Method for manufacturing Type IV collagen, a biosynthesized human structural material

Abstract

This specification provides a method for biosynthesizing type IV collagen, a structural material of the human body. This specification provides recombinant collagen comprising sequences represented by SEQ ID NO: 1, etc. The recombinant collagen of this application exhibits excellent cell adhesion activity. The method of this application overcomes the disadvantages of existing technologies by producing recombinant collagen using genetic engineering technology.

Description

Technology Field

[0001] This application claims priority to Chinese invention patent application No. 202311391711.1, titled "Method for producing biosynthetic human structural material type IV collagen," filed on October 25, 2023.

[0002] Technology field

[0003] The present disclosure relates to the field of biomedicine and relates to recombinant type IV humanized collagen, and methods for manufacturing and uses thereof. Background Technology

[0004] Collagen (abbreviated as COL) is a helical fibrous functional protein composed of three peptide chains. As a major component of the extracellular matrix, collagen is abundant and widely distributed. In the human body, collagen accounts for 25 to 30% of total protein content and is primarily found in the skin, tendons, and bones. Collagen plays a crucial role in protecting and connecting various tissues and exerts important physiological functions within the body. Type IV collagen is a key protein that, along with laminin, constitutes the basement membrane. Its structural unit is a trimer with a helical structure composed of three α-chains. While the subunits of the trimer vary by tissue, in numerous tissues such as the liver, the trimer consists of two α1 chains and one α2 chain. Type IV collagen molecules possess unique structures 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.

[0005] In recent years, collagen has garnered significant attention as a research hotspot due to its favorable biocompatibility, degradability, low antigenicity, and unique biological structure. It is widely applied in various fields, including biomedicine, cosmetics, health products, and food.

[0006] There are 28 types of collagen in the human body, which can be classified into two main categories—fibrous collagen and non-fibrous collagen—depending on whether their structure is fibrous. Fibrous collagen primarily serves as a cellular scaffold to fix cell positions and act as an anchor, while simultaneously providing tensile strength and stiffness to tissues. On the other hand, non-fibrous collagen is subdivided into basal collagen, short-chain collagen, and transmembrane collagen, each performing different functions.

[0007] Conventional methods for manufacturing collagen involve purifying animal tissues through acid hydrolysis, alkaline hydrolysis, or enzymatic hydrolysis to extract collagen derivatives. However, collagen obtained by these methods often loses its inherent biological activity, is poorly soluble in water, struggles to bind to the human body, presents difficulties in avoiding risks of viral infection and sensitization, and cannot fully perform its intended functions. While some research institutions have attempted to induce human collagen in vitro using existing recombinant expression methods, this approach is costly, time-consuming, and unsuitable for mass production.

[0008] Therefore, there is an urgent market demand for collagen materials that possess excellent biomaterial properties, have amino acid sequences very similar to the human body, and can be mass-produced within industrial systems.

[0009] The inventors performed a large-scale screening of functional regions of human type IV collagen and identified 11 recombinant collagens. These recombinant collagens are Escherichia coli ( Escherichia coli It can be purified after being expressed in ). In addition, the inventors discovered that this recombinant collagen exhibits a higher yield, higher purity collagen after fine purification, and higher activity compared to a positive control (bovine type I collagen) in a cell adhesion activity assay.

[0010] In one aspect, the present disclosure provides a recombinant collagen comprising one or more repeat units linked via a linker or directly, wherein the repeat unit comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (Gakgdkgskgevgfpglagspgipgskgeq) or SEQ ID NO: 28 (Gptgpagqkgepgsdgipgsagekgepglp) or a variant thereof, said variant being (1) an amino acid sequence having one or more amino acid residue mutations in said 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 with said amino acid sequence.

[0011] In one embodiment, the number of repeating units is 2 to 50 repeating 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 repeating units. For example, the number of repeating units may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, or 50, or a range in between.

[0012] In one embodiment, the linker comprises one or more amino acid residues, for example, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid residues.

[0013] In one embodiment, mutations are selected from a group consisting of substitutions, additions, insertions, or deletions.

[0014] In one embodiment, the substitution is a conservative amino acid substitution.

[0015] In one embodiment, the recombinant collagen is recombinant human type IV collagen or recombinant type IV humanized collagen.

[0016] In one embodiment, recombinant collagen has cell adhesion activity.

[0017] In one embodiment, a variant of SEQ NO: 1 comprises the following mutations: adding Gfpgfp (SEQ NO: 34) or an N-terminal truncated fragment of SEQ NO: 34 of length 1 to 5 amino acids to the N-terminus of the amino acid sequence of SEQ NO: 1, and / or adding GFMGPPGPQGQPGLP (SEQ NO: 35) or a C-terminal truncated fragment of SEQ NO: 35 of length 1 to 14 amino acids to the C-terminus of the amino acid sequence of SEQ NO: 1, or truncating the amino acid sequence of SEQ NO: 1 at the C-terminus by 1 to 5 amino acid residues.

[0018] In one embodiment, a variant of SEQ ID NO: 28 comprises the following mutation: addition of Glpgtp (SEQ ID NO: 36) or an N-terminal truncated fragment of SEQ ID NO: 36 of length 1 to 5 amino acid residues to the N-terminus of the amino acid sequence of SEQ ID NO: 28.

[0019] In one embodiment, variants of sequence number: 1 are sequence number: 4 (Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq), sequence number: 7 (GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP), sequence number: 10 (Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq), sequence number: 13 (Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm), sequence number: 16 (Gfpgfpgakgdkgskgevgfpglagspgipgskgeq), sequence number: 19 (Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp), sequence number: It is selected from the group consisting of 22 (Gakgdkgskgevgfpglagspgipgskgeqgfm), or sequence number: 31 (Gfpgfpgakgdkgskgevgfpglagspgipgsk).

[0020] In one embodiment, a variant of sequence number: 28 is sequence number: 25 (Glpgtpgptgpagqkgepgsdgipgsagekgepglp).

[0021] In one embodiment, the recombinant collagen comprises an amino acid sequence selected from the group consisting of sequence numbers: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 and 32 or a variant thereof, wherein the variant is (1) an amino acid sequence having one or more amino acid residue mutations 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 with the amino acid sequence.

[0022] In one embodiment, the mutation is selected from a group consisting of substitutions, additions, insertions, or deletions. In one embodiment, the substitution is a conservative amino acid substitution.

[0023] In another aspect, a nucleic acid encoding the recombinant collagen described herein is provided. In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. In one embodiment, the nucleotide sequence is a codon-optimized nucleotide sequence for expression in a eukaryotic host cell, such as yeast or E. coli, or a prokaryotic host cell. In one embodiment, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ No. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33.

[0024] In another aspect, a vector comprising the nucleic acid described herein is provided. In one embodiment, the vector comprises an expression control element operatively linked to the nucleic acid, a nucleotide of a purification tag, and / or a nucleotide of a leader sequence. In one embodiment, the expression control element is selected from the group consisting of a promoter, a terminator, or an enhancer. In one embodiment, the purification tag is selected from the group consisting of 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 include an HIS, thrombin, or T7 protein tag at the N-terminus and a His tag at the C-terminus. In this application, the recombinant collagen may include an enzymatic cleavage site at the N-terminus to facilitate purification.

[0025] In another aspect, a host cell comprising the nucleic acid or vector described herein is provided. 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 Escherichia coli cell, e.g., E. coli BL21.

[0026] In another aspect, a composition comprising one or more of the recombinant collagen, nucleic acids, vectors, and host cells described herein is provided. In one embodiment, the composition is a kit. In one embodiment, the composition is one or more of a biological dressing, a human bionic material, a plastic surgery material or beauty material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, an obstetrics and gynecology biomaterial, a nerve repair and regeneration material, a liver tissue material and a blood vessel repair and regeneration material, a 3D-printed artificial organ biomaterial, a cosmetic ingredient, a pharmaceutical excipient, and a food additive. In one embodiment, the composition is a composition for topical use, injectable use, or oral use. In one embodiment, the composition is a composition in the form of a solution, a freeze-dried powder, a gel, a sponge, or a fiber.

[0027] In another aspect, the recombinant collagen, nucleic acid, vector, host cell and / or composition of the present application is provided for use in one or more of biological dressings, human bioengineering materials, plastic surgery or cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetrics and gynecology biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

[0028] In another aspect, a method for promoting cell adhesion is provided, comprising the step of bringing the recombinant collagen, nucleic acid, vector, host cell and / or composition of the present application into contact with a cell. In one embodiment, the cell is an animal cell. The animal cell may be a mammalian cell or a human cell. It is provided to use the recombinant collagen, nucleic acid, vector, host cell and / or composition of the present application in the manufacture of a kit for promoting cell adhesion.

[0029] In another aspect, a beauty method is provided comprising administering the recombinant collagen described in this specification to a subject, preferably the administration is topical, oral, or injection, and preferably the subject is a human.

[0030] In another aspect, a method for producing recombinant collagen as described herein is provided, comprising the following steps:

[0031] (1) A step of incubating the host cells described in this specification under appropriate culture conditions;

[0032] (2) A step of harvesting host cells and / or culture medium containing recombinant collagen; and

[0033] (3) Step of purifying recombinant collagen.

[0034] In one embodiment, the host cell is an E. coli cell, preferably an E. coli BL21(DE3) cell.

[0035] In one embodiment, step (1) includes culturing E. coli cells in LB medium and inducing expression with IPTG.

[0036] In one embodiment, step (2) comprises harvesting E. coli cells, resuspending them in an equilibrium working solution, homogenizing the E. coli cells (preferably 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 at pH 7-9.

[0037] In one embodiment, step (3) includes crude purification, enzymatic digestion, fine purification and / or reverse nickel column purification.

[0038] In one embodiment, the crude purification comprises purifying the supernatant with a Ni-agarose resin column to obtain an eluent containing a target protein, wherein the eluent preferably contains 100-500 mM sodium chloride, 10-50 mM Tris, and 100-500 mM imidazole at pH 7-9.

[0039] In one embodiment, the fine purification comprises gradient eluting an eluent containing a target protein using a strong anion exchange chromatography column, preferably the gradient elution comprises flowing a 0-15% solution B for 1-5 minutes and holding for 3 times the column volume, flowing a 15-30% solution B for 1-5 minutes and holding for 3 times the column volume, flowing a 30-50% solution B for 1-5 minutes and holding for 3 times the column volume, and flowing a 50-100% solution B for 1-5 minutes and holding for 3 times the column volume; the solution B comprises 10-50 mM Tris and 0.5-5 M sodium chloride at pH 7-9. In one embodiment, step (3) comprises purifying the recombinant collagen using a purification column such as a nickel column and / or cleaving the recombinant collagen using a collagen-processing enzyme.

[0040] The advantages of the present disclosure include the following:

[0041] 1. The recombinant collagen of the present disclosure is derived from human type IV collagen and is recombinant type IV humanized collagen;

[0042] 2. The recombinant collagen of the present disclosure is suitable for production by E. coli and can be isolated and purified;

[0043] 3. The recombinant collagen of the present disclosure is produced in high yield and is suitable for subsequent purification (Ni column or strong anion column purification);

[0044] 4. The recombinant collagen of the present disclosure has cell adhesion activity. The recombinant collagen of the present disclosure (e.g., C4P7Ch) has higher cell adhesion activity compared to a positive control. Brief explanation of the drawing

[0045] Figure 1 shows the electrophoretic detection results of C4P7Ca. Figure 2 shows the electrophoretic detection results of C4P7Cb. Figure 3 shows the electrophoretic detection results of C4P7Cc. Figure 4 shows the electrophoretic detection results of C4P7Cd. Figure 5 shows the electrophoretic detection results of C4P7Ce. Figure 6 shows the electrophoretic detection results of C4P7Cf. Figure 7 shows the electrophoretic detection results of C4P7Cg. Figure 8 shows the electrophoretic detection results of C4P7Ch. Figure 9 shows the electrophoretic detection results of C4P7Ea. Figure 10 shows the electrophoretic detection results of C4P7Eb. Figure 11 shows the electrophoretic detection results of C4P7Ec. Figure 12 shows the effect of collagen C4P7Ch on cell adhesion. Figure 13 shows the effect of collagen C4P7Cf on cell adhesion. Specific details for implementing the invention

[0046] As used herein, “recombinant collagen” means an amino acid sequence encoded by a specific gene that is engineered or modified and produced by recombinant DNA technology, or a fragment thereof, or a combination of such functional amino acid sequence fragments. The gene encoding sequence or amino acid sequence of recombinant collagen may have low homology to the gene encoding sequence or amino acid sequence of human collagen. “Recombinant humanized collagen” means a combination comprising a full-length or partial amino acid sequence fragment encoded by a specific type of gene of human collagen produced using recombinant DNA technology, or functional fragments of human collagen. As used herein, recombinant collagen comprises one or more repeat units. The repeat units may be derived from human type IV collagen. Thus, recombinant collagen may be recombinant type IV humanized collagen. Multiple repeat units may be connected via a linker, and the linker may be natural amino acid residue(s) of the repeat unit of human type IV collagen, for example, 1 to 50 amino acid residues. The repeat unit may be sequence number: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, or 33. The recombinant collagen may be sequence number: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32.

[0047] As used herein, the term “variant” means a recombinant collagen having cell adhesion activity comprising a modification / mutation (i.e., substitution, addition, insertion, and / or deletion) at one or more positions. Substitution means changing an amino acid occupying a specific position to another amino acid, deletion means removing an amino acid occupying a specific position, and insertion means adding an amino acid immediately following and adjacent to an amino acid occupying a specific position. Addition means 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 in which one or more amino acid residues of sequence number: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, or 33 are altered or mutated (i.e., substituted, added, inserted, and / or deleted). Variants of recombinant collagen may be sequences in which one or more amino acid residues of sequence number: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 or 32 are altered or mutated (i.e., substituted, added, inserted and / or deleted).

[0048] For example, a variant of the repeat unit of SEQ ID NO: 1 may be a variant comprising the following mutations: Gfpgfp (SEQ ID NO: 34) or an N-terminal truncated fragment of SEQ ID NO: 34 (e.g., 1 to 5 amino acid residues at the N-terminus of SEQ ID NO: 34, e.g., 1, 2, 3, 4, or 5 amino acid residues cleaved to correspond to fpgfp (SEQ ID NO: 58), pgfp (SEQ ID NO: 59), gfp, fp, and p residues, respectively) added to the N-terminus of the amino acid sequence of SEQ ID NO: 1 and / or GFMGPPGPQGQPGLP (SEQ ID NO: 35) or a C-terminal truncated fragment of SEQ ID NO: 35 (1 to 14 amino acid residues at the C-terminus of SEQ ID NO: 35, e.g., 1, 2, 3, 4, 5, 6, 7, Addition of 8, 9, 10, 11, 12, 13, or 14 amino acid residues (cleaved) to the C-terminus of the amino acid sequence of SEQ No. 1. Variants of the repeat unit of SEQ No. 1 may be variants containing the following mutations: Gfpgfp (SEQ No. 34) or its N-terminal cleaved fragment (e.g., 1, 2, 3, 4, or 5 amino acid residues cleaved at the N-terminus of SEQ No. 34) to the N-terminus of the amino acid sequence of SEQ No. 1, and / or cleavage of 1-5 amino acid residues, e.g., 1, 2, or 3 amino acid residues, at the C-terminus of the amino acid sequence of SEQ No. 1.

[0049] A variant of the repeat unit of SEQ No. 28 may be a variant containing the following mutation: Glpgtp (SEQ No. 36) or an N-terminal truncated fragment thereof (e.g., 1 to 5 amino acid residues at the N-terminus of SEQ No. 36, e.g., 1, 2, 3, 4, or 5 amino acid residues truncated) added to the N-terminus of the amino acid sequence of SEQ No. 28.

[0050] In the context of the present disclosure, conservative substitutions may be defined as substitutions within one or more amino acid classes reflected in one or more of the following tables.

[0051] Conservative classes of amino acid residues:

[0052] Acid residues D and E

[0053] Basic residues K, R, and H

[0054] Hydrophilic uncharged residues S, T, N, and Q

[0055] Aliphatic uncharged residues G, A, V, L and I

[0056] Nonpolar uncharged residues C, M, and P

[0057] Aromatic residues F, Y, and W.

[0058] Physical and Functional Classification of Substitute Amino Acid Residues:

[0059] Alcohol-containing residues S and T

[0060] Aliphatic residues I, L, V and M

[0061] Cycloalkenyl-related residues F, H, W and Y

[0062] Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y

[0063] Negative charge residues D and E

[0064] Polar residues C, D, E, H, K, N, Q, R, S and T

[0065] Positively charged residues H, K, and R

[0066] Small residues A, C, D, G, N, P, S, T and V

[0067] Minimum remaining A, G and S

[0068] Residues A, C, D, E, G, H, K, N, Q, R, S, P, and T involved in turn formation

[0069] Flexible residues Q, T, K, S, G, P, D, E and R.

[0070] As used herein, "cell adhesion" refers to adhesion between a cell and collagen. Collagen, such as the recombinant collagen described herein, can facilitate adhesion between a cell and a container in which the cell is cultured.

[0071] As used herein, the term "expression" includes all steps related to the production of recombinant collagen, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0072] As used herein, the term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a recombinant collagen protein, which is operatively linked to a control sequence provided for its expression.

[0073] As used herein, the term "host cell" refers to any cell type suitable for transformation, transfection, transduction, etc. using a nucleic acid construct or expression vector containing the polynucleotide of the present disclosure. The term "host cell" includes all offspring of a parent cell that are not identical to the parent cell due to mutations occurring during replication.

[0074] As used herein, the term “nucleic acid” means a single-stranded or double-stranded nucleic acid molecule isolated from a naturally occurring gene, modified in a manner not found in nature to include nucleic acid segments, or synthesized, and the nucleic acid molecule may include one or more control sequences. The nucleic acid may be sequence number: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. The nucleic acid may be a codon-optimized nucleic acid, e.g., a codon-optimized nucleic acid for expression in E. coli cells.

[0075] The term "operationally linked" refers to a configuration in which a control sequence is positioned at an appropriate location relative to the encoding sequence of a polynucleotide so that the control sequence induces the expression of the encoding sequence.

[0076] The degree of association between two amino acid sequences or two nucleotide sequences is described by a parameter called "sequence identity." For the purposes of this disclosure, sequence identity between two amino acids is defined in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends Genet . 16: 276-277)'s Needle program (Needleman and Wunsch, 1970, J. Mol. Biol. It is determined by the Needleman-Wunsch algorithm implemented by 48: 443-453) (version 5.0.0 or higher is recommended). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) permutation matrix. The Needle output labeled "Longest Identity" (obtained using the desimplification option) is used as the identity percentage and is calculated as follows:

[0077] (Identical residues x 100) / (Alignment length - Total number of gaps in alignment)

[0078] For the purposes of this disclosure, sequence identity between two deoxyribonucleotides is determined by the Needleman-Wunsch algorithm implemented by the Needle program (Needleman and Wunsch, 1970) of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000) (use of version 5.0.0 or higher is recommended). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "Longest Identity" (obtained using the desimplification option) is used as the identity percentage and is calculated as follows:

[0079] (Identical deoxyribonucleotides x 100) / (Alignment length - Total number of gaps in alignment)

[0080] Recombinant collagen

[0081] The present disclosure provides a recombinant collagen comprising one or more repeat units connected via a linker or directly, wherein the repeat unit comprises an amino acid sequence selected from SEQ ID NOs: 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 in the amino acid sequence of SEQ No. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31 or 33 are mutated, 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 with the amino acid sequence of SEQ No. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31 or 33. In relation to the recombinant collagen described herein, the mutation may be selected from the group consisting of substitutions, additions, insertions or deletions. Preferably, the substitution is a conservative amino acid substitution.

[0082] The recombinant collagen described in this specification may comprise a plurality of repeating units, for example, 2 to 50 repeating units, for example, 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, or 50 repeating units.

[0083] The linker of the recombinant collagen described herein may include one or more amino acid residues, for example, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid residues.

[0084] Recombinant collagen is recombinant human type IV collagen or recombinant type IV humanized collagen, preferably having cell adhesion activity. The recombinant collagen described herein may be derived from humans and is therefore recombinant type IV humanized collagen.

[0085] The recombinant collagen described herein may also comprise an amino acid sequence selected from the group consisting of sequence numbers: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29 and 32, or a variant thereof, wherein the variant is (1) an amino acid sequence having one or more amino acid residue mutations 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 with the amino acid sequence.

[0086] nucleic acid composition

[0087] The present disclosure also relates to a nucleic acid construct comprising the nucleic acid of the present disclosure, wherein the nucleic acid construct is operatively linked to one or more control sequences to induce expression of an encoding sequence in a suitable host cell under conditions compatible with the control sequence. A vector may comprise the nucleic acid construct.

[0088] Nucleic acids can be modified in various ways to enable the expression of recombinant collagen. Depending on the expression vector, it may be desirable or necessary to modify the nucleic acid before insertion into the vector. Techniques for modifying nucleic acids using recombinant DNA methods are well known in the field.

[0089] The control sequence may be a promoter, that is, recognized by a host cell for the expression of a polynucleotide encoding the recombinant collagen of the present disclosure. The promoter comprises a transcriptional regulatory sequence that mediates the expression of the recombinant collagen. The promoter may be any nucleic acid exhibiting transcriptional activity in the host cell, including modified, cleaved, or hybrid promoters, and may be obtained from a gene encoding the extracellular or intracellular recombinant collagen that is homologous or heterologous to the host cell.

[0090] An example of a promoter suitable for inducing transcription of the vector or nucleic acid construct of the present disclosure in bacterial host cells is a promoter obtained from the following: Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) Alpha-amylase gene( amyQ ), Bacillus licheniformis( Bacillus licheniformis ) Alpha-amylase gene( amyL ), Bacillus licheniformis penicillinase gene( penP ), Bacillus stearothermophilus( Bacillus stearothermophilus ) Maltoamylase gene( amyM ), Bacillus subtilis( Bacillus subtilis ) Fructan sucrase gene( sacB ), Bacillus subtilis xylA and xylB Gene, Bacillus thuringiensis ( Bacillus thuringiensis ) cryIIIA Gene, E. coli lac operon, and E. coli trc promoter.

[0091] In yeast hosts, useful promoters are obtained from the following gene: Saccharomyces cerevisiae ( Saccharomyces cerevisiae) Enolase (ENO-1), galactokinase (GAL1) for Saccharomyces cerevisiae, alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP) for Saccharomyces cerevisiae, triosephosphate isomerase (TPI) for Saccharomyces cerevisiae, metallothionein (CUP1) for Saccharomyces cerevisiae, and 3-phosphoglycerate kinase for Saccharomyces cerevisiae.

[0092] The control sequence may also be a transcription terminator recognized by the host cell to terminate transcription. The terminator may be operatively linked to the 3' end of a polynucleotide encoding recombinant collagen. In this disclosure, any terminator functioning in the host cell may be used.

[0093] The preferred terminator for bacterial host cells is Bacillus clausii ( Bacillus clausii ) Alkaline protease( aprH ), Bacillus licheniformis alpha-amylase( amyL ) and this. E. coli ribosomal RNA ( rrnB It is obtained from the gene of ).

[0094] The preferred terminators in yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other terminators useful in yeast host cells are described in Romanos et al. (1992).

[0095] The control sequence can also be an mRNA stabilizer region located downstream of the promoter and upstream of the gene's encoding sequence that increases gene expression.

[0096] A suitable mRNA stabilization region can be obtained, for example, from the following gene: Bacillus thuringiensis cryIIIAGene (WO 94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

[0097] The control sequence can also be a leader sequence, that is, a non-translating region of mRNA important for translation in the host cell. The leader sequence is operatively linked to the 5' end of a polynucleotide encoding recombinant collagen. Any leader sequence that functions in the host cell can be used.

[0098] Reader sequences suitable for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0099] The control sequence may be a polyadenylation sequence, which operatively links to the 3' end of a polynucleotide and is recognized by the host cell as a signal to add polyadenylate residues to the transcribed mRNA upon transcription. Any polyadenylation sequence that functions in the host cell can be used.

[0100] Polyadenylation sequences useful for yeast host cells are from the literature (reference: Guo and Sherman, 1995, Mol. Cellular Biol. 15: It is explained in 5983-5990).

[0101] The control sequence may also be a signal peptide encoding region that encodes a signal peptide linked to the N-terminus of the recombinant collagen and directs the recombinant collagen into the cell's secretory pathway. The 5'-terminus of the polynucleotide encoding sequence may contain itself a signal peptide encoding sequence that is naturally linked as an open reading frame to the segment of the encoding sequence encoding the recombinant collagen. Alternatively, the 5'-terminus of the encoding sequence may contain a signal peptide encoding sequence that is heterogeneous to the encoding sequence. If the encoding sequence does not naturally contain a signal peptide encoding sequence, an exogenous signal peptide encoding sequence may be required. Alternatively, the natural signal peptide encoding sequence may simply be replaced with an exogenous signal peptide encoding sequence to enhance the secretion of recombinant collagen. However, any signal peptide encoding sequence that directs the expressed recombinant collagen into the host cell's secretory pathway may be used.

[0102] The signal peptide encoding sequences effective against bacterial host cells are signal peptide encoding sequences obtained from the following genes: Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, and Bacillus stearothermophilus neutral protease ( nprT , nprS , nprM ) and Bacillus subtilis prsA . Additional signal peptides are referenced in the literature (see Simonen and Palva, 1993, Microbiological Reviews 57: It is explained in 109-137).

[0103] Signal peptides useful to yeast host cells are obtained from the following genes: Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Sequences encoding other useful signal peptides are described in the literature (see Romanos et al., Yeast 8: 423-488).

[0104] Expression vector

[0105] The present disclosure also relates to a recombinant expression vector comprising the nucleic acid of the present disclosure, a promoter, a transcription and translation termination signal. A recombinant expression vector may be constructed by linking a nucleic acid and a regulatory sequence, and the vector may include one or more convenient restriction sites for inserting or replacing a polynucleotide encoding recombinant collagen at the corresponding site. Alternatively, the polynucleotide may be expressed by inserting the nucleic acid, or by inserting a nucleic acid construct containing the nucleic acid into a vector suitable for expression. When the expression vector is constructed, the encoding sequence is positioned within the vector to be operatively linked to a suitable control sequence for expression.

[0106] A recombinant expression vector can be any vector (e.g., plasmids or viruses) that can be conveniently applied to recombinant DNA procedures and enable the expression of polynucleotides. The selection of the vector will generally depend on the compatibility of the vector with the host cell into which it will be introduced. The vector can be a linear chain or a closed circular plasmid.

[0107] The vector may be an autonomously replicating vector, that is, a vector existing as an extrachromosomal entity that replicates independently of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may include means to ensure self-replication. Alternatively, the vector may be one that integrates into the genome upon introduction into a host cell and replicates along with one or more integrated chromosomes. Additionally, a separate vector or plasmid or two or more vectors or plasmids containing the entire DNA to be introduced into the host cell genome may be used, or a transposon may be used.

[0108] The vector preferably includes one or more selectable markers so that the transformed cells, transfected cells, transduced cells, etc., can be conveniently selected. The selectable marker is a gene whose product provides resistance to biocides, resistance to viruses, resistance to heavy metals, or a transition from prototrophy to auxotrophy.

[0109] Examples of bacterial screening markers include Bacillus licheniformis or Bacillus subtilis dal There are genes, or markers that confer antibiotic resistance, such as resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline. Markers suitable for yeast host cells include, but are not limited to, the following: ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0110] The screening marker may be a dual screening marker system as described in WO 2010 / 039889. In one aspect, the dual screening marker is hph-tk It is a dual screening marker system.

[0111] The vector may include elements that integrate the vector into the host cell's genome or enable autonomous replication of the vector within the cell independently of the genome.

[0112] The vector may rely on a polynucleotide sequence encoding recombinant collagen to be incorporated into the host cell genome, or any other element of the vector that enables integration into the genome via homologous or non-homologous recombination. Alternatively, the vector may include additional polynucleotides to induce integration via homologous recombination at specific location(s) of chromosomes within the host cell genome. To increase the likelihood of integration at the precise location, the integration element must contain a sufficient number of nucleic acids having a high level of sequence identity with the corresponding target sequence to increase the likelihood of homologous recombination, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, or 800 to 10,000 base pairs. The integration element may be any sequence that has homology with the target sequence within the host cell genome. Furthermore, the integration element may be a non-encoding polynucleotide or an encoding polynucleotide. In another aspect, the vector may be incorporated into the host cell genome via non-homologous recombination.

[0113] Vectors may additionally include an origin of replication to enable autonomous replication, thereby allowing the vector to replicate autonomously within the corresponding host cell. The origin of replication can be any plasmid replicator that functions to mediate autonomous replication within the cell. The terms "origin of replication" or "plasmid replicator" refer to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0114] Examples of bacterial replication origins include the replication origins of plasmids pBR322, pUC19, pACYC177, and pACYC184 that allow replication in E. coli, and the replication origins of plasmids pUB110, pE194, pTA1060, and pAMβ1 that allow replication in Bacillus.

[0115] Examples of replication origins available in yeast host cells include the 2-micron replication origin, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0116] The production of recombinant collagen can be increased by inserting two or more copies of the polynucleotide of the present disclosure into a host cell. To increase the number of copies of the polynucleotide, at least one additional copy of the sequence can be incorporated into the host cell genome or an amplifiable screening marker gene can be included along with the polynucleotide, in which case cells containing an amplified copy of the screening marker gene and a correspondingly added copy of the polynucleotide can be selected by culturing the cells in an environment where a suitable screening agent is present.

[0117] The procedure for linking the elements described above to construct the recombinant expression vector of the present disclosure is well known to those skilled in the art (see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)).

[0118] host cell

[0119] The present disclosure also relates to a recombinant host cell comprising the polynucleotide of the present disclosure, said polynucleotide being operatively linked to one or more control sequences that induce the production of the recombinant collagen of the present disclosure. An construct or vector comprising the polynucleotide is introduced into a host cell such that the construct or vector is maintained as a chromosomal conjugate or an extrachromosomal vector that replicates autonomously, as previously described. The term "host cell" includes all offspring of a parent cell that are not identical to the parent cell due to mutations occurring during replication. The selection of the host cell will depend primarily on the gene encoding the recombinant collagen and its source.

[0120] The host cell may be any cell useful for the recombinant production of the recombinant collagen of the present disclosure, for example, a prokaryotic cell or a eukaryotic cell.

[0121] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include Bacillus, Clostridium ( Clostridium ), Enterococcus( Enterococcus ), Geobacillus( Geobacillus ), Lactobacillus( Lactobacillus ), Lactococcus( Lactococcus ), Oceanobacillus( Oceanobacillus ), Staphylococcus( Staphylococcus ), Streptococcus( Streptococcus ), and Streptomyces ( Streptomyces ...is included, but not limited to. Gram-negative bacteria include Campylobacter ( Campylobacter ), Escherichia coli, Flavobacterium( Flavobacterium ), Fusobacterium( Fusobacterium ), Helicobacter ( Helicobacter ), Iliobacter( Ilyobacter ), Neisseria( Neisseria ), Pseudomonas ( Pseudomonas ), Salmonella( Salmonella ), and ureaplasma( Ureaplasma ) is included, but is not limited to.

[0122] The host cell may be a cell of a eukaryotic organism such as a mammal, insect, plant, or fungus. The plant cells described herein do not include plant cells that can be regenerated into a whole plant. The animal cells do not include cells that can form an animal body.

[0123] The host cell is a basidiomycete ( Basidiomycota ), choroid fungi ( Chytridiomycota ), Zygomycetes( Zygomycota ), oomycetes( Oomycota It can be a fungal cell such as ), etc. The host cell is an ascospore-forming yeast ( ascosporogenous yeast)(Endomycetales( Endomycetales )), Basidiomycete yeast( basidiosporogenous yeast) and imperfect fungi (Blastomycetes) Blastomycetes It may be a yeast cell, including yeasts belonging to )). The yeast host cell is Candida ( Candida ), Hansenula( Hansenula ), Cluiberomyces( Hansenula ), Peachia( Pichia ), Saccharomyces, Schizocaromyces( Schizosaccharomyces ) or Yarowia( Yarrowia It can be a ) cell, for example, Cluiberomyces lactis( Kluyveromyces lactis ), Saccharomyces carlsbergensis( Saccharomyces carlsbergensis ), Saccharomyces cerevisiae, Saccharomyces diastaticus( Saccharomyces cerevisiae ), Saccharomyces duglaci ( Saccharomyces douglasii ), Saccharomyces cluyberry ( Saccharomyces kluyveri ), Saccharomyces norvensis( Saccharomyces norbensis ), Saccharomyces orbiformis( Saccharomyces oviformis ) or Yarowia Lipolitika( Yarrowia lipolytica It can be a cell.

[0124] Manufacturing method

[0125] The present disclosure also relates to a method for producing recombinant collagen as described herein, the method comprising the following steps:

[0126] (1) A step of incubating the host cells described in this specification under appropriate culture conditions;

[0127] (2) A step of harvesting host cells and / or culture medium containing recombinant collagen; and

[0128] (3) Step of purifying recombinant collagen.

[0129] Host cells are cultured in a nutrient medium suitable for the production of recombinant collagen using methods known in the art. For example, cells may be cultured in shaking flasks or cultured via small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in laboratory or industrial fermenters, wherein the culture is performed in a suitable medium under conditions that allow for the expression and / or isolation of recombinant collagen. The culture is performed using procedures known in the art in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media may be purchased from commercial suppliers or prepared according to disclosed compositions (e.g., the American Type Culture Collection catalog). If recombinant collagen is secreted into the nutrient medium, the recombinant collagen may be recovered directly from the medium. If recombinant collagen is not secreted, the recombinant collagen may be recovered from the cell lysate.

[0130] Recombinant collagen can be detected using methods known in the art that are specific to recombinant collagen. Such detection methods include, but are not limited to, the use of specific antibodies. For example, an adhesion assay may be used to confirm the activity of recombinant collagen.

[0131] Recombinant collagen can be recovered using methods known in the art. For example, recombinant collagen can be recovered from a nutrient medium using conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, recombinant collagen is recovered from a fermentation broth containing recombinant collagen.

[0132] Recombinant collagen can be purified through 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., isoelectric point focusing electrophoresis for preparation), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure recombinant collagen.

[0133] Step (1) may include one or more of the following steps: a step of constructing an expression plasmid, e.g., a step of obtaining a recombinant expression plasmid by inserting an encoding nucleotide sequence into a pET-28a-Trx-His expression vector. The successfully constructed expression plasmid may be transformed into E. coli cells (e.g., E. coli competent cell BL21(DE3)). The specific process is as follows: (1) take the plasmid to be transformed and place it into E. coli competent cell BL21(DE3); (2) place the mixture on ice in an ice bath (e.g., 10 to 60 minutes, e.g., for 30 minutes), then apply heat shock in a water bath (e.g., 40 to 50°C, e.g., 45 to 90 seconds at 42°C), remove the mixture, and place it on ice in an ice bath (e.g., 1 to 5 minutes, e.g., for 2 minutes); (3) Add liquid LB medium and incubate (e.g., 35 to 40°C, e.g., 37°C, 150 to 300 rpm, e.g., 220 rpm for 40 to 80 minutes, e.g., 60 minutes); (4) spread the bacterial solution and collect a single colony. For example, take the bacterial solution and spread it evenly on an LB plate containing ampicillin sodium, and incubate the plate in an incubator at 37°C for 15 to 17 hours until a colony of uniform size grows on the plate.

[0134] Step (2) may include culturing a single colony in LB medium containing antibiotic stock (e.g., at a constant temperature of 35 to 40°C, e.g., 37°C, in a shaker at 150 to 300 rpm, e.g., 220 rpm, for 5 to 10 hours, e.g., 7 hours). Then, the cultured shake flask is cooled to 10 to 20°C, e.g., 16°C, IPTG is added to induce expression for a certain period of time, and then the cells are collected (e.g., by centrifugation).

[0135] Step (3) may include resuspending the bacterial cells in an equilibrium working solution, cooling the bacterial liquid to a temperature of 15°C or lower, performing homogenization (e.g., high-pressure homogenization, e.g., 1 to 5 times, e.g., 2 times), and separating the homogenized bacterial liquid to obtain the supernatant. The equilibrium working solution may contain 100-500 mM sodium chloride, 10-50 mM Tris, and 10-50 mM imidazole at pH 7-9. 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.

[0136] Step (3) may include purifying and enzymatically digesting the recombinant collagen. The purification may be a crude purification comprising purifying the supernatant with a Ni-agarose column to obtain an eluent containing the target protein. The crude purification may include washing the column material with water, for example, 2 to 10 column volumes (CV), for example, 5 CV. The column material may be equilibrated using an equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole at pH 8.0) of, for example, 2 to 10 CV, for example, 5 CV. The equilibration solution may contain 100-500 mM sodium chloride, 10-50 mM Tris, and 10-50 mM imidazole at pH 7-9. 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.

[0137] Step (3) may include loading the supernatant onto a column material and washing the impurity proteins with a washing solution. The washing solution may contain 100-500 mM sodium chloride, 10-50 mM Tris, and 10-50 mM imidazole at pH 7-9. 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 can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH can be 7, 7.5, 8, 8.5, or 9. Then, add the eluent and collect the flow-through liquid. The eluent may contain 100-500 mM sodium chloride, 10-50 mM Tris, and 100-500 mM imidazole at pH 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 can 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.

[0138] Enzymatic hydrolysis may include adding a collagen-treated enzyme for enzymatic hydrolysis (a ratio of total protein amount to total collagen-treated enzyme amount of 10-100:1, e.g., 50:1, at 10 to 20°C, e.g., 16°C for 2 to 8 hours, e.g., 4 hours). The enzymatically hydrolyzed protein solution is dialyzed, e.g., in a dialysis bag at 1 to 6°C, e.g., 4°C for 1 to 8 hours, e.g., 2 hours, then transferred to a new dialysate and dialyzed overnight at 1 to 6°C, e.g., 4°C.

[0139] Purification may include fine purification (e.g., for proteins with an isoelectric point greater than 8.0). Preferably, fine purification involves using a strong anion exchange chromatography column (e.g., pH may be 7, 7.5, 8, 8.5, or 9). The eluent containing the target protein or product after enzymatic digestion (e.g., enzymatic digestion and dialyzed product) undergoes gradient elution. Gradient elution comprises flowing a 0 to 15% solution B for 1 to 5 minutes and then holding it for 1 to 5 times the column volume, e.g., 3 times the column volume; flowing a 15 to 30% solution B for 1 to 5 minutes and then holding it for 1 to 5 times the column volume, e.g., 3 times the column volume; flowing a 30 to 50% solution B for 1 to 5 minutes and then holding it for 1 to 5 times the column volume, e.g., 3 times the column volume; and flowing a 50 to 100% solution B for 1 to 5 minutes and then holding it for 1 to 5 times the column volume, e.g., 3 times the column volume. Solution B may contain 10-50 mM Tris and 0.5-5 M sodium chloride at pH 7-9. For example, the concentration of Tris is 15, 20, 25, 30, 35, 40, or 45 mM. The concentration of sodium chloride is 1, 2, 3, or 4 M. The pH can be 7, 7.5, 8, 8.5, or 9. Precision purification may involve equilibrating the column material with solution A, loading it, and then performing gradient elution. Solution A may contain 10-50 mM Tris and 10-50 mM sodium chloride at pH 7-9. For example, the concentration of Tris is 15, 20, 25, 30, 35, 40, or 45 mM. The concentration of sodium chloride is 15, 20, 25, 30, 35, 40, or 45 mM. pH can be 7, 7.5, 8, 8.5, or 9.

[0140] To clarify the purpose, technical solution, and advantages of the present disclosure, the technical solution shown in the following examples will be described clearly and completely. Of course, the described examples are only some embodiments of the present disclosure, not all. All other examples that can be obtained by a person skilled in the art without any creative work based on the examples of the present disclosure are also within the scope of protection of the present disclosure.

[0141] The following examples are further provided to illustrate the present disclosure.

[0142] Examples

[0143] The present disclosure is further explained by the following examples, but no example or combination thereof should be construed as limiting the scope of the present disclosure. The scope of the present disclosure is defined by the claims below. Those skilled in the art can clearly identify the scope defined by the claims by combining the present specification with general knowledge of the art. Those skilled in the art may modify the technical solution of the present disclosure without departing from the spirit and scope of the present disclosure. Such modifications are also within the scope of the present disclosure.

[0144] Example 1: Construction, expression, and screening of recombinant type IV humanized collagen fragments

[0145] 1. Large-scale screening of functional regions was performed to obtain the following various target gene functional regions of recombinant type IV humanized collagen.

[0146] 1) The amino acid sequence of C4P7Ch (the amino acid sequence of the repeat unit is Gakgdkgskgevgfpglagspgipgskgeq, sequence number: 1, and the number of repeat units is 10; the amino acid sequence of C4P7Ch is sequence number: 2):

[0147] Gakgdkgskgevgfpglagspgipgskgeq

[0148] Gakgdkgskgevgfpglagspgipgskgeq

[0149] Gakgdkgskgevgfpglagspgipgskgeq

[0150] Gakgdkgskgevgfpglagspgipgskgeq

[0151] Gakgdkgskgevgfpglagspgipgskgeq

[0152] Gakgdkgskgevgfpglagspgipgskgeq

[0153] Gakgdkgskgevgfpglagspgipgskgeq

[0154] Gakgdkgskgevgfpglagspgipgskgeq

[0155] Gakgdkgskgevgfpglagspgipgskgeq

[0156] Gakgdkgskgevgfpglagspgipgskgeq (서열 번호: 2)

[0157] C4P7Ch 염기 서열(서열 번호: 3):

[0158] GGGGCTAAAGGAGACAAGGGCAGCAAGGGCGAAGTCGGTTTCCCAGGTCTGGCTGGTAGCCCGGGCATCCCGGGTTCAAAGGGTGAACAAGGTGCTAAAGGCGACAAAGGCAGCAAGGGTGAGGTTGGTTTCCCGGGTCTGGCGGGTTCTCCAGGCATCCCGGGTAGCAAAGGAGAACAAGGTGCGAAAGGCGATAAAGGCTCCAAGGGTGAAGTGGGCTTCCCGGGTTTAGCCGGTAGCCCAGGTATTCCGGGTAGCAAAGGCGAACAGGGTGCGAAAGGCGACAAAGGGAGTAAGGGCGAGGTGGGTTTTCCGGGTTTGGCTGGCTCGCCGGGTATTCCGGGTTCAAAGGGCGAACAGGGCGCGAAAGGTGATAAAGGCAGCAAAGGCGAGGTTGGCTTCCCGGGTCTGGCAGGTAGCCCGGGTATCCCGGGTAGCAAGGGTGAGCAGGGTGCCAAAGGCGACAAAGGTAGCAAGGGGGAAGTGGGTTTTCCGGGACTGGCAGGTAGCCCGGGTATCCCGGGTTCTAAGGGCGAGCAGGGTGCGAAAGGTGACAAAGGTAGCAAGGGCGAGGTTGGCTTTCCGGGCTTGGCGGGTAGCCCGGGCATTCCGGGCTCCAAGGGTGAACAAGGTGCGAAAGGTGATAAAGGCTCTAAGGGTGAGGTTGGTTTTCCGGGTCTGGCGGGTTCCCCGGGCATTCCGGGCTCGAAGGGCGAGCAAGGTGCTAAAGGTGATAAGGGCTCCAAGGGCGAGGTGGGTTTCCCGGGCCTGGCAGGCTCTCCGGGCATCCCGGGTTCGAAGGGCGAACAGGGTGCGAAAGGCGATAAAGGTTCCAAGGGCGAAGTCGGATTCCCTGGCCTCGCCGGTAGCCCGGGCATCCCTGGCTCCAAGGGCGAGCAG

[0159] 2) C4P7Cf amino acid sequence (the amino acid sequence of the repeat unit is Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq, sequence number: 4, and the number of repeat units is 8; the amino acid sequence of C4P7Cf is sequence number: 5):

[0160] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0161] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0162] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0163] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0164] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0165] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0166] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0167] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpq (Sequence No.: 5)

[0168] C4P7Cf nucleotide sequence (sequence number: 6):

[0169] GGAGCTAAAGGGGACAAAGGCTCCAAAGGCGAAGTCGGTTTCCCGGGCCTGGCGGGTAGCCCGGGTATCCCGGGTAGCAAGGGTGAGCAGGGGTTCATGGGTCCACCGGGCCCACAGGGTGCCAAAGGTGATAAAGGTTCTAAGGGCGAGGTGGGTTTCCCGGGGCTGGCGGGTTCTCCGGGCATTCCGGGAAGCAAGGGTGAACAGGGCTTTATGGGTCCGCCAGGTCCGCAGGGTGCGAAAGGTGATAAAGGCAGCAAGGGAGAAGTTGGCTTCCCGGGCCTGGCAGGCAGCCCGGGCATTCCGGGGTCGAAGGGCGAACAAGGTTTCATGGGTCCGCCTGGTCCGCAAGGTGCGAAAGGTGATAAGGGTAGCAAGGGTGAAGTGGGTTTTCCGGGATTAGCGGGTTCTCCGGGCATTCCGGGTTCAAAAGGTGAACAAGGCTTTATGGGTCCGCCTGGCCCGCAGGGTGCTAAAGGCGACAAGGGTAGCAAAGGCGAGGTAGGTTTCCCGGGTTTGGCGGGCAGCCCGGGCATTCCGGGTTCCAAGGGCGAGCAGGGTTTTATGGGCCCACCGGGCCCGCAAGGCGCAAAAGGTGATAAGGGCAGCAAAGGCGAGGTGGGCTTCCCGGGACTGGCAGGTTCTCCGGGTATCCCGGGTTCCAAGGGTGAGCAGGGTTTCATGGGCCCACCGGGTCCGCAGGGTGCGAAAGGCGACAAAGGTAGCAAGGGCGAAGTTGGTTTTCCGGGCCTGGCTGGTTCGCCGGGCATCCCGGGCTCCAAGGGCGAGCAAGGCTTCATGGGTCCACCGGGTCCGCAAGGTGCCAAAGGCGACAAAGGTAGCAAGGGCGAGGTTGGTTTTCCGGGCTTGGCTGGTAGCCCTGGCATCCCGGGGTCCAAGGGTGAACAGGGCTTTATGGGTCCGCCGGGCCCTCAA

[0170] 3) C4P7Ca amino acid sequence (the amino acid sequence of the repeat unit is GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP, SEQ No. 7, and the number of repeat units is 6; the amino acid sequence of C4P7Ca is SEQ No. 8):

[0171] GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP

[0172] GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP

[0173] GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP

[0174] GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP

[0175] GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP

[0176] GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLP (Sequence No.: 8)

[0177] C4P7Ca nucleotide sequence (sequence number: 9):

[0178] GGATTTCCCGGGTTCCCGGGTGCCAAAGGGGATAAAGGTTCAAAGGGCGAAGTGGGTTTCCCGGGTTTGGCTGGTAGCCCGGGTATCCCGGGTAGCAAAGGCGAACAGGGCTTTATGGGTCCGCCAGGACCGCAGGGTCAACCGGGACTGCCGGGTTTTCCGGGCTTCCCGGGTGCGAAAGGCGATAAAGGTTCCAAGGGTGAAGTTGGTTTTCCGGGTCTTGCAGGCAGCCCGGGTATTCCGGGTTCCAAGGGTGAACAGGGTTTCATGGGTCCACCGGGCCCACAAGGTCAGCCGGGTCTGCCTGGTTTCCCGGGCTTCCCGGGTGCCAAAGGCGACAAAGGTAGCAAGGGCGAAGTTGGCTTTCCGGGTCTGGCGGGTTCGCCGGGCATTCCGGGCTCGAAGGGCGAGCAGGGTTTCATGGGCCCACCGGGTCCGCAGGGTCAGCCTGGCCTGCCGGGATTCCCAGGTTTTCCGGGAGCGAAAGGCGACAAGGGTAGTAAGGGTGAGGTCGGTTTTCCAGGCTTGGCGGGCTCTCCCGGTATCCCGGGCTCTAAGGGCGAGCAAGGCTTTATGGGTCCACCGGGTCCGCAAGGTCAACCTGGATTACCGGGATTCCCAGGCTTTCCGGGCGCGAAAGGCGATAAAGGCAGCAAGGGTGAGGTGGGCTTCCCGGGCCTCGCGGGTAGCCCGGGCATCCCGGGTAGCAAGGGTGAGCAGGGCTTCATGGGTCCTCCGGGTCCGCAGGGCCAACCGGGCCTGCCGGGATTCCCGGGTTTCCCGGGCGCTAAAGGCGACAAAGGCAGCAAGGGTGAGGTTGGTTTTCCGGGTCTGGCAGGTAGCCCGGGCATTCCGGGCTCCAAGGGCGAACAGGGTTTTATGGGTCCACCGGGCCCTCAAGGTCAGCCGGGCCTGCCG

[0179] 4) C4P7Cb amino acid sequence (the amino acid sequence of the repeat unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq, sequence number: 10, and the number of repeat units is 8; the amino acid sequence of C4P7Cb is sequence number: 11):

[0180] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0181] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0182] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0183] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0184] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0185] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0186] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq

[0187] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfmgppgpq (Sequence No.: 11)

[0188] C4P7Cb nucleotide sequence (sequence number: 12):

[0189]

[0190] 5) C4P7Cc amino acid sequence (the amino acid sequence of the repeat unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm, sequence number: 13, and the number of repeat units is 8; the amino acid sequence of C4P7Cc is sequence number: 14):

[0191] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm

[0192] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm

[0193] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm

[0194] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm

[0195] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm

[0196] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm

[0197] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm

[0198] Gfpgfpgakgdkgskgevgfpglagspgipgskgeqgfm (Sequence No.: 14)

[0199] C4P7Cc nucleotide sequence (sequence number: 15):

[0200] GGATTTCCCGGGTTCCCGGGTGCGAAAGGTGATAAAGGCAGCAAGGGTGAAGTCGGTTTTCCGGGTCTGGCAGGCAGCCCGGGTATCCCGGGTAGCAAAGGCGAACAGGGCTTTATGGGTTTCCCGGGCTTCCCAGGTGCGAAGGGCGATAAAGGTTCGAAAGGTGAGGTAGGTTTCCCGGGTTTAGCAGGTTCCCCGGGCATTCCGGGCAGCAAGGGTGAACAGGGTTTCATGGGCTTTCCGGGCTTCCCAGGAGCTAAAGGCGACAAAGGTTCTAAGGGTGAAGTGGGCTTCCCGGGTCTGGCTGGTAGCCCGGGCATCCCGGGCTCCAAGGGTGAGCAGGGTTTCATGGGTTTTCCGGGCTTCCCAGGCGCGAAAGGCGACAAAGGCAGCAAGGGCGAGGTGGGTTTTCCGGGTTTGGCGGGTAGCCCGGGTATTCCGGGTTCGAAGGGTGAACAAGGTTTCATGGGTTTTCCGGGATTCCCAGGCGCGAAAGGCGATAAGGGCAGCAAGGGCGAGGTTGGCTTCCCGGGACTGGCCGGAAGCCCGGGTATCCCGGGATCTAAGGGCGAACAAGGCTTTATGGGTTTCCCGGGTTTTCCTGGTGCGAAAGGCGATAAAGGCTCCAAGGGCGAGGTTGGTTTTCCAGGCCTGGCTGGCTCTCCGGGCATTCCGGGTAGTAAGGGTGAGCAGGGTTTTATGGGTTTTCCGGGCTTCCCGGGTGCAAAGGGTGACAAAGGTAGCAAGGGTGAAGTTGGCTTTCCGGGTCTGGCGGGTTCCCCGGGCATTCCGGGTAGCAAAGGTGAGCAAGGTTTTATGGGTTTTCCGGGCTTCCCGGGTGCCAAAGGCGACAAAGGTAGCAAGGGAGAGGTGGGCTTCCCGGGATTGGCGGGTTCCCCGGGCATCCCGGGCTCAAAGGGTGAACAGGGTTTCATG

[0201] 6) C4P7Cd amino acid sequence (the amino acid sequence of the repeat unit is Gfpgfpgakgdkgskgevgfpglagspgipgskgeq, sequence number: 16, and the number of repeat units is 10; the amino acid sequence of C4P7Cd is sequence number: 17):

[0202] Gfpgfpgakgdkgskgevgfpglagspgipgskgeq

[0203] Gfpgfpgakgdkgskgevgfpglagspgipgskgeq

[0204] Gfpgfpgakgdkgskgevgfpglagspgipgskgeq

[0205] Gfpgfpgakgdkgskgevgfpglagspgipgskgeq

[0206] Gfpgfpgakgdkgskgevgfpglagspgipgskgeq

[0207] Gfpgfpgakgdkgskgevgfpglagspgipgskgeq

[0208] Gfpgfpgakgdkgskgevgfpglagspgipgskgeq

[0209] Gfpgfpgakgdkgskgevgfpglagspgipgskgeq

[0210] Gfpgfpgakgdkgskgevgfpglagspgipgskgeq

[0211] Gfpgfpgakgdkgskgevgfpglagspgipgskgeq (Sequence No.: 17)

[0212] C4P7Cd nucleotide sequence (sequence number: 18):

[0213]

[0214] 7) C4P7Ce amino acid sequence (the amino acid sequence of the repeat unit is Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp, sequence number: 19, and the number of repeat units is 8; the amino acid sequence of C4P7Ce is sequence number: 20):

[0215] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp

[0216] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp

[0217] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp

[0218] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp

[0219] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp

[0220] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp

[0221] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp

[0222] Gakgdkgskgevgfpglagspgipgskgeqgfmgppgpqgqpglp (Sequence No.: 20)

[0223] C4P7Ce nucleotide sequence (sequence number: 21):

[0224]

[0225] 8) C4P7Cg amino acid sequence (the amino acid sequence of the repeat unit is Gakgdkgskgevgfpglagspgipgskgeqgfm, sequence number: 22, and the number of repeat units is 10; the amino acid sequence of C4P7Cg is sequence number: 23):

[0226] Gakgdkgskgevgfpglagspgipgskgeqgfm

[0227] Gakgdkgskgevgfpglagspgipgskgeqgfm

[0228] Gakgdkgskgevgfpglagspgipgskgeqgfm

[0229] Gakgdkgskgevgfpglagspgipgskgeqgfm

[0230] Gakgdkgskgevgfpglagspgipgskgeqgfm

[0231] Gakgdkgskgevgfpglagspgipgskgeqgfm

[0232] Gakgdkgskgevgfpglagspgipgskgeqgfm

[0233] Gakgdkgskgevgfpglagspgipgskgeqgfm

[0234] Gakgdkgskgevgfpglagspgipgskgeqgfm

[0235] Gakgdkgskgevgfpglagspgipgskgeqgfm (Sequence No.: 23)

[0236] C4P7Cg nucleotide sequence (sequence number: 24):

[0237] GGGGCTAAAGGAGACAAAGGTTCGAAAGGCGAGGTGGGCTTCCCAGGTCTGGCCGGTTCCCCGGGCATTCCGGGTAGCAAAGGCGAACAAGGTTTCATGGGTGCTAAAGGCGATAAAGGTAGCAAGGGTGAGGTTGGCTTCCCAGGCCTGGCTGGTTCGCCGGGCATTCCGGGCTCTAAGGGTGAACAAGGTTTCATGGGTGCAAAAGGTGATAAGGGTAGCAAGGGAGAAGTCGGTTTTCCGGGATTGGCGGGTAGCCCGGGTATCCCGGGCAGCAAGGGCGAGCAGGGTTTTATGGGTGCAAAGGGCGACAAAGGTAGCAAGGGTGAGGTGGGCTTTCCGGGCCTCGCGGGTAGCCCTGGCATCCCGGGTTCCAAAGGTGAGCAAGGCTTCATGGGTGCTAAAGGTGATAAAGGCTCCAAAGGTGAAGTGGGTTTTCCGGGCCTGGCGGGTAGCCCGGGCATTCCGGGAAGCAAGGGCGAACAGGGTTTTATGGGCGCGAAGGGTGATAAAGGTAGTAAGGGCGAAGTTGGTTTCCCGGGCCTGGCTGGCTCTCCGGGTATCCCGGGCTCCAAAGGCGAGCAGGGTTTCATGGGTGCGAAAGGTGACAAGGGTAGCAAGGGTGAGGTGGGTTTCCCAGGTTTGGCGGGTAGCCCGGGCATTCCGGGTAGCAAGGGTGAACAAGGTTTCATGGGTGCGAAAGGTGACAAAGGCAGCAAGGGCGAGGTTGGTTTCCCGGGTCTGGCGGGTAGCCCGGGCATCCCGGGCTCTAAGGGCGAGCAGGGTTTTATGGGTGCCAAAGGCGACAAGGGCTCAAAGGGTGAAGTCGGTTTTCCGGGTTTAGCCGGTTCCCCGGGCATCCCGGGTTCTAAGGGTGAACAGGGCTTCATGGGCGCGAAAGGAGATAAAGGCAGCAAAGGGGAAGTTGGTTTTCCAGGCCTGGCAGGCTCGCCGGGTATCCCGGGTTCCAAGGGCGAGCAGGGTTTTATG

[0238] 9) C4P7Ea amino acid sequence (the amino acid sequence of the repeat unit is Glpgtpgptgpagqkgepgsdgipgsagekgepglp, sequence number: 25, and the number of repeat units is 10; the amino acid sequence of C4P7Ea is sequence number: 26):

[0239] Glpgtpgptgpagqkgepgsdgipgsagekgepglp

[0240] Glpgtpgptgpagqkgepgsdgipgsagekgepglp

[0241] Glpgtpgptgpagqkgepgsdgipgsagekgepglp

[0242] Glpgtpgptgpagqkgepgsdgipgsagekgepglp

[0243] Glpgtpgptgpagqkgepgsdgipgsagekgepglp

[0244] Glpgtpgptgpagqkgepgsdgipgsagekgepglp

[0245] Glpgtpgptgpagqkgepgsdgipgsagekgepglp

[0246] Glpgtpgptgpagqkgepgsdgipgsagekgepglp

[0247] Glpgtpgptgpagqkgepgsdgipgsagekgepglp

[0248] Glpgtpgptgpagqkgepgsdgipgsagekgepglp (Sequence No.: 26)

[0249] C4P7Ea nucleotide sequence (sequence number: 27):

[0250]

[0251] 10) C4P7Eb amino acid sequence (the amino acid sequence of the repeat unit is Gptgpagqkgepgsdgipgsagekgepglp, sequence number: 28, and the number of repeat units is 10; the amino acid sequence of C4P7Eb is sequence number: 29):

[0252] Gptgpagqkgepgsdgipgsagekgepglp

[0253] Gptgpagqkgepgsdgipgsagekgepglp

[0254] Gptgpagqkgepgsdgipgsagekgepglp

[0255] Gptgpagqkgepgsdgipgsagekgepglp

[0256] Gptgpagqkgepgsdgipgsagekgepglp

[0257] Gptgpagqkgepgsdgipgsagekgepglp

[0258] Gptgpagqkgepgsdgipgsagekgepglp

[0259] Gptgpagqkgepgsdgipgsagekgepglp

[0260] Gptgpagqkgepgsdgipgsagekgepglp

[0261] Gptgpagqkgepgsdgipgsagekgepglp (Sequence No.: 29)

[0262] C4P7Eb nucleotide sequence (sequence number: 30):

[0263] GGTCCCACAGGACCGGCAGGCCAGAAAGGTGAGCCGGGTTCCGACGGCATCCCGGGTTCGGCGGGTGAGAAAGGCGAGCCGGGTTTACCGGGTCCGACCGGTCCCGCGGGTCAAAAGGGCGAGCCGGGTAGCGATGGCATTCCGGGTTCTGCGGGTGAAAAGGGCGAACCGGGCCTCCCGGGTCCTACCGGTCCGGCGGGTCAGAAAGGCGAACCGGGCAGCGATGGCATCCCGGGCAGCGCGGGCGAGAAAGGCGAACCGGGCCTGCCGGGCCCGACCGGACCAGCTGGGCAAAAAGGTGAACCGGGCAGCGACGGCATCCCGGGTTCTGCAGGCGAGAAAGGTGAACCAGGCCTGCCGGGACCGACCGGTCCGGCAGGCCAGAAAGGTGAGCCTGGCAGTGATGGTATTCCGGGTTCTGCCGGTGAAAAAGGTGAGCCGGGCCTGCCGGGGCCAACGGGCCCAGCCGGACAAAAAGGTGAGCCGGGTTCCGACGGCATCCCGGGCTCCGCCGGTGAAAAGGGTGAGCCGGGCCTGCCTGGCCCAACGGGTCCGGCTGGCCAAAAGGGCGAGCCGGGTAGCGACGGCATTCCGGGCAGCGCGGGTGAGAAGGGTGAGCCGGGATTGCCGGGTCCGACTGGTCCTGCGGGCCAGAAGGGTGAACCGGGTTCCGACGGCATCCCCGGCTCGGCGGGTGAAAAGGGCGAACCGGGTCTGCCTGGTCCGACCGGCCCAGCGGGTCAGAAGGGTGAACCGGGTAGCGATGGAATCCCGGGTAGCGCTGGTGAAAAGGGCGAGCCGGGCCTGCCGGGTCCGACCGGTCCGGCAGGCCAGAAGGGTGAACCGGGTAGCGATGGTATTCCGGGTAGCGCGGGCGAAAAAGGTGAGCCGGGCTTGCCG

[0264] 11) C4P7Ec amino acid sequence (the amino acid sequence of the repeat unit is Gfpgfpgakgdkgskgevgfpglagspgipgsk, sequence number: 31, and the number of repeat units is 10; the amino acid sequence of C4P7Ec is sequence number: 32):

[0265] Gfpgfpgakgdkgskgevgfpglagspgipgsk

[0266] Gfpgfpgakgdkgskgevgfpglagspgipgsk

[0267] Gfpgfpgakgdkgskgevgfpglagspgipgsk

[0268] Gfpgfpgakgdkgskgevgfpglagspgipgsk

[0269] Gfpgfpgakgdkgskgevgfpglagspgipgsk

[0270] Gfpgfpgakgdkgskgevgfpglagspgipgsk

[0271] Gfpgfpgakgdkgskgevgfpglagspgipgsk

[0272] Gfpgfpgakgdkgskgevgfpglagspgipgsk

[0273] Gfpgfpgakgdkgskgevgfpglagspgipgsk

[0274] Gfpgfpgakgdkgskgevgfpglagspgipgsk (Sequence No.: 32)

[0275] C4P7Ec nucleotide sequence (sequence number: 33):

[0276] GGATTTCCCGGGTTCCCAGGCGCAAAAGGTGATAAAGGCAGCAAGGGCGAGGTTGGTTTTCCAGGTTTAGCTGGTAGCCCGGGTATCCCGGGTAGCAAGGGCTTCCCGGGTTTTCCGGGTGCTAAAGGCGACAAAGGCTCCAAGGGCGAAGTCGGTTTCCCGGGTTTGGCGGGTAGCCCGGGTATCCCGGGTAGTAAGGGCTTTCCGGGATTCCCAGGCGCGAAAGGTGACAAAGGTAGCAAGGGCGAAGTTGGCTTCCCGGGTTTGGCGGGTTCCCCGGGTATCCCGGGGTCCAAGGGCTTCCCCGGATTCCCGGGCGCGAAAGGCGATAAAGGTAGCAAGGGTGAAGTGGGTTTTCCGGGTCTCGCTGGCAGCCCGGGTATTCCGGGCTCCAAGGGCTTTCCAGGCTTTCCGGGTGCGAAAGGCGATAAAGGTAGCAAGGGTGAGGTGGGTTTTCCGGGTCTGGCAGGTAGCCCTGGCATCCCGGGCTCGAAGGGGTTCCCGGGCTTCCCGGGAGCCAAGGGTGATAAAGGTTCTAAGGGTGAGGTCGGTTTTCCGGGCCTGGCCGGTAGCCCTGGTATCCCGGGGAGCAAGGGTTTCCCGGGTTTTCCGGGTGCCAAAGGCGATAAAGGCTCTAAGGGCGAGGTGGGCTTCCCCGGTCTGGCGGGTAGCCCGGGTATTCCGGGTTCTAAGGGCTTCCCGGGTTTTCCGGGTGCGAAAGGTGACAAGGGCTCCAAGGGTGAAGTTGGTTTTCCGGGTCTGGCTGGTAGCCCGGGTATCCCGGGTAGCAAGGGCTTCCCGGGTTTCCCGGGCGCGAAAGGCGACAAAGGTTCAAAGGGTGAAGTTGGTTTTCCTGGCCTGGCAGGCAGCCCGGGCATTCCGGGTTCCAAAGGTTTTCCGGGCTTCCCGGGTGCGAAAGGTGACAAAGGCTCGAAGGGTGAGGTGGGCTTCCCGGGTCTGGCAGGTTCTCCTGGCATTCCGGGTTCGAAA

[0277] Each of the aforementioned encoding nucleotide sequences was commercially synthesized. Subsequently, each of the above encoding nucleotide sequences (with a collagen-treated enzyme cleavage site added at the 5' end having the amino acid sequence of ENLYFQ labeled SEQ No. 56 and the nucleotide sequence of GAAAACCTGTATTTCCAG labeled SEQ No. 57) was inserted between the KpnI and XhoI cleavage sites of the pET-28a-Trx-His expression vector to generate a recombinant expression plasmid.

[0278] 3. The successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3). Specifically, the procedure was as follows: (1) The E. coli competent cells BL21(DE3) were removed from the ultra-low temperature refrigerator and placed on ice. When the cells were half-thawed, 2 μl of the plasmid to be transformed was transferred to the E. coli competent cells BL21(DE3) using a pipette and lightly mixed 2 to 3 times. (2) The mixture was placed on ice in an ice bath for 30 minutes, heat shock was applied in a 42°C water bath for 45 to 90 seconds, the mixture was removed, and the mixture was placed on ice in an ice bath for 2 minutes. (3) The mixture was transferred to a biosafety cabinet, 700 μl of liquid LB medium was added, and the mixture was incubated at 37°C and 220 rpm for 60 minutes. (4) Take 200 μl of the bacterial solution and spread it evenly on an LB plate containing ampicillin sodium. (5) Incubate the plate in an incubator at 37°C for 15 to 17 hours until colonies of uniform size grow.

[0279] 4. Five to six single colonies were taken from LB plates containing transformed cells and placed in a shaking flask containing LB medium supplemented with antibiotic stock solution, and then incubated in a shaker at a speed of 220 rpm and a constant temperature of 37°C for 7 hours. The cultured shaking flask was cooled to 16°C, and then IPTG was added to induce expression for a specific period. The bacterial solution was dispensed into a centrifuge bottle and centrifuged at 8000 rpm and 4°C for 10 minutes. The bacterial cells were collected, the weight of the bacterial cells was recorded, and then a sample (labeled "bacterial solution") was taken for detection by electrophoresis.

[0280] 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), and the bacterial solution was cooled to a temperature of 15°C or lower. The solution was homogenized by repeating high-pressure homogenization for two rounds (a sample was taken after each round and labeled "Homogenization 1" and "Homogenization 2," respectively), and the bacterial solution was collected after homogenization. The homogenized bacterial solution was distributed into a centrifuge bottle and centrifuged at 17,000 rpm at 4°C for 30 minutes. The supernatant was collected, and both the supernatant (labeled "Supernatant") and the pellet were detected by electrophoresis.

[0281] 6. Recombinant humanized type IV collagen is purified and enzymatically digested. Specifically, the process was as follows: (1) Crude Purification: a. The column material (Ni6FF, Cytiva) is washed with 5 CV water. b. The column material is equilibrated with a 5 CV equilibrium solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0). c. Loading: The supernatant obtained after centrifugation is loaded onto the column material to allow the liquid to completely flow out of the column material, after which the pass-through liquid (labeled "pass-through liquid") is taken and detected by electrophoresis. d. Impurity Protein Wash: 25 mL of the wash solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) is added to allow the liquid to completely flow out, after which the impurity-wash pass-through liquid (labeled "impurity-wash") is taken and detected by electrophoresis. e. Target protein collection: Add 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0), collect the pass-through (labeled "Elution"), detect the protein concentration, calculate the protein amount, and perform electrophoretic detection. f. Wash the column material with 1 M imidazole working solution (labeled "Wash with 1 M"). g. Wash the column material with purified water. (2) Enzymatic digestion: Add TEV enzyme so that the ratio of total protein amount to total TEV enzyme amount is 50:1, digest at 16°C for 4 hours, then sample and detect by electrophoresis (labeled "After digestion"). Place the enzymatically digested protein solution in a dialysis bag and dialyze at 4°C for 2 hours, then transfer it to fresh dialysate and dialyze overnight at 4°C (labeled "Liquid exchange").

[0282] (3) Precision Purification: a. Equilibration of column material (Capto Q, Cytiva): Equilibrate the column material using solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0) at a flow rate of 10 ml / min. b. Loading: Load the sample at a flow rate of 5 ml / min, collect the pass-through (indicated as "QFL"), and perform electrophoretic detection. c. Gradient Elution: Run 0 to 15% solution B (20 mM Tris, 1 M sodium chloride, pH 8.0) for 2 minutes and hold for 3 CV, run 15 to 30% solution B for 2 minutes and hold for 3 CV, run 30 to 50% solution B for 2 minutes and hold for 3 CV, collect peaks for each, and perform electrophoretic detection (indicated as "Wash with solution B"). d. Wash the column material. The protein was stored at 4℃.

[0283] 7. Electrophoresis detection

[0284] Specifically, the procedure was as follows. 40 μL of sample solution was taken, and 10 μL of 5× protein loading buffer (250 mM Tris-HCl at pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol) was added. The mixture was heated in boiling water at 100°C for 10 minutes, and then 10 μL was added to each well of the SDS-PAGE protein gel. After running the gel at 80V for 2 hours, it was stained for 20 minutes using Coomassie brilliant blue staining solution (0.1% Coomassie brilliant blue R-250, 25% isopropanol, 10% glacial acetic acid), and then destained using protein destaining solution (10% acetic acid, 5% ethanol).

[0285] Figure 1 shows the electrophoretic detection results of C4P7Ca. Figure 2 shows the electrophoretic detection results of C4P7Cb. Figure 3 shows the electrophoretic detection results of C4P7Cc. Figure 4 shows the electrophoretic detection results of C4P7Cd. Figure 5 shows the electrophoretic detection results of C4P7Ce. Figure 6 shows the electrophoretic detection results of C4P7Cf. Figure 7 shows the electrophoretic detection results of C4P7Cg. Figure 8 shows the electrophoretic detection results of C4P7Ch. Figure 9 shows the electrophoretic detection results of C4P7Ea. Figure 10 shows the electrophoretic detection results of C4P7Eb. Figure 11 shows the electrophoretic detection results of C4P7Ec. Figures 1 to 11 show that the actual molecular weights of the isolated proteins (C4P7Ca, C4P7Cb, C4P7Cc, C4P7Cd, C4P7Ce, C4P7Cf, C4P7Cg, C4P7Ch, C4P7Ea, C4P7Eb, and C4P7Ec) match the corresponding expected molecular weights, which proves that the proteins were correctly expressed.

[0286] Example 2: Mass Spectrometry Detection of Recombinant Type IV Humanized Collagen

[0287] [Table 1] Experimental Method

[0288]

[0289] Protein samples (collagen C4P7Cf and C4P7Ch) were reduced with DTT and alkylated with iodoacetamide, then trypsin was added for enzymatic digestion and left overnight. The peptide fragments obtained after enzymatic digestion were desalted using C18ZipTip, mixed with matrix α-cyano-4-hydroxycinnamic acid (CHCA), and spotted onto plates. Finally, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF / TOF Ulraflextreme) TMBruker, Germany) was used for the analysis (for peptide fingerprinting techniques, see Protein J. 2016;35:212-7).

[0290] Data retrieval was performed using the MS / MS ion search page of the local Mascot website. Protein identification results were obtained based on primary mass spectrometry of peptide fragments generated after enzymatic digestion. Detection parameters: trypsin enzymatic digestion, allowing two missing cleavage sites. Cysteine ​​alkylation was set as a fixed modification, and methionine oxidation was set as a variable modification. The database used for identification was NCBprot.

[0291] [Table 2]

[0292] The coverage rate of the detected polypeptide segment is relative to the theoretical sequence (GAK GDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGA KGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQ, SEQ ID NO: 5 The detection result was very reliable, with 99.04% compared to (the covered part in this sequence is underlined).

[0293] [Table 3]

[0294] The coverage rate of the detected polypeptide segment is the theoretical sequence (GAKGDK GSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAK GDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQ, SEQ ID NO: 2 The detection result was very reliable, with 98% compared to (the covered part in this sequence is underlined).

[0295] Example 3: Detection of biological activity of recombinant type IV humanized collagen

[0296] Methods for detecting collagen activity can be found in the literature [Reference: Juming Yao, Satoshi Yanagisawa, 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)]. The specific method was performed as follows:

[0297] (1) Ultraviolet absorption method was used to measure the concentration of the protein sample to be detected. The protein sample included bovine type I collagen (National Institutes for Food and Drug Control, No. 380002), recombinant humanized collagen C4P7Cf and C4P7Ch provided herein.

[0298] Specifically, the UV absorbance of the samples was measured at 215 nm and 225 nm, respectively, and the protein concentration was calculated using the empirical formula C(μg / mL) = 144 × (A215-A225). Detection should be performed when A215 < 1.5. The principle of this method is to detect the characteristic absorption of peptide bonds in extreme ultraviolet light, which is unaffected by chromophore content and has minimal interfering substances. Because this method is easy to operate, it is suitable for detecting human collagen and its analogs that do not develop color with Coomassie Brilliant Blue (see reference Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45). After detecting the protein concentration, the concentration of all target proteins was adjusted to 0.5 mg / mL using PBS.

[0299] (2) Sample preparation: The experiment was conducted using the sample stock solution as is. The positive control, bovine type 1 collagen (PC), was diluted to 1 mg / ml using D-PBS for future use, and the negative control was D-PBS buffer (NC).

[0300] (3) Coating: 100 μL of collagen (C4P7Cf or C4P7Ch) at various concentrations, positive control, and negative control was added to each well of an ELISA plate, and 5 repeat wells were made for each group and incubated overnight at 4°C.

[0301] (4) Blocking: Discard the supernatant, add 100 μL of 1% BSA (heat-inactivated at 56°C for 30 minutes), and incubate at 37°C for 60 minutes. Discard the supernatant and wash the plate three times with D-PBS solution.

[0302] (5) Cell inoculation: 10 5 Well-cultured 3T3 / NIH cells were suspended in D-PBS and placed in each well, and incubated at 37°C for 120 minutes. Each well was washed 3 times with D-PBS solution.

[0303] (6) Detection: Absorbance at OD450nm was detected using a CCK8 detection kit (manufacturer Beyotime Biotech Inc., product catalog number C0038). The degree of cell adhesion was calculated according to 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 provided to the cells in a short period of time, which can help the cells adhere.

[0304]

[0305] In the above formula,

[0306] P: Relative cell adhesion ratio;

[0307] OD1: Average UV absorbance at 450 nm of all replicate wells of the tested collagen sample;

[0308] OD2: Average UV absorbance at 450 nm of all replicate wells of the control collagen sample;

[0309] OD0: Average UV absorbance at 450 nm of all replicate wells in the blank control group.

[0310] (7) Statistical analysis: Statistical differences between targeted recombinant humanized collagen and negative control were statistically analyzed using a two-sided t-test, where *, P < 0.05; * *, P < 0.01; * * *, P < 0.001.

[0311] The results are shown in Figures 11 and 12. The positive control group had a significant effect in promoting cell adhesion compared to the D-PBS group, and recombinant humanized collagen C4P7Cf and C4P7Ch also promoted cell adhesion.

[0312] Although the present disclosure has been described with reference to exemplary embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions are possible, and that elements of the described embodiments may be replaced by substantial equivalents without departing from the spirit and scope of the present disclosure. Furthermore, many modifications may be made to adapt specific situations or materials to the teachings of the present disclosure without departing from the scope of the present disclosure. Accordingly, the present specification is not intended to limit the present disclosure to the specific embodiments disclosed to carry out the present disclosure, but rather to include all embodiments within the scope of the appended claims.

Claims

Claim 1 A recombinant collagen, wherein the amino acid sequence of the recombinant collagen is SEQ ID NO: 2 or SEQ ID NO:

5. Claim 2 Nucleic acid encoding recombinant collagen as described in claim 1. Claim 3 In paragraph 2, the nucleic acid is of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO:

6. Claim 4 A vector comprising the nucleic acid described in paragraph 2 or 3. Claim 5 A host cell containing the nucleic acid described in paragraph 2. Claim 6 In paragraph 5, the host cell is a eukaryotic cell or a prokaryotic cell, and the eukaryotic cell is a yeast cell, an animal cell, or an insect cell and / or the prokaryotic cell is E. coli ( E. coli ) A host cell that is a cell. Claim 7 A composition comprising the recombinant collagen described in claim 1. Claim 8 In claim 7, the composition is one of a biological dressing, a human bionic material, a plastic surgery or beauty material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, an obstetrics and gynecology biomaterial, a nerve repair material, a nerve regeneration material, a liver tissue material, a blood vessel repair material, a blood vessel regeneration material, a 3D-printed artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient, and a food additive. Claim 9 In claim 7, the composition is a composition for topical use, injectable use, or oral use. Claim 10 In claim 7, the composition is a composition in the form of a solution, freeze-dried powder, gel, sponge, or fiber. Claim 11 A kit containing recombinant collagen as described in claim 1. Claim 12 A method for manufacturing one of a biological dressing, human bioengineering material, plastic surgery or cosmetic material, organoid culture material, cardiovascular stent material, coating material, tissue injection filling material, ophthalmic material, gynecological biomaterial, nerve repair material, nerve regeneration material, liver tissue material, blood vessel repair material, blood vessel regeneration material, 3D printed artificial organ biomaterial, cosmetic raw material, pharmaceutical excipient, and food additive, comprising using recombinant collagen described in claim 1. Claim 13 An in vitro method for promoting cell adhesion to a culture vessel for non-therapeutic purposes, wherein the method comprises the step of contacting a cell with the recombinant collagen described in claim 1 to cause the cell to adhere to the culture vessel. Claim 14 In paragraph 13, the above cell is an animal cell, in vitro method. Claim 15 A beauty method comprising administering the recombinant collagen described in claim 1 to a subject. Claim 16 A cosmetic method according to claim 15, wherein the administration is local administration, oral administration, or injection administration. Claim 17 A method for producing recombinant collagen as described in claim 1, wherein the method comprises: (1) incubating a host cell containing a nucleic acid encoding the recombinant collagen under appropriate culture conditions; (2) harvesting the host cell and / or culture medium containing the recombinant collagen; and (3) purifying the recombinant collagen. Claim 18 A method of preparation according to claim 17, wherein the host cell is an E. coli cell.

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