Method for biosynthesizing type IV collagen, a structural material for the human body

Abstract

A method for biosynthesizing the human structural material type IV collagen is provided. Provided herein is a recombinant collagen comprising a sequence such as that shown in SEQ ID NO:1. The recombinant collagen has good cell adhesion activity. The method herein utilizes genetic engineering technology to produce recombinant collagen, overcoming the deficiencies of the prior art.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from a Chinese patent application filed on October 25, 2023, bearing application number 202311391711.1, and entitled "Method for biosynthesizing type IV collagen, a human body structural material."

[0002] The present invention belongs to the field of biomedicine, and relates to recombinant type IV humanized collagen and its preparation method and use. [Background technology]

[0003] Collagen (COL), abbreviated as "collagen" in Chinese, is a helical fibrous functional protein composed of three peptide chains. It is also a major component of the extracellular matrix, abundant in abundance and with a wide range of applications. Collagen accounts for 25% to 30% of the total protein content in the human body and is primarily found in skin, tendons, and bones, where it plays an important role in protecting and connecting various tissues and performs important physiological functions in the body. Type IV collagen, along with laminin, is the main protein that constitutes basement membranes. It consists of a trimer with a helical structure composed of three α chains. The subunits that make up the trimer vary depending on the tissue, but in many tissues, such as the liver, the trimer is composed of two α1 chains and one α2 chain. Type IV collagen molecules have a unique structure at both ends of the TH domain that forms the helical structure: the N-terminal structure is called the 7S domain, and the C-terminal structure is called the NC1 domain.

[0004] Collagen has good biocompatibility, degradability, and low antigenicity, and its unique biostructure has made it the focus of research in recent years and it has been widely applied in many fields such as biomedicine, cosmetics, health foods, and food.

[0005] The human body contains 28 different types of collagen, which can be classified into two types: fibrous and non-fibrous, depending on whether their structure is fibrous or not. Fibrous collagen mainly functions as a cell scaffold, fixing the position of cells and exerting an anchoring effect, as well as providing tensile strength and rigidity to tissues. Non-fibrous collagen is further subdivided into basal collagen, short-chain collagen, transmembrane collagen, etc., each of which has different functions.

[0006] Conventional collagen production methods involve purifying animal tissues using acid, alkali, or enzymatic hydrolysis to extract collagen derivatives. However, the collagen obtained by these methods has already lost its original biological activity, is poorly water-soluble, does not easily bind to the human body, and is susceptible to viral infection and sensitization, making it unable to exert its true function. Some research institutions have used conventional recombinant expression methods to express human collagen in vitro, but this method is expensive, requires a long production cycle, and does not allow for large-scale production.

[0007] Therefore, there is an urgent need in the market for collagen materials that have excellent biomaterial properties, whose amino acid sequences are highly homologous to those of the human body, and that can be produced in large quantities in an industrialized system. Summary of the Invention

[0008] The inventors conducted a large-scale screening of human type IV collagen for functional domains and discovered 11 recombinant collagens. These recombinant collagens can be expressed in E. coli and purified. Furthermore, the inventors discovered that these recombinant collagens have high yields, high purity of the target protein after precision purification, and higher activity than the positive control (bovine type I collagen) in cell adhesion assays.

[0009] In one aspect, the present invention provides a recombinant collagen comprising one or more repeat units, the repeat units being linked directly or via a linker, and the repeat units comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (Gakgdkgskgevgfpglagspgipgskgeq) or 28 (Gptgpagqkgepgsdgipgsagekgepglp) or a variant thereof, wherein the variant is either (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence.

[0010] In one embodiment, the plurality of repeat units is 2 to 50 repeat units, e.g., 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 4 to 10, or 6 to 10 repeat units. For example, the number of repeat units is 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, or any range therebetween.

[0011] In one embodiment, the linker comprises one or more amino acid residues, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acid residues.

[0012] In one embodiment, the mutation is selected from a substitution, addition, insertion or deletion.

[0013] In one embodiment, the substitutions are conservative amino acid substitutions.

[0014] In one embodiment, the recombinant collagen is recombinant human type IV collagen or recombinant humanized type IV collagen.

[0015] In one embodiment, the recombinant collagen has cell adhesion activity.

[0016] In one embodiment, the variant of SEQ ID NO:1 includes mutations such as the addition of Gfpgfp (SEQ ID NO:34) to the N-terminus of the amino acid sequence of SEQ ID NO:1 or truncating the fragment to the N-terminus of SEQ ID NO:34 by 1 to 5 amino acid residues in length, and / or the addition of GFMGPPGPQGQPGLP (SEQ ID NO:35) to the C-terminus of the amino acid sequence of SEQ ID NO:1 or truncating the fragment to the C-terminus of SEQ ID NO:35 by 1 to 14 amino acid residues in length, or truncating the C-terminus of the amino acid sequence of SEQ ID NO:1 by 1 to 5 amino acid residues.

[0017] In one embodiment, the variant of SEQ ID NO:28 includes a mutation that adds Glpgtp (SEQ ID NO:36) to the N-terminus of the amino acid sequence of SEQ ID NO:28 or truncates it to an N-terminal fragment of 1 to 5 amino acid residues in length.

[0018] In one embodiment, the variants of SEQ ID NO: 1 are SEQ ID NOs: 4 (Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq), 7 (GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP), 10 (Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq), 13 (Gfpgfpgakgdkgskgevgfpglagspgipg skgeqgfm), 16(Gfpgfpgakgdkgskgevgfpglagspgipgskgeq), 19(Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp), 22(Gakgdkgskgevgfpglagspgipgskgeqgfm) or 31(Gfpgfpgakgdkgskgevgfpglagspgipgsk).

[0019] In one embodiment, the variant of SEQ ID NO:28 is SEQ ID NO:25 (Glpgtpgptgpagqkgepgsdgipgsagekgepglp).

[0020] In one embodiment, the recombinant collagen comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or a variant thereof, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence.

[0021] In one embodiment, the mutation is selected from a substitution, addition, insertion or deletion, hi one embodiment, the substitution is a conservative amino acid substitution.

[0022] In another aspect, a nucleic acid is provided that encodes the recombinant collagen described herein. In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. In one embodiment, the nucleotide sequence is codon-optimized for expression in a eukaryotic or prokaryotic host cell, e.g., yeast or E. coli. In one embodiment, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33.

[0023] In another aspect, a vector is provided comprising the nucleic acid described herein. In one embodiment, the vector comprises an expression control element, a purification tag nucleotide, and / or a leader sequence nucleotide operably linked to the nucleic acid. In one embodiment, the expression control element is selected from a promoter, a terminator, or an enhancer. In one embodiment, the purification tag is selected from a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag. In one embodiment, the vector is an expression vector or a cloning vector, preferably pET-28a(+). pET-28a(+) may comprise an N-terminal His tag, a Thrombin tag, and a T7 protein tag, as well as a C-terminal His tag. The recombinant collagen herein may comprise an enzyme cleavage site at the N-terminus to facilitate purification.

[0024] In another aspect, a host cell is provided comprising a nucleic acid or vector described herein. In one embodiment, the host cell is a eukaryotic cell or a prokaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell, and in one embodiment, the prokaryotic cell is an E. coli cell, such as E. coli BL21.

[0025] In another aspect, a composition is provided comprising one or more of the recombinant collagen, nucleic acid, vector, and host cell described herein. In one embodiment, the composition is a kit. In one embodiment, the composition is one or more of a bio-covering material, a human biomimetic material, a cosmetic cosmetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, an obstetric and gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material, a vascular repair and regeneration material, a 3D printing artificial organ biomaterial, a cosmetic ingredient, a pharmaceutical auxiliary material, and a food additive. In one embodiment, the composition is a surface composition, an injectable composition, or an oral composition. In one embodiment, the composition is in the form of a solution, a lyophilized powder, a gel, a sponge, or a fiber.

[0026] In another aspect, there is provided a use of the recombinant collagen, nucleic acid, vector, host cell and / or composition herein in one or more of bio-dressing materials, human biomimetic materials, cosmetic and reconstructive materials, organoid culture materials, cardiovascular stents, coating materials, tissue injection and filling materials, ophthalmic materials, gynecological and obstetric biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, medicinal supplements and food additives.

[0027] In another aspect, there is provided a method for promoting cell adhesion, comprising contacting a cell with the recombinant collagen, nucleic acid, vector, host cell, and / or composition herein. In one embodiment, the cell is an animal cell. The animal cell may be a mammalian cell or a human cell. There is also provided a use of the recombinant collagen, nucleic acid, vector, host cell, and / or composition herein in the manufacture of a kit, wherein the agent is for promoting cell adhesion.

[0028] In another aspect, the cosmetic method comprises administering to a subject a recombinant collagen as described herein, preferably the administration is topical, oral or injectable, and preferably the subject is a human.

[0029] In another aspect, (1) culturing a host cell described herein under suitable culture conditions; (2) harvesting the host cells and / or medium containing the recombinant collagen; and (3) purifying the recombinant collagen.

[0030] In one embodiment, the host cell is an E. coli cell, preferably an E. coli BL21(DE3) cell.

[0031] In one embodiment, step (1) comprises culturing E. coli cells in LB medium and inducing expression with IPTG.

[0032] In one embodiment, step (2) comprises harvesting the E. coli cells, resuspending them in an equilibrium working solution, homogenizing the E. coli cells, preferably by high-pressure homogenization, and separating the supernatant. In one embodiment, the equilibrium working solution comprises 100-500 mM sodium chloride, 10-50 mM Tris, and 10-50 mM imidazole, and has a pH of 7-9.

[0033] In one embodiment, step (3) comprises crude purification, enzymatic cleavage, precision purification, and / or reversed-phase nickel column purification.

[0034] In one embodiment, the crude purification includes purifying the supernatant on a Ni-agarose gel column to obtain an eluate containing the target protein, the eluate containing 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 100 to 500 mM imidazole, preferably at a pH of 7 to 9.

[0035] In one embodiment, the micropurification comprises gradient elution of an eluent containing the target protein using a strong anion exchange chromatography column. In one embodiment, the gradient elution comprises eluting with 0-15% solution B for 1-5 minutes and then retaining for three column volumes, eluting with 15-30% solution B for 1-5 minutes and then retaining for three column volumes, eluting with 30-50% solution B for 1-5 minutes and then retaining for three column volumes, and eluting with 50-100% solution B for 1-5 minutes and then retaining for three column volumes, where solution B comprises 10-50 mM Tris, 0.5-5 M sodium chloride, and a pH of 7-9. In one embodiment, step (3) comprises purifying the recombinant collagen on a purification column, such as a nickel column, and / or cleaving the recombinant collagen with a collagen tool enzyme.

[0036] Advantages of the present invention include the following: 1. The recombinant collagen of the present invention is derived from human type IV collagen and is a recombinant humanized type IV collagen. 2. The recombinant collagen of the present invention is suitable for production in E. coli and can be separated and purified. 3. The recombinant collagen of the present invention has a higher yield and is suitable for subsequent purification (purification by Ni column or strong anion column). 4. The recombinant collagen of the present invention has cell adhesion activity. The recombinant collagen of the present invention (eg, C4P7Ch) has higher cell adhesion activity than the positive control. [Brief explanation of the drawings]

[0037] [Figure 1] The results of electrophoretic detection of C4P7Ca are shown. [Figure 2] The results of electrophoretic detection of C4P7Cb are shown. [Figure 3] The results of electrophoretic detection of C4P7Cc are shown. [Figure 4] The results of electrophoretic detection of C4P7Cd are shown. [Figure 5] The results of electrophoretic detection of C4P7Ce are shown. [Figure 6] The results of electrophoretic detection of C4P7Cf are shown. [Figure 7] The results of electrophoretic detection of C4P7Cg are shown. [Figure 8] The results of electrophoretic detection of C4P7Ch are shown. [Figure 9] The results of electrophoretic detection of C4P7Ea are shown. [Figure 10] The results of electrophoretic detection of C4P7Eb are shown. [Figure 11] The results of electrophoretic detection of C4P7Ec are shown. [Figure 12] 1 shows the effect of collagen C4P7Ch on cell adhesion. [Figure 13] 1 shows the effect of collagen C4P7Cf on cell adhesion. DETAILED DESCRIPTION OF THE INVENTION

[0038] As used herein, "recombinant collagen" refers to an amino acid sequence or a fragment thereof encoded by a specific gene that has been designed and modified and produced by DNA recombinant technology, or a combination of fragments of such functional amino acid sequences. The gene coding sequence or amino acid sequence of a recombinant collagen may have low homology to the gene coding sequence or amino acid sequence of a human collagen. Recombinant humanized collagen refers to a fragment of a full-length or partial amino acid sequence encoded by a specific human collagen gene produced by DNA recombinant technology, or a combination of functional fragments containing human collagen. As used herein, recombinant collagen comprises one or more repeat units. The repeat unit may be derived from human type IV collagen. Thus, recombinant collagen may be recombinant type IV humanized collagen. Multiple repeat units may be connected by a linker, which may be a natural amino acid residue in the human type IV collagen repeat unit, e.g., 1 to 50 amino acid residues. The repeat unit may be SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, or 33. The recombinant collagen may be SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32.

[0039] As used herein, the term "variant" refers to a recombinant collagen that has cell adhesion activity and contains changes / mutations (i.e., substitutions, additions, insertions, and / or deletions) at one or more positions. Substitutions refer to replacing an amino acid occupying a certain position with a different amino acid; deletions refer to removing an amino acid occupying a certain position; and insertions refer to adding an amino acid adjacent to and immediately following the amino acid occupying a certain position. Additions refer to adding one or more amino acid residues to the C-terminus and / or N-terminus of the amino acid sequence. Substitutions may be conservative substitutions. A variant of a repeat unit may be a sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, or 33 after one or more amino acid residues have been changed or mutated (i.e., substituted, added, inserted, and / or deleted). The variant of recombinant collagen may be the sequence of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32 after one or more amino acid residues have been changed or mutated (ie, substituted, added, inserted and / or deleted).

[0040] For example, a variant of the repeat unit of SEQ ID NO:1 may be a variant comprising a mutation in which Gfpgfp (SEQ ID NO:34) is added to the N-terminus of the amino acid sequence of SEQ ID NO:1 or a truncation of the fragment at the N-terminus of SEQ ID NO:34 (i.e., truncation of 1 to 5 amino acid residues from the N-terminus of SEQ ID NO:34, for example, 1, 2, 3, 4 or 5 amino acid residues corresponding to the fpgfp, pgfp, gfp, fp and p residues, respectively), and / or a mutation in which GFMGPPGPQGQPGLP (SEQ ID NO:35) is added to the C-terminus of the amino acid sequence of SEQ ID NO:1 or a truncation of the fragment at the C-terminus of SEQ ID NO:35 (i.e., truncation of 1 to 14 amino acid residues from the C-terminus of SEQ ID NO:35, for example, truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid residues). Variants of the repeat unit of SEQ ID NO:1 may include mutations that add Gfpgfp (SEQ ID NO:34) to the N-terminus of the amino acid sequence of SEQ ID NO:1 or that truncate the fragment at the N-terminus (i.e., by 1, 2, 3, 4 or 5 amino acid residues from the N-terminus of SEQ ID NO:34), and / or truncate the amino acid sequence of SEQ ID NO:1 by 1 to 5 amino acid residues, for example 1, 2 or 3 amino acid residues, at the C-terminus.

[0041] A variant of the repeat unit of SEQ ID NO:28 may be a variant that includes a mutation in which Glpgtp (SEQ ID NO:36) is added to the N-terminus of the amino acid sequence of SEQ ID NO:28 or a truncated fragment at the N-terminus (i.e., truncated by 1 to 5 amino acid residues, for example 1, 2, 3, 4 or 5 amino acid residues from the N-terminus of SEQ ID NO:36).

[0042] In the context of the present invention, conservative substitutions may be defined by substitutions within one or more of the amino acid types reflected below. Conservative amino acid residues: Acidic residues D and E Basic residues K, R and H Hydrophilic uncharged residues S, T, N and Q Aliphatic uncharged residues G, A, V, L and I Non-polar uncharged residues C, M and P Aromatic residues F, Y and W. Physical and functional classification of candidate amino acid residues: Alcohol group-containing residues S and T Aliphatic residues I, L, V and M Cycloalkenyl-related residues F, H, W and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S and T Positively charged residues H, K and R Small residues A, C, D, G, N, P, S, T and V Smallest residues A, G and S Residues involved in the formation of reverse turns: A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q, T, K, S, G, P, D, E and R.

[0043] As used herein, "cell adhesion" refers to adhesion between cells and collagen. Collagen (e.g., the recombinant collagen described herein) can promote adhesion between cells and the container in which they are cultured.

[0044] As used herein, the term "expression" includes any step involved in the production of recombinant collagen, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0045] As used herein, the term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding a recombinant collagen, operably linked to a control sequence that provides for its expression.

[0046] As used herein, the term "host cell" means any type of cell that is readily transformed, transfected, transduced, etc., with a nucleic acid construct or expression vector containing a polynucleotide of the invention. The term "host cell" encompasses the progeny of any parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0047] As used herein, the term "nucleic acid" refers to a single- or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or that has been modified to include a segment of nucleic acid in a manner not naturally occurring in nature, or that is synthetic, and that includes one or more regulatory sequences. The nucleic acid may be SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. The nucleic acid may be a codon-optimized nucleic acid, for example, a nucleic acid that has been codon-optimized for expression in E. coli cells.

[0048] The term "operably linked" refers to a configuration in which a control sequence is positioned in appropriate relation to a coding sequence of a polynucleotide so as to direct the expression of the coding sequence.

[0049] The degree of relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity." For the purposes of the present invention, sequence identity between two amino acid sequences is determined using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented by the Needle program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or an updated version. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output marked "longest identity" (obtained using the unabbreviated option) is used as the percentage identity, calculated as follows: (Identical residues × 100) / (alignment length - total number of gaps in the alignment)

[0050] For the purposes of the present invention, the sequence identity between two deoxynucleotide sequences is determined using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) as implemented by the Needle program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000), preferably version 5.0.0 or an updated version. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version in NCBI NUC4.4) substitution matrix. The output of Needle marked "longest identity" (obtained using the unabbreviated option) is used as the percentage identity and is calculated as follows: (Identical deoxyribonucleotides × 100) / (alignment length - total number of gaps in the alignment)

[0051] Recombinant collagen The recombinant collagen of the present invention comprises one or more repeat units, said repeat units being linked directly or via a linker, said repeat units comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31 or 33 or a variant thereof. The variant may be (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, or 33, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, or 33. For the recombinant collagens described herein, the mutation may be selected from a substitution, addition, insertion, or deletion. Preferably, the substitution is a conservative amino acid substitution.

[0052] The recombinant collagen described herein may comprise a plurality of repeating units, e.g., 2 to 50 repeating units, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 repeating units.

[0053] The linker in the recombinant collagen described herein may contain one or more amino acid residues, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acid residues.

[0054] The recombinant collagen is recombinant human type IV collagen or recombinant type IV humanized collagen, and preferably has cell adhesion activity. The recombinant collagen described herein is derived from humans and may therefore be human recombinant type IV humanized collagen.

[0055] The recombinant collagen described herein may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or a variant thereof, wherein the variant is either (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence.

[0056] nucleic acid construct The invention also relates to nucleic acid constructs comprising a nucleic acid of the invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. A vector may comprise the nucleic acid construct.

[0057] Nucleic acids can be manipulated in a variety of ways to provide for expression of recombinant collagen. Depending on the expression vector, it may be desirable or necessary to manipulate the nucleic acid prior to its insertion into the vector. Techniques for modifying nucleic acids using recombinant DNA methods are well known in the art.

[0058] The control sequence may be a promoter recognized by a host cell for expression of a polynucleotide encoding the recombinant collagen of the present invention. The promoter comprises a transcriptional control sequence that mediates expression of the recombinant collagen. The promoter may be any nucleic acid that exhibits transcriptional activity in the host cell, including mutant promoters, truncated promoters, and hybrid promoters, and may be derived from an extracellular or intracellular gene encoding the recombinant collagen, either homologous or heterologous to the host cell.

[0059] Illustrative examples of promoters suitable for directing transcription of the vectors or nucleic acid constructs of the invention in bacterial host cells are promoters obtained from the Bacillus amyloliquefaciens α-amylase gene (amyQ), the Bacillus licheniformis α-amylase gene (amyL), the Bacillus licheniformis penicillinase gene (penP), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and xylB genes, the Bacillus thuringiensis cryIIIA gene, the E. coli lac operon, and the E. coli trc promoter.

[0060] In yeast hosts, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.

[0061] The control sequence may be a transcription terminator recognized by a host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the recombinant collagen. Any terminator that functions in the host cell may be used in the present invention.

[0062] Preferred terminators in bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and E. coli ribosomal RNA (rrnB).

[0063] Preferred terminators in yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al. (1992).

[0064] The regulatory sequence may be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of the gene, which increases expression of the gene.

[0065] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0066] The control sequence may be a leader sequence, which is a non-translated region of an mRNA that is important for translation in a host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the recombinant collagen. Any leader sequence that functions in the host cell may be used.

[0067] Suitable leader sequences in yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0068] The control sequence may also be a polyadenylation sequence, which is operably linked to the 3' end of a polynucleotide and is recognized by a host cell as a signal for adding polyadenosine residues to transcribed mRNA during transcription. Any polyadenylation sequence that functions in the host cell may be used.

[0069] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0070] The control sequence may be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the recombinant collagen, directing the recombinant collagen into the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide may inherently contain the signal peptide coding sequence originally linked in translation reading frame with the segment of the coding sequence encoding the recombinant collagen. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence foreign to the coding sequence. A foreign signal peptide coding sequence may be required when the coding sequence does not originally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the recombinant collagen. However, any signal peptide coding sequence that directs the expressed recombinant collagen into the secretory pathway of the host cell may be used.

[0071] Useful signal peptide coding sequences for bacterial host cells include those obtained from the genes encoding Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0072] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al. (Yeast 8:423-488).

[0073] Expression vector The present invention also relates to recombinant expression vectors comprising the nucleic acids of the present invention, promoters, and transcriptional and translational termination signals. The nucleic acids and control sequences can be ligated together to produce a recombinant expression vector, which may contain one or more convenient restriction sites, such that a polynucleotide encoding the recombinant collagen is inserted or substituted into such sites. Alternatively, the polynucleotide can be expressed by inserting the nucleic acid or a nucleic acid construct containing the nucleic acid into an appropriate vector for expression. When an expression vector is generated, the coding sequence is placed in the vector so that the coding sequence is operably linked to appropriate control sequences for expression.

[0074] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be readily programmed with recombinant DNA and cause expression of a polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector will be introduced. The vector may be a linear or closed circular plasmid.

[0075] The vector may be an autonomously replicating vector, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome, which exists as an extrachromosomal entity and whose replication is independent of chromosomal replication. The vector may include any means for ensuring self-replication. Alternatively, the vector may be a vector that is integrated into the genome when introduced into a host cell and replicated together with one or more integrated chromosomes. Also, a single vector or plasmid, or two or more vectors or plasmids, which together contain the total DNA to be introduced into the host cell genome, may be used, or a transposon may be used.

[0076] Vectors preferably contain one or more selectable markers which allow for easy selection of transformed, transfected, transduced, etc. cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0077] Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance (ampicillin resistance, chloramphenicol resistance, kanamycin resistance, neomycin resistance, spectinomycin resistance, tetracycline resistance, etc.) Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0078] The selectable marker may be a dual selectable marker system as described in WO2010 / 039889. Alternatively, the dual selectable marker may be the hph-tk dual selectable marker system.

[0079] The vector may contain elements that allow the vector to integrate into the genome of the host cell or to replicate autonomously within the cell independently of the genome.

[0080] When integrating into the genome of a host cell, the vector may rely on the polynucleotide sequence encoding the recombinant collagen or any other element of the vector to integrate into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration into a precise chromosomal location in the genome of the host cell by homologous recombination. To increase the likelihood of integration at a precise location, the integration element should contain a sufficient number of nucleic acids, e.g., 100-10,000 base pairs, 400-10,000 base pairs, or 800-10,000 base pairs, which have high sequence identity with the corresponding target sequence to increase the probability of homologous recombination. The integration element may be any sequence homologous to the target sequence in the genome of the host cell. Alternatively, the integration element may be a non-coding or coding polynucleotide. Alternatively, the vector may integrate into the genome of the host cell by non-homologous recombination.

[0081] For autonomous replication, the vector may further comprise an origin of replication that allows autonomous replication in the host cell under consideration. The origin of replication may be any plasmid replicon that mediates autonomous replication and functions in the cell. The term "origin of replication" or "plasmid replicon" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0082] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177 and pACYC184 that can replicate in E. coli, and the origins of replication of plasmids pUB110, pE194, pTA1060 and pAMβ1 that can replicate in Bacillus.

[0083] Examples of origins of replication for use in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0084] One or more copies of the polynucleotides of the invention can be inserted into host cells to improve recombinant collagen production. Increased copy numbers of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell's genome or by including an amplifiable selectable marker gene along with the polynucleotide, and cells containing an amplified copy of the selectable marker gene and additional copies of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selection agent.

[0085] Procedures for ligating the above elements to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)).

[0086] host cell The present invention also relates to recombinant host cells comprising the polynucleotide of the present invention operably linked to one or more control sequences that direct the production of the recombinant collagen of the present invention. As described above, by introducing a construct or vector comprising the polynucleotide into a host cell, the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector. The term "host cell" encompasses the progeny of any parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will largely depend on the gene encoding the recombinant collagen and its source.

[0087] The host cell can be any cell useful for the recombinant production of the recombinant collagen of the present invention, for example, a prokaryote or a eukaryote.

[0088] The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oenobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Labobacterium, Fusobacterium, Helicobacter, Lysobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0089] The host cell may be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell. As used herein, a plant cell does not include a plant cell capable of regenerating a plant. An animal cell also does not include a cell capable of producing an animal.

[0090] The host cell may be a fungal cell of the phylum Basidiomycota, Chytridiomycota, Zygomycota, and Oomycota. The fungal host cell may also be a yeast cell, including ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes). The yeast host cell may be a cell of the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces crivelii, kluyveri, Saccharomyces norbensis, Saccharomyces oviformis or Yarrowia lipolytica cells.

[0091] Production Method The present invention also relates to a method for producing the recombinant collagen described herein, the method comprising: (1) culturing a host cell described herein under suitable culture conditions; (2) harvesting the host cells and / or medium containing the recombinant collagen; and (3) purifying the recombinant collagen.

[0092] The host cells are cultured in a suitable nutrient medium for producing recombinant collagen using methods known in the art. For example, the cells may be cultured in shake flasks or by small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in laboratory or industrial fermentors, under conditions that allow the expression and / or isolation of the recombinant collagen in a suitable medium. The cells are cultured in a suitable nutrient medium containing a carbon source, a nitrogen source, and inorganic salts, using procedures known in the art. Suitable media can be obtained from commercial suppliers or prepared according to disclosed compositions (e.g., in the catalog of the American Type Culture Collection). If the recombinant collagen is secreted into the nutrient medium, it can be recovered directly from the medium. If the recombinant collagen is not secreted, it can be recovered from cell lysates.

[0093] Recombinant collagen may be detected using methods known in the art that are specific for recombinant collagen. These detection methods include, but are not limited to, the use of specific antibodies. For example, adhesion assays may be used to determine the activity of recombinant collagen.

[0094] The recombinant collagen may be recovered using methods known in the art. For example, the recombinant collagen may be recovered from the nutrient medium by conventional procedures including, but not limited to, harvesting, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Alternatively, the fermentation broth containing the recombinant collagen is recovered.

[0095] To obtain substantially pure recombinant collagen, the recombinant collagen may be purified by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focusing chromatography, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing), differential lysis (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.

[0096] Step (1) may include one or more of the following steps: Construct an expression plasmid, for example, by inserting the coding nucleotide sequence into a pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid; Transform the successfully constructed expression plasmid into E. coli cells (e.g., E. coli competent cells BL21(DE3)). A specific process may be as follows: (1) Add the plasmid to be transformed to E. coli competent cells BL21(DE3); (2) Incubate the mixture on ice (e.g., for 10 to 60 minutes, e.g., 30 minutes), then heat-shock in a water bath (e.g., at 40 to 50°C, e.g., 42°C, for 45 to 90 seconds), remove the mixture, and then incubate it on ice (e.g., for 1 to 5 minutes, e.g., 2 minutes); (3) Add liquid LB medium, and then culture (e.g., at 35 to 40°C, e.g., 37°C, at 150 to 300 rpm, e.g., 220 rpm, for 40 to 80 minutes, e.g., 60 minutes). (4) Spread the bacterial suspension and select single colonies. For example, spread the bacterial suspension evenly on an LB plate containing sodium ampicillin, and then incubate the plate in an incubator at 37°C for 15 to 17 hours to allow colonies of uniform size to grow.

[0097] Step (2) may include culturing a single colony in LB medium containing an antibiotic stock solution (e.g., culturing at 150 to 300 rpm, e.g., 220 rpm, in a constant temperature shaker at 35 to 40°C, e.g., 37°C, for 5 to 10 hours, e.g., 7 hours), and further cooling the shake flask after culturing to 10 to 20°C, e.g., 16°C, adding IPTG to induce expression for a certain period of time, and then collecting the bacterial cells (e.g., by centrifugation).

[0098] Step (3) may include resuspending the bacterial cells in an equilibrium working solution, cooling the bacterial solution to ≦15°C, homogenizing (e.g., high-pressure homogenization 1 to 5 times, e.g., 2 times), and separating the homogenized bacterial solution to obtain a supernatant. The equilibrium working solution may contain 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 10 to 50 mM imidazole, and may have a pH of 7 to 9. For example, the concentration of sodium chloride may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH may be 7, 7.5, 8, 8.5 or 9.

[0099] Step (3) may include purifying the recombinant collagen and enzymatically cleaving it. The purification may be crude purification, which involves purifying the supernatant through a Ni-agarose gel column to obtain an eluate containing the target protein. The crude purification may include washing the column with water, for example, 2 to 10 column volumes (CV), for example, 5 CV. The column may be equilibrated with an equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0), for example, 2 to 10 CV, for example, 5 CV. The equilibration solution may contain 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 10 to 50 mM imidazole, and have a pH of 7 to 9. For example, the concentration of sodium chloride may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH may be 7, 7.5, 8, 8.5 or 9.

[0100] Step (3) may include adding the supernatant to a column and washing off contaminating proteins with a scrubbing solution, which may contain 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 10 to 50 mM imidazole, and have a pH of 7 to 9. For example, the concentration of sodium chloride may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH may be 7, 7.5, 8, 8.5, or 9. An eluent may then be added and the flow-through liquid may be collected. The eluent may contain 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 100 to 500 mM imidazole, and have a pH of 8.0. For example, the concentration of sodium chloride may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480 or 490 nM. The pH may be 7, 7.5, 8, 8.5 or 9.

[0101] The enzymatic cleavage may include adding a collagen tool enzyme and performing enzymatic cleavage (at a ratio of the total amount of protein to the total amount of collagen tool enzyme of 10 to 100:1, for example 50:1, at 10 to 20°C, for example 16°C, for 2 to 8 hours, for example 4 hours). The enzymatically cleaved protein solution is dialyzed, for example, by placing it in a dialysis bag and dialyzing it at 1 to 6°C, for example 4°C, for 1 to 8 hours, for example 2 hours, and then transferring it to fresh dialysis solution and dialyzing it at 1 to 6°C, for example 4°C, overnight.

[0102] Purification may include micropurification (e.g., protein isoelectric point > 8.0). Preferably, micropurification involves gradient elution of an eluate containing the target protein or an enzymatically cleaved product (e.g., a dialyzed product of enzymatic cleavage) using a strong anion exchange chromatography column (e.g., pH 7, 7.5, 8, 8.5, or 9). Gradient elution may include elution with 0-15% solution B for 1-5 minutes followed by retention for 1-5, e.g., 3 column volumes, elution with 15-30% solution B for 1-5 minutes followed by retention for 1-5, e.g., 3 column volumes, elution with 30-50% solution B for 1-5 minutes followed by retention for 1-5, e.g., 3 column volumes, and elution with 50-100% solution B for 1-5 minutes followed by retention for 1-5, e.g., 3 column volumes. Solution B may contain 10-50 mM Tris, 0.5-5 M sodium chloride, and a pH of 7-9. For example, the Tris concentration is 15, 20, 25, 30, 35, 40, or 45 mM. The sodium chloride concentration is 1, 2, 3, or 4 M. The pH may be 7, 7.5, 8, 8.5, or 9. Precision purification may include equilibrating a column with solution A, loading the column, and then performing gradient elution. Solution A may contain 10 to 50 mM Tris and 10 to 50 mM sodium chloride, and may have a pH of 7 to 9. For example, the Tris concentration is 15, 20, 25, 30, 35, 40, or 45 mM. For example, the sodium chloride concentration is 15, 20, 25, 30, 35, 40, or 45 mM. The pH may be 7, 7.5, 8, 8.5, or 9.

[0103] In order to clarify the purpose, technical means and advantages of the present invention, the following will clearly and completely describe the technical means in the embodiments of the present invention with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only some of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be obtained by those skilled in the art based on the embodiments of the present invention without creative labor fall within the scope of protection of the present invention.

[0104] To further illustrate the present invention, the following examples are provided. [Example]

[0105] The present invention will be further described by the following examples, but any of the examples or combinations thereof should not be understood to limit the scope or embodiments of the present invention. The scope of the present invention is limited by the appended claims, and those skilled in the art can clearly understand the scope limited by the claims by combining this specification and general knowledge in the art. Without departing from the spirit and scope of the present invention, those skilled in the art can make any modifications or changes to the technical means of the present invention, and these modifications and changes are also included in the scope of the present invention.

[0106] Example 1. Construction, expression and screening of recombinant type IV humanized collagen fragments 1. A large-scale screening of functional regions was conducted to obtain the target functional regions of the following different recombinant humanized type IV collagen genes:

[0107] 1) Amino acid sequence of C4P7Ch (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeq, SEQ ID NO: 1, the number of repeating units is 10, and the amino acid sequence of C4P7Ch is SEQ ID NO: 2): Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Gakgdkgskgevgfpglagspgipgskgeq Nucleotide sequence of C4P7Ch (SEQ ID NO:3): GGGGCTAAAGGAGACAAGGGCAGCAAGGGCGAAGTCGGTTTCCCAGGTCTGGCTGGTAGCCCGGGCATCCCGGGTTCAAAGGGTGAACAAGGTGCTAAAGGCGACAAAGGCAGCAAGGGTGAGGTTGGTTTCCCGGGTCTGGCGGGTTCTCCAGGCATCCCGGGTAGCAAAGGAGAACAAGGTGCGAAAGGCGATAAAGGCTCCAAGGGTGAAGTGGGCTTCCCGGGTTTAGCCGGTAGCCCAGGTATTCCGGGTAGCAAAGGCGAACAGGGTGCGAAAGGCGACAAAGGGAGTAAGGGCGAGGTGGGTTTTCCGGGTTTGGCTGGCTCGCCGGGTATTCCGGGTTCAAAGGGCGAACAGGGCGCGAAAGGTGATAAAGGCAGCAAAGGCGAGGTTGGCTTCCCGGGTCTGGCAGGTAGCCCGGGTATCCCGGGTAGCAAGGGTGAGCAGGGTGCCAAAGGCGACAAAGGTAGCAAGGGGGAAGTGGGTTTTCCGGGACTGGCAGGTAGCCCGGGTATCCCGGGTTCTAAGGGCGAGCAGGGTGCGAAAGGTGACAAAGGTAGCAAGGGCGAGGTTGGCTTTCCGGGCTTGGCGGGTAGCCCGGGCATTCCGGGCTCCAAGGGTGAACAAGGTGCGAAAGGTGATAAAGGCTCTAAGGGTGAGGTTGGTTTTCCGGGTCTGGCGGGTTCCCCGGGCATTCCGGGCTCGAAGGGCGAGCAAGGTGCTAAAGGTGATAAGGGCTCCAAGGGCGAGGTGGGTTTCCCGGGCCTGGCAGGCTCTCCGGGCATCCCGGGTTCGAAGGGCGAACAGGGTGCGAAAGGCGATAAAGGTTCCAAGGGCGAAGTCGGATTCCCTGGCCTCGCCGGTAGCCCGGGCATCCCTGGCTCCAAGGGCGAGCAG

[0108] 2) C4P7Cf (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq, SEQ ID NO: 4, the number of repeating units is 8, and the amino acid sequence of C4P7Cf is SEQ ID NO: 5): Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Nucleotide sequence of C4P7Cf (SEQ ID NO:6): GGAGCTAAAGGGGACAAAGGCTCCAAAGGCGAAGTCGGTTTCCCGGGCCTGGCGGGTAGCCCGGGTATCCCGGGTAGCAAGGGTGAGCAGGGGTTCATGGGTCCACCGGGCCCACAGGGTGCCAAAGGTGATAAAGGTTCTAAGGGCGAGGTGGGTTTCCCGGGGCTGGCGGGTTCTCCGGGCATTCCGGGAAGCAAGGGTGAACAGGGCTTTATGGGTCCGCCAGGTCCGCAGGGTGCGAAAGGTGATAAAGGCAGCAAGGGAGAAGTTGGCTTCCCGGGCCTGGCAGGCAGCCCGGGCATTCCGGGGTCGAAGGGCGAACAAGGTTTCATGGGTCCGCCTGGTCCGCAAGGTGCGAAAGGTGATAAGGGTAGCAAGGGTGAAGTGGGTTTTCCGGGATTAGCGGGTTCTCCGGGCATTCCGGGTTCAAAAGGTGAACAAGGCTTTATGGGTCCGCCTGGCCCGCAGGGTGCTAAAGGCGACAAGGGTAGCAAAGGCGAGGTAGGTTTCCCGGGTTTGGCGGGCAGCCCGGGCATTCCGGGTTCCAAGGGCGAGCAGGGTTTTATGGGCCCACCGGGCCCGCAAGGCGCAAAAGGTGATAAGGGCAGCAAAGGCGAGGTGGGCTTCCCGGGACTGGCAGGTTCTCCGGGTATCCCGGGTTCCAAGGGTGAGCAGGGTTTCATGGGCCCACCGGGTCCGCAGGGTGCGAAAGGCGACAAAGGTAGCAAGGGCGAAGTTGGTTTTCCGGGCCTGGCTGGTTCGCCGGGCATCCCGGGCTCCAAGGGCGAGCAAGGCTTCATGGGTCCACCGGGTCCGCAAGGTGCCAAAGGCGACAAAGGTAGCAAGGGCGAGGTTGGTTTTCCGGGCTTGGCTGGTAGCCCTGGCATCCCGGGGTCCAAGGGTGAACAGGGCTTTATGGGTCCGCCGGGCCCTCAA

[0109] 3) Amino acid sequence of C4P7Ca (the amino acid sequence of the repeating unit is GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP, SEQ ID NO: 7, the number of repeating units is 6, and the amino acid sequence of C4P7Ca is SEQ ID NO: 8): GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP Nucleotide sequence of C4P7Ca (SEQ ID NO:9): GGATTTCCCGGGTTCCCGGGTGCCAAAGGGGATAAAGGTTCAAAGGGCGAAGTGGGTTTCCCGGGTTTGGCTGGTAGCCCGGGTATCCCGGGTAGCAAAGGCGAACAGGGCTTTATGGGTCCGCCAGGACCGCAGGGTCAACCGGGACTGCCGGGTTTTCCGGGCTTCCCGGGTGCGAAAGGCGATAAAGGTTCCAAGGGTGAAGTTGGTTTTCCGGGTCTTGCAGGCAGCCCGGGTATTCCGGGTTCCAAGGGTGAACAGGGTTTCATGGGTCCACCGGGCCCACAAGGTCAGCCGGGTCTGCCTGGTTTCCCGGGCTTCCCGGGTGCCAAAGGCGACAAAGGTAGCAAGGGCGAAGTTGGCTTTCCGGGTCTGGCGGGTTCGCCGGGCATTCCGGGCTCGAAGGGCGAGCAGGGTTTCATGGGCCCACCGGGTCCGCAGGGTCAGCCTGGCCTGCCGGGATTCCCAGGTTTTCCGGGAGCGAAAGGCGACAAGGGTAGTAAGGGTGAGGTCGGTTTTCCAGGCTTGGCGGGCTCTCCCGGTATCCCGGGCTCTAAGGGCGAGCAAGGCTTTATGGGTCCACCGGGTCCGCAAGGTCAACCTGGATTACCGGGATTCCCAGGCTTTCCGGGCGCGAAAGGCGATAAAGGCAGCAAGGGTGAGGTGGGCTTCCCGGGCCTCGCGGGTAGCCCGGGCATCCCGGGTAGCAAGGGTGAGCAGGGCTTCATGGGTCCTCCGGGTCCGCAGGGCCAACCGGGCCTGCCGGGATTCCCGGGTTTCCCGGGCGCTAAAGGCGACAAAGGCAGCAAGGGTGAGGTTGGTTTTCCGGGTCTGGCAGGTAGCCCGGGCATTCCGGGCTCCAAGGGCGAACAGGGTTTTATGGGTCCACCGGGCCCTCAAGGTCAGCCGGGCCTGCCG

[0110] 4) Amino acid sequence of C4P7Cb (the amino acid sequence of the repeating unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq, SEQ ID NO: 10, the number of repeating units is 8, and the amino acid sequence of C4P7Cb is SEQ ID NO: 11): Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq Nucleotide sequence of C4P7Cb (SEQ ID NO:12):

[0111] 5) Amino acid sequence of C4P7Cc (the amino acid sequence of the repeating unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm, SEQ ID NO: 13, the number of repeating units is 8, and the amino acid sequence of C4P7Cc is SEQ ID NO: 14): Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm Nucleotide sequence of C4P7Cc (SEQ ID NO: 15): GGATTTCCCGGGTTCCCGGGTGCGAAAGGTGATAAAGGCAGCAAGGGTGAAGTCGGTTTTCCGGGTCTGGCAGGCAGCCCGGGTATCCCGGGTAGCAAAGGCGAACAGGGCTTTATGGGTTTCCCGGGCTTCCCAGGTGCGAAGGGCGATAAAGGTTCGAAAGGTGAGGTAGGTTTCCCGGGTTTAGCAGGTTCCCCGGGCATTCCGGGCAGCAAGGGTGAACAGGGTTTCATGGGCTTTCCGGGCTTCCCAGGAGCTAAAGGCGACAAAGGTTCTAAGGGTGAAGTGGGCTTCCCGGGTCTGGCTGGTAGCCCGGGCATCCCGGGCTCCAAGGGTGAGCAGGGTTTCATGGGTTTTCCGGGCTTCCCAGGCGCGAAAGGCGACAAAGGCAGCAAGGGCGAGGTGGGTTTTCCGGGTTTGGCGGGTAGCCCGGGTATTCCGGGTTCGAAGGGTGAACAAGGTTTCATGGGTTTTCCGGGATTCCCAGGCGCGAAAGGCGATAAGGGCAGCAAGGGCGAGGTTGGCTTCCCGGGACTGGCCGGAAGCCCGGGTATCCCGGGATCTAAGGGCGAACAAGGCTTTATGGGTTTCCCGGGTTTTCCTGGTGCGAAAGGCGATAAAGGCTCCAAGGGCGAGGTTGGTTTTCCAGGCCTGGCTGGCTCTCCGGGCATTCCGGGTAGTAAGGGTGAGCAGGGTTTTATGGGTTTTCCGGGCTTCCCGGGTGCAAAGGGTGACAAAGGTAGCAAGGGTGAAGTTGGCTTTCCGGGTCTGGCGGGTTCCCCGGGCATTCCGGGTAGCAAAGGTGAGCAAGGTTTTATGGGTTTTCCGGGCTTCCCGGGTGCCAAAGGCGACAAAGGTAGCAAGGGAGAGGTGGGCTTCCCGGGATTGGCGGGTTCCCCGGGCATCCCGGGCTCAAAGGGTGAACAGGGTTTCATG

[0112] 6) Amino acid sequence of C4P7Cd (the amino acid sequence of the repeating unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeq, SEQ ID NO: 16, the number of repeating units is 10, and the amino acid sequence of C4P7Cd is SEQ ID NO: 17): Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Gfpgfpgakgdkgskgevgfpglagspgipgskgeq Nucleotide sequence of C4P7Cd (SEQ ID NO: 18):

[0113] 7) Amino acid sequence of C4P7Ce (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp, SEQ ID NO: 19, the number of repeating units is 8, and the amino acid sequence of C4P7Ce is SEQ ID NO: 20): Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp Nucleotide sequence of C4P7Ce (SEQ ID NO:21):

[0114] 8) Amino acid sequence of C4P7Cg (the amino acid sequence of the repeating unit is Gakgdkgskgevgfpglagspgipgskgeqgfm, SEQ ID NO: 22, the number of repeating units is 10, and the amino acid sequence of C4P7Cg is SEQ ID NO: 23): Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Gakgdkgskgevgfpglagspgipgskgeqgfm Nucleotide sequence of C4P7Cg (SEQ ID NO:24): GGGGCTAAAGGAGACAAAGGTTCGAAAGGCGAGGTGGGCTTCCCAGGTCTGGCCGGTTCCCCGGGCATTCCGGGTAGCAAAGGCGAACAAGGTTTCATGGGTGCTAAAGGCGATAAAGGTAGCAAGGGTGAGGTTGGCTTCCCAGGCCTGGCTGGTTCGCCGGGCATTCCGGGCTCTAAGGGTGAACAAGGTTTCATGGGTGCAAAAGGTGATAAGGGTAGCAAGGGAGAAGTCGGTTTTCCGGGATTGGCGGGTAGCCCGGGTATCCCGGGCAGCAAGGGCGAGCAGGGTTTTATGGGTGCAAAGGGCGACAAAGGTAGCAAGGGTGAGGTGGGCTTTCCGGGCCTCGCGGGTAGCCCTGGCATCCCGGGTTCCAAAGGTGAGCAAGGCTTCATGGGTGCTAAAGGTGATAAAGGCTCCAAAGGTGAAGTGGGTTTTCCGGGCCTGGCGGGTAGCCCGGGCATTCCGGGAAGCAAGGGCGAACAGGGTTTTATGGGCGCGAAGGGTGATAAAGGTAGTAAGGGCGAAGTTGGTTTCCCGGGCCTGGCTGGCTCTCCGGGTATCCCGGGCTCCAAAGGCGAGCAGGGTTTCATGGGTGCGAAAGGTGACAAGGGTAGCAAGGGTGAGGTGGGTTTCCCAGGTTTGGCGGGTAGCCCGGGCATTCCGGGTAGCAAGGGTGAACAAGGTTTCATGGGTGCGAAAGGTGACAAAGGCAGCAAGGGCGAGGTTGGTTTCCCGGGTCTGGCGGGTAGCCCGGGCATCCCGGGCTCTAAGGGCGAGCAGGGTTTTATGGGTGCCAAAGGCGACAAGGGCTCAAAGGGTGAAGTCGGTTTTCCGGGTTTAGCCGGTTCCCCGGGCATCCCGGGTTCTAAGGGTGAACAGGGCTTCATGGGCGCGAAAGGAGATAAAGGCAGCAAAGGGGAAGTTGGTTTTCCAGGCCTGGCAGGCTCGCCGGGTATCCCGGGTTCCAAGGGCGAGCAGGGTTTTATG

[0115] 9) Amino acid sequence of C4P7Ea (the amino acid sequence of the repeating unit is Glpgtpgptgpagqkgepgsdgipgsagekgepglp, SEQ ID NO: 25, the number of repeating units is 10, and the amino acid sequence of C4P7Ea is SEQ ID NO: 26): Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Glpgtpgptgpagqkgepgsdgipgsagekgepglp Nucleotide sequence of C4P7Ea (SEQ ID NO:27):

[0116] 10) Amino acid sequence of C4P7Eb (the amino acid sequence of the repeating unit is Gptgpagqkgepgsdgipgsagekgepglp, SEQ ID NO: 28, the number of repeating units is 10, and the amino acid sequence of C4P7Eb is SEQ ID NO: 29): Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Gptgpagqkgepgsdgipgsagekgepglp Nucleotide sequence of C4P7Eb (SEQ ID NO:30): GGTCCCACAGGACCGGCAGGCCAGAAAGGTGAGCCGGGTTCCGACGGCATCCCGGGTTCGGCGGGTGAGAAAGGCGAGCCGGGTTTACCGGGTCCGACCGGTCCCGCGGGTCAAAAGGGCGAGCCGGGTAGCGATGGCATTCCGGGTTCTGCGGGTGAAAAGGGCGAACCGGGCCTCCCGGGTCCTACCGGTCCGGCGGGTCAGAAAGGCGAACCGGGCAGCGATGGCATCCCGGGCAGCGCGGGCGAGAAAGGCGAACCGGGCCTGCCGGGCCCGACCGGACCAGCTGGGCAAAAAGGTGAACCGGGCAGCGACGGCATCCCGGGTTCTGCAGGCGAGAAAGGTGAACCAGGCCTGCCGGGACCGACCGGTCCGGCAGGCCAGAAAGGTGAGCCTGGCAGTGATGGTATTCCGGGTTCTGCCGGTGAAAAAGGTGAGCCGGGCCTGCCGGGGCCAACGGGCCCAGCCGGACAAAAAGGTGAGCCGGGTTCCGACGGCATCCCGGGCTCCGCCGGTGAAAAGGGTGAGCCGGGCCTGCCTGGCCCAACGGGTCCGGCTGGCCAAAAGGGCGAGCCGGGTAGCGACGGCATTCCGGGCAGCGCGGGTGAGAAGGGTGAGCCGGGATTGCCGGGTCCGACTGGTCCTGCGGGCCAGAAGGGTGAACCGGGTTCCGACGGCATCCCCGGCTCGGCGGGTGAAAAGGGCGAACCGGGTCTGCCTGGTCCGACCGGCCCAGCGGGTCAGAAGGGTGAACCGGGTAGCGATGGAATCCCGGGTAGCGCTGGTGAAAAGGGCGAGCCGGGCCTGCCGGGTCCGACCGGTCCGGCAGGCCAGAAGGGTGAACCGGGTAGCGATGGTATTCCGGGTAGCGCGGGCGAAAAAGGTGAGCCGGGCTTGCCG

[0117] 11) Amino acid sequence of C4P7Ec (the amino acid sequence of the repeating unit is Gfpgfpgakgdkgskgevgfpglagspgipgsk, SEQ ID NO: 31, the number of repeating units is 10, and the amino acid sequence of C4P7Ec is SEQ ID NO: 32): Gfpgfpgakgdkgskgevgfpglagspgipgsk Gfpgfpgakgdkgskgevgfpglagspgipgsk Gfpgfpgakgdkgskgevgfpglagspgipgsk Gfpgfpgakgdkgskgevgfpglagspgipgsk Gfpgfpgakgdkgskgevgfpglagspgipgsk Gfpgfpgakgdkgskgevgfpglagspgipgsk Gfpgfpgakgdkgskgevgfpglagspgipgsk Gfpgfpgakgdkgskgevgfpglagspgipgsk Gfpgfpgakgdkgskgevgfpglagspgipgsk Gfpgfpgakgdkgskgevgfpglagspgipgsk Nucleotide sequence of C4P7Ec (SEQ ID NO:33): GGATTTCCCGGGTTCCCAGGCGCAAAAGGTGATAAAGGCAGCAAGGGCGAGGTTGGTTTTCCAGGTTTAGCTGGTAGCCCGGGTATCCCGGGTAGCAAGGGCTTCCCGGGTTTTCCGGGTGCTAAAGGCGACAAAGGCTCCAAGGGCGAAGTCGGTTTCCCGGGTTTGGCGGGTAGCCCGGGTATCCCGGGTAGTAAGGGCTTTCCGGGATTCCCAGGCGCGAAAGGTGACAAAGGTAGCAAGGGCGAAGTTGGCTTCCCGGGTTTGGCGGGTTCCCCGGGTATCCCGGGGTCCAAGGGCTTCCCCGGATTCCCGGGCGCGAAAGGCGATAAAGGTAGCAAGGGTGAAGTGGGTTTTCCGGGTCTCGCTGGCAGCCCGGGTATTCCGGGCTCCAAGGGCTTTCCAGGCTTTCCGGGTGCGAAAGGCGATAAAGGTAGCAAGGGTGAGGTGGGTTTTCCGGGTCTGGCAGGTAGCCCTGGCATCCCGGGCTCGAAGGGGTTCCCGGGCTTCCCGGGAGCCAAGGGTGATAAAGGTTCTAAGGGTGAGGTCGGTTTTCCGGGCCTGGCCGGTAGCCCTGGTATCCCGGGGAGCAAGGGTTTCCCGGGTTTTCCGGGTGCCAAAGGCGATAAAGGCTCTAAGGGCGAGGTGGGCTTCCCCGGTCTGGCGGGTAGCCCGGGTATTCCGGGTTCTAAGGGCTTCCCGGGTTTTCCGGGTGCGAAAGGTGACAAGGGCTCCAAGGGTGAAGTTGGTTTTCCGGGTCTGGCTGGTAGCCCGGGTATCCCGGGTAGCAAGGGCTTCCCGGGTTTCCCGGGCGCGAAAGGCGACAAAGGTTCAAAGGGTGAAGTTGGTTTTCCTGGCCTGGCAGGCAGCCCGGGCATTCCGGGTTCCAAAGGTTTTCCGGGCTTCCCGGGTGCGAAAGGTGACAAAGGCTCGAAGGGTGAGGTGGGCTTCCCGGGTCTGGCAGGTTCTCCTGGCATTCCGGGTTCGAAA

[0118] The above coding nucleotide sequences were commercially synthesized. Each coding nucleotide sequence (containing a collagen tool enzyme cleavage site at the 5' end, with the amino acid sequence ENLYFQ and the nucleotide sequence GAAAACCTGTATTTCCAG) was inserted between the KpnI and XhoI enzyme cleavage sites of the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid.

[0119] 3. The successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3). The specific process was as follows: (1) E. coli competent cells BL21(DE3) were removed from the ultra-low temperature refrigerator and placed on ice. When partially thawed, 2 μl of the target plasmid was added to the E. coli competent cells BL21(DE3) and mixed thoroughly 2-3 times. (2) The mixture was then placed on ice for 30 minutes, heat-shocked in a 42°C water bath for 45-90 seconds, removed, and placed on ice for 2 minutes. (3) The mixture was transferred to a biological safety cabinet, and 700 μl of liquid LB medium was added. The mixture was then cultured at 37°C and 220 rpm for 60 minutes. (4) 200 μl of the bacterial suspension was evenly spread on an LB plate containing sodium ampicillin. (5) The plate was then cultured in a 37°C incubator for 15-17 hours to allow uniformly sized colonies to grow.

[0120] Five to six single colonies were selected from the transformed LB plate, placed in a shake flask containing LB medium containing antibiotic stock solution, and cultured for 7 hours at 220 rpm and 37°C in a constant temperature shaker. After further culture, the shake flask was cooled to 16°C, and IPTG was added to induce expression for a certain period. The bacterial solution was then dispensed into centrifuge flasks and centrifuged at 8000 rpm and 4°C for 10 minutes to collect the bacterial cells. The bacterial weight was recorded, and a sample (denoted as "bacterial solution") was then subjected to electrophoresis.

[0121] 5. The collected bacterial cells were resuspended in an equilibrium working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0), the solution was cooled to ≤15°C, homogenized, and then homogenized twice under high pressure (the samples homogenized twice were labeled "homogenized" and "homogenized twice," respectively). The homogenized solution was then collected after completion. The homogenized solution was dispensed into centrifuge flasks and centrifuged at 17,000 rpm at 4°C for 30 minutes. The supernatant (labeled "supernatant") and the precipitate were subjected to electrophoresis detection.

[0122] 6. Recombinant type IV humanized collagen was purified and enzymatically cleaved. The specific processes are as follows (1) to (4). (1) Crude purification was performed as follows: a. The column was washed with water for 5 CV (Ni6FF, Cytiva). b. The column was equilibrated with an equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) for 5 CV. c. For loading, the supernatant from centrifugation was added to the column until the liquid flowed through, and the flow-through was subjected to electrophoretic detection (referred to as "flow-through"). d. For washing of impurity proteins, 25 mL of scrubbing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) was added until the liquid flowed through, and the scrubbed flow-through was subjected to electrophoretic detection (referred to as "scrubbing"). e) To collect the target protein, 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0) was added, and the flow-through (labeled "Elution") was collected. The protein concentration was measured to calculate the protein amount, and electrophoretic detection was performed. f) The column was washed with 1 M imidazole working solution (labeled "1 M Wash"). g) The column was washed with purified water. (2) Enzymatic cleavage was performed as follows: TEV enzyme was added at a ratio of 50:1 between the total protein and the total TEV enzyme, and enzymatic cleavage was performed at 16°C for 4 hours. Samples were then sampled and electrophoretically detected (labeled "After Cleavage"). The enzymatically cleaved protein solution was placed in a dialysis bag and dialyzed at 4°C for 2 hours. It was then transferred to fresh dialysate and dialyzed overnight at 4°C (labeled "Replacement"). (3) The following purification procedures were performed: a) Column equilibration (Capto Q, Cytiva) was performed with Solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0) at a flow rate of 10 mL / min. b) Loading was performed at a flow rate of 5 mL / min. The flow-through was collected (denoted "QFL") and subjected to electrophoretic detection. c) Gradient elution was performed as follows: 0-15% Solution B (20 mM Tris, 1 M sodium chloride, pH 8.0) for 2 min and held for three CVs; 15-30% Solution B for 2 min and held for three CVs; 30-50% Solution B for 2 min and held for three CVs; and 50-100% Solution B for 2 min and held for three CVs. Peaks were collected when they appeared and subjected to electrophoretic detection (denoted "B wash"). d. The column was washed and the protein was stored at 4°C.

[0123] 7. Electrophoretic detection The specific process was as follows: 10 μl of 5x protein loading buffer (250 mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol) was added to 40 μl of sample solution and boiled in 100°C water for 10 minutes. Then, 10 μl of each well was loaded onto an SDS-PAGE protein gel and electrophoresed at 80 V for 2 hours. The proteins were stained with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20 minutes, and then destained with protein destaining solution (10% acetic acid, 5% ethanol).

[0124] FIG. 1 shows the electrophoretic detection results for C4P7Ca. FIG. 2 shows the electrophoretic detection results for C4P7Cb. FIG. 3 shows the electrophoretic detection results for C4P7Cc. FIG. 4 shows the electrophoretic detection results for C4P7Cd. FIG. 5 shows the electrophoretic detection results for C4P7Ce. FIG. 6 shows the electrophoretic detection results for C4P7Cf. FIG. 7 shows the electrophoretic detection results for C4P7Cg. FIG. 8 shows the electrophoretic detection results for C4P7Ch. FIG. 9 shows the electrophoretic detection results for C4P7Ea. FIG. 10 shows the electrophoretic detection results for C4P7Eb. FIG. 11 shows the electrophoretic detection results for C4P7Ec. Figures 1 to 11 show that the actual molecular weights of each isolated protein (C4P7Ca, C4P7Cb, C4P7Cc, C4P7Cd, C4P7Ce, C4P7Cf, C4P7Cg, C4P7Ch, C4P7Ea, C4P7Eb, and C4P7Ec) were consistent with their corresponding predicted molecular weights, indicating that the proteins were accurately expressed.

[0125] Example 2. Mass spectrometry detection of recombinant type IV humanized collagen Experimental Method [Table 1]

[0126] Protein samples (collagen C4P7Cf and C4P7Ch) were reduced with DTT, alkylated with iodoacetamide, and then enzymatically digested overnight with trypsin. The resulting peptide fragments were further desalted using a C18 ZipTip, mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA), and then dropped onto a target plate. Finally, a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF / TOF) Ulraflextreme mass spectrometer was used. TM , Brucker, Germany (for peptide mass fingerprinting techniques see Protein J. 2016;35:212-7).

[0127] Data searches are performed from the MS / MS Ion Search page on the local masco site. Protein identification results are obtained from the primary mass spectra of peptide fragments generated after enzymatic digestion. For detection parameters, two uncleaved sites are set for trypsin enzymatic digestion. Cysteine ​​alkylation is set as a fixed modification. Methionine oxidation is set as a variable modification. The database used for identification is NCBprot.

[0128] Table 1. Molecular weights and corresponding polypeptides of recombinant type IV humanized collagen C4P7Cf detected by mass spectrometry. [Table 2]

[0129] The polypeptide fragments showed a coverage of 99.04% ( gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgakgdkgskgevgfpglagspgipgskgeqgfmgppgpqGakgdkgskgevgfpglagspgipgskgeqgfmgppgpq , the underlined residues are the cover portion), and the detection results are very reliable.

[0130] Table 2. Molecular weights and corresponding polypeptides of recombinant type IV humanized collagen C4P7Ch as detected by mass spectrometry. [Table 3]

[0131] The polypeptide fragments showed a coverage of 98% (Gakgdk gskgevgfpglagspgipgskgeqgakgdkgskgevgfpglagspgipgskgeqgakgdkgskgevgfpglagspgipgskgeqgakgdkgskgevgfpglagspgipgskgeqgakgdkgskgevgfpglagspgipgskgeqgak gdkgskgevgfpglagspgipgskgeqgakgdkgskgevgfpglagspgipgskgeqgakgdkgskgevgfpglagspgipgskgeqgakgdkgskgevgfpglagspgipgskgeqgakgdkgskgevgfpglagspgipgskgeq , the underlined residues are the cover portion), and the detection results are very reliable.

[0132] Example 3: Bioactivity detection of recombinant type IV humanized collagen For a method for detecting collagen activity, see Juming Yao, Satoshi Yanagisawa, and Tetsuo Asakura, "Design, Expression, and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens," J. Biochem. 136, 643-649 (2004). Specific implementation methods are as follows:

[0133] (1) Using ultraviolet absorption method, the concentrations of target protein samples, including bovine type I collagen (China Food and Drug Administration, No. 380002) and the recombinant humanized collagens C4P7Cf and C4P7Ch according to the present invention, were detected.

[0134] Specifically, the UV absorption of the sample at 215 nm and 225 nm was measured, and the protein concentration was calculated according to the empirical formula C (μg / mL) = 144 × (A215 - A225), noting that detection is required when A215 < 1.5. The principle of this method is as follows: It measures the characteristic absorption of peptide bonds in the far UV, is not affected by chromophore content, has few disturbing substances, is easy to operate, and is suitable for detecting human collagen and its analogs that do not react with Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition. Humana Press. pp. 43-45.) After protein concentration detection, the concentration of all target proteins was adjusted to 0.5 mg / mL with PBS.

[0135] (2) Sample preparation: The sample stock solution was used directly for the experiment. The positive control bovine type I collagen (PC) was diluted to 1 mg / ml with D-PBS for use. For the negative control, D-PBS buffer (NC) was used.

[0136] (3) Coating: 100 μL each of different concentrations of collagen (C4P7Cf or C4P7Ch) and positive and negative controls was added to each well of the enzyme standard plate, and five duplicate wells were set up for each group. The plates were then incubated overnight at 4°C.

[0137] (4) Blocking: The supernatant was discarded, and 100 μL of 1% BSA (heat-inactivated at 56° C. for 30 minutes) was added, followed by incubation at 37° C. for 60 minutes. The supernatant was discarded, and the plate was washed three times with D-PBS solution.

[0138] (5) Cell inoculation: 10 well-cultured cells resuspended in D-PBS were inoculated into each well. 5 3T3 / NIH cells were added and incubated for 120 minutes at 37° C. Each well was washed three times with D-PBS solution.

[0139] (6) Detection: OD 300 using a CCK8 detection kit (manufacturer: Beyotime, product catalog number: C0038). 450 The absorbance at 100 nm was detected. The degree of cell adhesion was calculated using the following formula. The cell adhesion rate can reflect the cell adhesion ability of collagen. The higher the cell adhesion ability, the better the external environment can be provided to cells in a short time, which helps cell adhesion.

number

[0140] (7) Statistical analysis: The statistical difference between the target recombinant humanized collagen and the negative control was analyzed using a two-tailed t-test, where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0141] The results are shown in Figures 11 and 12. Compared with the D-PBS group, the positive control clearly has the effect of promoting cell adhesion, and the recombinant humanized collagens C4P7Cf and C4P7Ch also have the effect of promoting cell adhesion.

[0142] While the present invention has been described with reference to illustrative embodiments, those skilled in the art will recognize that various other modifications, omissions, and / or additions may be made, and that elements of the described embodiments may be substituted with substantially equivalent elements, without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is not intended that the specification limit the invention to the particular embodiments disclosed for carrying out this invention, but rather, the invention is intended to cover all embodiments falling within the scope of the appended claims.

Claims

1. A recombinant collagen comprising one or more repeat units, the repeat units being linked directly or via a linker, the repeat units comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or 28 or a variant thereof, the variant being either (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence; Preferably, the plurality of repeat units is 2 to 50 repeat units, for example, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 4 to 10, or 6 to 10 repeat units; Preferably, the linker comprises one or more amino acid residues, for example 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 amino acid residues; Preferably, the mutation is selected from a substitution, addition, insertion or deletion, Preferably, the substitutions are conservative amino acid substitutions, Preferably, the recombinant collagen is recombinant humanized collagen, preferably recombinant type IV collagen, preferably recombinant human type IV collagen or recombinant type IV humanized collagen; Preferably, the recombinant collagen has cell adhesion activity, Preferably, the variant of SEQ ID NO: 1 comprises the following mutations: an addition of GFPGFP (SEQ ID NO: 34) to the N-terminus of the amino acid sequence of SEQ ID NO: 1 or a truncation of the N-terminal fragment of SEQ ID NO: 34 by 1 to 5 amino acid residues in length; and / or an addition of GFMGPPGPQGQPGLP (SEQ ID NO: 35) to the C-terminus of the amino acid sequence of SEQ ID NO: 1 or a truncation of the C-terminal fragment of SEQ ID NO: 35 by 1 to 14 amino acid residues in length; or a truncation of the C-terminus of the amino acid sequence of SEQ ID NO: 1 by 1 to 5 amino acid residues in length; Preferably, the variant of SEQ ID NO: 28 comprises a mutation in which Glpgtp (SEQ ID NO: 36) is added to the N-terminus of the amino acid sequence of SEQ ID NO: 28 or a fragment of 1 to 5 amino acid residues in length is truncated to the N-terminus thereof; Preferably, the variant of SEQ ID NO: 1 is selected from SEQ ID NO: 4, 7, 10, 13, 16, 19, 22 or 31, and / or the variant of SEQ ID NO: 28 is SEQ ID NO:

25.

2. The present invention relates to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or a variant thereof, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence; Preferably, the mutation is selected from a substitution, addition, insertion or deletion, The recombinant collagen of claim 1, wherein the substitutions are preferably conservative amino acid substitutions.

3. Encoding the recombinant collagen of claim 1 or 2, Preferably, it comprises a codon-optimized nucleotide sequence, Preferably, the nucleotide sequence is a nucleotide sequence that is codon-optimized for expression in a eukaryotic or prokaryotic host cell, such as yeast or E. coli; Preferably, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 or 33.

4. A vector comprising the nucleic acid of claim 3, Preferably, the nucleic acid comprises an expression control element, a purification tag nucleotide and / or a leader sequence nucleotide operably linked to the nucleic acid, Preferably, the expression control element is selected from a promoter, a terminator, or an enhancer; Preferably, the purification tag is selected from a His tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag; Preferably, the vector is an expression vector or a cloning vector, preferably pET-28a(+).

5. A host cell comprising a nucleic acid according to claim 3 or a vector according to claim 4, preferably a eukaryotic or prokaryotic cell, preferably wherein the eukaryotic cell is a yeast cell, an animal cell and / or an insect cell, and / or wherein the prokaryotic cell is an E. coli cell, e.g. E. coli BL21.

6. The composition comprises one or more of the recombinant collagen of claim 1 or 2, the nucleic acid of claim 3, the vector of claim 4, and the host cell of claim 5, and is preferably a kit, and is preferably one or more of a bio-covering material, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, an obstetric and gynecological bio-material, a nerve repair and regeneration material, a liver tissue material, and a vascular repair and regeneration material, a 3D printed artificial organ bio-material, a cosmetic raw material, a medicinal auxiliary material, and a food additive, and is preferably a surface composition, an injectable composition, or an oral composition, and is preferably a composition in the form of a solution, a freeze-dried powder, a gel, a sponge, or a fiber.

7. Use of the recombinant collagen according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4, the host cell according to claim 5 and / or the composition according to claim 6 in one or more of the following: a bio-covering material, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, an obstetric and gynecological biological material, a nerve repair and regeneration material, a liver tissue material and a vascular repair and regeneration material, a 3D printed artificial organ biological material, a cosmetic raw material, a medicinal auxiliary material and a food additive.

8. A method for promoting cell adhesion, comprising the step of contacting cells with a recombinant collagen according to claim 1 or 2, a nucleic acid according to claim 3, a vector according to claim 4, a host cell according to claim 5 and / or a composition according to claim 6, wherein the cells are preferably animal cells, preferably mammalian cells, preferably human cells.

9. A cosmetic method comprising the step of administering the recombinant collagen of claim 1 or 2 to a subject, preferably by topical administration, oral administration or injection, and preferably the subject is a human.

10. (1) culturing the host cell of claim 5 under suitable culture conditions; (2) harvesting the host cells and / or medium containing the recombinant collagen; (3) purifying the recombinant collagen; Preferably, the host cell is an E. coli cell, preferably an E. coli BL21(DE3) cell; 3. The method for producing recombinant collagen according to claim 1 or 2, wherein step (3) preferably comprises purifying the recombinant collagen on a purification column, e.g., a nickel column, and / or cleaving the recombinant collagen with a collagen tool enzyme.

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