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

Abstract

A method for biosynthesizing the human structural material type VIII collagen is provided. The present disclosure provides collagen comprising one or more repeating units, linked directly or via a linker, each repeating unit comprising an amino acid sequence selected from SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28, or a variant thereof. The collagen can be used in bio-dressings, human biomimetic materials, or cosmetic and reconstructive materials. The method herein produces type VIII collagen using genetic engineering techniques, overcoming the deficiencies of the prior art.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to a Chinese patent application filed on July 18, 2023, bearing application number 202310883772.3 and entitled "Method for Biosynthesis of Human Body Structural Material Type VIII Collagen," the contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of collagen, and in particular to a method for biosynthesizing the human structural material type VIII collagen. [Background technology]

[0003] Collagen is the most abundant and widely distributed protein in animal bodies and is the major component of connective tissue. Type VIII collagen is a short-chain, nonfibrinogenic collagen and is the main component of the retinal membrane and the corneal endothelial cell sublayer (the basement membrane separating the corneal endothelial cells from the corneal stroma). Type VIII collagen is produced by endothelial cells and forms a unique hexagonal lattice structure. Type VIII collagen is present around chondrocytes in the heart, brain, liver, lungs, muscle, and cartilage. Type VIII collagen has also been found around actively growing blood vessels in brain tumors and in the clumped fibrotic blood vessels of hemangiomas.

[0004] Type VIII collagen, also known as short-chain collagen or network-forming collagen, contains two similar α chains, α1(VIII) and α2(VIII), and two homotrimeric subtypes, [α1(VIII)]3 and [α2(VIII)]3, which are considered to be the major molecular species. Heterotrimeric forms may also exist. Descemet's membrane in bovine eyes has a hexagonal network structure containing thin type VIII collagen fibrils. Immunohistochemical analysis and electron microscopy have shown that type VIII collagen is the main component of the structural framework. Rotary shadow analysis has also shown that type VIII collagen may form tetrahedral supramolecular structures that can provide structural support and regulate cellular behavior.

[0005] Mice double-knockout of two types of type VIII collagen genes, Col8a1 and Col8a2, exhibit a pronounced anterior segment dysgenesis phenotype, with a spherical anterior chamber and a spherically protruding cornea. The corneal stroma is diffusely thinned, similar to that seen in human keratoconjunctiva. Descemet's membrane is clearly thin and the anterior cingulate region is absent. Corneal endothelial cells are enlarged and reduced in number. Finally, the in vitro response of mutant corneal endothelial cells to different growth factors demonstrates impaired proliferative capacity, suggesting that type VIII collagen may be an enhancer of growth factor-induced cell proliferation.

[0006] Type VIII collagen was previously described as a heterotrimer consisting of two α1 chains and one α2 chain, but in vitro studies have shown that either α1 or α2 homotrimers can be formed. These homotrimers are pepsin-resistant, and immunohistochemical studies have shown that these homotrimers do not always co-localize in the cornea, optic nerve, aorta, and umbilical cord. α1 has a total length of 744 amino acids (aa), with the first 27 amino acids at the N-terminus being a signal peptide.

[0007] The NC1 domain (aa 572-744) of type VIII collagen is a protein called vastatin, which has been shown to be a potent antiangiogenic agent with apoptosis-inducing activity in aortic endothelial cells. The α2 procollagen has a chain length of 703 aa and a signal peptide at the N-terminus of α1-28. The α1 and α2 procollagen genes each contain four exons. One of their distinctive features is that the largest exon encodes the entire triple-helical domain (COL1) and the C-terminal non-triple-helical domain (NC1). Type VIII collagen shares high sequence homology with type X collagen and a similar intron-exon structure. This indicates that the two collagens originate from the same progenitor gene and belong to the same collagen subclass. Type VIII collagen, like type X collagen, can form a hexagonal lattice structure.

[0008] Type VIII collagen is a glycoprotein highly sensitive to neutrophil elastase and, unlike other vascular collagens, such as type I, type II, type IV, and type V, is completely degraded within 4 hours. Type VIII collagen is synthesized by aortic endothelial cells, corneal endothelial cells, pulmonary artery endothelial cells, and microvascular endothelial cells. Because this collagen is absent in large and small blood vessels, not all endothelial cells express type VIII collagen. Furthermore, monocytes and macrophages have been shown to express type VIII collagen in vitro and in vivo. Human mast cells have also been shown to produce type VIII collagen under normal and pathological conditions. Type VIII collagen has been shown to contribute to angiogenesis, tissue remodeling, fibrosis, and cancer. Type VIII-expressing mast cells are found in perivascular spaces. Type VIII collagen is also expressed in smooth muscle cells and stimulates cell migration.

[0009] The corneal endothelium secretes type VIII collagen, which is assembled into a hexagonal lattice structure within the endothelial membrane through the interaction of the α1 and α2 polypeptides. Mutations in the COL8A2 gene, but not COL8A1, are associated with early Fuchs endothelial corneal dystrophy. Type VIII collagen is thought to be involved in endothelial cell differentiation and organization. During cardiac development, type VIII collagen plays an important role in angiogenesis and is immunolocalized in the subendothelial layer of capillaries and arterioles. Type VIII collagen expression is elevated in early atherosclerosis and is thought to be associated with thrombus formation and monocyte infiltration. Type VIII collagen accumulates in atherosclerotic lesions, and its distribution pattern suggests its role in plaque stability. Type VIII collagen is present around actively proliferating brain tumor vessels and in the clumped fibrotic vessels of hemangiomas. Finally, type VIII collagen is expressed in human diabetic nephropathy but not in other kidney diseases.

[0010] To date, most collagens used in various research studies are derived from animal tissues and skin extracts. Collagen extracted from animal bodies has poor water solubility and poor processability, which directly limits the development of many potential applications. Collagen produced by genetic engineering technology can effectively overcome these drawbacks. Summary of the Invention

[0011] The present invention is based in part on the following discovery by the inventors. The present inventors conducted a large-scale screening of functional domains in human type VIII collagen to obtain target gene functional domains of different recombinant humanized type VIII collagens, which showed higher cell adhesion-promoting activity compared to the positive control (human type I collagen). The present invention also demonstrates that the collagens or polypeptides of the present invention are suitable for expression and purification. Furthermore, the inventors discovered that the expression levels and purification efficiency of C8a and C8c are better than those of other collagens (C8b and C8d-j), making them suitable for industrial production. The C8a and C8c of the present invention also have a triple helix structure.

[0012] In one aspect, the present invention provides a collagen or polypeptide comprising one or more repeat units, the repeat units being linked directly or via a linker, and the repeat units comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28, or a variant thereof, wherein the variant is either (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence.

[0013] In one embodiment, the plurality of repeat units is 2 to 50 repeat units, for example, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, or 2 to 8 repeat units.

[0014] In one embodiment, the linker comprises one or more amino acid residues, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acid residues.

[0015] In one embodiment, the mutation is selected from a substitution, addition, insertion or deletion.

[0016] In one embodiment, the substitutions are conservative amino acid substitutions.

[0017] In one embodiment, the collagen or polypeptide is recombinant collagen. In one embodiment, the collagen or polypeptide is recombinant type VIII collagen. In one embodiment, the collagen or polypeptide is human recombinant type VIII collagen.

[0018] In one embodiment, the collagen or polypeptide has cell adhesion activity or a triple helix structure.

[0019] In one embodiment, the collagen or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, or a variant thereof, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence.

[0020] In one embodiment, the mutation is selected from a substitution, addition, insertion or deletion, hi one embodiment, the substitution is a conservative amino acid substitution.

[0021] In another aspect, a nucleic acid is provided that encodes a collagen or polypeptide 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: 3, 6, 9, 12, 15, 18, 21, 24, 27, or 30.

[0022] In another aspect, a vector is provided comprising the nucleic acid described herein. In one embodiment, the vector comprises an expression control element, a purification tag nucleotide, and / or a leader sequence nucleotide operably linked to the nucleic acid. In one embodiment, the expression control element is selected from a promoter, a terminator, or an enhancer. In one embodiment, the purification tag is selected from a His tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag. In one embodiment, the vector is an expression vector or a cloning vector, preferably pET-28a(+). pET-28a(+) may comprise an N-terminal His tag, a Thrombin tag, and a T7 protein tag, as well as a C-terminal His tag. The collagen or polypeptide herein may comprise an enzyme cleavage site, such as a TEV enzyme cleavage site, at the N-terminus to facilitate purification.

[0023] In another aspect, a host cell is provided comprising a nucleic acid or vector described herein. In one embodiment, the host cell is a eukaryotic cell or a prokaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell, and in one embodiment, the prokaryotic cell is an E. coli cell, such as E. coli BL21.

[0024] In another aspect, a composition is provided, comprising one or more of the collagen or polypeptide, nucleic acid, vector, and host cell described herein.In one embodiment, the composition is a kit.In one embodiment, the composition is one or more of the following: bio-covering material, human biomimetic material, cosmetic cosmetic material, organoid culture material, cardiovascular stent, coating material, tissue injection filling material, ophthalmic material, gynecological biomaterial, nerve repair and regeneration material, liver tissue material and vascular repair and regeneration material, 3D printing artificial organ biomaterial, cosmetic raw material, pharmaceutical auxiliary material, and food additive.In one embodiment, the composition is an injectable composition or an oral composition.

[0025] In another aspect, there is provided a use of the collagen or polypeptide, nucleic acid, vector, host cell and / or composition herein in one or more of a bio-dressing material, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection and filling material, an ophthalmic material, an obstetric and gynecological biomaterial, a nerve repair and regeneration material, a liver tissue material and a vascular repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, a medicinal supplement, and a food additive.

[0026] In another aspect, there is provided a method of promoting cell adhesion comprising contacting a cell with a collagen or polypeptide, nucleic acid, vector, host cell and / or composition herein. In one embodiment, the cell is an animal cell. The animal cell may be a mammalian cell or a human cell.

[0027] In another aspect, there is provided a method of cosmetic surgery, tissue injection filling, ophthalmic treatment, nerve repair, or vascular repair in a subject in need thereof, comprising administering to the subject a collagen or polypeptide described herein. In one embodiment, administration is oral or by injection. In one embodiment, the subject suffers from a disease or condition associated with a type VIII collagen deficiency, e.g., anterior segment hypoplasia.

[0028] In another aspect, (1) culturing a host cell described herein under suitable culture conditions; (2) harvesting the host cells and / or medium containing the collagen or polypeptide; and (3) purifying the collagen or polypeptide.

[0029] In one embodiment, the host cell is an E. coli cell, preferably an E. coli BL21(DE3) cell.

[0030] In one embodiment, step (1) comprises culturing E. coli cells in LB medium and inducing expression with IPTG.

[0031] In one embodiment, step (2) comprises harvesting the E. coli cells, resuspending them in an equilibrium working solution, homogenizing the E. coli cells, preferably under high pressure, and separating the supernatant. In one embodiment, the equilibrium working solution comprises 100-500 mM sodium chloride, 10-50 mM Tris, and 10-50 mM imidazole, and has a pH of 7-9.

[0032] In one embodiment, step (3) comprises crude purification, enzymatic cleavage, precision purification, and / or reversed-phase nickel column purification. In one embodiment, step (3) comprises crude purification and one or more of enzymatic cleavage, precision purification, and / or reversed-phase nickel column purification.

[0033] In one embodiment, the crude purification includes purifying the supernatant on a Ni-agarose gel column to obtain an eluate containing the target protein, the eluate containing 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 100 to 500 mM imidazole, preferably at a pH of 7 to 9.

[0034] In one embodiment, the refinement comprises gradient elution of the enzymatically cleaved product using a strong anion exchange chromatography column, in which the gradient elution comprises eluting with 0-15% Solution B for 1-5 minutes followed by holding for three column volumes, eluting with 15-30% Solution B for 1-5 minutes followed by holding for three column volumes, eluting with 30-50% Solution B for 1-5 minutes followed by holding for three column volumes, and eluting with 50-100% Solution B for 1-5 minutes followed by holding for three column volumes, where Solution B comprises 10-50 mM Tris, 0.5-5 M sodium chloride, and a pH of 7-9.

[0035] In one embodiment, reverse-phase nickel column purification involves purifying the enzymatically cleaved product on a Ni-agarose gel column, preferably using an eluent containing 10-50 mM Tris, 10-50 mM sodium chloride, and 0.5-5 M imidazole, at a pH of 7-9.

[0036] In this specification, the enzymatic cleavage may be TEV enzymatic cleavage.

[0037] Advantages of the present invention include the following: 1. The collagen (e.g., C8a-C8j) of the present invention is derived from type VIII collagen and is recombinant type VIII collagen, particularly derived from human type VIII collagen and is human recombinant type VIII collagen. 2. The collagen of the present invention (e.g., C8a-C8j) is suitable for production in E. coli and can be separated and purified. 3. The collagens of the present invention (eg, C8a and C8c) have higher expression levels and are suitable for subsequent purification. 4. The collagen of the present invention has cell adhesion activity. The collagen of the present invention (e.g., C8a and C8c) has higher cell adhesion activity than the positive control. The collagen of the present invention has a triple helix structure. [Brief explanation of the drawings]

[0038] [Figure 1] The results of C8a purification are shown below. After precision purification, C8a yield was high and the purity of the target protein was good. [Figure 2] The results of C8b purification are shown below. Some non-specific bands were found in the crudely purified C8b target protein, and some of the target protein was found in the 1M wash. The yield after precision purification was low, but the purity of the target protein was good. [Figure 3] The results of C8c purification are shown below. C8c has high purity of the target protein, and some of the target protein was not cleaved by enzymatic cleavage at a ratio of 20:1. [Figure 4]The results of C8d purification are shown below. C8d was purified in high yield, and a non-specific band at 75 kDa was not removed after reversed-phase nickel treatment. [Figure 5] The results of C8e purification are shown below. The crudely purified target protein contained non-specific bands, and the yield was low. [Figure 6] The results of C8f purification are shown below. The crudely purified C8f target protein contained many non-specific bands. [Figure 7] The results of C8g purification are shown below. C8g had a high crude purification yield, and the target protein appeared as two bands. [Figure 8] The results of purification of C8h are shown below. The crude purification yield of C8h was low. [Figure 9] The results of C8i purification are shown below. For C8i, most of the protein was not cleaved by enzymatic cleavage at a ratio of 20:1, and was still not cleaved by enzymatic cleavage at a ratio of 5:1. [Figure 10] The results of purification of C8j are shown below. The crude purification yield of C8j was low. [Figure 11] The results of detecting C8a and C8c cell adhesion activities are shown. [Figure 12] 1 shows the results of C8a circular dichroism spectral scanning analysis. [Figure 13] 1 shows the results of C8c circular dichroism spectral scanning analysis. DETAILED DESCRIPTION OF THE INVENTION

[0039] In order to clarify the purpose, technical means and advantages of the present invention, the following will clearly and completely describe the technical means in the embodiments of the present invention with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only some of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments that a person skilled in the art can come up with without any creative effort are all within the protection scope of the present invention.

[0040] Recombinant collagen is produced by screening and manufacturing using advanced structural biology, genetic engineering and other technologies, using the genetic code of the functional domain of a specific type of human collagen as a template, to obtain novel biomaterials with amino acid sequences identical or similar to those of human collagen.

[0041] As used herein, type VIII collagen, also known as short-chain collagen or network-forming collagen, has two similar α chains, α1(VIII) and α2(VIII), and two homotrimeric subtypes, [α1(VIII)]3 and [α2(VIII)]3, which are considered to be the major molecular species, although heterotrimers may also exist. Descemet's membrane from bovine eyes has a hexagonal network structure containing thin type VIII collagen fibrils. Immunohistochemical analysis and electron microscopy have shown that type VIII collagen is the main component of the structural framework. Rotary shadow analysis has also shown that type VIII collagen may form tetrahedral supramolecular structures that can provide structural support and regulate cellular behavior.

[0042] As used herein, a "polypeptide" refers to multiple amino acid residues linked via peptide bonds. As used herein, a polypeptide includes one or more repeat units. The repeat unit may be derived from human type VIII collagen. Thus, the polypeptide may be human recombinant type VIII collagen. Multiple repeat units may be linked by a linker, which may be a natural amino acid residue in the repeat unit of human type VIII collagen, for example, 1 to 50 amino acid residues. The repeat unit may be SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28. The polypeptide may be SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, or 29.

[0043] As used herein, "human recombinant type VIII collagen" refers to a recombinant protein consisting of or consisting essentially of a sequence derived from human type VIII collagen. As used herein, human recombinant type VIII collagen may consist of or consist essentially of a fragment or multiple repeats of a fragment derived from human type VIII collagen. As used herein, recombinant collagen, human recombinant type VIII collagen, collagen, or polypeptide are used interchangeably.

[0044] As used herein, the term "variant" refers to a collagen or polypeptide that has cell adhesion activity and contains an alteration (i.e., substitution, addition, insertion, and / or deletion) at one or more positions. A substitution refers to replacing an amino acid occupying a position with a different amino acid, a deletion refers to removing an amino acid occupying a position, and an insertion refers to adding an amino acid adjacent to and immediately following the amino acid occupying a position. An addition refers to adding one or more amino acid residues to the C-terminus and / or N-terminus of the amino acid sequence. A substitution may be a conservative substitution. A variant of a repeat unit may be a sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28 after one or more amino acid residues have been changed or mutated (i.e., substituted, added, inserted, and / or deleted). A collagen or polypeptide variant may be the sequence of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26 or 29 after one or more amino acid residues have been altered or mutated (ie, substituted, added, inserted and / or deleted).

[0045] In the context of the present invention, conservative substitutions may be defined by substitutions within one or more of the amino acid types reflected below. Conservative amino acid residues: Acidic residues D and E Basic residues K, R and H Hydrophilic uncharged residues S, T, N and Q Aliphatic uncharged residues G, A, V, L and I Non-polar uncharged residues C, M and P Aromatic residues F, Y and W. Physical and functional classification of candidate amino acid residues: Alcohol group-containing residues S and T Aliphatic residues I, L, V and M Cycloalkenyl-related residues F, H, W and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S and T Positively charged residues H, K and R Small residues A, C, D, G, N, P, S, T and V Smallest residues A, G and S Residues involved in the formation of reverse turns: A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q, T, K, S, G, P, D, E and R.

[0046] As used herein, "cell adhesion" refers to adhesion between cells and collagen. Collagen (e.g., a polypeptide described herein) can promote adhesion between cells and a container in which the cells are cultured.

[0047] As used herein, the term "expression" includes any step involved in the production of a collagen or polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0048] As used herein, the term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding a collagen or polypeptide, operably linked to control sequences that provide for its expression.

[0049] As used herein, the term "host cell" means any type of cell that is readily transformed, transfected, transduced, etc., with a nucleic acid construct or expression vector containing a polynucleotide of the invention. The term "host cell" encompasses the progeny of any parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0050] As used herein, the term "nucleic acid" refers to a single- or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or that has been modified to include a segment of nucleic acid in a manner not naturally occurring in nature, or that is synthetic, and that includes one or more regulatory sequences. The nucleic acid may be SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, or 30. The nucleic acid may be a codon-optimized nucleic acid, for example, a nucleic acid that has been codon-optimized for expression in E. coli cells.

[0051] The term "operably linked" refers to a configuration in which a control sequence is positioned in appropriate relation to a coding sequence of a polynucleotide so as to direct the expression of the coding sequence.

[0052] The degree of relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity." For purposes of the present invention, sequence identity between two amino acid sequences is determined using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented by the Needle program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or an updated version. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output marked "longest identity" (obtained using the unabbreviated option) is used as the percentage identity, calculated as follows: (Identical residues × 100) / (alignment length - total number of gaps in the alignment)

[0053] For the purposes of the present invention, the sequence identity between two deoxynucleotide sequences is determined using, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, as above) implemented by the Needle program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, as above), preferably version 5.0.0 or an updated version. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version in NCBI NUC4.4) substitution matrix. The output of Needle marked "longest identity" (obtained using the unabbreviated option) is used as the percentage identity, calculated as follows: (Identical deoxyribonucleotides × 100) / (alignment length - total number of gaps in the alignment)

[0054] collagen In the collagen or polypeptide comprising one or more repeat units according to the present invention, the repeat units are linked directly or via a linker, and the repeat units comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28 or a variant thereof. A variant may be (1) an amino acid sequence in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28 have been mutated, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28. For collagens or polypeptides described herein, mutations may be selected from substitutions, additions, insertions, or deletions. Preferably, the substitutions are conservative amino acid substitutions.

[0055] The collagen or polypeptides described herein may comprise a plurality of repeating units, e.g., 2 to 50 repeating units, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 repeating units.

[0056] The linker in the collagen or polypeptide described herein may contain one or more amino acid residues, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acid residues.

[0057] The collagen or polypeptide described herein is preferably a recombinant collagen, particularly recombinant type VIII collagen, having cell adhesion activity. The collagen or polypeptide described herein is derived from a human, and may therefore be human recombinant type VIII collagen.

[0058] The collagen or polypeptide described herein may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, or a variant thereof, wherein the variant is either (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence.

[0059] The collagens described herein may have a triple helical structure or three identical chains (i.e., trimeric form), and the sequence of each chain may be the sequence of a collagen or polypeptide described herein.

[0060] nucleic acid construct The invention also relates to nucleic acid constructs comprising a nucleic acid of the invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. A vector may comprise the nucleic acid construct.

[0061] Nucleic acids can be manipulated in a variety of ways to provide for collagen or polypeptide expression. 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 utilizing recombinant DNA methods are well known in the art.

[0062] The control sequence may be a promoter, which is a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a collagen or polypeptide of the present invention. The promoter comprises a transcriptional control sequence that mediates expression of the collagen or polypeptide. The promoter may be any nucleic acid that exhibits transcriptional activity in the host cell, including mutant promoters, truncated promoters, and hybrid promoters, and may be derived from extracellular or intracellular collagen or polypeptide-encoding genes that are homologous or heterologous to the host cell.

[0063] Illustrative examples of promoters suitable for directing transcription of the vectors or nucleic acid constructs of the invention in bacterial host cells are promoters obtained from the Bacillus amyloliquefaciens α-amylase gene (amyQ), the Bacillus licheniformis α-amylase gene (amyL), the Bacillus licheniformis penicillinase gene (penP), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and xylB genes, the Bacillus thuringiensis cryIIIA gene, the E. coli lac operon, and the E. coli trc promoter.

[0064] 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.

[0065] The control sequence may be a transcription terminator recognized by a host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the collagen or polypeptide. Any terminator that functions in the host cell may be used in the present invention.

[0066] Preferred terminators in bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and E. coli ribosomal RNA (rrnB).

[0067] Preferred terminators in yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described in Romanos et al. (1992, supra).

[0068] The regulatory sequence may be an mRNA stabilizing region downstream of the promoter and upstream of the coding sequence of the gene, which increases expression of the gene.

[0069] Examples of suitable mRNA stabilization regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0070] The control sequence may be a leader sequence, a non-translated region of an mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the collagen or polypeptide. Any leader sequence that functions in the host cell may be used.

[0071] Suitable leader sequences in yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0072] The control sequence may be a polyadenylation sequence, which is operably linked to the 3' end of the polynucleotide and is recognized by the host cell as a signal for adding polyadenosine residues to the transcribed mRNA during transcription. Any polyadenylation sequence that functions in the host cell may be used.

[0073] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0074] The control sequence may be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the collagen or polypeptide, directing the collagen or polypeptide into the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide may inherently contain the signal peptide coding sequence originally linked in translation reading frame with the segment of the coding sequence encoding the collagen or polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence foreign to the coding sequence. A foreign signal peptide coding sequence may be required when the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the collagen or polypeptide. However, any signal peptide coding sequence that directs the expressed collagen or polypeptide into the secretory pathway of the host cell may be used.

[0075] Useful signal peptide coding sequences for bacterial host cells include those obtained from the genes encoding Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0076] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described in Romanos et al. (1992, supra).

[0077] Expression vector The present invention also relates to recombinant expression vectors comprising the nucleic acids of the invention, promoters, and transcription and translation termination signals. The nucleic acids and control sequences can be ligated together to produce a recombinant expression vector, which may contain one or more convenient restriction sites, such that a polynucleotide encoding the collagen or polypeptide is inserted or substituted at such sites. Alternatively, the polynucleotide may be expressed by inserting the nucleic acid or a nucleic acid construct containing the nucleic acid into an appropriate vector for expression. When an expression vector is generated, the coding sequence is placed in the vector so that the coding sequence is operably linked to appropriate control sequences for expression.

[0078] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be readily programmed with recombinant DNA and cause expression of a polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector will be introduced. The vector may be a linear or closed circular plasmid.

[0079] The vector may be an autonomously replicating vector, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome, which exists as an extrachromosomal entity and whose replication is independent of chromosomal replication. The vector may include any means for ensuring self-replication. Alternatively, the vector may be a vector that is integrated into the genome when introduced into a host cell and replicated together with one or more integrated chromosomes. Also, a single vector or plasmid, or two or more vectors or plasmids, which together contain the total DNA to be introduced into the host cell genome, may be used, or a transposon may be used.

[0080] Vectors preferably contain one or more selectable markers which allow for easy selection of transformed, transfected, transduced, etc. cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0081] Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance (such as ampicillin resistance, chloramphenicol resistance, kanamycin resistance, neomycin resistance, spectinomycin resistance, or tetracycline resistance.) Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0082] The selectable marker may be a dual selectable marker system as described in WO2010 / 039889. Alternatively, the dual selectable marker may be the hph-tk dual selectable marker system.

[0083] The vector may contain elements that allow the vector to integrate into the genome of the host cell or to replicate autonomously within the cell independently of the genome.

[0084] When integrating into the genome of a host cell, the vector may rely on the collagen or polypeptide-encoding polynucleotide sequence or any other element of the vector to integrate into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration into a precise chromosomal location in the host cell genome by homologous recombination. To increase the likelihood of integration at a precise location, the integration element should contain a sufficient number of nucleic acids, e.g., 100-10,000 base pairs, 400-10,000 base pairs, or 800-10,000 base pairs, which have high sequence identity with the corresponding target sequence to increase the probability of homologous recombination. The integration element may be any sequence homologous to the target sequence in the genome of the host cell. Alternatively, the integration element may be a non-coding or coding polynucleotide. Alternatively, the vector may integrate into the genome of the host cell by non-homologous recombination.

[0085] For autonomous replication, the vector may further comprise an origin of replication that allows autonomous replication in the host cell under consideration. The origin of replication may be any plasmid replicon that mediates autonomous replication and functions in the cell. The term "origin of replication" or "plasmid replicon" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0086] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177 and pACYC184 that can replicate in E. coli, and the origins of replication of plasmids pUB110, pE194, pTA1060 and pAMβ1 that can replicate in Bacillus.

[0087] Examples of origins of replication for use in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0088] One or more copies of the polynucleotides of the invention can be inserted into a host cell to improve collagen or polypeptide production. Increased copy numbers of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell's genome or by including an amplifiable selectable marker gene along with the polynucleotide, and cells containing an amplified copy of the selectable marker gene and additional copies of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selection agent.

[0089] Procedures for ligating the above elements to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989).

[0090] host cell The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a collagen or polypeptide of the present invention. As described above, by introducing a construct or vector comprising a polynucleotide into a host cell, the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector. The term "host cell" encompasses the progeny of any parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will largely depend on the gene encoding the collagen or polypeptide and its source.

[0091] The host cell can be any cell useful for the recombinant production of the collagen or polypeptides of the invention, for example, prokaryotic or eukaryotic.

[0092] The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oenobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Labobacterium, Fusobacterium, Helicobacter, Lysobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0093] The host cell may be a eukaryotic cell, such as a mammalian, insect, plant or fungal cell.

[0094] The host cell may be a fungal cell of the phylum Basidiomycota, Chytridiomycota, Zygomycota, and Oomycota. The fungal host cell may also be a yeast cell, including ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes). The yeast host cell may be a cell of the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces crivelii, kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cells.

[0095] Production Method The present invention also relates to a method for producing a collagen or polypeptide as described herein, the method comprising: (1) culturing a host cell described herein under suitable culture conditions; (2) harvesting the host cells and / or medium containing the collagen or polypeptide; and (3) purifying the collagen or polypeptide.

[0096] The host cells are cultured in an appropriate nutrient medium to produce collagen or polypeptide using methods known in the art. For example, the cells may be cultured in shake flask cultures or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in laboratory or industrial fermentors under conditions that allow for expression and / or isolation of the collagen or polypeptide in the appropriate medium. Using procedures known in the art, the culture is carried out in an appropriate nutrient medium containing a carbon source, a nitrogen source, and inorganic salts. Suitable media can be obtained from commercial suppliers or prepared according to disclosed compositions (e.g., in the catalog of the American Type Culture Collection). If the collagen or polypeptide is secreted into the nutrient medium, the collagen or polypeptide can be recovered directly from the medium. If the collagen or polypeptide is not secreted, it can be recovered from cell lysates.

[0097] Collagen or polypeptide may be detected using methods known in the art that are specific for collagen or 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 may be used to determine the activity of collagen or polypeptide.

[0098] The collagen or polypeptide may be recovered using methods known in the art. For example, the collagen or polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. Alternatively, the fermentation broth containing the collagen or polypeptide is recovered.

[0099] To obtain substantially pure collagen or polypeptide, the collagen or polypeptide may be purified by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focusing chromatography, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing), differential lysis (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.

[0100] Step (1) may include one or more of the following steps: Construct an expression plasmid, for example, by inserting the coding nucleotide sequence into a pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid; Transform the successfully constructed expression plasmid into E. coli cells (e.g., E. coli competent cells BL21(DE3)). A specific process may be as follows: (1) Add the plasmid to be transformed to E. coli competent cells BL21(DE3); (2) Incubate the mixture on ice (e.g., for 10 to 60 minutes, e.g., 30 minutes), then heat shock in a water bath (40 to 50°C, e.g., 42°C, 45 to 90 seconds), remove the mixture, and then incubate it on ice (1 to 5 minutes, e.g., 2 minutes); (3) Add liquid LB medium, and then culture (e.g., at 35 to 40°C, e.g., 37°C, at 150 to 300 rpm, e.g., 220 rpm, for 40 to 80 minutes, e.g., 60 minutes). (4) Spread the bacterial suspension and select single colonies. For example, spread the bacterial suspension evenly on an LB plate containing sodium ampicillin, and incubate the plate in an incubator at 37°C for 15 to 17 hours to allow colonies of uniform size to grow.

[0101] Step (2) may include culturing a single colony in LB medium containing an antibiotic stock solution (e.g., culturing at 150 to 300 rpm, e.g., 220 rpm, in a constant temperature shaker at 35 to 40°C, e.g., 37°C, for 5 to 10 hours, e.g., 7 hours), and further comprising the steps of lowering the temperature of the shake flask after culturing to 10 to 20°C, e.g., 16°C, adding IPTG to induce expression for a certain period of time, and then collecting the bacterial cells (e.g., by centrifugation).

[0102] Step (3) may include resuspending the bacterial cells in an equilibrium working solution, cooling the bacterial solution to ≦15°C, homogenizing (e.g., 1 to 5 times, e.g., 2 times under high pressure), and separating the homogenized bacterial solution to obtain a supernatant. The equilibrium working solution may contain 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 10 to 50 mM imidazole, and have a pH of 7 to 9. For example, the concentration of sodium chloride may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH may be 7, 7.5, 8, 8.5 or 9.

[0103] Step (3) may include purifying and enzymatically cleaving the collagen or polypeptide. The purification may include crude purification, which involves purifying the supernatant through a Ni-agarose gel column to obtain an eluate containing the target protein. The crude purification may include washing the column with water, for example, 2 to 10 column volumes (CV), for example, 5 CV. The column may be equilibrated with an equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0), for example, 2 to 10 CV, for example, 5 CV. The equilibration solution may contain 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 10 to 50 mM imidazole, and have a pH of 7 to 9. For example, the concentration of sodium chloride may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH may be 7, 7.5, 8, 8.5 or 9.

[0104] Step (3) may include adding the supernatant to the column and washing the contaminating proteins with a wash solution. The wash solution may contain 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 10 to 50 mM imidazole, and have a pH of 7 to 9. For example, the concentration of sodium chloride may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The Tris concentration may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The imidazole concentration may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH may be 7, 7.5, 8, 8.5, or 9. An eluent may then be added, and the flow-through liquid may be collected. The eluent may contain 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 100 to 500 mM imidazole, and have a pH of 8.0. For example, the concentration of sodium chloride may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480 or 490 nM. The pH may be 7, 7.5, 8, 8.5 or 9.

[0105] The enzymatic cleavage may include adding TEV enzyme (at a ratio of the total amount of protein to the total amount of TEV enzyme of 10 to 100:1, for example 50:1, at 10 to 20°C, for example 16°C, for 2 to 8 hours, for example 4 hours). The enzymatically cleaved protein solution is dialyzed, for example, by placing it in a dialysis bag and dialyzing it at 1 to 6°C, for example 4°C, for 1 to 8 hours, for example 2 hours, and then transferring it to fresh dialysate and dialyzing it at 1 to 6°C, for example 4°C, overnight.

[0106] Purification may include micropurification (e.g., protein isoelectric point > 8.0). Preferably, micropurification involves gradient elution of an eluate containing the target protein or an enzymatically cleaved product (e.g., a dialyzed product of enzymatic cleavage) using a strong anion exchange chromatography column (e.g., pH 7, 7.5, 8, 8.5, or 9). Gradient elution includes elution with 0-15% solution B for 1-5 minutes, followed by retention for 1-5, e.g., 3, column volumes; elution with 15-30% solution B for 1-5 minutes, followed by retention for 1-5, e.g., 3, column volumes; elution with 30-50% solution B for 1-5 minutes, followed by retention for 1-5, e.g., 3, column volumes; and elution with 50-100% solution B for 1-5 minutes, followed by retention for 1-5, e.g., 3, column volumes. Solution B may contain 10 to 50 mM Tris and 0.5 to 5 M sodium chloride, and may have a pH of 7 to 9. For example, the Tris concentration is 15, 20, 25, 30, 35, 40, or 45 mM. The sodium chloride concentration is 1, 2, 3, or 4 M. The pH may be 7, 7.5, 8, 8.5, or 9. Precision purification may include a step of equilibrating a column with solution A, loading the column, and then performing gradient elution. Solution A may contain 10 to 50 mM Tris and 10 to 50 mM sodium chloride, and may have a pH of 7 to 9. For example, the Tris concentration is 15, 20, 25, 30, 35, 40, or 45 mM. The sodium chloride concentration is 15, 20, 25, 30, 35, 40, or 45 mM. The pH may be 7, 7.5, 8, 8.5, or 9.

[0107] Purification may include reverse-phase nickel column purification (e.g., protein isoelectric point <8.0). Reverse-phase nickel column purification involves purifying the enzymatically cleaved product (e.g., the dialyzed product) on a Ni-agarose gel column. The eluent may contain 10-50 mM (e.g., 15, 20, 25, 30, 35, 40, or 45 mM) Tris, 10-50 mM (e.g., 15, 20, 25, 30, 35, 40, or 45 mM) sodium chloride, and 0.5-5 M (e.g., 1, 2, 3, or 4 M) imidazole, at a pH of 7-9 (e.g., 7, 7.5, 8, 8.5, or 9).

[0108] To further illustrate the present invention, the following examples are provided. [Example]

[0109] The present invention will be further described by the following examples, but any of the examples or combinations thereof should not be understood to limit the scope or embodiments of the present invention. The scope of the present invention is limited by the appended claims, and those skilled in the art can clearly understand the scope limited by the claims by combining this specification and general knowledge in the art. Without departing from the spirit and scope of the present invention, those skilled in the art can make any modifications or changes to the technical means of the present invention, and these modifications and changes are also included in the scope of the present invention.

[0110] Example 1. Construction, expression and screening of type VIII collagen fragments 1. A large-scale screening of functional regions was conducted to obtain the following target gene functional regions of different recombinant humanized type VIII collagen genes:

[0111] 1) C8a amino acid sequence: gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp(SEQ ID NO:1) gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp (wherein the amino acid sequence of the repeating unit of C8a is represented by SEQ ID NO:1, the number of repeats is 6, and the amino acid sequence of C8a is represented by SEQ ID NO:2) Nucleotide sequence: (SEQ ID NO:3)

[0112] 2) C8b amino acid sequence: gKpggpglpgqpgpKgDRgpKglpgpqglRgpKgDK(SEQ ID NO:7) gKpggpglpgqpgpKgDRgpKglpgpqglRgpKgDK gKpggpglpgqpgpKgDRgpKglpgpqglRgpKgDK gKpggpglpgqpgpKgDRgpKglpgpqglRgpKgDK gKpggpglpgqpgpKgDRgpKglpgpqglRgpKgDK gKpggpglpgqpgpKgDRgpKglpgpqglRgpKgDK (However, the amino acid sequence of the repeating unit of C8b is represented by SEQ ID NO:7, the number of repeats is 6, and the amino acid sequence of C8b is represented by SEQ ID NO:8.) Nucleotide sequence: (SEQ ID NO:9)

[0113] 3) C8c amino acid sequence: gKpgvtgfpgpqgplgKpgapgEpgpqgpigvpgvqgppgip(SEQ ID NO:4) gKpgvtgfpgpqgplgKpgapgEpgpqgpigvpgvqgppgip gKpgvtgfpgpqgplgKpgapgEpgpqgpigvpgvqgppgip gKpgvtgfpgpqgplgKpgapgEpgpqgpigvpgvqgppgip gKpgvtgfpgpqgplgKpgapgEpgpqgpigvpgvqgppgip gKpgvtgfpgpqgplgKpgapgEpgpqgpigvpgvqgppgip (However, the amino acid sequence of the repeating unit of C8c is represented by SEQ ID NO:4, the number of repeats is 6, and the amino acid sequence of C8c is represented by SEQ ID NO:5.) Nucleotide sequence: (SEQ ID NO:6)

[0114] 4) C8d amino acid sequence: gKpgqDgipgqpgfpggKgEqglpglpgppglp(SEQ ID NO:10) gKpgqDgipgqpgfpggKgEqglpglpgppglp gKpgqDgipgqpgfpggKgEqglpglpgppglp gKpgqDgipgqpgfpggKgEqglpglpgppglp gKpgqDgipgqpgfpggKgEqglpglpgppglp gKpgqDgipgqpgfpggKgEqglpglpgppglp (wherein the amino acid sequence of the repeating unit of C8d is represented by SEQ ID NO: 10, the number of repeats is 6, and the amino acid sequence of C8d is represented by SEQ ID NO: 11) Nucleotide sequence: (SEQ ID NO:12)

[0115] 5) C8e amino acid sequence: gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEpglpgipgpmgppgaigfpgpKgEggivgpqgppgpKgEpglqgfpgKpgflgEvgppgmRglpgpigpKgEagqKgvpglpgvpgllgpKgEpgipgDqglqgppgipgiggpsgpigppgipgpKgEpglpgppgfp(SEQ ID NO:13) gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEpglpgipgpmgppgaigfpgpKgEggivgpqgppgpKgEpglqgfpgKp gflgEvgppgmRglpgpigpKgEagqKgvpglpgvpgllgpKgEpgipgDqglqgppgipgiggpsgpigppgipgpKgEpglpgppgfp (wherein the amino acid sequence of the repeating unit of C8e is represented by SEQ ID NO: 13, the number of repeats is 2, and the amino acid sequence of C8e is represented by SEQ ID NO: 14) Nucleotide sequence:

[0116] 6) C8f amino acid sequence: gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEpglpgipgpmgppgaigfpgpKgEggivgpq gppgpKgEpglqgfpgKpgflgEvgppgmRglpgpigpKgEagqKgvpglpgvpgllgpKgEpgipgDq(SEQ ID NO:16) gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEpglpgipgpmgppgaigfpgpKgEggivgpqgppgpKgEpglqgfpgKpgflgEvgppgmRglpgpigpKgEagqKgvpglpgvpgllgpKgEpgipgDq (wherein the amino acid sequence of the C8f repeating unit is shown in SEQ ID NO: 16, the number of repeats is 2, and the amino acid sequence of C8f is shown in SEQ ID NO: 17) Nucleotide sequence: (SEQ ID NO:18)

[0117] 7) C8g amino acid sequence: gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp(SEQ ID NO:19) gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp (wherein the amino acid sequence of the repeating unit of C8g is shown in SEQ ID NO: 19, the number of repeats is 6, and the amino acid sequence of C8g is shown in SEQ ID NO: 20) Nucleotide sequence: (SEQ ID NO:21)

[0118] 8) C8h amino acid sequence: gpKgEggivgpqgppgpKgEpglqgfpgKpgflgEvgppgmR(SEQ ID NO:22) gpKgEggivgpqgppgpKgEpglqgfpgKpgflgEvgppgmR gpKgEggivgpqgppgpKgEpglqgfpgKpgflgEvgppgmR gpKgEggivgpqgppgpKgEpglqgfpgKpgflgEvgppgmR gpKgEggivgpqgppgpKgEpglqgfpgKpgflgEvgppgmR gpKgEggivgpqgppgpKgEpglqgfpgKpgflgEvgppgmR (wherein the amino acid sequence of the repeating unit of C8h is shown in SEQ ID NO: 22, the number of repeats is 6, and the amino acid sequence of C8h is shown in SEQ ID NO: 23) Nucleotide sequence: (SEQ ID NO:24)

[0119] 9) C8i amino acid sequence: gpKgEagqKgvpglpgvpgllgpKgEpgipgDq(SEQ ID NO:25) gpKgEagqKgvpglpgvpgllgpKgEpgipgDq gpKgEagqKgvpglpgvpgllgpKgEpgipgDq gpKgEagqKgvpglpgvpgllgpKgEpgipgDq gpKgEagqKgvpglpgvpgllgpKgEpgipgDq gpKgEagqKgvpglpgvpgllgpKgEpgipgDq (wherein the amino acid sequence of the repeating unit of C8i is shown in SEQ ID NO: 25, the number of repeats is 6, and the amino acid sequence of C8i is shown in SEQ ID NO: 26) Nucleotide sequence: (SEQ ID NO:27)

[0120] 10) C8j amino acid sequence: gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp(SEQ ID NO:28) gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp (wherein the amino acid sequence of the repeating unit of C8j is shown in SEQ ID NO:28, the number of repeats is 6, and the amino acid sequence of C8j is shown in SEQ ID NO:29) Nucleotide sequence: (SEQ ID NO:30)

[0121] 2. The above coding nucleotide sequences were commercially synthesized. The above coding nucleotide sequences (containing a collagenase enzyme cleavage site at the 5' end, the amino acid sequence of which is ENLYFQ and the nucleotide sequence is GAAAACCTGTATTTCCAG) were inserted between the KpnI and XhoI enzyme cleavage sites of the pET-28a-Trx-His expression vector to obtain recombinant expression plasmids.

[0122] 3. The successfully constructed expression plasmid was transformed into E. coli competent cells BL21(DE3). The specific process was as follows: (1) E. coli competent cells BL21(DE3) were removed from the ultra-low temperature refrigerator and placed on ice. When partially thawed, 2 μl of the target plasmid was added to the E. coli competent cells BL21(DE3) and mixed thoroughly 2-3 times. (2) The mixture was then placed on ice for 30 minutes, heat-shocked in a 42°C water bath for 45-90 seconds, and then removed and placed on ice for 2 minutes. (3) The mixture was transferred to a biological safety cabinet, and 700 μl of liquid LB medium was added. The mixture was then cultured at 37°C and 220 rpm for 60 minutes. (4) 200 μl of the bacterial suspension was evenly spread on an LB plate containing sodium ampicillin. (5) The plate was then cultured in an incubator at 37°C for 15-17 hours to allow uniformly sized colonies to grow.

[0123] Five to six single colonies were selected from the transformed LB plate, placed in a shake flask containing LB medium containing antibiotic stock solution, and cultured for 7 hours at 220 rpm and 37°C in a constant temperature shaker. After further culture, the shake flask was cooled to 16°C, and IPTG was added to induce expression for a certain period. The bacterial solution was then dispensed into centrifuge flasks and centrifuged at 8000 rpm and 4°C for 10 minutes to collect the bacterial cells. The bacterial weight was recorded, and a sample (denoted as "bacterial solution") was then subjected to electrophoresis.

[0124] 5. The collected bacterial cells were resuspended in an equilibrium working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0), the solution was cooled to ≤15°C, homogenized, and then homogenized twice under high pressure (the samples homogenized twice were labeled "homogenized" and "homogenized twice," respectively). The homogenized bacterial solution was then collected after completion. The homogenized bacterial solution was dispensed into centrifuge flasks and centrifuged at 17,000 rpm at 4°C for 30 minutes. The supernatant was collected, and the supernatant (labeled "supernatant") and precipitate (labeled "precipitate") were subjected to electrophoresis detection.

[0125] 6. Recombinant humanized type VIII collagen was purified and enzymatically cleaved. The specific processes are as follows: (1) (1) Crude purification was performed as follows: a) The column was washed with water for 5 CV (Ni6FF, Cytiva). b) The column was equilibrated with an equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0) for 5 CV. c) For loading, the supernatant was added to the column after centrifugation until the liquid flowed through, and the flow-through was subjected to electrophoretic detection (denoted as "flow-through"). d) For washing of contaminating proteins, 25 mL of washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) was added until the liquid flowed through, and the washed flow-through was subjected to electrophoretic detection (denoted as "washing"). e) To collect the target protein, 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0) was added, and the flow-through solution (labeled "Elution") was collected. The protein concentration was measured to calculate the protein amount, and electrophoretic detection was performed. f) The column was washed with 1 M imidazole working solution (labeled "1 M Wash"). g) The column was washed with purified water. (2) Enzymatic cleavage was performed as follows: TEV enzyme was added at a ratio of 50:1 between the total amount of protein and the total amount of TEV enzyme (if no cleavage was performed, the enzyme concentration could be increased, e.g., to a ratio of 20:1 or 5:1). Enzymatic cleavage was performed at 16°C for 4 hours, and a sample (labeled "After cleavage") was sampled and electrophoretically detected. The cleaved protein solution was placed in a dialysis bag and dialyzed at 4°C for 2 hours. It was then transferred to fresh dialysate and dialyzed overnight at 4°C (labeled "Replacement"). (3) The following purification procedures were used (protein isoelectric point > 8.0): a) Column equilibration (Capto Q, Cytiva) was performed using solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0) at a flow rate of 10 ml / min. b) Loading was performed using a flow rate of 5 ml / min. The flow-through solution (denoted as QFL) was collected and subjected to electrophoretic detection. c) Gradient elution consisted of 2 min elution with 0-15% B (20 mM Tris, 1 M sodium chloride, pH 8.0) followed by three CV retention (denoted as "0-15% B wash"); 2 min elution with 15-30% B followed by three CV retention; 2 min elution with 30-50% B followed by three CV retention; and 2 min elution with 50-100% B followed by three CV retention. Peaks were collected when they appeared and electrophoretic detection was performed. d) The column was washed. Proteins were stored at 4°C. (4) The column was treated with reversed-phase nickel (Ni6FF, Cytiva) (protein isoelectric point <8.0) as follows: a) Column equilibration: Five CVs were performed with solution A (20 mM Tris, 20 mM sodium chloride, 20 mM imidazole, pH 8.0). b) Loading: The enzyme-cleaved and solution-exchanged protein was loaded onto the column until the liquid flowed through, and electrophoretic detection was performed on the flow-through solution (denoted as "reverse-phase nickel treatment"). c) The column was washed with 1 M imidazole working solution (20 mM Tris, 20 mM sodium chloride, 1 M imidazole, pH 8.0) (denoted as "1 M wash"). d) The column was washed with purified water. The protein was stored at 4°C.

[0126] 7. Concentration detection An appropriate amount of sample was accurately taken, diluted 10-50 times with the eluent, and thoroughly stirred with a glass rod to ensure uniformity. The absorbance was measured at 280 nm using a UV-Vis spectrophotometer, and the protein concentration was calculated using the formula C (mg / ml) = A280 × extinction coefficient × dilution factor (Note: The extinction coefficient is determined based on the amino acid sequence, and the absorbance value should be between 0.1 and 1).

[0127] The concentration detection results are as follows: [Table 1]

[0128] The protein expression levels were C8a>C8i>C8b>C8g>C8d>C8c>C8f>C8e>C8j>C8h.

[0129] 8. Electrophoretic detection The specific process was as follows: 40 μl of sample protein solution was taken, 10 μl of 5x protein loading buffer (250 mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol) was added, and the mixture was boiled in 100°C water for 10 minutes. After that, 10 μl of SDS-PAGE protein gel was added to each well and electrophoresed at 80 V for 2 hours. The proteins were stained with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20 minutes, and then destained with protein destaining solution (10% acetic acid, 5% ethanol).

[0130] The electrophoretic detection results are shown below. Figure 1 shows the purification results for C8a. C8a had a high yield after precision purification and good target protein purity. Figure 2 shows the purification results for C8b. C8b had some non-specific bands in the crudely purified target protein, some target protein in the 1M wash, and low yield after precision purification, but good target protein purity. Figure 3 shows the purification results for C8c. C8c had good target protein purity, and some target protein was not cleaved by 20:1 enzymatic cleavage. Figure 4 shows the purification results for C8d. C8d had a high yield, and a non-specific band at 75 kDa was not removed after reversed-phase nickel treatment. Figure 5 shows the purification results for C8e. C8e had non-specific bands in the crudely purified target protein and low yield. Figure 6 shows the purification results for C8f. C8f had many non-specific bands in the crudely purified target protein. Figure 7 shows the purification results for C8g. C8g had a high crude purification yield, and the target protein consisted of two bands. Figure 8 shows the purification results for C8h. The crude purification yield for C8h was low. Figure 9 shows the purification results for C8i. For C8i, most of the protein was not cleaved by enzymatic cleavage at 20:1, and was still not cleaved by enzymatic cleavage at 5:1. Figure 10 shows the purification results for C8j. The crude purification yield for C8j was low.

[0131] The electrophoresis results show that 1. C8e, C8h, and C8j have low crude purification yields and thin target protein bands; 2. C8i has one obvious non-specific band at 35 kDa of crudely purified target protein, which basically cannot be enzymatically cleaved; 3. C8b and C8f have many non-specific bands of the target protein, resulting in low yields after precision purification; 4. C8g has high yields, but the target protein has two bands and low purity; 5. C8d has high yields, but many non-specific bands after reversed-phase treatment on a Ni column and low purity; 5. C8a and C8c have high yields and good target protein purity. C8a and C8c are selected for subsequent detection.

[0132] Example 2. Mass spectrometry detection of recombinant humanized collagen type VIII Experimental Method [Table 2]

[0133] Protein samples were reduced with DTT, alkylated with iodoacetamide, and then enzymatically digested overnight with trypsin. The resulting peptide fragments were desalted using a C18 ZipTip, mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA), and subjected to thin-layer chromatography. Finally, the samples were analyzed using a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF / TOF) Ulraflextreme mass spectrometer. TM , Brucker, Germany (for peptide mass fingerprinting techniques, see Protein J. 2016;35:212-7).

[0134] Data searches were performed using the MS / MS Ion Search page on the local masco site. Protein identification results were obtained from the primary mass spectra of peptide fragments generated after enzymatic digestion. Regarding detection parameters, two uncleaved sites were set for trypsin enzymatic digestion. Cysteine ​​alkylation was set as a fixed modification. Methionine oxidation was set as a variable modification. The database used for identification was NCBprot.

[0135] Table 1. Recombinant type VIII humanized collagen C8a mass spectrometry detected molecular weight and corresponding polypeptide [Table 3]

[0136] The detected polypeptide fragments have a coverage rate of 100% compared with the theoretical sequence, making the detection results highly reliable.

[0137] Example 3: Bioactivity detection of recombinant humanized collagen VIII For a method for detecting collagen activity, see Juming Yao, Satoshi Yanagisawa, and Tetsuo Asakura, "Design, Expression, and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens," J. Biochem. 136, 643-649 (2004). Specific implementation methods are as follows:

[0138] (1) Using ultraviolet absorption method, the concentration of the target protein sample containing bovine type I collagen (China Food and Drug Administration, No.: 380002) and the recombinant humanized proteins C8a and C8c of the present invention is detected.

[0139] Specifically, the UV absorption of the sample at 215 nm and 225 nm is measured, and the protein concentration is calculated according to the empirical formula C (μg / mL) = 144 × (A215 - A225), with the caveat that detection is required when A215 is less than 1.5. The principle of this method is as follows: It measures the characteristic absorption of peptide bonds in the far UV, is not affected by chromophore content, has few disturbing substances, is easy to operate, and is suitable for detecting human collagen and its analogs that do not react with Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition, Humana Press, pp. 43-45.) After protein concentration detection, the concentration of all target proteins is adjusted to 0.5 mg / mL with PBS.

[0140] (2) Sample preparation: The experiment was carried out directly using the sample stock solution. The positive control, human type I collagen (PC), was diluted to 1 mg / ml with D-PBS for use, and the negative control was D-PBS buffer (NC).

[0141] (3) Coating: 100 μL of collagen at different concentrations, positive control, and negative control were added to each well of the enzyme standard plate, and five duplicate wells were set up for each group, followed by incubation at 4°C overnight.

[0142] (4) Blocking: The supernatant was discarded, and 100 μL of 1% BSA (heat-inactivated at 56° C. for 30 minutes) was added, followed by incubation at 37° C. for 60 minutes. The supernatant was discarded, and the plate was washed three times with D-PBS solution.

[0143] (5) Cell inoculation: 10 well-cultured cells resuspended in D-PBS were inoculated into each well. 5 3T3 / NIH cells were added and incubated for 120 minutes at 37° C. Each well was washed three times with D-PBS solution.

[0144] (6) Detection: OD 300 using a CCK8 detection kit (manufacturer: Beyotime, product catalog number: C0038). 450 The absorbance at 100 nm was detected. The degree of cell adhesion was calculated using the following formula. The cell adhesion rate can reflect the cell adhesion ability of collagen. The higher the cell adhesion ability, the better the external environment can be provided to cells in a short time, which helps cell adhesion.

number

[0145] (7) Statistical analysis: The statistical difference between the target recombinant humanized collagen and the negative control was analyzed using a two-tailed t-test, where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0146] The results are shown in FIG. 11. Compared with the D-PBS group, the positive control clearly has the effect of promoting cell adhesion, and the recombinant humanized collagens C8a and C8c also have the effect of promoting cell adhesion.

[0147] Example 4. Circular dichroism spectral ultraviolet scanning analysis of recombinant humanized collagen type VIII Experimental Method (1) Setting the device parameters Band width: 1.0 nm Step: 1.0 nm Measurement range: 190-260nm (scanning in the far UV region) / 250-340nm (scanning in the near UV region) Time-per-point: 0.5s Repeats: 3 times Cell length: 10mm|0.5mm Temperature: room temperature (2) Far-UV and near-UV scans of standard products Set the scanning wavelength to 180 to 340 nm to perform a background test and a buffer solution blank test, and obtain the circular dichroism (far-ultraviolet, near-ultraviolet) absorbance in the 180 to 340 nm range of a 1 mg / mL CSA standard solution. (3) Sample processing The precisely purified C8a and C8c protein samples are concentrated using a 10 KD ultrafiltration filter (Millipore) until the protein concentration reaches 1 mg / ml. (4) Far-UV scanning of the sample Soak the cuvette in 2M HNO3 overnight, wash with deionized water, dry, collect background, then collect blank buffer, add an appropriate amount of test substance to the cuvette, and perform a far-UV scan from 190 to 260 nm based on the above parameters to acquire data. (5) Near-UV scanning of the sample Soak the cuvette in 2M HNO3 overnight, wash with deionized water, dry, collect background, then collect blank buffer, add an appropriate amount of test substance to the cuvette, and perform near-ultraviolet scanning from 250 to 340 nm based on the above parameters to acquire data. (6) Scanning spectrum processing All spectra after scanning are subjected to baseline subtraction and smoothing using the software Pro-Data Viewer.

[0148] Experimental results and analysis

[0149] The results showed that both C8a and C8c exhibited positive peaks at 221 nm, indicating that these proteins both possess triple helical structures (i.e., the general structural characteristics of active collagen). The results are shown in Figures 12 and 13.

[0150] The above examples are preferred embodiments of the present invention, and the embodiments of the present invention are not limited to the above examples. Any changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention are all equivalent substitutions and fall within the protection scope of the present invention.

Claims

1. A collagen comprising one or more repeat units, the repeat units being linked directly or via a linker, the repeat units comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28, or a variant thereof, wherein the variant is either (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence; Preferably, the plurality of repeat units is 2 to 50 repeat units, for example, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, or 2 to 8 repeat units; Preferably, the linker comprises one or more amino acid residues, for example 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 amino acid residues; Preferably, the mutation is selected from a substitution, addition, insertion or deletion, Preferably, the substitutions are conservative amino acid substitutions, Preferably, the collagen is recombinant collagen, preferably recombinant type VIII collagen, preferably human recombinant type VIII collagen; Preferably, the collagen has cell adhesion activity and has a triple helix structure or is in a trimeric form.

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

3. Encoding the collagen of claim 1 or 2, Preferably, it comprises a codon-optimized nucleotide sequence, Preferably, the nucleotide sequence is codon-optimized for E. coli expression, Preferably, the nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30.

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

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

6. A composition comprising one or more of the collagen of claim 1 or 2, the nucleic acid of claim 3, the vector of claim 4, and the host cell of claim 5, preferably in the form of a kit, and preferably one or more of a biological covering material, a human biomimetic material, a plastic and cosmetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, an obstetric and gynecological biological material, a nerve repair and regeneration material, a liver tissue material, and a vascular repair and regeneration material, a 3D printed artificial organ biological material, a cosmetic raw material, a medicinal auxiliary material, and a food additive, preferably an injectable composition or an oral composition.

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

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

9. 1. A method for cosmetic surgery, tissue injection filling, ophthalmic treatment, nerve repair or vascular repair in a subject in need thereof, comprising administering to the subject the collagen of claim 1 or 2, preferably the administration being oral or by injection, preferably the subject suffering from a disease or condition associated with a type VIII collagen deficiency, such as anterior ocular hypoplasia, and preferably the subject is a human.

10. (1) culturing the host cell of claim 5 under suitable culture conditions; (2) harvesting the host cells and / or medium containing collagen; (3) purifying the collagen, Preferably, the host cell is an E. coli cell, preferably an E. coli BL21(DE3) cell; Preferably, step (1) comprises culturing E. coli cells in LB medium and inducing expression with IPTG; Preferably, step (2) comprises harvesting the E. coli cells, resuspending them in a balanced working solution, homogenizing the E. coli cells, preferably homogenizing them under high pressure, and separating the supernatant, preferably the balanced working solution comprising 100-500 mM sodium chloride, 10-50 mM Tris, 10-50 mM imidazole, and having a pH of 7-9; Preferably, step (3) comprises crude purification and one or more of enzymatic cleavage, precision purification, and reversed-phase nickel column purification; Preferably, the crude purification comprises purifying the supernatant on a Ni-agarose gel column to obtain an eluate containing the target protein, the eluate comprising 100 to 500 mM sodium chloride, 10 to 50 mM Tris, and 100 to 500 mM imidazole, preferably at a pH of 7 to 9; Preferably, the enzymatic cleavage comprises cleavage with TEV enzyme, preferably for 2 to 8 hours at a ratio of the total amount of protein to the total amount of TEV enzyme of 10 to 100:1; Preferably, the precision purification comprises a step of gradient eluting the enzymatically cleaved product using a strong anion exchange chromatography column, and preferably, the gradient elution comprises the steps of eluting with 0-15% solution B for 1-5 minutes and then holding for 3 column volumes, eluting with 15-30% solution B for 1-5 minutes and then holding for 3 column volumes, eluting with 30-50% solution B for 1-5 minutes and then holding for 3 column volumes, and eluting with 50-100% solution B for 1-5 minutes and then holding for 3 column volumes, wherein solution B comprises 10-50 mM Tris, 0.5-5 M sodium chloride, and has a pH of 7-9; The method for producing collagen according to claim 1 or 2, wherein the reversed-phase nickel column purification preferably comprises purifying the enzymatically cleaved product on a Ni-agarose gel column, and the eluent preferably comprises 10 to 50 mM Tris, 10 to 50 mM sodium chloride, and 0.5 to 5 M imidazole, and has a pH of 7 to 9.

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