Method for biosynthesizing type VI collagen, a structural material for human body

Abstract

A method for biosynthesizing type VI collagen as a human structural material is provided. The polypeptide described herein comprises an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21. The recombinant human type VI collagen herein has cell adhesion activity, is easy to separate and purify, and can be used as a biological dressing, a human bionic material, a plastic surgery material, and the like.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of July 18, 2023, application number: 202310883890.4, and invention name “Method for biosynthesizing type VI collagen as human structural material”. Technical Field

[0002] The present application relates to the field of biosynthesis, and in particular to a method for preparing type VI collagen, a biosynthetic human structural material. Background Art

[0003] Collagen is an important component of the extracellular matrix in animal bodies, and plays an important role in cell migration, cell metabolism, cell signaling pathway response, platelet aggregation, and the maintenance, regulation, and injury repair of normal physiological functions of cells, tissues, and organs. As an important natural biological protein, collagen has good biocompatibility, bioactivity, and degradability, and can be widely used in many fields such as chemical industry, medicine, food, and cosmetics.

[0004] At present, the main way to obtain collagen is to use acid, alkali, and enzymatic hydrolysis to treat animal tissues to obtain collagen extracts. The technology for extracting collagen from animal tissues is relatively mature, but the collagen peptides extracted by this method during the production process are uneven in nature, have large batch differences, and have safety risks of viral infection. In addition, the amino acid sequence of animal collagen is quite different from that of human protein, which can easily lead to immunogenicity and allergic reactions. In addition, collagen is obtained through genetic engineering technology expression. At present, it is mainly expressed and produced using systems including Escherichia coli, Pichia pastoris, mammalian cells, insect cells, and plants. There are also some shortcomings in the above expression systems, such as the difficulty of subsequent purification of the expression products of the Escherichia coli system; the high cost and low yield of mammalian cell expression; the insect cell expression system is also costly and low in yield, and there are large differences with human cells after translation; the plant expression cycle is long and is not suitable for factory production; although the Pichia pastoris expression system has the advantages of large-scale industrial production such as high-density fermentation production, extremely low culture cost, short cycle, and high expression, it is impossible to determine whether it can be applied to all types of collagen expression.

[0005] At present, the recombinant human collagen that has been studied more is mainly type I, II, and III, and there is little research on type VI collagen. Human type VI collagen is a type of collagen present in all extracellular matrices (ECM). It can bind to different substances in the ECM, thereby bridging cells to the surrounding connective tissue and organizing the three-dimensional tissue structure of skeletal muscle, tendons, bones, and cartilage. By binding to type IV collagen and other pearl albumin in the basal layer, type VI collagen can establish a close connection between muscle cells and ECM. As the main component of the extracellular matrix, type VI collagen can promote the adhesion of fibroblasts in wound repair and enhance the regeneration of chondrocytes, playing an important role in wound healing.

[0006] There is a need in the art for methods of biosynthesizing Type VI collagen, a structural material of the human body. Summary of the invention

[0007] The inventors have conducted a large-scale screening of human type VI collagen and selected 11 types of human recombinant type VI collagen. These recombinant type VI collagens are suitable for separation and purification. After the activity assay of these human recombinant type VI collagens, the inventors found that the recombinant type VI collagen has cell adhesion activity and can be used in industry. The inventors also found that some recombinant type VI collagens (C6c, C6g, C6i, C6k proteins) have higher yields, yields and / or purity than other recombinant type VI collagens. The present invention provides a method for efficiently obtaining type VI collagen, a structural material for the human body.

[0008] In one aspect, the present invention provides a polypeptide comprising one or more repeating units, wherein the repeating units are connected directly or via linkers, and the repeating units comprise an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21, 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 that has 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.

[0009] The repeating sequence is as follows:

[0010] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGREGPVGVPGDPGEAGPIGPKG YRGDEGPPGSEGARGAPGPAGPPGDPGLMGERGEDGPA(SEQ ID NO:1)

[0011] GCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRP(SEQ ID NO:3)

[0012] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGRE(SEQ ID NO:5)

[0013] GTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDD(SEQ ID NO:7)

[0014] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDLGPVGYQGMKGEKGSRGEK GSRGPKGYKGEKGKRGIDGVDGVKGEMGYPGLPGCKGSPGFDGIQGPPGPKGDPGAF GLKGEKGEPGADGEAGRPGSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP(SEQ ID NO:9)

[0015] GIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRPGSSGPSGDEGQPGEPGPPGEKG EAGDEGNPGPDGAPGERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGREGPVGVPG DPGEAGPIGPKGYRGDEGPP(SEQ ID NO:11)

[0016] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDL(SEQ ID NO:13)

[0017] GMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEM(SEQ ID NO:15)

[0018] GSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP(SEQ ID NO:17)

[0019] GDPGEAGPIGPKGYRGDEGPPGSEGARGAPGPAGPPGDPGLMGERGEDGPA(SEQ ID NO:19)

[0020] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDLGPVGYQGMKGEKGSRGEKGSR

[0021] GPKGYKGEKGKRGIDGVDGVKGEMGYPGLPGCKGSPGFDGIQGPPGPKGDPGAFGLK

[0022] GEKGEPGADGEAGRPGSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAPGERGGP

[0023] GERGPRGTPGTRGPRGDPGEAGPQGDQGREGPVGVPGDPGEAGPIGPKGYRGDEGPP

[0024] GSEGARGAPGPAGPPGDPGLMGERGEDGPAGNGTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDDNNDIAPRGVKGAKGYRGPEGPQGPPGHQGPPGPD (SEQ ID NO: 21).

[0025] In one embodiment, the plurality of repeating units is 2-80 repeating units, such as 2-60, 2-70, 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15 or 2-10 repeating units, such as 2, 3, 4, 5, 6, 7, 8, 9.

[0026] In one embodiment, the linker comprises one or more amino acid residues, e.g., 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid residues.

[0027] In one embodiment, the mutation is selected from a substitution, an addition, an insertion or a deletion.

[0028] In one embodiment, the substitutions are conservative amino acid substitutions.

[0029] In one embodiment, the polypeptide is recombinant collagen. In one embodiment, the polypeptide is recombinant type VI collagen or human recombinant type VI collagen.

[0030] In one embodiment, the polypeptide has cell adhesion activity.

[0031] In one embodiment, the polypeptide comprises an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21, 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 that has 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.

[0032] In one embodiment, the mutation is selected from a substitution, addition, insertion or deletion. In one embodiment, the substitution is a conservative amino acid substitution.

[0033] In another aspect, a nucleic acid is provided that encodes a polypeptide as described herein. In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. In one embodiment, the nucleotide sequence is codon-optimized for E. coli expression. In one embodiment, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 22-32.

[0034] In another aspect, a vector is provided, which comprises a nucleic acid as described herein. In one embodiment, the vector comprises an expression control element operably connected to the nucleic acid, nucleotides of a purification tag and / or nucleotides of a leader sequence. 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 N-terminal His, Thrombin and T7 protein tags, and a C-terminal His tag. In this article, the N-terminus of the polypeptide may comprise an enzyme cleavage site for ease of purification, such as a TEV enzyme cleavage site.

[0035] In another aspect, a host cell is provided, comprising a nucleic acid or a vector as 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 Escherichia coli cell, such as Escherichia coli BL21.

[0036] In another aspect, a composition is provided, comprising one or more of the polypeptides, nucleic acids, vectors, and host cells described herein. In one embodiment, the composition is a kit. In one embodiment, the composition is a biological dressing, a human bionic material, a plastic surgery material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material, and a vascular repair and regeneration material, 3D printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients, and food additives. One or more. In one embodiment, the composition is an injectable composition or an oral composition.

[0037] In another aspect, provided is the use of the polypeptides, nucleic acids, vectors, host cells and / or compositions of the present invention in one or more of biological dressings, human bionic materials, plastic surgery 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.

[0038] In another aspect, a method of promoting cell adhesion is provided, comprising the step of contacting the polypeptide, nucleic acid, vector, host cell and / or composition herein with a cell (eg, an animal cell, a mammalian cell or a human cell).

[0039] In another aspect, a method for performing cosmetic surgery, tissue injection filling, ophthalmic treatment, nerve repair or vascular repair on a subject in need is provided, which comprises administering the polypeptide herein to the subject. In one embodiment, the administration is oral administration or injection administration. In one embodiment, the subject has a disease or condition associated with type VIII collagen deficiency, such as anterior segment hypoplasia.

[0040] In another aspect, there is provided a method for producing a polypeptide as described herein, comprising:

[0041] (1) culturing the host cell described herein under appropriate culture conditions;

[0042] (2) harvesting host cells and / or culture medium containing the polypeptide; and

[0043] (3) Purification of polypeptides.

[0044] In one embodiment, the host cell is an E. coli cell, preferably an E. coli BL21 (DE3) cell.

[0045] In one embodiment, step (1) comprises culturing the E. coli cells in LB medium and inducing expression by IPTG.

[0046] In one embodiment, step (2) comprises harvesting the E. coli cells, resuspending them in a balanced working solution, homogenizing the E. coli cells, preferably high pressure homogenizing, and separating the supernatant. In one embodiment, the balanced working solution comprises 100-500 mM sodium chloride, 10-50 mM Tris, 10-50 mM imidazole, pH 7-9.

[0047] In one embodiment, step (3) comprises crude purification, enzyme cleavage, fine purification and / or inverse nickel column purification. In one embodiment, step (3) comprises crude purification, and one or more of the following: enzyme cleavage, fine purification and / or inverse nickel column purification.

[0048] In one embodiment, the crude purification comprises subjecting the supernatant to Ni-Sepharose column purification to obtain an eluate containing the target protein, wherein the eluate comprises 100-500 mM sodium chloride, 10-50 mM Tris and 100-500 mM imidazole, preferably at pH 7-9.

[0049] In one embodiment, the purification comprises gradient elution of the eluate containing the target protein or the product after enzymatic cleavage with a strong anion exchange chromatography column; preferably, the gradient elution comprises 0-15% B solution for 1-5 minutes and then maintained for 3 column volumes, 15-30% B solution for 1-5 minutes and then maintained for 3 column volumes, 30-50% B solution for 1-5 minutes and then maintained for 3 column volumes, 50-100% B solution for 1-5 minutes and then maintained for 3 column volumes; wherein B solution contains 10-50mM Tris, 0.5-5M sodium chloride, pH7-9.

[0050] In one embodiment, the inverted nickel column purification includes purifying the product after enzyme cleavage on a Ni-agarose gel column; preferably, the eluent contains 10-50 mM Tris, 10-50 mM sodium chloride, 0.5-5 M imidazole, pH 7-9.

[0051] Herein, the enzymatic cleavage may be enzymatic cleavage by TEV enzyme.

[0052] The advantages of the present invention include: (1) providing human recombinant type VI collagen, which has cell adhesion activity and can be used in industry; (2) providing a method for preparing human recombinant type VI collagen with high yield, yield and / or purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The electropherogram of C6a is shown.

[0054] Figure 2 The electropherogram of C6d is shown.

[0055] Figure 3 The electropherogram of C6e is shown.

[0056] Figure 4 The electropherogram of C6h is shown.

[0057] Figure 5 The electropherogram of C6j is shown.

[0058] Figure 6 The electropherogram of C6f is shown.

[0059] Figure 7 The electropherogram of C6b is shown.

[0060] Figure 8 The electropherogram of C6c is shown.

[0061] Fig. 9 The electropherogram of C6i is shown.

[0062] Fig.10 The electropherogram of C6g is shown.

[0063] Fig.11 The electropherogram of C6k is shown.

[0064] Fig.12 Cell adhesion results are shown. DETAILED DESCRIPTION

[0065] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0066] As used herein, "type VI collagen" is a type of collagen present in all extracellular matrices (ECM) that is able to bind different substances of the ECM, thereby bridging cells to surrounding connective tissue and organizing the three-dimensional tissue structure of skeletal muscle, tendon, bone and cartilage.

[0067] As used herein, "polypeptide" refers to a plurality of amino acid residues connected by peptide bonds. In this article, a polypeptide comprises one or more repeating units. The repeating unit may be derived from human type VI collagen. Therefore, the polypeptide may be human recombinant type VI collagen. Multiple repeating units may be connected by a linker, which may be a natural amino acid residue of the repeating unit on human type VI collagen, such as 1-80 amino acid residues, such as 1, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80. The repeating unit may be SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21. The polypeptide may be SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21.

[0068] As used herein, "human recombinant type VI collagen" refers to a recombinant protein consisting of or substantially consisting of a sequence derived from human type VI collagen. Herein, human recombinant type VI collagen may consist of or substantially consist of a fragment or multiple repeats of a fragment derived from human type VI collagen.

[0069] As used herein, the term "variant" means a polypeptide having cell adhesion activity that includes changes (i.e., substitutions, additions, insertions and / or deletions) at one or more positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following an amino acid occupying a position. Addition refers to adding one or more amino acid residues at the C-terminus and / or N-terminus of an amino acid sequence. Substitution may be a conservative substitution. A variant of a repeating unit may be a sequence after one or more amino acid residues are changed or mutated (i.e., substituted, added, inserted and / or deleted) in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21. A variant of a polypeptide may be a sequence after one or more amino acid residues are changed or mutated (i.e., substituted, added, inserted and / or deleted) in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21.

[0070] In the context of the present invention, conservative substitutions may be defined by substitutions within the amino acid classes reflected in one or more of the following tables:

[0071] Conservative categories of amino acid residues:

[0072] Acidic residues D and E

[0073] Basic residues K, R, and H

[0074] Hydrophilic uncharged residues S, T, N and Q

[0075] Aliphatic uncharged residues G, A, V, L and I

[0076] Nonpolar uncharged residues C, M and P

[0077] Aromatic residues F, Y and W.

[0078] Physical and functional classification of candidate amino acid residues:

[0079] Containing alcohol residues S and T

[0080] Aliphatic residues I, L, V and M

[0081] Cycloalkenyl related residues F, H, W and Y

[0082] Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y

[0083] Negatively charged residues D and E

[0084] Polar residues C, D, E, H, K, N, Q, R, S, and T

[0085] Positively charged residues H, K and R

[0086] Small residues A, C, D, G, N, P, S, T, and V

[0087] Minimal residues A, G, and S

[0088] Residues involved in turn formation: 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.

[0089] As used herein, "cell adhesion" refers to the adhesion between cells and collagen. Collagen (such as the polypeptides described herein) can promote the adhesion between cells and the container in which the cells are cultured.

[0090] As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0091] As used herein, the term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

[0092] As used herein, the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0093] As used herein, the term "nucleic acid" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified to contain a nucleic acid segment in a manner that does not originally exist in nature, or is synthetic, and the nucleic acid molecule may contain one or more control sequences. The nucleic acid may be SEQ ID NO: 22-32. The nucleic acid may be a codon-optimized nucleic acid, such as a codon-optimized nucleic acid for expression in E. coli cells.

[0094] The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0095] The degree of association between two amino acid sequences or between two nucleotide sequences is described by parameter " sequence identity ".For purposes of the present invention, use as in EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Genetics Trend] 16:276-277) (preferred 5.0.0 version or updated version) Niederman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [J.Molecular Biology] 48:443-453) determine the sequence identity between two amino acid sequences.The parameter used is gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.The output (using non-simplified option to obtain) of Nieder marked as "longest identity" is used as identity percentage and is calculated as follows:

[0096] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0097] For the purposes of the present invention, the sequence identity between two deoxynucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented by the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferably 5.0.0 version or updated version). The parameters used are gap opening penalty 10, gap extension penalty 0.5, and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle, labeled "longest identity", (obtained using the non-simplified option) is used as identity percentage and is calculated as follows:

[0098] (number of identical deoxyribonucleotides x 100) / (alignment length - total number of gaps in the alignment)

[0099] Peptides

[0100] The present invention provides a polypeptide comprising one or more repeating units, the repeating units being linked directly or through a linker, the repeating units comprising an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21. 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, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21, 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, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21. For the polypeptides described herein, the mutation may be selected from substitution, addition, insertion or deletion. Preferably, the substitutions are conservative amino acid substitutions.

[0101] The polypeptides described herein may comprise a plurality of repeating units, e.g., 2-80 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.

[0102] 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 amino acid residues.

[0103] The polypeptide described herein is a recombinant collagen, in particular a recombinant type VI collagen, preferably having cell adhesion activity. In this context, the recombinant type VI collagen is a human recombinant type VI collagen.

[0104] The polypeptide described herein may also comprise an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21, 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 that has 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.

[0105] Nucleic acid construct

[0106] The present invention also relates to nucleic acid constructs comprising a nucleic acid of the present invention operably linked to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.A vector may comprise a nucleic acid construct.

[0107] Nucleic acids can be manipulated in a variety of ways to provide expression of polypeptides. 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.

[0108] The control sequence may be a promoter, i.e., a polynucleotide that is recognized by the host cell for expression of a polypeptide encoding the invention. The promoter comprises a transcriptional control sequence that mediates the expression of the polypeptide. The promoter may be any nucleic acid that exhibits transcriptional activity in the host cell, including variants, truncated and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0109] Examples of suitable promoters for directing transcription of the vector or nucleic acid construct of the present invention in a bacterial host cell are those obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus licheniformis penicillinase gene (penP), the Bacillus stearothermophilus maltoamylase gene (amyM), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and xylB genes, the Bacillus thuringiensis cryIIIA gene, the Escherichia coli lac operon, the Escherichia coli trc promoter.

[0110] 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.

[0111] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator may be operably linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.

[0112] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0113] Preferred terminators for 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, supra).

[0114] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0115] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0116] The control sequence may also be a leader sequence, i.e., a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell may be used.

[0117] Suitable leaders for yeast host cells are obtained from the genes for 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).

[0118] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the polynucleotide and, when transcribed, recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0119] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0120] The control sequence can also be a signal peptide coding region that encodes a signal peptide connected to the N-terminus of the polypeptide and directs the polypeptide to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide itself can contain a signal peptide coding sequence that is naturally connected to the coding sequence segment of the coded polypeptide in the translation reading frame. Alternatively, the 5'-end of the coding sequence can contain a signal peptide coding sequence that is exogenous to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide to enter the secretory pathway of the host cell can be used.

[0121] Effective signal peptide coding sequences for bacterial host cells are those obtained from the genes for 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 by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0122] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al. (1992, supra).

[0123] Type VI collagen

[0124] Collagen plays an important role in maintaining the structure and function of the cell matrix. Type VI collagen is a fine mesh structure, distributed between type I, III, and V collagen fibers, connecting type I, III, and V collagen to the basement membrane. It is the anchor point between collagens and the attachment site of cells in the matrix, and plays an important role in the self-stabilization of the matrix. Under an electron microscope, type VI collagen is dumbbell-shaped, with a rod-shaped part in the middle of about 150nm and two spherical wings. Type VI collagen is composed of three different peptide chains α1, α2, and α3. The genes of the α1 and α2 chains of human type VI collagen are both located at the 21g 22.3 site, with a length of 36kb and composed of 30 exons. The gene of α3 collagen is located at the 2q 37 site. Each peptide chain contains a helical region, and there are large globular regions at the N and C terminals. The two globular regions mainly contain the von Willebrand factor A-type repeat region. The von Willebrand factor A-type repeat region has a large molecular weight of about 21kDa, and the number of NC chain ends is different in α1, α2, and α3. Studies have shown that the center of the A region is a β-fold, with 3α helices on both sides. Type VI collagen contains 18 non-helical von Willebrand factor A-type repeat regions, each of which has a repeat sequence of 200 amino acid residues, of which α3 contains 12, and α1 and α2 each contain 3. In microfibrils, discontinuous Gly-XY turns the helical structure into multiple segments. Type VI collagen forms monomers, dimers, and tetramers under the action of disulfide bonds. Dimers are formed by two monomers in an anti-parallel manner, with an overlapping region of 75mm, and the globular regions are still at both ends. The C-terminus can be stably combined with its adjacent helical structure by disulfide bonds. Two dimers form a tetramer in parallel. Recent studies have shown that although the C-terminal region is a non-helical region of type VI collagen, it plays an important role and is required for the formation of dimers and tetramers. Outside the cell, the terminals of the tetramers are connected to form a microfibril structure, and the tetramers are bound to each other by non-covalent bonds. This binding is assumed to be the interaction of the A region. Type II collagen and aggrecan bind to type VI collagen through a proteoglycan complex rich in leucine repeat sequences. In the early stages of tissue formation or in the process of repairing trauma or fractures, through this binding, type VI collagen plays a leading role in tissue formation, construction and self-stabilization. Type VI collagen can also be intertwined with type V collagen between fibers, and combined with mucopolysaccharides to maintain the structure of the epidermal matrix. In addition to the above-mentioned collagens, type VI collagen can also combine with other macromolecules, such as type IV collagen, type XIV collagen, core protein polysaccharide, microfibril-associated glycoprotein 1, hyaluronic acid, 1B2 and ot2pl binding proteins, and cell surface proteoglycan NG. Type VI collagen stabilizes the structure of the extracellular matrix by combining with these macromolecules.By maintaining the structure and function of the extracellular matrix, type VI collagen can maintain the integrity of tissues such as blood vessels, lungs, cartilage, muscles and skin. If the gene encoding type VI collagen mutates, Bethlem myopathy and Ullrich syndrome will occur, leading to muscle weakness and emaciation. The loss of type VI collagen can also lead to the loss of mitochondrial function and cell apoptosis. On the other hand, the increase or accumulation of type VI collagen can also lead to the occurrence of diseases such as superficial fibroma, neurofibroma, keloid, pulmonary fibrosis, liver fibrosis, diabetic kidney damage and rheumatoid arthritis. Type VI collagen also affects cell differentiation, adhesion, migration, proliferation and survival. When myocardial fibroblasts were cultured in a matrix containing type VI collagen, it was found that myofibroblasts differentiated due to the induction of type VI, and this result was also confirmed by in vivo experiments. In the study of the function of type VI collagen, type VI collagen has a strong adhesion to various directional hematopoietic stem cells, and the location of this adhesion is limited to the helical region of the three peptide chains. Collagen VI can also promote the proliferation of various cells, which can be blocked by the single peptide chain of collagen VI. The enhancement of fibroblast proliferation and migration is achieved by combining collagen VI with cell surface proteoglycan NG, core protein glycan, syndecan, hyaluronic acid and other types of collagen.

[0125] Expression vector

[0126] The present invention further relates to a recombinant expression vector comprising nucleic acid of the present invention, promoter and transcription and translation termination signals. Nucleic acid and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to insert or replace a polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting a nucleic acid or a nucleic acid construct comprising the nucleic acid into a suitable vector for expression. When producing an expression vector, the encoding sequence is located in the vector so that the encoding sequence is operably connected to a suitable control sequence for expression.

[0127] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0128] The vector can be an autonomously replicating vector, i.e. a vector existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome when it is introduced into the host cell and replicates with one or more chromosomes into which it has been integrated. Moreover, a single vector or plasmid or two or more vectors or plasmids can be used, which contain the total DNA to be introduced into the host cell genome together, or a transposon can be used.

[0129] The vector preferably contains one or more selectable markers that permit easy selection of transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0130] Examples of bacterial selectable markers are the dal genes of Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0131] The selectable marker may be a dual selectable marker system as described in WO 2010 / 039889. In one aspect, the dual selectable marker is the hph-tk dual selectable marker system.

[0132] The vector may contain elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0133] For being integrated into the host cell genome, the vector can rely on the polynucleotide sequence encoding the polypeptide or any other element of the vector for being integrated into the genome by homologous or non-homologous recombination.Alternately, the vector can contain other polynucleotides at the precise position for guiding the chromosome to be integrated into the host cell genome by homologous recombination.In order to improve the possibility of integration at the precise position, the integration element should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs and 800 to 10,000 base pairs, and these nucleic acids have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination.The integration element can be any sequence homologous to the target sequence in the host cell genome.And, the integration element can be a non-coding or coded polynucleotide.On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0134] For autonomous replication, the vector may further comprise an origin of replication, which enables autonomous replication of the vector in the host cell in question. The origin of replication may be any plasmid replicon mediating autonomous replication that functions in the cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0135] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

[0136] Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0137] More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the production of the polypeptide. The increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene together with the polynucleotide, wherein cells containing amplified copies of the selectable marker gene and thus additional copies of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selective agent.

[0138] The procedures used to ligate the above-mentioned elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989).

[0139] Host cells

[0140] The present invention also relates to recombinant host cells comprising polynucleotides of the present invention operably linked to one or more control sequences that direct the production of polypeptides of the present invention. A construct or vector comprising the polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier. The term "host cell" encompasses any parent cell progeny that is not identical to the parent cell due to mutations that occur during replication. The selection of the host cell will depend to a large extent on the gene encoding the polypeptide and its source.

[0141] The host cell can be any cell useful in the recombinant production of the polypeptides of the present invention, eg, a prokaryotic or a eukaryotic cell.

[0142] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella and Ureaplasma.

[0143] Host cells can also be eukaryotic organisms, such as mammalian, insect, plant or fungal cells. Plant cells herein do not include plant cells that can be regenerated into plants. Animal cells also do not include cells that can produce animal bodies.

[0144] The host cell may be a fungal cell, such as Basidiomycota, Chytridiomycota, Zygomycota, and Oomycota, etc. The fungal host cell may be a yeast cell, including ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to Fungi Imperfecti (Blastomycetes). The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or a Yarrowia lipolytica cell.

[0145] Production method

[0146] The present invention also relates to the production of a polypeptide as described herein, comprising:

[0147] (1) culturing the host cell described herein under appropriate culture conditions;

[0148] (2) harvesting host cells and / or culture medium containing the polypeptide; and

[0149] (3) Purification of polypeptides.

[0150] Host cells are cultured in a suitable nutrient medium for producing polypeptides using methods known in the art. For example, cells can be cultured by shaking flasks or in small or large-scale fermentations (including continuous, batch, fed-batch or solid-state fermentation) in a laboratory or industrial fermenter, in a suitable medium and under conditions that allow polypeptide expression and / or separation. Using procedures as known in the art, cultivation occurs in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable culture medium can be obtained from commercial suppliers or can be prepared according to disclosed compositions (for example, in the catalog of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be directly recovered from the culture medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

[0151] The polypeptide can be detected using methods known in the art that are specific for the polypeptide. These detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay can be used to determine the activity of a polypeptide.

[0152] The polypeptide can be recovered using methods known in the art. For example, the polypeptide can be recovered from the nutrient medium by conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the fermentation broth containing the polypeptide is recovered.

[0153] The polypeptide can 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 electrophoresis), differential solubilization (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction, in order to obtain a substantially pure polypeptide.

[0154] Step (1) may include one or more of the following steps: constructing an expression plasmid, for example, inserting the encoding nucleotide sequence into the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid. The successfully constructed expression plasmid can be transformed into Escherichia coli cells (for example, Escherichia coli competent cells BL21 (DE3)). The specific process may be: (1) taking the plasmid to be transformed and adding it to the Escherichia coli competent cells BL21 (DE3); (2) placing the mixture in an ice bath on ice (for example, 10-60 minutes, for example, 30 minutes), then heat shocking in a water bath (for example, at 40-50°C, for example, 42°C, 45-90 seconds), taking it out and placing it in an ice bath on ice (for example, 1-5 minutes, for example, 2 minutes); (3) adding liquid LB culture medium, and then culturing (for example, culturing at 35-40°C, for example, 37°C, 150-300rpm, for example, 220rpm for 40-80 minutes, for example, 60 minutes); (4) applying the bacterial solution and selecting a single colony. For example, take the bacterial solution and spread it evenly on an LB plate containing ampicillin sodium, and culture the plate in a 37°C incubator for 15-17 hours until colonies of uniform size grow.

[0155] Step (2) may include culturing a single colony in LB medium containing an antibiotic stock solution (e.g., at 150-300 rpm, e.g., 220 rpm, 35-40° C., e.g., 37° C. in a constant temperature shaker for 5-10 h, e.g., 7 h). The cultured shake flask is then cooled to 10-20° C., e.g., 16° C., and IPTG is added to induce expression for a period of time before collecting the bacteria (e.g., by centrifugation).

[0156] Step (3) may include resuspending the bacterial cells with a balanced working solution, cooling the bacterial solution to ≤15°C, homogenizing (e.g., high pressure homogenization, e.g., 1-5 times, e.g., 2 times), and separating the homogenized bacterial solution to obtain a supernatant. The balanced working solution may contain 100-500 mM sodium chloride, 10-50 mM Tris, and 10-50 mM imidazole, pH 7-9. For example, the concentration of sodium chloride can 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 may be 7, 7.5, 8, 8.5 or 9.

[0157] Step (3) may include purifying and enzymatically cleaving the polypeptide. Purification may be crude purification, including Ni-agarose gel column purification of the supernatant to obtain an eluent containing the target protein. Crude purification may include washing the column material with water, for example, 2-10 column volumes (CV), for example, 5 CV. The column material may be balanced with a balance solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0), for example, 2-10 CV, for example, 5 CV. The balance solution may contain 100-500 mM sodium chloride, 10-50 mM Tris and 10-50 mM imidazole, pH 7-9. For example, the concentration of sodium chloride can 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 may be 7, 7.5, 8, 8.5 or 9.

[0158] Step (3) may include adding the supernatant to the column material and washing the impurity protein with a washing solution. The washing solution may contain 100-500 mM sodium chloride, 10-50 mM Tris and 10-50 mM imidazole, 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, an eluent can be added and the flow-through can be collected. The eluent can contain 100-500 mM sodium chloride, 10-50 mM Tris, 100-500 mM imidazole, pH 8.0. For example, the concentration of sodium chloride can 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 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 can be 7, 7.5, 8, 8.5, or 9.

[0159] Enzymatic digestion may include adding TEV enzyme for digestion (the ratio of total protein to total TEV enzyme is 10-100:1, such as 50:1, and digestion is performed at 10-20°C, such as 16°C for 2-8h, such as 4h). The protein solution after digestion is dialyzed, for example, placed in a dialysis bag, dialyzed at 1-6°C, such as 4°C for 1-8h, such as 2h, and then transferred to a new dialysate for overnight dialysis at 1-6°C, such as 4°C.

[0160] Purification may include fine purification (e.g., protein isoelectric point>8.0). Preferably, fine purification includes gradient elution of the eluent containing the target protein or the product after enzymatic cleavage (e.g., the product after enzymatic cleavage and dialysis) using a strong anion exchange chromatography column (e.g., pH 7, 7.5, 8, 8.5, or 9). Gradient elution includes 0-15% B solution for 1-5 minutes and then kept for 1-5, such as 3 column volumes, 15-30% B solution for 1-5 minutes and then kept for 1-5, such as 3 column volumes, 30-50% B solution for 1-5 minutes and then kept for 1-5, such as 3 column volumes, 50-100% B solution for 1-5 minutes and then kept for 1-5, such as 3 column volumes. B solution may contain 10-50mM Tris, 0.5-5M sodium chloride, pH7-9. For example, the concentration of Tris is 15, 20, 25, 30, 35, 40, or 45mM. The concentration of sodium chloride is 1, 2, 3 or 4 M. The pH can be 7, 7.5, 8, 8.5 or 9. Purification can include balancing the column with liquid A and loading the sample, followed by gradient elution. Liquid A can contain 10-50 mM Tris, 10-50 mM sodium chloride, 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. The pH can be 7, 7.5, 8, 8.5 or 9.

[0161] Purification can include reverse hanging nickel column purification (e.g., protein isoelectric point <8.0). Reverse hanging nickel column purification can include Ni-agarose gel column purification of the product after enzyme cleavage (e.g., the product after dialysis). The eluent can contain 10-50mM (e.g., 15, 20, 25, 30, 35, 40, or 45mM) Tris, 10-50mM (e.g., 15, 20, 25, 30, 35, 40, or 45mM) sodium chloride, 0.5-5M (e.g., 1, 2, 3, or 4M) imidazole, pH 7-9 (e.g., 7, 7.5, 8, 8.5, or 9).

[0162] Advantages of the present invention: 1. The polypeptide of the present invention is derived from type VI collagen and is recombinant type VI collagen; 2. The polypeptide of the present invention is suitable for the preparation of Escherichia coli and can be separated and purified; 3. The polypeptide of the present invention has a large expression amount and is suitable for subsequent purification.

[0163] The following examples are provided to further illustrate the present invention.

[0164] Example

[0165] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims. In combination with this specification and common knowledge in the art, a person of ordinary skill in the art can clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, a person of ordinary skill in the art can make any modification or change to the technical solution of the present invention, and such modification and change are also included in the scope of the present invention.

[0166] Example 1: Construction, expression and screening of type VI collagen fragments

[0167] 1. Perform large-scale functional region screening to obtain the following different target gene functional regions of recombinant humanized type VI collagen

[0168] Amino acid sequence

[0169] (1) C6a

[0170] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDLGPVGYQGMKGEK

[0171] GSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEMGYPGLPGCKGSPGFD

[0172] GIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRPGSSGPSGDEGQPGEPGP

[0173] PGEKGEAGDEGNPGPDGAPGERGGPGERGPRGTPGTRGPRGDPGEAGPQ

[0174] GDQGREGPVGVPGDPGEAGPIGPKGYRGDEGPPGSEGARGAPGPAGPPG

[0175] DPGLMGERGEDGPAGNGTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDDNNDIAPRGVKGAKGYRGPEGPQGPPGHQGPPGPD(SEQ ID NO:21)

[0176] (2) C6b

[0177] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDLGPVGYQGMKG

[0178] EKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEMGYPGLPGCKGSPG

[0179] FDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRPGSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP(SEQ ID NO:9)

[0180] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDLGPVGYQGMKG

[0181] EKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEMGYPGLPGCKGSPG

[0182] FDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRPGSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP (the amino acid sequence of the repeating unit of C6b is SEQ ID NO: 9, the number of repeating units is 2, and the amino acid sequence of C6b is SEQ ID NO: 10)

[0183] (3)C6c

[0184] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGREGPVGVPGDPG

[0185] EAGPIGPKGYRGDEGPPGSEGARGAPGPAGPPGDPGLMGERGEDGPA(SEQ ID NO:1)

[0186] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGREGPVGVPGDPG

[0187] EAGPIGPKGYRGDEGPPGSEGARGAPGPAGPPGDPGLMGERGEDGPA

[0188] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGREGPVGVPGDPG

[0189] EAGPIGPKGYRGDEGPPGSEGARGAPGPAGPPGDPGLMGERGEDGPA (the amino acid sequence of the repeating unit of C6c is SEQ ID NO: 1, the number of repeating units is 3, and the amino acid sequence of C6c is SEQ ID NO: 2)

[0190] (4)C6d

[0191] GIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRPGSSGPSGDEGQPGE

[0192] PGPPGEKGEAGDEGNPGPDGAPGERGGPGERGPRGTPGTRGPRGDPGEA GPQGDQGREGPVGVPGDPGEAGPIGPKGYRGDEGPP(SEQ ID NO:11)

[0193] GIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRPGSSGPSGDEGQPGE

[0194] PGPPGEKGEAGDEGNPGPDGAPGERGGPGERGPRGTPGTRGPRGDPGEA GPQGDQGREGPVGVPGDPGEAGPIGPKGYRGDEGPP (the amino acid sequence of the repeating unit of C6d is SEQ ID NO: 11, the number of repeating units is 2, and the amino acid sequence of C6d is SEQ ID NO: 12)

[0195] (5)C6e

[0196] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDL(SEQ ID NO:13)

[0197] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDL

[0198] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDL

[0199] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDL

[0200] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDL

[0201] GPPGLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDL (the amino acid sequence of the repeating unit of C6e is SEQ ID NO: 13, the number of repeating units is 6, and the amino acid sequence of C6e is SEQ ID NO: 14)

[0202] (6)C6f

[0203] GMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEM(SEQ ID NO:15)

[0204] GMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEM

[0205] GMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEM

[0206] GMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEM

[0207] GMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEM

[0208] GMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGVDGVKGEM (the amino acid sequence of the repeating unit of C6f is SEQ ID NO: 15, the number of repeating units is 6, and the amino acid sequence of C6f is SEQ ID NO: 16)

[0209] (7)C6g

[0210] GCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRP(SEQ ID NO:3)

[0211] GCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRP

[0212] GCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRP

[0213] GCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRP

[0214] GCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRP

[0215] GCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAGRP (the amino acid sequence of the repeating unit of C6g is SEQ ID NO: 3, the number of repeating units is 6, and the amino acid sequence of C6g is SEQ ID NO: 4)

[0216] (8)C6h

[0217] GSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP(SEQ ID NO:17)

[0218] GSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP

[0219] GSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP

[0220] GSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP

[0221] GSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP

[0222] GSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGAP (the amino acid sequence of the repeating unit of C6h is SEQ ID NO: 17, the number of repeating units is 6, and the amino acid sequence of C6h is SEQ ID NO: 18)

[0223] (9)C6i

[0224] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGRE(SEQ ID NO:5)

[0225] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGRE

[0226] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGRE

[0227] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGRE

[0228] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGRE

[0229] GERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGRE (the amino acid sequence of the repeating unit of C6i is SEQ ID NO: 5, the number of repeating units is 6, and the amino acid sequence of C6i is SEQ ID NO: 6)

[0230] (10)C6j

[0231] GDPGEAGPIGPKGYRGDEGPPGSEGARGAPGPAGPPGDPGLMGERGE DGPA(SEQ ID NO:19)

[0232] GDPGEAGPIGPKGYRGDEGPPGSEGARGAPGPAGPPGDPGLMGERGE

[0233] DGPA

[0234] GDPGEAGPIGPKGYRGDEGPPGSEGARGAPGPAGPPGDPGLMGERGE

[0235] DGPA

[0236] GDPGEAGPIGPKGYRGDEGPPGSEGARGAPGPAGPPGDPGLMGERGEDGPA (the amino acid sequence of the repeating unit of C6j is SEQ ID NO: 19, the number of repeating units is 4, and the amino acid sequence of C6j is SEQ ID NO: 20)

[0237] (11)C6k

[0238] GTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDD(SEQ ID NO:7)

[0239] GTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDD

[0240] GTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDD

[0241] GTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDD

[0242] GTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDD

[0243] GTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDD (the amino acid sequence of the repeating unit of C6k is SEQ ID NO: 7, the number of repeating units is 6, and the amino acid sequence of C6k is SEQ ID NO: 8)

[0244] Nucleotide sequence

[0245] 1.C6a

[0246]

[0247] 2.C6b

[0248] GGACCACCCGGGCTCCGTGGTGACCCGGGCTTCGAAGGCGAGCGCGGCAAACCGGGTTTGCCGGGTGAAAAGGGTGAAGCAGGCGACCCGGGTCGCCCAGGCGACCTGGGTCCGGTTGGTTACCAAGGTATGAAAGGGGAGAAGGGATCTAGAGGCGAAAAAGGCTCCCGCGGTCCGAAAGGCTATAAGGGCGAAAAGGGCAAGCGTGGCATTGATGGTGTCGATGGTGTTAAAGGCGAAATGGGTTATCCGGGTCTGCCGGGCTGCAAAGGTAGCCCGGGTTTTGATGGTATCCAGGGTCCGCCTGGTCCGAAGGGCGACCCGGGTGCGTTTGGTCTGAAAGGTGAGAAGGGCGAGCCGGGCGCGGATGGTGAGGCGGGTCGTCCGGGCAGCAGCGGTCCGAGCGGTGACGAAGGTCAGCCGGGCGAGCCGGGTCCGCCTGGTGAAAAGGGTGAGGCCGGTGACGAGGGCAACCCGGGTCCGGATGGCGCGCCGGGCCCACCGGGCTTACGTGGTGACCCGGGCTTCGAAGGTGAACGTGGTAAGCCGGGTTTGCCGGGTGAGAAGGGCGAAGCTGGCGACCCGGGCCGTCCGGGCGACCTGGGTCCGGTGGGTTATCAGGGTATGAAAGGCGAGAAGGGTTCTCGTGGCGAAAAGGGTTCCCGTGGTCCGAAAGGCTACAAAGGTGAAAAGGGAAAGCGCGGCATTGATGGTGTGGATGGCGTGAAAGGTGAGATGGGTTACCCGGGTCTGCCTGGTTGTAAAGGTTCCCCGGGATTCGACGGCATCCAGGGTCCGCCCGGTCCGAAAGGTGACCCGGGCGCGTTTGGCCTGAAAGGCGAAAAGGGCGAGCCGGGTGCCGATGGCGAGGCAGGACGTCCGGGGTCGAGCGGCCCAAGCGGTGATGAAGGCCAACCGGGTGAGCCGGGCCCACCGGGCGAGAAGGGTGAGGCTGGCGACGAAGGTAATCCGGGTCCGGATGGCGCGCCG(SEQ IDNO:23)

[0249] 3.C6c

[0250] GGAGAAAGGGGGGGCCCGGGCGAGCGCGGCCCGCGTGGCACCCCGGGGACCCGTGGCCCGCGTGGTGACCCGGGCGAGGCTGGCCCGCAAGGTGATCAAGGTCGTGAAGGTCCGGTGGGCGTGCCGGGTGATCCGGGTGAGGCGGGCCCCATCGGTCCGAAAGGTTACCGTGGCGATGAGGGCCCTCCGGGTAGCGAAGGCGCGCGTGGCGCTCCGGGTCCGGCGGGTCCGCCAGGCGACCCGGGCCTGATGGGTGAACGCGGCGAAGATGGTCCGGCGGGCGAACGCGGTGGTCCGGGCGAGCGCGGTCCGCGTGGCACGCCGGGCACTCGCGGCCCACGTGGTGATCCGGGTGAGGCGGGTCCGCAGGGTGACCAGGGTCGTGAAGGTCCAGTTGGTGTTCCGGGCGACCCGGGTGAAGCCGGTCCGATTGGTCCGAAGGGTTACCGCGGCGACGAAGGCCCACCGGGTAGCGAAGGTGCCCGTGGCGCACCGGGTCCGGCAGGCCCACCGGGCGACCCGGGCTTGATGGGCGAGCGCGGCGAGGACGGCCCGGCTGGCGAGCGCGGTGGTCCGGGTGAGCGCGGCCCGCGTGGCACCCCGGGCACCCGTGGTCCGCGTGGTGACCCGGGTGAGGCAGGTCCTCAAGGTGATCAGGGCAGAGAAGGTCCGGTCGGTGTTCCGGGCGACCCGGGAGAGGCGGGTCCGATCGGCCCGAAGGGTTATCGTGGTGACGAAGGTCCGCCTGGTAGCGAAGGCGCGCGTGGCGCGCCAGGCCCTGCCGGTCCACCGGGTGACCCGGGCCTGATGGGTGAGCGTGGTGAGGATGGTCCGGCG(SEQ ID NO:24)

[0251] 4.C6d

[0252] GGAATACAAGGGCCACCGGGGCCGAAGGGCGACCCGGGTGCTTTCGGCCTGAAAG

[0253] GCGAGAAAGGCGAGCCGGGCGCGGATGGTGAGGCGGGTCGTCCGGGTAGCAGCG

[0254] GTCCGTCTGGCGACGAGGGCCAGCCGGGTGAACCGGGTCCGCCAGGAGAAAAGG

[0255] GTGAGGCCGGCGACGAAGGTAATCCGGGTCCGGATGGCGCGCCTGGCGAGCGCGG

[0256] CGGCCCGGGCGAACGCGGACCGCGTGGCACGCCGGGCACCCGTGGTCCGCGTGGT

[0257] GATCCGGGCGAAGCAGGTCCCCAAGGCGATCAGGGTCGCGAAGGCCCGGTTGGTG

[0258] TGCCGGGCGACCCGGGCGAGGCGGGTCCGATTGGTCCGAAAGGCTACCGCGGCGA

[0259] CGAAGGTCCACCGGGTATCCAGGGTCCACCGGGTCCCAAGGGCGACCCGGGTGCG

[0260] TTTGGTTTGAAAGGCGAGAAGGGTGAACCGGGTGCAGATGGTGAGGCGGGCAGAC

[0261] CTGGCAGCTCGGGCCCGTCCGGTGACGAAGGCCAACCGGGCGAACCGGGTCCGCC

[0262] AGGTGAGAAGGGCGAGGCCGGTGACGAGGGCAACCCGGGTCCGGATGGTGCACC

[0263] GGGGGAGCGCGGTGGTCCGGGCGAGCGTGGTCCGCGTGGCACCCCGGGCACCCGT

[0264] GGTCCGCGTGGTGACCCTGGTGAGGCGGGTCCGCAGGGCGATCAAGGCCGTGAAG

[0265] GTCCGGTGGGTGTTCCGGGCGACCCGGGCGAAGCTGGCCCGATCGGTCCGAAAGGTTATCGTGGTGATGAAGGTCCGCCA(SEQ ID NO:25)

[0266] 5.C6e

[0267] GGGCCCCCAGGATTGCGCGGCGATCCGGGTTTCGAGGGCGAGCGCGGCAAGCCGG

[0268] GTCTGCCGGGTGAAAAAGGTGAGGCTGGTGACCCGGGCCGTCCGGGCGACCTGGG

[0269] TCCGCCTGGCCTGCGTGGTGATCCGGGTTTTGAAGGTGAACGTGGCAAGCCGGGTC

[0270] TGCCGGGTGAAAAAGGTGAGGCCGGTGATCCGGGCCGTCCGGGCGACCTTGGCCC

[0271] ACCGGGCTTGCGTGGCGACCCGGGCTTCGAGGGCGAGCGCGGTAAGCCGGGCCTG

[0272] CCCGGTGAAAAGGGTGAGGCAGGCGATCCGGGACGCCCAGGCGACCTGGGTCCGC

[0273] CAGGCCTGCGTGGTGACCCGGGTTTCGAAGGCGAACGCGGTAAACCGGGTTTGCC

[0274] GGGTGAGAAAGGTGAGGCGGGTGATCCGGGCCGTCCGGGCGACCTGGGTCCGCCT

[0275] GGATTACGTGGTGACCCGGGCTTTGAAGGTGAGCGCGGAAAACCGGGTCTGCCAG

[0276] GCGAGAAGGGCGAAGCGGGTGACCCGGGTCGTCCGGGCGACCTGGGCCCACCGG

[0277] GTTTGAGAGGTGATCCGGGCTTTGAAGGTGAGCGTGGTAAGCCGGGTCTGCCGGG

[0278] TGAGAAAGGCGAAGCGGGCGATCCGGGTCGTCCGGGCGATCTC(SEQ ID NO:26)

[0279] 6.C6f

[0280] GGGATGAAAGGAGAGAAGGGCTCCCGTGGTGAAAAGGGCAGCCGTGGTCCGAAA

[0281] GGTTATAAAGGTGAGAAGGGCAAACGCGGCATTGATGGTGTTGATGGCGTGAAGG

[0282] GCGAGATGGGTATGAAAGGTGAGAAGGGCAGCCGTGGTGAAAAAGGTAGCCGTG

[0283] GCCCAAAGGGTTACAAAGGTGAAAAGGGCAAACGCGGGATCGATGGTGTTGACG

[0284] GCGTGAAAGGCGAGATGGGTATGAAAGGCGAGAAGGGTTCTCGTGGTGAAAAGG

[0285] GCAGCCGCGGTCCGAAAGGCTACAAAGGCGAGAAGGGTAAACGTGGGATCGACG

[0286] GCGTTGATGGCGTCAAGGGCGAAATGGGCATGAAGGGCGAAAAGGGTAGCCGTG

[0287] GTGAAAAGGGTTCACGTGGTCCGAAAGGTTATAAAGGTGAAAAGGGCAAACGTG

[0288] GTATTGATGGTGTGGACGGCGTCAAAGGAGAGATGGGTATGAAAGGTGAAAAGG

[0289] GTAGCCGTGGTGAGAAGGGTTCTCGCGGTCCGAAAGGTTATAAAGGTGAGAAGGG

[0290] CAAACGTGGCATCGACGGTGTGGATGGCGTTAAAGGCGAGATGGGTATGAAGGGC

[0291] GAGAAGGGTTCCCGCGGTGAGAAGGGTAGCCGTGGTCCGAAAGGCTACAAGGGT

[0292] GAAAAGGGCAAGCGCGGTATTGACGGCGTTGACGGCGTGAAAGGCGAAATG(SEQ

[0293] ID NO:27)

[0294] 7.C6g

[0295] GGGTGTAAAGGATCGCCGGGCTTTGACGGCATCCAAGGTCCGCCTGGTCCGAAAG

[0296] GAGATCCGGGTGCGTTCGGTCTGAAAGGTGAAAAGGGCGAGCCGGGTGCCGACGG

[0297] CGAAGCAGGCCGTCCTGGCTGTAAAGGTAGCCCGGGTTTCGACGGCATTCAAGGT

[0298] CCTCCGGGTCCGAAAGGCGACCCGGGTGCGTTTGGCTTGAAGGGCGAAAAGGGCG

[0299] AACCGGGTGCTGATGGCGAGGCAGGTCGCCCAGGCTGCAAAGGTTCTCCGGGTTT

[0300] TGATGGTATTCAGGGTCCACCGGGCCCCAAGGGCGATCCGGGCGCGTTCGGTCTG

[0301] AAGGGTGAAAAGGGCGAACCGGGCGCGGACGGTGAGGCGGGTCGTCCGGGCTGT

[0302] AAAGGTAGCCCGGGTTTTGATGGTATCCAGGGTCCGCCAGGCCCGAAAGGCGACC

[0303] CGGGTGCGTTTGGTTTAAAGGGCGAGAAAGGCGAGCCGGGTGCGGACGGCGAGG

[0304] CTGGTCGTCCGGGGTGCAAAGGCAGCCCGGGCTTCGATGGCATTCAGGGTCCACC

[0305] GGGTCCGAAGGGTGACCCGGGTGCGTTCGGCCTGAAAGGTGAAAAGGGCGAGCC

[0306] GGGTGCTGATGGTGAAGCCGGTCGCCCTGGCTGCAAGGGCAGCCCGGGATTCGAC

[0307] GGCATCCAGGGCCCGCCGGGTCCGAAGGGCGATCCGGGTGCCTTCGGCCTGAAAG

[0308] GGGAGAAGGGTGAGCCGGGTGCGGACGGTGAAGCAGGTCGTCCG(SEQ ID NO:28)

[0309] 8.C6h

[0310] GGGTCAAGTGGACCGTCTGGTGATGAAGGTCAGCCGGGCGAACCGGGTCCGCCGG

[0311] GTGAAAAAGGTGAGGCGGGTGACGAAGGTAACCCGGGTCCGGATGGCGCCCCTG

[0312] GCTCCAGCGGTCCGTCAGGCGACGAGGGTCAGCCGGGTGAACCGGGCCCTCCGGG

[0313] CGAGAAGGGCGAAGCTGGCGACGAGGGAAACCCGGGCCCGGACGGTGCTCCGGG

[0314] TAGCAGCGGTCCGTCTGGCGATGAGGGTCAACCGGGTGAGCCGGGCCCACCGGGT

[0315] GAAAAGGGTGAGGCGGGTGACGAGGGCAATCCGGGTCCGGATGGCGCGCCGGGC

[0316] AGCAGCGGCCCATCCGGCGACGAGGGCCAACCGGGCGAACCGGGCCCACCGGGC

[0317] GAGAAGGGTGAAGCGGGTGATGAGGGGAACCCGGGTCCGGACGGCGCACCGGGC

[0318] AGCAGCGGTCCGTCTGGTGACGAAGGCCAGCCGGGCGAGCCGGGTCCGCCGGGCG

[0319] AGAAAGGTGAGGCCGGTGACGAAGGTAATCCGGGTCCGGATGGTGCGCCAGGTTC

[0320] GAGCGGTCCGTCCGGCGATGAAGGTCAACCGGGCGAGCCTGGTCCACCGGGTGAA

[0321] AAAGGCGAGGCAGGCGACGAAGGCAACCCGGGTCCGGATGGAGCGCCA(SEQ ID

[0322] NO:29)

[0323] 9.C6i

[0324] GGAGAAAGGGGGGGTCCGGGCGAGCGCGGCCCACGTGGCACCCCCGGCACTCGC

[0325] GGTCCGCGCGGCGATCCGGGTGAAGCAGGCCCACAGGGTGATCAAGGTCGCGAGG

[0326] GCGAGCGTGGCGGTCCGGGTGAACGCGGGCCGCGTGGCACCCCGGGCACCCGTGG

[0327] TCCGCGTGGTGATCCGGGTGAAGCGGGTCCGCAGGGTGATCAGGGTCGTGAAGGC

[0328] GAGCGCGGAGGCCCAGGCGAGCGTGGTCCGCGTGGCACCCCGGGTACGCGTGGTC

[0329] CGCGTGGTGACCCGGGCGAGGCGGGTCCGCAAGGTGACCAGGGCCGTGAAGGTG

[0330] AACGTGGTGGCCCGGGCGAGCGCGGCCCGCGTGGCACCCCGGGCACCCGTGGTCC

[0331] GCGCGGCGACCCGGGTGAGGCCGGTCCGCAGGGCGACCAAGGTCGTGAAGGTGA

[0332] ACGCGGCGGTCCGGGTGAGCGCGGCCCACGTGGCACGCCGGGTACGCGTGGCCCG

[0333] AGAGGCGACCCTGGTGAGGCTGGCCCTCAAGGTGATCAGGGCCGTGAAGGTGAGA

[0334] GAGGTGGTCCGGGCGAGCGCGGTCCGAGAGGCACCCCGGGCACCCGTGGTCCGCGTGGTGACCCGGGTGAAGCGGGTCCGCAAGGCGACCAGGGTCGTGAA(SEQ ID NO:30)

[0335] 10.C6j

[0336] GGAGATCCCGGAGAGGCAGGTCCGATTGGTCCGAAAGGCTATCGTGGTGATGAAG

[0337] GCCCGCCAGGCTCCGAGGGCGCGCGTGGTGCCCCGGGCCCGGCTGGTCCGCCGGG

[0338] CGACCCGGGCCTGATGGGCGAGCGTGGCGAGGATGGTCCGGCGGGTGACCCGGGT

[0339] GAAGCCGGTCCGATTGGCCCCAAGGGCTATCGTGGCGACGAAGGTCCGCCGGGGT

[0340] CTGAAGGTGCGCGTGGTGCTCCGGGTCCGGCTGGCCCGCCGGGCGATCCGGGCCT

[0341] GATGGGTGAGCGCGGTGAAGATGGTCCGGCAGGTGATCCGGGCGAGGCCGGTCCG

[0342] ATCGGCCCGAAGGGTTACCGCGGTGATGAAGGTCCGCCTGGCAGCGAAGGTGCGC

[0343] GTGGTGCGCCTGGTCCAGCAGGCCCGCCGGGCGACCCGGGCCTGATGGGTGAGCG

[0344] CGGCGAAGATGGTCCGGCGGGTGACCCGGGTGAGGCAGGTCCGATCGGTCCGAAA

[0345] GGTTACCGCGGTGACGAGGGTCCGCCTGGCAGCGAAGGTGCGAGAGGCGCGCCAG

[0346] GCCCGGCTGGCCCACCGGGCGACCCGGGCTTGATGGGTGAACGTGGTGAGGACGGCCCGGCG(SEQ IDNO:31)

[0347] 11.C6k

[0348] AGGGACAGAAGGATTCCCGGGCTTCCCAGGTTATCCGGGCAACCGCGGTGCGCCA

[0349] GGCATTAACGGCACCAAAGGTTATCCGGGTTTGAAGGGCGACGAAGGCGAGGCGG

[0350] GTGATCCGGGAGACGACGGCACCGAAGGTTTTCCGGGCTTTCCGGGCTACCCGGG

[0351] TAATCGTGGTGCACCGGGGATCAACGGCACCAAGGGTTACCCGGGCCTGAAAGGT

[0352] GATGAGGGCGAGGCGGGTGATCCGGGCGATGATGGCACCGAGGGCTTCCCGGGGT

[0353] TCCCGGGTTATCCGGGTAACCGTGGCGCCCCCGGCATTAATGGTACGAAGGGTTAC

[0354] CCGGGCCTGAAAGGTGATGAAGGTGAAGCGGGTGACCCGGGGGACGACGGCACC

[0355] GAAGGTTTTCCGGGCTTCCCGGGTTACCCGGGAAACCGCGGTGCGCCAGGCATCA

[0356] ATGGCACCAAGGGTTACCCGGGTCTGAAAGGTGACGAGGGCGAAGCGGGTGATCC

[0357] GGGTGACGACGGTACGGAGGGTTTTCCGGGTTTCCCGGGTTACCCGGGTAATCGTG

[0358] GTGCACCAGGGATCAACGGCACCAAAGGCTATCCGGGTTTGAAGGGTGATGAAGG

[0359] CGAGGCCGGTGACCCGGGCGACGATGGTACTGAGGGTTTCCCTGGCTTTCCGGGCT

[0360] ACCCGGGAAACCGTGGTGGCTCCGGGCATTAACGGTACGAAAGGCTATCCTGGCCTGAAGGGCGACGAGGGTGAAGCTGGTGACCCCGGGTGATGAT (SEQ ID NO: 32) plasmid clone

[0361] Each of the above coding nucleotide sequences is commercially synthesized. Each of the above coding nucleotide sequences (with a collagenase cleavage site added at the 5' end, the amino acid sequence of the collagenase cleavage site is ENLYFQ, and the nucleotide sequence is GAAAACCTGTATTTCCAG) is inserted between the KpnI and XhoI cleavage sites of the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid.

[0362] Host cell transformation

[0363] The successfully constructed expression plasmid was transformed into E. coli competent cells BL21 (DE3). The specific process is as follows: (1) Take out the E. coli competent cells BL21 (DE3) from the ultra-low temperature refrigerator and place them on ice. When they are half melted, take 2 μl of the plasmid to be transformed and add it to the E. coli competent cells BL21 (DE3), and mix it slightly 2-3 times. (2) Place the mixture on ice for 30 minutes, then heat shock it in a 42°C water bath for 45-90 seconds, take it out and place it on ice for 2 minutes. (3) Transfer it to a biological safety cabinet, add 700 μl of liquid LB culture medium, and then culture it at 37°C and 220 rpm for 60 minutes. (4) Take 200 μl of the bacterial solution and evenly spread it on an LB plate containing ampicillin sodium. (5) Culture the plate in a 37°C incubator for 15-17 hours until colonies of uniform size grow. Pick 5-6 single colonies from the transformed LB plate and place them in a shake flask containing LB medium containing antibiotic stock solution, and incubate in a constant temperature shaker at 220rpm and 37℃ for 7h. Then cool the shake flask after culture to 16℃, add IPTG to induce expression for a period of time, divide the bacterial solution into centrifuge bottles, centrifuge at 8000rpm and 4℃ for 10min, collect the bacterial cells, record the bacterial cell weight, and take samples (marked: bacterial solution) for electrophoresis detection.

[0364] Peptide isolation and purification

[0365] The collected bacteria were resuspended with a balanced working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0), the bacterial solution was cooled to ≤ 15 °C, homogenized, and high pressure homogenized twice, and the bacterial solution was collected after completion. The homogenized bacterial solution was divided into centrifuge bottles, centrifuged at 17000 rpm and 4 °C for 30 min, the supernatant was collected, and the supernatant (marked: supernatant) and the precipitate were taken for electrophoresis detection.

[0366] Recombinant type VI humanized collagen was purified and digested by enzymes. The specific process was as follows: (1) Crude purification: a. Wash the column material (Ni6FF, Cytiva) with water for 5 CVs. b. Equilibrate the column material with equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) for 5 CVs. c. Loading: Add the supernatant after centrifugation to the column material until the liquid flows out, and take the flow-through for electrophoresis inspection (marked as flow-through). d. Wash impurities: Add 25 mL of washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) until the liquid flows out, and take the wash impurities flow-through for electrophoresis inspection (marked as wash impurities). e. Collect the target protein: add 20 mL of elution solution (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0), collect the flow-through (marked as elution), detect the protein concentration, calculate the protein amount, and perform electrophoresis detection. f. Wash the column with 1 M imidazole working solution (marked as 1 M wash). g. Wash the column with purified water. (2) Enzyme digestion: add TEV enzyme at a ratio of 50:1 between the total amount of protein and the total amount of TEV enzyme, digest at 16°C for 4 hours, and take samples for electrophoresis detection (after digestion). Put the protein solution after enzyme digestion into a dialysis bag, dialyze at 4°C for 2 hours, and then transfer to new dialysis solution for overnight dialysis at 4°C (marked as changing A solution).

[0367] (3) Purification (protein isoelectric point > 8.0): a. Equilibrate the column (Capto Q, Cytiva): Use solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0) to equilibrate the column at a flow rate of 10 ml / min. b. Loading: The flow rate is 5 ml / min, load the sample and collect the flow-through (marked as QFL), and perform electrophoresis detection. c. Gradient elution: Set 0-15% solution B (20 mM Tris, 1 M sodium chloride, pH 8.0) for 2 min and then keep for 3 CVs, 15-30% solution B for 2 min and then keep for 3 CVs, 30-50% solution B for 2 min and then keep for 3 CVs, 50-100% solution B for 2 min and then keep for 3 CVs, collect the peaks and perform electrophoresis detection (marked as B wash). d. Clean the column. Store the protein at 4°C.

[0368] (4) Nickel reverse mounting (Ni6FF, Cytiva) (protein isoelectric point <8.0): a. Equilibrate the column: Use solution A (20mMTris, 20mM sodium chloride, 20mM imidazole, pH8.0) to equilibrate the column for 5 CVs. b. Loading: Add the protein after enzyme switching solution to the column, and wait until the liquid has flowed out, then take the flow-through for electrophoresis detection (marked as nickel reverse mounting). c. Wash the column with 1M imidazole working solution (20mMTris, 20mM sodium chloride, 1M imidazole, pH8.0) (marked as 1M wash). d. Wash the column with purified water. Store the protein at 4°C.

[0369] Concentration detection

[0370] Accurately measure an appropriate amount of sample, dilute 10-50 times with eluent, and stir thoroughly with a glass rod. Use a UV-visible spectrophotometer to measure the absorbance at 280nm, and calculate the protein concentration according to the formula C (mg / ml) = A280 × absorbance coefficient × dilution factor (Note: the absorbance coefficient can be obtained based on the amino acid sequence; the absorbance value must be between 0.1-1).

[0371] The concentration test results are as follows:

[0372] Plasmids Absorption coefficient A280 Dilution multiple concentration Elution protein volume Protein Amount C6a 1.91 0.665 1 1.27mg / ml 20ml 25.40mg C6b 2.01 0.330 5 3.32mg / ml 20ml 66.33mg C6c 2.18 0.121 5 1.32mg / ml 20ml 26.38mg C6D 2.28 0.238 2 1.08mg / ml 20ml 21.66mg C6e 2.48 0.595 1 1.48mg / ml 20ml 29.51mg C6F 1.69 0.360 10 6.08mg / ml 20ml 121.68mg C6 2.65 0.291 5 3.94mg / ml 20ml 78.71mg C6h 2.37 0.130 5 1.54mg / ml 20ml 30.81mg C6i 2.49 0.277 5 3.45mg / ml 20ml 68.97mg C6J 1.68 0.329 5 2.76mg / ml 20ml 55.27mg C6k 1.25 0.392 5 2.46mg / ml 20ml 49.25mg

[0373] Protein expression: C6f>C6g>C6i>C6b>C6j>C6k>C6h>C6e>C6c>C6a>C6d

[0374] Electrophoresis detection

[0375] The specific process is: take 40μl of sample solution, add 10μl 5× protein loading buffer (250mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), place it in 100℃ boiling water for 10min, then add 10μl to each well of SDS-PAGE protein gel, run at 80V for 2h, use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 20min, and then use protein decolorization solution (10% acetic acid, 5% ethanol) for decolorization.

[0376] Figure 1 The electropherogram of C6a is shown. Figure 2 The electropherogram of C6d is shown. Figure 3 The electropherogram of C6e is shown. Figure 4 The electropherogram of C6h is shown. Figure 5 The electropherogram of C6j is shown. Figure 6 The electropherogram of C6f is shown. Figure 7 The electropherogram of C6b is shown. Figure 8 The electropherogram of C6c is shown. Fig. 9 The electropherogram of C6i is shown. Fig.10 The electropherogram of C6g is shown. Fig.11 The electropherogram of C6k is shown.

[0377] The electrophoresis results showed that the crude yields of C6a, C6d and C6h were low ( Figure 1 , 2 , 4); the crude yield of C6e is low and the enzyme digestion effect is poor ( Figure 3); C6j had a lower crude yield and more impurities ( Figure 5 ); there are some impurities after C6f elution, and the amount of protein is small after enzyme digestion and purification ( Figure 6 ); there are some impurities after C6b elution, and the enzyme digestion is incomplete ( Figure 7 ); The crude yields of C6c, C6g, C6i, and C6k were high, and the purity of the target protein was high after nickel hanging ( Figure 8 , 9 , 10, 11).

[0378] According to the purification results, the recombinant type VI humanized collagen prepared by the two plasmids C6c, C6g, C6i, and C6k is better, so mass spectrometry detection is performed on C6c, C6g, C6i, and C6k proteins.

[0379] Example 2: Mass spectrometry detection of recombinant humanized type VI collagen

[0380] Experimental methods

[0381]

[0382] After DTT reduction and iodoacetamide alkylation, the protein sample was enzymatically digested overnight with trypsin. The peptides obtained after enzymatic hydrolysis were desalted by C18ZipTip and mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA) and spotted on the plate. Finally, the analysis was performed using a matrix-assisted laser desorption ionization-time of flight mass spectrometer MALDI-TOF / TOF UlraflextremeTM, Brucker, Germany (the technology of peptide fingerprinting can be referred to Protein J.2016;35:212-7).

[0383] Data retrieval was processed from the MS / MSIon Search page on the local masco website. Protein identification results were obtained based on the primary mass spectra of the peptides produced after enzymatic digestion. Detection parameters: Trypsin digestion, two missed cleavage sites. Alkylation of cysteine ​​was set as a fixed modification. Oxidation of methionine was set as a variable modification. The database used for identification was NCBprot.

[0384] Table 1: Molecular weight and corresponding peptides detected by mass spectrometry of recombinant type VI humanized collagen C6c

[0385]

[0386] The coverage of the detected polypeptide fragments compared with the theoretical sequence is 100%, and the detection result is very reliable.

[0387] Table 2: Molecular weight and corresponding peptides detected by mass spectrometry of recombinant type VI humanized collagen C6g

[0388]

[0389]

[0390] The coverage of the detected polypeptide fragments compared with the theoretical sequence is 100%, and the detection result is very reliable.

[0391] Table 3: Molecular weight and corresponding peptides detected by mass spectrometry of recombinant type VI humanized collagen C6i

[0392]

[0393] The coverage of the detected polypeptide fragments compared with the theoretical sequence is 100%, and the detection result is very reliable.

[0394] Table 4: Molecular weight and corresponding peptides detected by mass spectrometry of recombinant type VI humanized collagen C6k

[0395]

[0396] The coverage of the detected polypeptide fragments compared with the theoretical sequence was 78.57%, and the detection result was very reliable.

[0397] Example 3: Detection of biological activity of recombinant humanized type VI collagen

[0398] The method for detecting the activity of collagen can refer to the literature 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 implementation method is as follows:

[0399] (1) The concentration of the protein sample to be tested is detected by ultraviolet absorption method, including bovine type I collagen (PC, China Food and Drug Inspection Institute, No.: 380002), and the recombinant type VI humanized collagen C6c, C6g, C6i, and C6k provided by the present invention.

[0400] Specifically, the ultraviolet absorption of the sample at 215nm and 225nm is measured respectively, and the protein concentration is calculated using the empirical formula C (μg / mL) = 144×(A215-A225). Note that the test must be performed when A215 < 1.5. The principle of this method is: to measure the characteristic absorption of peptide bonds under far-ultraviolet light, it is not affected by the chromophore content, has few interfering substances, is easy to operate, and is suitable for detecting human collagen and its analogs that do not develop color with Coomassie Brilliant Blue. (The reference is Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45.). After the protein concentration is detected, the concentration of all proteins to be tested is adjusted to 1 mg / mL with PBS.

[0401] (2) Add different concentrations of collagen and positive and negative controls to the ELISA plate, 100 μL per well, 5 replicate wells per group, and incubate at 4°C overnight.

[0402] (3) Discard the supernatant, add 100 μL 1% BSA (heat-inactivated at 56°C for 30 min), and incubate at 37°C for 60 min. Discard the supernatant, and wash three times with D-PBS solution (NC).

[0403] (4) Add 10 5 Resuspend 3T3 / NIH cells in good culture state in D-PBS and incubate at 37℃ for 120min. Wash each well with D-PBS solution for 3 times.

[0404] (5) Detect OD using CCK8 detection kit 450 The absorbance of the cells is 100 nm. Based on the value of the blank control, the cell adhesion rate can be calculated. The calculation formula is as follows: The cell adhesion rate can reflect the activity of collagen. The higher the activity of the protein, the better the external environment it can provide to the cells in a short time, helping the cells to adhere to the wall.

[0405] The results are as follows Fig.12 As shown, compared with the D-PBS group, the positive control group had a significant effect on promoting cell adhesion, and the recombinant humanized collagen also promoted cell adhesion at the experimental concentration (the vertical axis is the cell adhesion activity % relative to the D-PBS group), and the results were statistically significant, among which *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0406] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0407] The present invention provides the following contents:

[0408] 1. A polypeptide comprising one or more repeating units, wherein the repeating units are connected directly or through a linker, wherein the repeating units comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21, 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 with the amino acid sequence;

[0409] Preferably, the plurality of repeating units is 2-50 repeating units, such as 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-8 or 2-6 repeating units;

[0410] Preferably, wherein the linker comprises one or more amino acid residues, such as 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acid residues;

[0411] Preferably, wherein the mutation is selected from substitution, addition, insertion or deletion;

[0412] Preferably, wherein the substitution is a conservative amino acid substitution;

[0413] Preferably, the polypeptide is recombinant collagen; preferably recombinant type VI collagen; preferably human recombinant type VI collagen;

[0414] Preferably, the polypeptide has cell adhesion activity.

[0415] 2. A polypeptide according to claim 1, comprising an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21, 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 with the amino acid sequence;

[0416] Preferably, wherein the mutation is selected from substitution, addition, insertion or deletion;

[0417] Preferably, the substitution is a conservative amino acid substitution.

[0418] 3. A nucleic acid encoding a polypeptide according to item 1 or 2,

[0419] Preferably, wherein the nucleic acid comprises a codon-optimized nucleotide sequence,

[0420] Preferably, the nucleotide sequence is codon-optimized for E. coli expression;

[0421] Preferably, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 22-32.

[0422] 4. A vector comprising a nucleic acid according to item 3,

[0423] Preferably, the vector comprises an expression control element, a purification tag nucleotide and / or a leader sequence nucleotide operably linked to the nucleic acid,

[0424] Preferably, wherein the expression control element is selected from a promoter, a terminator or an enhancer;

[0425] Preferably, the purification tag is selected from a His tag, a GST tag, a MBP tag, a SUMO tag or a NusA tag;

[0426] Preferably, the vector is an expression vector or a cloning vector, preferably pET-28a(+).

[0427] 5. A host cell comprising the nucleic acid according to item 3 or the vector according to item 4; wherein preferably, the host cell is a eukaryotic cell or a prokaryotic cell; wherein preferably, the eukaryotic cell is a yeast cell, an animal cell and / or an insect cell, and / or the prokaryotic cell is an Escherichia coli cell, such as Escherichia coli BL21.

[0428] 6. A composition comprising one or more of the polypeptide according to item 1 or 2, the nucleic acid according to item 3, the vector according to item 4 and the host cell according to item 5, preferably, the composition is a kit; preferably, the composition is one or more of biological dressings, human bionic materials, plastic surgery 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 printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives, preferably, the composition is an injectable composition or an oral composition.

[0429] 7. Use of the polypeptide according to item 1 or 2, the nucleic acid according to item 3, the vector according to item 4, the host cell according to item 5 and / or the composition according to item 6 in one or more of biological dressings, human bionic materials, plastic surgery 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.

[0430] 8. A method for promoting cell adhesion, comprising the step of contacting a cell with the polypeptide according to item 1 or 2, the nucleic acid according to item 3, the vector according to item 4, the host cell according to item 5 and / or the composition according to item 6, wherein the cell is an animal cell, preferably a mammalian cell, preferably a human cell.

[0431] 9. A method for performing cosmetic surgery, tissue injection filling, ophthalmic treatment, nerve repair or vascular repair on a subject in need thereof, comprising administering the polypeptide according to item 1 or 2 to the subject, preferably orally or by injection; preferably, the subject is a human.

[0432] 10. Producing a polypeptide according to item 1 or 2, comprising:

[0433] (1) culturing the host cell according to item 5 under suitable culture conditions;

[0434] (2) harvesting host cells and / or culture medium containing the polypeptide; and

[0435] (3) purifying the polypeptide and optionally removing the tag;

[0436] Preferably, the host cell is an Escherichia coli cell, preferably an Escherichia coli BL21 (DE3) cell;

[0437] Preferably, step (1) comprises culturing E. coli cells in LB medium and inducing expression by IPTG;

[0438] Preferably, step (2) comprises harvesting the E. coli cells, resuspending them in a balanced working solution, homogenizing the E. coli cells, preferably high pressure homogenization, and separating the supernatant; preferably, the balanced working solution comprises 100-500 mM sodium chloride, 10-50 mM Tris, 10-50 mM imidazole, pH 7-9;

[0439] Preferably, step (3) comprises crude purification, enzyme digestion, fine purification and / or reverse hanging nickel column purification;

[0440] Preferably, the crude purification comprises subjecting the supernatant to Ni-agarose gel column purification to obtain an eluate containing the target protein, wherein the eluate comprises 100-500 mM sodium chloride, 10-50 mM Tris and 100-500 mM imidazole, preferably at pH 7-9;

[0441] Preferably, the enzymatic digestion includes digestion with TEV enzyme, preferably with a ratio of total protein to total TEV enzyme of 10-100:1 for 2-8 hours;

[0442] Preferably, the purification comprises gradient elution of the eluate containing the target protein or the product after enzyme cleavage with a strong anion exchange chromatography column; preferably, the gradient elution comprises 0-15% B solution for 1-5 minutes and then maintained for 3 column volumes, 15-30% B solution for 1-5 minutes and then maintained for 3 column volumes, 30-50% B solution for 1-5 minutes and then maintained for 3 column volumes, 50-100% B solution for 1-5 minutes and then maintained for 3 column volumes; wherein B solution contains 10-50mM Tris, 0.5-5M sodium chloride, pH7-9;

[0443] Preferably, the inverted nickel column purification comprises purifying the product after enzyme cleavage on a Ni-agarose gel column; preferably, the eluent comprises 10-50 mM Tris, 10-50 mM sodium chloride, 0.5-5 M imidazole, pH 7-9.

Claims

1. A recombinant collagen, the amino acid sequence of which is SEQ ID NO:

4.

2. A nucleic acid encoding the recombinant collagen according to claim 1.

3. The nucleic acid of claim 2, comprising a codon-optimized nucleotide sequence.

4. The nucleic acid of claim 3, comprising a nucleotide sequence codon-optimized for E. coli expression.

5. The nucleic acid according to claim 4, comprising the nucleotide sequence shown in SEQ ID NO:

28.

6. A vector comprising the nucleic acid according to any one of claims 2 to 5.

7. The vector according to claim 6, comprising an expression control element, nucleotides of a purification tag and / or nucleotides of a leader sequence operably linked to the nucleic acid.

8. The vector according to claim 7, wherein the expression control element is selected from a promoter, a terminator or an enhancer.

9. The vector according to claim 7, wherein the purification tag is selected from a His tag, a GST tag, a MBP tag, a SUMO tag or a NusA tag.

10. The vector according to claim 6, which is an expression vector or a cloning vector. The vector according to claim 10 , which is pET-28a(+).

12. A host cell comprising a nucleic acid according to any one of claims 2 to 5 or a vector according to any one of claims 6 to 11; wherein the host cell is a eukaryotic cell or a prokaryotic cell; wherein the eukaryotic cell is a yeast cell or an animal cell.

13. The host cell according to claim 12, wherein the animal cell is an insect cell. The host cell according to claim 12 , wherein the prokaryotic cell is an Escherichia coli cell. The host cell according to claim 14 , wherein the E. coli cell is E. coli BL21.

16. A composition comprising one or more of the recombinant collagen according to claim 1, the nucleic acid according to any one of claims 2-5, the vector according to any one of claims 6-11, and the host cell according to any one of claims 12-15.

17. The composition of claim 16, wherein the composition is a kit.

18. The composition according to claim 16, The composition is one or more of biological dressings, human bionic materials, plastic surgery 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 printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

19. The composition according to claim 16, wherein the composition is an injectable composition or an oral composition.

20. Use of the recombinant collagen according to claim 1, the nucleic acid according to any one of claims 2-5, the vector according to any one of claims 6-11 and / or the host cell according to any one of claims 12-15 in the preparation of one or more of biological dressings, human bionic materials, plastic surgery 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 printing artificial organ biomaterials, cosmetic raw materials, pharmaceutical excipients and food additives.

21. A method for promoting cell adhesion in vitro, comprising the step of contacting the recombinant collagen according to claim 1, the nucleic acid according to any one of claims 2 to 5, the vector according to any one of claims 6 to 11 and / or the host cell according to any one of claims 12 to 15 with a cell in vitro.

22. The method of claim 21, wherein the cell is an animal cell.

23. The method of claim 22, wherein the animal cell is a mammalian cell.

24. The method of claim 23, wherein the mammalian cell is a human cell.

25. A method for producing the recombinant collagen according to claim 1, comprising: (1) culturing the host cell according to any one of claims 12 to 15 under suitable culture conditions; (2) harvesting host cells and / or culture medium containing the recombinant collagen; and (3) Purify the recombinant collagen and optionally remove the tag.

26. The method of claim 25, wherein the host cell is an E. coli cell.

27. The method according to claim 26, wherein the E. coli cells are E. coli BL21 (DE3) cells.

28. The method according to claim 25, wherein step (1) comprises culturing the E. coli cells in LB medium and inducing expression by IPTG.

29. The method according to claim 25, wherein step (2) comprises harvesting the Escherichia coli cells, resuspending them in a balanced working solution, homogenizing the Escherichia coli cells, and separating the supernatant.

30. The method of claim 29, wherein the homogenization is high pressure homogenization.

31. The method according to claim 29, wherein the equilibrium working solution comprises 100-500 mM sodium chloride, 10-50 mM Tris, 10-50 mM imidazole, pH 7-9.

32. The method according to claim 25, wherein step (3) comprises crude purification, enzyme digestion, fine purification and / or nickel column purification.

33. The method according to claim 32, wherein the crude purification comprises subjecting the supernatant to Ni-agarose gel column purification to obtain an eluate containing the target protein, wherein the eluate comprises 100-500 mM sodium chloride, 10-50 mM Tris and 100-500 mM imidazole.

34. The method of claim 33, wherein the pH of the eluent is 7-9.

35. The method of claim 32, wherein the enzymatic cleavage comprises enzymatic cleavage with TEV enzyme.

36. The method according to claim 35, wherein the ratio of the total amount of protein to the total amount of TEV enzyme is 10-100:1 and the digestion is carried out for 2-8 hours.

37. The method according to claim 32, wherein the purification comprises gradient elution of the eluate containing the target protein or the product after enzyme cleavage using a strong anion exchange chromatography column.

38. The method of claim 37, wherein the gradient elution comprises 0-15% B solution for 1-5 minutes and then maintained for 3 column volumes, 15-30% B solution for 1-5 minutes and then maintained for 3 column volumes, 30-50% B solution for 1-5 minutes and then maintained for 3 column volumes, 50-100% B solution for 1-5 minutes and then maintained for 3 column volumes; wherein B solution contains 10-50mM Tris, 0.5-5M sodium chloride, pH7-9.

39. The method according to claim 32, wherein the inverse nickel column purification comprises purifying the product after enzyme cleavage on a Ni-agarose gel column.

40. The method according to claim 39, wherein the eluent for Ni-Sepharose column purification comprises 10-50 mM Tris, 10-50 mM sodium chloride, 0.5-5 M imidazole, pH 7-9.

41. Use of the recombinant collagen according to claim 1, the nucleic acid according to any one of claims 2 to 5, the vector according to any one of claims 6 to 11 and / or the host cell according to any one of claims 12 to 15 in the preparation of a composition for promoting cell adhesion.

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