructed.
(1) Introduction of vector into hepatic stellate cells The vector obtained in Example 1 was isolated from rats with hepatic fibrosis and constantly activated hepatic stellate cells (5H cells) causing hepatic fibrosis and activation. Hepatic stellate cells (2G92 cells) were transfected. Specifically, it was introduced into cells using a transfection reagent (Fugne6). As shown in FIG. 7, green fluorescence was observed in unactivated hepatic stellate cells, indicating normal processing and secretion of collagen proteins (FIG. 7A). However, in constantly activated hepatic stellate cells (5H cells), only a yellow signal indicating abnormal collagen processing and secretion was observed, and no green fluorescence indicating normal collagen secretion was observed (Fig. 7C). ..

(2) Activation of normal hepatic stellate cells by TGF-β Furthermore, cells used for screening for drugs for hepatic fibrosis were prepared using 2G92 cells as follows. Unactivated hepatic stellate cells 2G92 were grown to semiconfluent on two dishes. One dish was added with TGF-β, which activates hepatic stellate cells at a final concentration of 1 ng / mL. As a result of detecting collagen protein processing and secretion with a confocal microscope 8 hours after the addition, only a yellow signal indicating abnormal collagen processing and secretion was observed in the dish to which TGF-β was added. , It was confirmed that the cells were transformed into activated hepatic stellate cells (activated 2G92 cells) that cause liver fibrosis (Fig. 7B).

<< Example 7 >>
In this example, a screening system for a candidate substance for a therapeutic agent for hepatic fibrosis using the activated hepatic stellate cells (activated 2G92 cells) and the stationary activated hepatic stellate cells (5H cells) according to Example 6. Was built.
SB431542 (0.5 μM) or DMSO solvent, which has a therapeutic effect on liver fibrosis, was added to 2G92 cells grown to semiconfluent and cultured for 1 hour. Then, TGF-β was added at a final concentration of 1 ng / mL, and the cells were cultured for 8 hours. As a result, the collagen protein was normally processed and secreted in the 2G92 cells supplemented with SB431542 (Fig. 8), whereas the 2G92 cells supplemented with DMSO had a yellow signal indicating abnormal processing and secretion of the collagen protein. Only seen (not shown). Activated hepatic stellate cells supplemented with SB431542 have normally altered collagen secretion. Therefore, 2G92 cells expressing collagen protein by the vector of the present invention were used as candidate substances for the treatment of liver fibrosis. It turns out that it can be screened.
In addition, SB431542 (0.5 μM) or solvent DMSO, which has a therapeutic effect on liver fibrosis, was added to 5H cells grown to semi-confluent, and the cells were cultured for 1 hour or longer. As a result, the collagen protein was normally processed and secreted in the 5H cells supplemented with SB431542 (Fig. 9), whereas the 5H cells supplemented with DMSO showed changes (normalization) in the processing and secretion of the collagen protein. Not seen (no figure). Constantly activated hepatic stellate cells supplemented with SB431542 have normally altered collagen secretion, and therefore, 5H cells expressing collagen protein by the vector of the present invention are used as candidates for therapeutic agents for liver fibrosis. It turns out that the substance can be screened.

<< Example 8 >>
In this example, pulmonary fibroblasts and renal tubular epithelial cells were used to prepare cells used for screening a therapeutic agent for pulmonary fibrosis and cells used for screening a therapeutic agent for renal fibrosis.
Example 6 (1), except that pulmonary fibroblasts (TIG-3-20 cells and A549 cells) and renal tubule epithelial cells (MDCK cells) were used instead of hepatic stellate cells. By repeating the operation, TIG-3-20 cells, A549 cells, and MDCK cells expressing the collagen protein by the vector of the present invention were prepared. As shown in FIG. 10, TIG-3-20 cells (FIG. 10A), A549 cells (FIG. 10C), and MDCK cells (FIG. 10D) were able to visualize collagen.
For TIG-3-20 cells, TGF-β was used to activate the cells. Except for the fact that lung fibroblasts (TIG-3-20 cells) were used instead of hepatic stellate cells, the operation of Example 6 (2) was repeated to activate TIG-3-20 cells. It was. As shown in FIG. 10B, it can be confirmed that the TIG-3-20 cells can be used as model cells for pulmonary fibrosis by observing only a yellow signal indicating abnormal collagen processing and secretion. It was. These cells can be used to screen pulmonary fibrosis drugs.

The vector of the present invention can be effectively used for analysis of the collagen formation process. This analysis of the collagen formation process can be used as an effective means for elucidating the causes of diseases caused by molecular abnormalities of collagen and developing therapeutic methods for them. In addition, the screening method of the present invention can be used for screening a drug for treating or preventing a collagen-related disease.
Although the present invention has been described above according to a specific aspect, modifications and improvements that are obvious to those skilled in the art are included in the scope of the present invention.

Claims (13)

A fusion protein of a collagen-related protein, which is a preprocollagen protein, a procollagen protein, a collagen protein, or a variant thereof, and a fluorescent protein or a photoprotein.
Insertion of the fluorescent protein or photoprotein from the C-terminal to the N-terminal side of the collagen protein from 30 amino acids,
Insertion of fluorescent protein or photoprotein from N-terminal of C-proprotein to C-terminal side from 30 amino acids, and insertion of fluorescent protein or photoprotein into N-proprotein,
A fusion protein that has an insertion of one or more fluorescent or photoproteins selected from the group consisting of and can be secreted extracellularly when expressed in animal cells. The collagen-related protein is a pre-procollagen protein or a pro-collagen protein, and the fluorescent protein or a luminescent protein is
(A) Is it inserted from the C-terminal of the collagen protein to the N-terminal side from the 30 amino acids, and from the N-terminal of the C-proprotein to the C-terminal side from the 30 amino acids?
(B) Is it inserted from the C-terminal of the collagen protein to the N-terminal side from 30 amino acids, and is inserted into the N-proprotein?
(C) It is inserted from the N-terminal of C-proprotein to the C-terminal side from 30 amino acids, and is inserted into N-proprotein.
The fusion protein according to claim 1. A step of contacting a collagen-related disease cell (excluding cells in a human body) expressing the fusion protein according to claim 1 or 2 with a test substance, and a step of analyzing the dynamics of the fusion protein.
A method for screening a therapeutic or prophylactic substance for collagen-related diseases including. A nucleic acid encoding the fusion protein according to claim 1 or 2. A vector containing the nucleic acid according to claim 4. A vector-containing host cell comprising the vector according to claim 5. The host cells are mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7679, galU, galK, rpsL (str ^r ), nupG, galE, The vector-containing host cell according to claim 6, which is an Escherichia coli having a gene mutation selected from the group consisting of hsdS and a combination of two or more thereof. The E. coli is DH10B, TOP10F', MC1061, XL1-Blue MRF', AG1, BL21 (DE3), DB3.1, DH1, DH5α, DH5α Turbo, DH12S, DM1, E. coli. Clone (r) 5 alpha, E.I. Clone (r) 10G, E.I. Clone (r) 10GF', ER2267, HB101, HMS174 (DE3), High-Control (tm) BL21 (DE3), High-Control (tm) 10G, IJ1126, JM108, JM109, Mach1, MC1061, MFDpir, (Tm) C41 (DE3), OverExpress (tm) C43 (DE3), Rosetta (DE3), SOLR, SS320, STBL2, STBL3, STBL4, SURE, SURE2, TG1, TOP10, XL2-Blue, XL2-BlueMRF', and The vector-containing host cell according to claim 7, which is selected from the group consisting of XL10-Gold. A vector obtained from the vector-containing host cell according to claim 7 or 8. The vector according to claim 5 is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7679, galU, galK, rpsL (str ^r ), nupG. , GalE, hsdS, and a method for producing vector-containing Escherichia coli, which comprises introducing into Escherichia coli having a gene mutation selected from the group consisting of two or more combinations thereof. The vector according to claim 6 is mcrA, Δ (mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 7679, galU, galK, rpsL (str ^r ), nupG. , GalE, hsdS, and a method for producing a vector, which comprises a step of introducing into Escherichia coli having a gene mutation selected from the group consisting of two or more combinations thereof, and a step of recovering the vector. (1) A step of introducing the vector according to claim 5 into an animal cell (excluding cells in a human body) , and (2) a step of secreting collagen from the animal cell into which the vector has been introduced.
A method for producing collagen, including. (1) A step of introducing the vector according to claim 5 into an animal cell (excluding cells in a human body) , and (2) a step of detecting fluorescence or luminescence in the animal cell into which the vector has been introduced.
A method for detecting fluorescence or luminescence of collagen, which comprises.

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