Method for biosynthesis of 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 the priority of a Chinese patent application with an application date of July 18, 2023, an application number of 202310883772.3, and an invention title of "Method for Biosynthesizing Type VIII Collagen of Human Structural Material", the content of which is incorporated herein by reference.

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

Background Art

[0003] Collagen is the most abundant and widely distributed protein in animals and is the main component of connective tissue. Type VIII collagen is a short - chain non - fibrinogen collagen, the main component of the membrane of the retina and the main component of the sub - endothelial basement membrane of the cornea (the basement membrane separating corneal endothelial cells and corneal stroma). Type VIII collagen is produced from endothelial cells and forms a unique hexagonal lattice structure. Type VIII collagen is present around chondrocytes in the heart, brain, liver, lungs, muscles, and cartilage. Also, type VIII collagen has been found around the actively proliferating blood vessels of brain tumors and in the massive fibrotic blood vessels of hemangiomas.

[0004] 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 main molecular species. Heterotrimers may also exist. Descemet's membrane of buvine eye has a hexagonal reticular structure and contains thin type VIII collagen fibrils. Immunohistochemical analysis and electron microscopy have shown that type VIII collagen is the main component of this structural framework. Furthermore, rotational shadow analysis has shown that type VIII collagen may form a tetrahedral supramolecular structure that supports the structure and can regulate cellular behavior.

[0005] Double knockout mice of two types of type VIII collagen genes, Col8a1 and Col8a2, exhibited a marked anterior segment hypoplasia phenotype, with a spherical anterior chamber and a spherical projection of the cornea. The corneal matrix was diffusely thinned, becoming thin like that seen in the human corneal sphere. Descemet's membrane was noticeably thin, and the anterior band was absent. Corneal endothelial cells were enlarged and decreased in number. Finally, the in vitro response of mutated corneal endothelial cells to different growth factors showed decreased proliferative capacity, suggesting that type VIII collagen may be an enhancer of growth factor-induced cell proliferation.

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

[0007] The NC1 domain (aa572-744) of type VIII collagen is a protein called vastatin, which has been proven to be an effective angiogenesis inhibitor with apoptosis-inducing activity in aortic endothelial cells. α2 has a chain length of 703aa and has one signal peptide at the n-terminus of α1-28. The α1 and α2 procollagen genes each contain four exons. One of their characteristics is that the largest exon encodes the entire triple helix domain (COL1) and the C-terminal non-triple helix domain (NC1). Type VIII collagen has high sequence homology with type X collagen, and their intron-exon structures are similar. This indicates that the two types of collagen originate from the same progenitor cell gene and belong to the same collagen subclass. Type VIII collagen can form a hexagonal lattice structure, like type X collagen.

[0008] Type VIII collagen is a glycoprotein highly sensitive to neutrophil elastase and, unlike other vascular collagens such as type I, II, IV, and 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. Not all endothelial cells express type VIII collagen because this type of collagen is not present in large and small blood vessels. Furthermore, it has been found that monocytes and macrophages express type VIII collagen both 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 regeneration, fibrosis, and cancer. Mast cells expressing type VIII collagen are found in the perivascular spaces. Type VIII collagen is also expressed in smooth muscle cells and stimulates cell migration.

[0009] Corneal endothelium secretes type VIII collagen, which is assembled within the endothelial membrane through the interaction of α1 and α2 polypeptides to form a hexagonal lattice structure. COL8A2 gene mutations are associated with early Fuchs endothelial corneal dystrophy but are not associated with COL8A1. Type VIII collagen is thought to be involved in endothelial cell differentiation and tissue formation. During cardiac development, type VIII collagen plays a crucial role in angiogenesis and is immune-localized 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 stabilization. Type VIII collagen is present around actively proliferating brain tumor blood vessels and in the fibrotic blood vessels of hemangiomas. Finally, type VIII collagen is expressed in human diabetic nephropathy but not in other kidney diseases.

[0010] To date, most collagen used in various studies is 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 using genetic engineering techniques can effectively overcome these drawbacks. [Overview of the Initiative]

[0011] Part of this invention is based on the following discoveries made by the inventors. The inventors conducted a large-scale functional region screening of human type VIII collagen and obtained target gene functional regions of different recombinant humanized type VIII collagens, which exhibited higher cell adhesion-promoting activity compared to a positive control (human type I collagen). The present invention also demonstrates that the collagen or polypeptide of the present invention is suitable for expression and purification. Furthermore, the inventors discovered that C8a and C8c have better expression levels and purification efficiency compared to other collagens (C8b and C8d-j), making them suitable for industrial production. The C8a and C8c of the present invention further possess a triple helix structure.

[0012] In one embodiment, the present invention provides collagen or polypeptide comprising one or more repeating units, wherein the repeating units are linked directly or via linkers, and the repeating units comprise an amino acid sequence or variant thereof selected from the group consisting of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28, 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.

[0013] In one embodiment, the number of repeating units is 2 to 50, 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 repeating 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 substitution, addition, insertion, or deletion.

[0016] In one embodiment, the substitution is a conservative amino acid substitution.

[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 has a triple helix structure.

[0019] In one embodiment, the collagen or polypeptide comprises an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, 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.

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

[0021] In another embodiment, nucleic acids encoding collagen or polypeptides described herein are provided. In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. In one embodiment, it is a nucleotide sequence that codon-optimizes expression in E. coli. In one embodiment, 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.

[0022] In another embodiment, a vector comprising the nucleic acid described herein is provided. In one embodiment, the vector comprises an expression regulatory element operably linked to the nucleic acid, a nucleotide of a purified tag, and / or a nucleotide of a leader sequence. In one embodiment, the expression regulatory element is selected from a promoter, a terminator, or an enhancer. In one embodiment, the purified 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 clone vector, preferably pET-28a(+). pET-28a(+) may include an N-terminal His tag, a thrombin tag, and a T7 protein tag, as well as a C-terminal His tag. In this specification, the collagen or polypeptide may include an enzymatic cleavage site, such as a TEV enzymatic cleavage site, at the N-terminus to facilitate purification.

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

[0024] In another embodiment, a composition comprising one or more of the collagen or polypeptides, nucleic acids, vectors and host cells described herein is provided. In one embodiment, the composition is a kit. In one embodiment, the composition is one or more of the biomimetic materials, human biomimetic materials, cosmetic materials, organoid culture materials, cardiovascular stents, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, medicinal auxiliary materials and food additives. In one embodiment, the composition is an injectable composition or an oral composition.

[0025] In another aspect, provided is the use of the collagen or polypeptide, nucleic acid, vector, host cell, and / or composition herein in one or more of the following: a biological coating material, a human biomimetic material, a plastic and aesthetic material, an organoid culture material, a cardiovascular stent, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological and obstetric biological material, a nerve repair and regeneration material, a liver tissue material, a blood vessel repair and regeneration material, a 3D printed artificial organ biological material, a cosmetic raw material, a pharmaceutical adjuvant material, and a food additive.

[0026] In another aspect, provided is a method for promoting cell adhesion, which includes the step of contacting cells with the collagen or polypeptide, nucleic acid, vector, host cell, and / or composition herein. In one embodiment, the cells are animal cells, which may be mammalian cells or human cells.

[0027] In another aspect, provided is a method for performing plastic and aesthetic treatment, tissue injection filling, ophthalmic treatment, nerve repair, or blood vessel repair on a subject who needs it, which includes the step of administering the collagen or polypeptide herein to the subject. In one embodiment, the administration is oral administration or injection administration. In one embodiment, the subject suffers from a disease or disorder associated with type VIII collagen deficiency, such as anterior segment dysgenesis.

[0028] In another aspect, step (1) of culturing the host cell described herein under appropriate culture conditions, step (2) of harvesting the host cell and / or medium containing collagen or polypeptide, step (3) of purifying the collagen or polypeptide, are included, and provided is a method for producing the collagen or polypeptide described herein.

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

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

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

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

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

[0034] In one embodiment, the precision purification includes the step of gradient eluting the enzymatically cleaved product using a strong anion exchange chromatography column. In one embodiment, the gradient elution includes the steps of eluting with 0-15% solution B for 1-5 minutes and holding three column volumes, eluting with 15-30% solution B for 1-5 minutes and holding three column volumes, eluting with 30-50% solution B for 1-5 minutes and holding three column volumes, and eluting with 50-100% solution B for 1-5 minutes and holding three column volumes, wherein solution B contains 10-50 mM Tris, 0.5-5 M sodium chloride, and has a pH of 7-9.

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

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

[0037] The advantages of the present invention include the following: 1. The collagen of the present invention (for example, C8a-C8j) is derived from type VIII collagen, is recombinant type VIII collagen, and in particular is 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 the production of E. coli and can be isolated and purified. 3. The collagen of the present invention (e.g., C8a and C8c) has a higher expression level and is 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 compared to the positive control. The collagen of the present invention has a triple helix structure. [Brief explanation of the drawing]

[0038] [Figure 1] The purification results for C8a are shown. After precise purification, C8a yielded a high yield and demonstrated good purity of the target protein. [Figure 2] The purification results for C8b are shown. C8b showed some non-specific bands in the crudely purified target protein, some target protein in the 1M wash, a low yield after fine purification, and good purity of the target protein. [Figure 3] The purification results for C8c are shown. C8c showed good purity of the target protein, and some of the target protein was not cleaved by the 20:1 enzymatic cleavage. [Figure 4]The purification results for C8d are shown. C8d yielded a high yield, and non-specific bands were not removed at the 75kDa region after reverse-phase nickel treatment. [Figure 5] The purification results for C8e are shown. C8e had non-specific bands in the crudely purified target protein, resulting in a low yield. [Figure 6] The purification results for C8f are shown. C8f had many non-specific bands in the crudely purified target protein. [Figure 7] The purification results for C8g are shown. C8g had a high crude purification yield, and the target protein was found as two bands. [Figure 8] The purification results for C8h are shown. The crude purification yield for C8h was low. [Figure 9] The purification results for C8i are shown. With C8i, the majority of the protein was not cleaved by 20:1 enzymatic cleavage, and still not cleaved by 5:1 enzymatic cleavage. [Figure 10] The purification results for C8j are shown. The crude purification yield for C8j was low. [Figure 11] The results of detecting C8a and C8c cell adhesion activity are shown. [Figure 12] The results of the C8a circular dichroism spectral scanning analysis are shown. [Figure 13] The results of the C8c circular dichroism spectral scanning analysis are shown. [Modes for carrying out the invention]

[0039] To further clarify the object, technical means, and advantages of the present invention, the technical means in the embodiments of the present invention will be described clearly and completely below with reference to the embodiments of the present invention, and obviously the embodiments described are some embodiments of the present invention, not all embodiments. All other embodiments that a person skilled in the art could conceive of based on the embodiments of the present invention without requiring creative effort are all within the scope of the protection of the present invention.

[0040] Recombinant collagen is produced by using advanced structural biology and genetic engineering techniques to screen and manufacture novel biomaterials that are identical or similar to the amino acid sequence of human collagen, using the genetic code of the functional region of specific human collagen as a template.

[0041] As used herein, type VIII collagen is also called short-chain collagen or network-forming collagen. Type VIII 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 main molecular species. Heterotrimers may also exist. Descemet's membrane of the bovine eye has a hexagonal reticular structure containing thin type VIII collagen fibrils, and immunohistochemical analysis and electron microscopy have shown that type VIII collagen is the main component of this structural framework. Furthermore, rotational shadow analysis has shown that type VIII collagen may form a tetrahedral supramolecular structure that supports the structure and can regulate cellular behavior.

[0042] As used herein, “polypeptide” refers to a plurality of amino acid residues linked together by peptide bonds. In this specification, a polypeptide comprises one or more repeating units, which may be derived from human type VIII collagen. Therefore, a polypeptide may be human recombinant type VIII collagen. The repeating units may be linked by linkers, which may consist of native amino acid residues in the human type VIII collagen of the repeating units, for example, 1 to 50 amino acid residues. The repeating units may have SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28. The polypeptide may have SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, or 29.

[0043] As used herein, “human recombinant type VIII collagen” means a recombinant protein consisting of or essentially consisting of a sequence derived from human type VIII collagen. In this specification, human recombinant type VIII collagen may consist of or essentially consisting of a fragment or multiple repeats of a fragment derived from human type VIII collagen. In this specification, recombinant collagen, human recombinant type VIII collagen, collagen, or polypeptides are used interchangeably.

[0044] As used herein, the term “variant” means a collagen or polypeptide having cell adhesion activity and containing alterations (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 or immediately following an amino acid occupying a position. Addition means adding one or more amino acid residues to the C-terminus and / or N-terminus of an amino acid sequence. Substitutions may be conservative substitutions. A variant of a repeating unit may be a sequence after one or more amino acid residues have been altered or mutated (i.e., substituted, added, inserted, and / or deleted) at SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28. The collagen or polypeptide variant may be a sequence in which one or more amino acid residues have been altered or mutated (i.e., substituted, added, inserted, and / or deleted) in SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, or 29.

[0045] In the context of the present invention, a conservative substitution may be defined by a substitution within one or more amino acid types as 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 Nonpolar 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 group-related residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Loaded electrical 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 Tiny residues A, G, and S Residues involved in reverse 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.

[0046] As used herein, “cell adhesion” refers to the adhesion between cells and collagen. Collagen (e.g., the polypeptides described herein) can promote adhesion between cells and the vessel in which the cells are cultured.

[0047] As used herein, the term “expression” includes, but is not limited to, any steps relating to the production of collagen or polypeptides, including 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 comprising a polynucleotide encoding collagen or polypeptide, operably ligated to a control sequence that provides expression thereof.

[0049] As used herein, the term “host cell” means any type of cell that readily undergoes transformation, transfection, transduction, etc., by the nucleic acid construct or expression vector comprising the polynucleotide of the present invention. The term “host cell” includes any offspring of a parent cell that are not identical to the parent cell due to mutations occurring during replication.

[0050] 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, modified to contain a nucleic acid segment in a manner not originally occurring in nature, or synthesized, and contains 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 codon-optimized for expression in E. coli cells.

[0051] The term "operably linked" refers to a configuration in which a control sequence is positioned appropriately relative to the coding sequence of a polynucleotide to direct the expression of the coding sequence, as follows:

[0052] The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity". For the purposes of this invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.[Molecular Biology Journal] 48:443-453), which is performed by the Needleman program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.[Genetic Trends] 16:276~277) (preferably version 5.0.0 or updated version). The parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and the use of the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needleman output (obtained using the unsimplified option) marked "longest identity" is used as the identity percentage and calculated as follows: (Same residue × 100) / (Alignment length - Total number of gaps in alignment)

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

[0054] collagen In the present invention, a collagen or polypeptide comprising one or more repeating units, wherein the repeating units are linked 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, 4, 7, 10, 13, 16, 19, 22, 25, or 28. The mutant may be (1) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28, or (2) an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28. For the collagen or polypeptide described herein, the mutation may be selected from substitution, addition, insertion, or deletion. Preferably, the substitution is a conservative amino acid substitution.

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

[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 recombinant collagen, and is particularly preferably recombinant type VIII collagen, which has cell adhesion activity. Since the collagen or polypeptide described herein is derived from humans, it may also be human recombinant type VIII collagen.

[0058] The collagen or polypeptides described herein may include an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, or 29, wherein the variant is (1) an amino acid sequence in which one or more amino acid residues are mutated in the above 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 above amino acid sequence.

[0059] The collagen described herein may have a triple helix structure or three identical chains (i.e., a trimer). The arrangement of each chain may be the arrangement of the collagen or polypeptide described herein.

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

[0061] Nucleic acids can be manipulated in various ways to provide collagen or polypeptide expression. Depending on the expression vector, it may be desirable or necessary to manipulate the nucleic acid before inserting it into the vector. Techniques for modifying nucleic acids using recombinant DNA are well known in this field.

[0062] The regulatory sequence may be a promoter, which is a polynucleotide recognized by the host cell for the expression of the polynucleotide encoding the collagen or polypeptide of the present invention. The promoter includes a transcriptional regulatory sequence that mediates the expression of 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 obtained from a gene encoding extracellular or intracellular collagen or polypeptide of the same or different species as the host cell.

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

[0064] In the yeast host, useful promoters can be 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 triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.

[0065] The regulatory sequence may be a transcriptional terminator that is recognized by the host cell and terminates transcription. The terminator is operably ligated to the 3' end of a polynucleotide encoding collagen or polypeptide. Any terminator that functions within a host cell may be used in this invention.

[0066] Preferred terminators within bacterial host cells are derived from the genes of Bacillus clausii alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia 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 in yeast host cells are described by Romanos et al. (1992, same as above).

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

[0069] Examples of suitable mRNA stabilization regions can be found in 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 regulatory sequence may be a leader sequence, which is an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operably ligated to the 5' end of a polynucleotide encoding collagen or polypeptide. Any leader sequence that functions in the host cell may be used.

[0071] Appropriate leader sequences in yeast host cells can be 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 regulatory sequence may be a polyadenylated sequence, which is operably ligated to the 3' end of a polynucleotide and recognized by the host cell as a signal to add a polyadenosine residue to the transcribed mRNA during transcription. Any polyadenylated sequence that functions within the host cell may be used.

[0073] Useful polyadenylated sequences in yeast host cells are described in Guo and Sherman, 1995, Mol. Cellular Biol. [Molecular Cell Biology] 15:5983-5990.

[0074] The regulatory sequence may be a signal peptide coding region that codes for a signal peptide linked to the N-terminus of collagen or polypeptide, thereby directing the collagen or polypeptide into the cell's secretory pathway. The 5'-terminus of the polynucleotide coding sequence may essentially contain a signal peptide coding sequence that is originally linked in the translational reading frame with the coding sequence segment encoding collagen or polypeptide. Alternatively, the 5'-terminus of the coding sequence may contain an exogenous signal peptide coding sequence relative to the coding sequence. An exogenous signal peptide coding sequence may be required if the coding sequence does not originally contain a signal peptide coding sequence. Alternatively, the exogenous signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance the secretion of collagen or polypeptide. However, any signal peptide coding sequence that directs expressed collagen or polypeptide into the host cell's secretory pathway may be used.

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

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

[0077] Expression vector The present invention also relates to a recombinant expression vector comprising the nucleic acid, promoter, and transcription and translation termination signals of the present invention. The nucleic acid and control sequence 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 a suitable vector for expression. When the expression vector is produced, the coding sequence is thus positioned in the vector so that the coding sequence is operably ligated to a suitable control sequence for expression.

[0078] The recombinant expression vector may be any vector (e.g., plasmid or virus) that can readily undergo recombinant DNA programming and induce polynucleotide expression. Vector selection typically depends on the compatibility between the vector and the host cell into which it is introduced. The vector may be a linear or closed circular plasmid.

[0079] The vector is an extrachromosomal entity, and may be an autonomously replicating vector whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector may include any means to ensure self-replication. Alternatively, the vector may be a vector that is integrated into the genome upon introduction into a host cell and replicates together with one or more integrated chromosomes. Alternatively, a single vector or plasmid or two or more vectors or plasmids containing the total DNA introduced into the host cell genome may be used, or a transposon may be used.

[0080] The vector preferably includes one or more selective markers that allow for easy selection of cells such as transformed cells, transfection cells, or transduced cells. The selective markers are genes whose products provide biocide resistance or viral resistance, heavy metal resistance, prototrophicity to trophic requirements, etc.

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

[0082] The selectivity marker may be a biselectivity marker system as described in WO2010 / 039889. On the other hand, the biselectivity marker is an hph-tk biselectivity marker system.

[0083] The vector may include elements that enable the vector to be incorporated into the genome of a host cell, or that enable the vector to replicate autonomously within the cell independently of the genome.

[0084] When integrated into the genome of a host cell, the vector may rely on a polynucleotide sequence encoding the collagen or polypeptide, or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration into a precise location on a chromosome in the host cell's genome by homologous recombination. To increase the likelihood of precise integration, 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, and these nucleic acids should 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 host cell's genome. The integration element may also be a non-coding or coding polynucleotide. On the other hand, the vector can be integrated into the genome of a host cell by non-homologous recombination.

[0085] For autonomous replication, the vector may further include a replication origin that enables autonomous replication in the host cell under consideration. The replication origin may be any plasmid replicon that functions in the cell and mediates autonomous replication. The terms “replication origin” or “plasmid replicon” refer to a polynucleotide that can replicate a plasmid or vector in the body.

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

[0087] Examples of replication origins used in yeast host cells include a 2-micrometer replication origin, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

[0088] One or more copies of the polynucleotide of the present invention can be inserted into host cells to improve collagen or polypeptide production. An increased copy number of the polynucleotide can be obtained by incorporating at least one other copy of the sequence into the host cell genome or by including a selectable marker gene that can be amplified together with the polynucleotide, and cells containing amplified copies of the selectable marker gene and other copies of the polynucleotide can be selected by culturing the cells in the presence of a suitable selective reagent.

[0089] The procedure for constructing the recombinant expression vector of the present invention by linking the above elements is 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 the polynucleotides of the present invention, operably linked to one or more control sequences that direct the production of the collagen or polypeptides of the present invention. As described previously, by introducing a construct or vector comprising polynucleotides into a host cell, the construct or vector is maintained as a chromosomal integration or as an autonomously replicating extrachromosomal vector. The term “host cell” encompasses any offspring of a parent cell that is not identical to the parent cell due to mutations occurring during replication. The selection of host cells depends largely on the genes encoding the collagen or polypeptide and their sources.

[0091] The host cell may be any cell useful for the recombinant production of collagen or polypeptide according to the present invention, for example, a prokaryote or a eukaryote.

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

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

[0094] The host cell may be a fungal cell belonging to the phyla Basidiomycota, Chytridiomycota, Zygomycota, or Oomycota. The fungal host cell may also be a yeast cell, including ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeasts belonging to the imperfect fungi (Blastomycetes). The yeast host cells may be from the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, for example, Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces crivelli These are cells of Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica.

[0095] Production method The present invention also relates to a method for producing collagen or polypeptide as described herein, the method being Step (1) of culturing the host cells described herein under appropriate culture conditions, (2) Harvesting host cells and / or culture medium containing collagen or polypeptides, The process includes (3) purifying collagen or polypeptide.

[0096] Host cells are cultured in a suitable nutrient medium for producing collagen or polypeptides using methods known in the art. For example, cells may be cultured by shaking flask culture or by small or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermenter, under conditions that allow for the expression and / or isolation of collagen or polypeptides in a suitable medium. Using procedures known in the art, the culture is carried out in a suitable nutrient medium containing a carbon source, a nitrogen source, and inorganic salts. Suitable media can be obtained from commercial suppliers or can be manufactured according to disclosed compositions (e.g., the catalog of the U.S. Center for the Preservation of Typical Cultures). If collagen or polypeptides are secreted into the nutrient medium, the collagen or polypeptides can be recovered directly from the medium. If collagen or polypeptides are not secreted, they can be recovered from the cell lysate.

[0097] Collagen or polypeptides may be detected using methods known in the art that are specific to collagen or polypeptides. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, the activity of collagen or polypeptides may be determined using enzyme assays.

[0098] Collagen or polypeptides may be recovered using methods known in this art. For example, collagen or polypeptides 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, a fermentation broth containing collagen or polypeptides may be recovered.

[0099] To obtain substantially pure collagen or polypeptides, the collagen or polypeptides may be purified by various procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focal chromatography, and size exclusion chromatography), electrophoresis (e.g., calibrated isoelectric focusing), differential dissolution (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 and obtain a recombinant expression plasmid by, for example, inserting the coding nucleotide sequence into a pET-28a-Trx-His expression vector. Transform E. coli cells (e.g., E. coli competent cells BL21(DE3)) with the successfully constructed expression plasmid. The specific process may be as follows: (1) Add the plasmid to be transformed to E. coli competent cells BL21(DE3). (2) After ice bathing the mixture on ice (e.g., 10-60 min, e.g., 30 min), heat shock in a water bath (40-50°C, e.g., 42°C, 45-90 s), remove and then ice bathing on ice (1-5 min, e.g., 2 min). (3) Add liquid LB medium and then culture (e.g., 35-40°C, e.g., 37°C, at 150-300 rpm, e.g., 220 rpm for 40-80 min, e.g., 60 min). (4) Apply the bacterial suspension and select a single colony. For example, apply the bacterial suspension uniformly to an LB plate containing ampicillin sodium, and incubate the plate in a 37°C incubator for 15-17 hours to grow a colony of uniform size.

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

[0102] Step (3) may include the steps of resuspending the bacterial cells in an equilibrium working solution, cooling the bacterial suspension to ≤15°C, homogenizing it (for example, 1 to 5 times, for example, 2 times under high pressure), and separating the homogenized bacterial suspension to obtain the 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. Purification may include crude purification, which includes purifying the supernatant on a Ni-agarose gel column to obtain an eluent containing the target protein. Crude purification may include washing the column with water for, for example, 2 to 10 column volumes (CV), for example, 5 CVs. The column may be equilibrated with, for example, 2 to 10 CVs, for example, 5 CVs of equilibrium solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0). The equilibrium 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 away contaminating proteins with a washing solution. The washing solution may contain 100-500 mM sodium chloride, 10-50 mM Tris, and 10-50 mM imidazole, with a pH of 7-9. For example, the concentration of sodium chloride may be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 nM. The concentration of Tris may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The concentration of imidazole may be 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM. The pH may be 7, 7.5, 8, 8.5, or 9. The eluent may then be added and the flow-through solution collected. The eluent may contain 100-500 mM sodium chloride, 10-50 mM Tris, and 100-500 mM imidazole, with 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 imidazole concentration 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] Enzymatic cleavage may include adding TEV enzyme (with a ratio of total protein to total TEV enzyme of 10-100:1, e.g., 50:1, at 10-20°C, e.g., 16°C, for 2-8 hours, e.g., 4 hours) to perform enzymatic cleavage. The enzymatically cleaved protein solution is dialyzed, for example, in a dialysis bag, dialyzed at 1-6°C, e.g., 4°C for 1-8 hours, e.g., 2 hours, and then transferred to fresh dialysate and dialyzed overnight at 1-6°C, e.g., 4°C.

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

[0108] To further illustrate the present invention, the following embodiments are provided. [Examples]

[0109] The present invention will be further illustrated by the following examples, but none of these examples or any combination thereof should be understood as limiting 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 will be able to clearly understand the scope limited by the claims by combining this specification and common sense 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 within the scope of the present invention.

[0110] Example 1: Construction, expression, and screening of type VIII collagen fragments 1. A large-scale functional region screening was performed, and the target gene functional regions of the following different recombinant humanized type VIII collagens were obtained.

[0111] 1) C8a amino acid sequence: gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp(SEQ ID NO:1) gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp gKpgmpgmpgKpgamgmpgaKgEigqKgEigpmgipgpqgppgphglp (However, the amino acid sequence of the C8a repeating unit is shown as SEQ ID NO:1, with 6 repeats, and the amino acid sequence of C8a is shown as SEQ ID NO:2.) Base 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 C8b repeating unit is indicated by SEQ ID NO:7, with 6 repeats, and the amino acid sequence of C8b is indicated by SEQ ID NO:8.) Base 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 C8c repeating unit is shown as SEQ ID NO:4, with 6 repeats, and the amino acid sequence of C8c is shown as SEQ ID NO:5.) Base sequence: (SEQ ID NO:6)

[0114] 4) C8d amino acid sequence: gKpgqDgipgqpgfpggKgEqglpglpgppglp(SEQ ID NO:10) gKpgqDgipgqpgfpggKgEqglpglpgppglp gKpgqDgipgqpgfpggKgEqglpglpgppglp gKpgqDgipgqpgfpggKgEqglpglpgppglp gKpgqDgipgqpgfpggKgEqglpglpgppglp gKpgqDgipgqpgfpggKgEqglpglpgppglp (However, the amino acid sequence of the repeating unit of C8d is shown as SEQ ID NO:10, with 6 repeats, and the amino acid sequence of C8d is shown as SEQ ID NO:11.) Base sequence: (SEQ ID NO:12)

[0115] 5) C8e amino acid sequence: gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEpglpgipgpmgppgaigfpgpKgEggivgpqgppgpKgEpglqgfpgKpgflgEvgppgmRglpgpigpKgEagqKgvpglpgvpgllgpKgEpgipgDqglqgppgipgiggpsgpigppgipgpKgEpglpgppgfp (SEQ ID NO:13) gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEpglpgipgpmgppgaigfpgpKgEggivgpqgppgpKgEpglqgfpgKp gflgEvgppgmRglpgpigpKgEagqKgvpglpgvpgllgpKgEpgipgDqglqgppgipgiggpsgpigppgipgpKgEpglpgppgfp (However, the amino acid sequence of the C8e repeating unit is shown as SEQ ID NO:13, with 2 repeats, and the amino acid sequence of C8e is shown as SEQ ID NO:14.) Base sequence:

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

[0117] 7) C8g amino acid sequence: gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp(SEQ ID NO:19) gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp gKpgfpgpKgDRgmggvpgalgpRgEKgpigapgiggppgEp (However, the amino acid sequence of the repeating unit of C8g is shown as SEQ ID NO:19, with 6 repeats, and the amino acid sequence of C8g is shown as SEQ ID NO:20) Base sequence: (SEQ ID NO:21)

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

[0119] 9) C8i amino acid sequence: gpKgEagqKgvpglpgvpgllgpKgEpgipgDq(SEQ ID NO:25) gpKgEagqKgvpglpgvpgllgpKgEpgipgDq gpKgEagqKgvpglpgvpgllgpKgEpgipgDq gpKgEagqKgvpglpgvpgllgpKgEpgipgDq gpKgEagqKgvpglpgvpgllgpKgEpgipgDq gpKgEagqKgvpglpgvpgllgpKgEpgipgDq (However, the amino acid sequence of the repeating unit of C8i is shown as SEQ ID NO:25, with 6 repeats, and the amino acid sequence of C8i is shown as SEQ ID NO:26) Base sequence: (SEQ ID NO:27)

[0120] 10) C8j amino acid sequence: gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp(SEQ ID NO:28) gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp gKpgvaglhgppgKpgalgpqgqpglpgppgppgppgpp (However, the amino acid sequence of the repeating unit of C8j is shown as SEQ ID NO:28, with 6 repeats, and the amino acid sequence of C8j is shown as SEQ ID NO:29) Base sequence: (SEQ ID NO:30)

[0121] 2. The above coding nucleotide sequences were commercially synthesized. Each of the above coding nucleotide sequences (with a collagen tool enzyme cleavage site added to the 5' end, the amino acid sequence of the collagen tool enzyme cleavage site being ENLYFQ, and the nucleotide sequence being GAAAACCTGTATTTCCAG) was inserted between the KpnI and XhoI enzyme cleavage sites of the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid.

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

[0123] 4. Five to six single colonies were selected from the transformed LB plates and placed in a shaking flask containing LB medium with antibiotic preservation solution. The cells were incubated for 7 hours at 220 rpm and 37°C using a constant temperature shaker. After incubation, the shaking flask was cooled to 16°C, IPTG was added to induce expression for a certain period of time, and the bacterial suspension was dispensed into a centrifuge flask. The cells were centrifuged at 8000 rpm and 4°C for 10 minutes to collect the cells, record the weight of the cells, and perform electrophoresis detection on the sampled cells (referred to as "bacterial suspension").

[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 bacterial suspension was cooled to ≤15°C and homogenized, then homogenized twice under high pressure (the two homogenized samples were labeled "homogeneous" and "homogeneous," respectively), and the bacterial suspension was collected after completion. The homogenized bacterial suspension was dispensed into centrifuge flasks and centrifuged at 17000 rpm at 4°C for 30 minutes, the supernatant was collected, and electrophoresis detection was performed on the supernatant (labeled "supernatant") and precipitate (labeled "precipitate").

[0125] 6. Recombinant humanized type VIII collagen was purified and enzymatically cleaved. The specific process is as follows (1) to (4): (1) Crude purification was performed as follows a to g. a. The column was washed with water using 5 CVs (Ni6FF, Cytiva). b. The column was equilibrated with an equilibrium solution for 5 CVs (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole, pH 8.0). c. For loading, the supernatant obtained by centrifugation was added to the column until the liquid had finished flowing, and electrophoresis detection was performed on the flow-through solution (indicated 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 had finished flowing, and electrophoresis detection was performed on the washed flow-through solution (indicated as "washing"). e. For the collection of the target protein, 20 mL of eluent (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole, pH 8.0) was added, the flow-through solution was collected (indicated as "elution"), the protein concentration was detected to calculate the amount of protein, and electrophoresis detection was performed. f. The column was washed with 1 M imidazole working solution (indicated as "1 M wash"). g. The column was washed with purified water. (2) Enzymatic digestion was performed as follows: The ratio of the total amount of protein to the total amount of TEV enzyme was 50:1 (if digestion was not performed, the ratio of the total amounts was set to 20:1 or 5:1 to consider increasing the enzyme concentration, for example), TEV enzyme was added, and enzymatic digestion was performed at 16°C for 4 hours, and a sample was taken (indicated as "after digestion") for electrophoresis detection. The enzyme-digested protein solution was placed in a dialysis bag, dialyzed at 4°C for 2 hours, and then transferred to a new dialysate and dialyzed overnight at 4°C (indicated as "fluid change"). (3) The samples were precisely purified as follows (protein isoelectric point > 8.0): a. For column equilibration (Capto Q, Cytiva), the column was equilibrated with solution A (20 mM Tris, 20 mM sodium chloride, pH 8.0) and the flow rate was set to 10 ml / min. b. For loading, the flow rate was set to 5 ml / min, the column was loaded, the flow-through solution was collected (denoted as QFL), and electrophoresis detection was performed. c. For gradient elution, each column was eluted for 2 minutes with 0-15% solution B (20 mM Tris, 1 M sodium chloride, pH 8.0) (indicated as "0-15% B wash"), and three CVs were retained. Then, eluted for 2 minutes with 15-30% solution B and three CVs were retained. Then, eluted for 2 minutes with 30-50% solution B and three CVs were retained. Then, eluted for 2 minutes with 50-100% solution B and three CVs were retained. When a peak appeared, the samples were collected and electrophoretic detection was performed. d. The column was washed. The protein was stored at 4°C. (4) The following reverse-phase nickel treatment was performed (Ni6FF, Cytiva) (protein isoelectric point < 8.0). a. For column equilibration, the column was equilibrated with solution A (20 mM Tris, 20 mM sodium chloride, 20 mM imidazole, pH 8.0) and 5 CVs were created. b. For loading, the enzyme-cleaved and solution-exchanged proteins were placed in the column until the liquid flowed out, and electrophoresis detection was performed on the flow-through solution (indicated 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) (indicated as "1 M wash"). d. The column was washed with purified water. The proteins were stored at 4°C.

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

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

[0128] Regarding protein expression levels, the order was C8a > C8i > C8b > C8g > C8d > C8c > C8f > C8e > C8j > C8h.

[0129] 8. Electrophoresis detection The specific process is as follows: Take 40 μl of sample protein solution, add 10 μl of 5× protein loading buffer (250 mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), boil in 100°C water for 10 minutes, then add 10 μl of SDS-PAGE protein gel to each well, perform electrophoresis at 80 V for 2 hours, stain the protein with Coomassie Brilliant Blue stain (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20 minutes, and then destain 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 fine purification and good purity of the target protein. Figure 2 shows the purification results for C8b. C8b had some non-specific bands in the crudely purified target protein, some of the target protein was present in the 1M wash, the yield after fine purification was low, and the purity of the target protein was good. Figure 3 shows the purification results for C8c. C8c had good purity of the target protein, and some of the target protein was not cleaved by 20:1 enzyme cleavage. Figure 4 shows the purification results for C8d. C8d had a high yield, and non-specific bands were not removed at the 75kDa region after reverse-phase nickel treatment. Figure 5 shows the purification results for C8e. C8e had non-specific bands in the crudely purified target protein and a 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 had two bands. Figure 8 shows the purification results for C8h. C8h had a low crude purification yield. Figure 9 shows the purification results for C8i. For C8i, most of the protein was not cleaved with 20:1 enzymatic cleavage, and still not cleaved with 5:1 enzymatic cleavage. Figure 10 shows the purification results for C8j. C8j had a low crude purification yield.

[0131] As can be seen from the electrophoresis detection results, 1. C8e, C8h, and C8j have low crude purification yields and narrow target protein bands; 2. C8i has one clear non-specific band in the crudely purified 35kDa target protein and is basically unable to be enzymatically cleaved; 3. C8b and C8f have many non-specific bands of the target protein and low yield after fine purification; 4. C8g has a high yield but the target protein has two bands and low purity; 5. C8d has a high yield but many non-specific bands after reverse-phase treatment on the Ni column and low purity; and 5. C8a and C8c have a high yield and good purity of the target protein. C8a and C8c will be selected for subsequent detection.

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

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

[0134] The data search was 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 degradation. For detection parameters, two uncleaved sites were defined in Trypsin enzymatic degradation. Cysteine ​​alkylation was considered a fixed modification. Methionine oxidation was considered a variable modification. The database used for identification was NCBprot.

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

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

[0137] Example 3: Detection of the bioactivity of recombinant type VIII humanized collagen For information on methods for detecting collagen activity, please 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 procedure is as follows.

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

[0139] Specifically, the amount of ultraviolet 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). Note that detection is necessary when A215 < 1.5. The principle of this method is as follows: It measures the characteristic absorption of peptide bonds in far ultraviolet light, is unaffected by the content of chromophore, has few disturbances, is easy to operate, and is suitable for detecting human collagen and its analogs that do not fluoresce with Coomassie Brilliant Blue. (Reference: Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45.) After detecting the protein concentration, the concentration of all target proteins is adjusted to 0.5 mg / mL with PBS.

[0140] (2) Sample preparation: Experiments were performed directly using the stock sample 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 each of collagen at different concentrations and positive and negative controls were added to each well of the enzyme standard plate, creating five overlapping wells for each group, and incubated overnight at 4°C.

[0142] (4) Blocking: Discard the supernatant, add 100 μL of 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.

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

[0144] (6) Detection: OD using a CCK8 detection kit (Manufacturer: Beyotime, Product Catalog Number: C0038) 450 Absorbance in nm was detected. The degree of cell adhesion was calculated using the following formula. Cell adhesion rate can reflect the cell adhesion capacity of collagen. The higher the cell adhesion capacity, the more quickly a favorable external environment can be provided to the cells, thus aiding cell adhesion.

number

[0145] (7) Statistical analysis: A two-sided t-test was used to statistically analyze the statistical difference between the target recombinant humanized collagen and the negative control, where P<0.05 for *, P<0.01 for **, and P<0.001 for ***.

[0146] The results are shown in Figure 11. Compared to the D-PBS group, the positive control clearly had a cell adhesion-promoting effect, and recombinant humanized collagen C8a and C8c also had a cell adhesion-promoting effect.

[0147] Example 4: Circular dichroism spectral ultraviolet scanning analysis of recombinant type VIII humanized collagen Experimental method (1) Setting of equipment parameters Bandwidth: 1.0nm Step: 1.0nm 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 ultraviolet scanning and near ultraviolet scanning of standard products The scanning wavelength was set to 180-340 nm, and background tests and buffer blank tests were performed to obtain the circular dichroism (far-ultraviolet, near-ultraviolet) absorbance of a 1 mg / mL CSA standard solution in the 180-340 nm range. (3) Sample processing The finely purified C8a and C8c protein samples are concentrated using a 10kD ultrafiltration filter (Millipore) until the protein concentration reaches 1 mg / ml. (4) Far ultraviolet scanning of the sample The cuvette is immersed overnight in 2M HNO3, washed with deionized water and dried, and the background is collected. Then, the blank buffer is collected, an appropriate amount of the test substance is added to the cuvette, and data is acquired by performing a far-ultraviolet scan at 190-260 nm based on the above parameters. (5) Near-ultraviolet scanning of the sample The cuvette is immersed overnight in 2M HNO3, washed with deionized water and dried, and the background is collected. Then, the blank buffer is collected, an appropriate amount of the test substance is added to the cuvette, and near-ultraviolet scanning is performed at 250-340 nm based on the above parameters to acquire data. (6) Scanning spectral processing After scanning, baseline subtraction and smoothing are performed on all spectra using the Pro-Data Viewer software.

[0148] Experimental results and analysis

[0149] The results showed that both C8a and C8c exhibited a positive peak at 221 nm, indicating that both proteins possess a triple helix structure (i.e., they have the general structural characteristics of active collagen). The results are shown in Figures 12 and 13.

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

Claims

1. Collagen comprising one repeating unit or comprising two to eight repeating units, wherein the repeating units are directly linked, and the repeating units comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 4 or a variant thereof, wherein the variant is (1) an amino acid sequence in which one to four amino acid residues are mutated in the amino acid sequence of SEQ ID NO: 1 or 4, or (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 1 or 4. The collagen is collagen that has cell adhesion promoting activity.

2. The collagen according to claim 1, wherein the mutation is selected from substitution, addition, insertion or deletion.

3. The collagen according to claim 1, wherein the collagen is human recombinant type VIII collagen.

4. The collagen according to claim 1, wherein the collagen has a triple helix structure or is in trimer form.

5. The collagen according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 2 or 5.

6. A nucleic acid encoding collagen as described in claim 1.

7. The nucleic acid according to claim 6, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 and 6.

8. A vector comprising the nucleic acid described in Claim 6.

9. The vector according to claim 8, comprising an expression control element operably linked to the nucleic acid, a nucleotide sequence encoding a purification tag and / or a nucleotide sequence encoding a leader sequence.

10. The expression control element is selected from a promoter, terminator or enhancer, or The vector according to claim 9, wherein the refined tag is selected from His tag, GST tag, MBP tag, SUMO tag, or NusA tag.

11. The vector according to claim 10, which is an expression vector or a clone vector.

12. The vector according to claim 10, which is pET-28a(+).

13. A host cell comprising the vector according to claim 8.

14. The host cell according to claim 13, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

15. The host cell according to claim 14, wherein 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.

16. A composition comprising the collagen described in claim 1.

17. The composition according to claim 16, which is one or more of the following: a bio-coating material, a human biomimetic material, a cosmetic orthopedic material, an organoid culture material, a cardiovascular stent material, 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 blood vessel repair and regeneration material, a 3D printed artificial organ biological material, a cosmetic raw material, a medicinal auxiliary material and a food additive, or an injectable composition or an oral composition.

18. Use of the collagen according to any one of claims 1 to 5, the nucleic acid according to claim 6 or 7, the vector according to any one of claims 8 to 12, the host cell according to any one of claims 13 to 15, and / or the composition according to claim 16 or 17, in the manufacture of one or more of the following: biomimetic materials, human biomimetic materials, cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling materials, ophthalmic materials, obstetric and gynecological biological materials, nerve repair and regeneration materials, liver tissue materials, blood vessel repair and regeneration materials, 3D printed artificial organ biological materials, cosmetic raw materials, medicinal auxiliary materials, and food additives.

19. An in vitro method for promoting cell adhesion, comprising the step of bringing cells into contact with collagen according to any one of claims 1 to 5 in vitro.

20. The in vitro method according to claim 19, wherein the cells are animal cells.

21. A composition comprising the collagen described in claim 1 or 2, for use in cosmetic surgery, tissue injection and filling, ophthalmic treatment, nerve repair or vascular repair in a subject requiring the use thereof, wherein the use comprises the step of administering the collagen to the subject.

22. The composition according to claim 21, wherein the administration is by oral administration or by injection.

23. The composition according to claim 21, wherein the subject suffers from a disease or condition related to type VIII collagen deficiency.

24. The composition according to claim 21, wherein the ophthalmic treatment includes administering the collagen to a subject suffering from anterior segment hypoplasia.

25. (1) A step of culturing host cells containing a vector having nucleic acid encoding collagen under appropriate culture conditions, Step (2) harvesting host cells and / or culture medium containing collagen, The process includes the step (3) of purifying collagen, A method for producing collagen according to any one of claims 1 to 5.

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

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