Recombinant II-type humanized collagen and application thereof

Abstract

The present disclosure provides recombinant type II humanized collagen and uses thereof. The recombinant II-type humanized collagen has a typical triple helix structure and excellent biological characteristics, can be used for cartilage repair, and does not generate immunological rejection and anaphylactic reaction when being applied to a human body.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biotechnology, and particularly relates to recombinant type II humanized collagen and its uses. Background Art

[0002] Collagen is a class of proteins widely distributed in human connective tissues and is also the most abundant protein in the human body, accounting for 25% - 35% of the total protein. Currently, at least 28 collagen subtypes have been found in the human body, which are located in different tissues and organs.

[0003] Type II collagen mainly exists in cartilage tissues, vitreous bodies, and corneas. It is a high-molecular-weight protein, and its filamentous collagen fibers are intertwined with elastin and proteoglycans to form a network structure, also known as composite ossein. Type II collagen is an essential component for cartilage and bone formation, bone growth, and maintenance of mature cartilage. Therefore, non-denatured collagen can be used as a structural and functional component of cartilage.

[0004] As the main component of articular cartilage matrix, type II collagen, together with lubricating components such as hyaluronic acid and proteoglycans, protects cartilage from wear. With the increase of age, the synthesis rate of collagen gradually decreases, the loss of collagen intensifies, articular cartilage degenerates, and the friction between bones increases, which will cause inflammation at the joints. Currently, the common treatment method for osteoarthritis is to consume collagen, but its protein utilization rate is low, resulting in a slow speed of cartilage regeneration, which is not conducive to the recovery of patients and affects the treatment process and efficiency. Due to the unsatisfactory curative effect of the current treatment methods, patients often have to choose artificial joint replacement surgery in the end, which not only costs a lot but also may have catastrophic complications such as postoperative thrombosis, infection after replacement, and even amputation.

[0005] The traditional method for producing type II collagen is to treat animal-derived tissues with acid, alkali, and enzymatic hydrolysis to extract collagen derivatives. The collagen extracted by these methods has lost its original biological activity and cannot be applied in the biomedical field to play its true function. With the development of modern technology, some extraction methods for obtaining non-denatured type II collagen by removing impurities and enzymatic hydrolysis of animal cartilage have emerged at home and abroad. However, these methods have a long time for impurity removal and extraction, low product purity, it is difficult to guarantee the triple helix structure of collagen, poor product stability, and are not conducive to large-scale production. In addition, the immunogenicity of animal-derived collagen cannot be eliminated, which is an important reason for the limitation of collagen in application. In view of the defects of the existing technology, a company has proposed to use Pichia pastoris as a host strain to prepare type II-like collagen for cartilage repair. However, this collagen does not belong to humanized collagen, so it has an immunogenic reaction.

[0006] Therefore, there is an urgent need for a recombinant type II humanized collagen that can be directly injected into the human body and does not cause an immunogenic reaction, for use as a human structural material in cartilage repair. Summary of the Invention

[0007] The present invention uses synthetic biology and structural biology techniques to develop a recombinant type II humanized collagen with a triple helix structure that can perform the functions of human collagen.

[0008] In a first aspect of the present invention, there is provided a recombinant type II humanized collagen, which comprises n repeating units, the repeating units being directly or connected through a linker, the repeating unit comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or a variant thereof, the variant being (1) an amino acid sequence obtained by substituting, deleting or inserting one or more amino acids on the basis of SEQ ID NO:1 or SEQ ID NO:3; 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% identity with SEQ ID NO:1 or SEQ ID NO:3.

[0009] In some embodiments, n is an integer from 1 to 20. In some embodiments, n is 1, 4, 8 or 12.

[0010] In some embodiments, the linker comprises one amino acid or multiple amino acid residues; preferably, the linker comprises 2, 3, 4, 5, 6, 7 or 8 amino acid residues.

[0011] In some embodiments, the collagen comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5.

[0012] In some embodiments, the collagen comprises 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 SEQ ID NO:4 or SEQ ID NO:5.

[0013] In some embodiments, the collagen comprises an amino acid sequence obtained by substituting, deleting or inserting one or more amino acids on the basis of SEQ ID NO:4 or SEQ ID NO:5.

[0014] In a second aspect of the present invention, there is provided a fusion protein, which comprises the recombinant type II humanized collagen described in the first aspect of the present invention and a purification tag and / or a leader.

[0015] In some embodiments, the purification tag is selected from His tag, GST tag, MBP tag, SUMO tag or NusA tag.

[0016] In some embodiments, the leader is selected from a synthetic leader sequence, a signal peptide or a leader peptide.

[0017] The third aspect of the present invention provides a polynucleotide encoding the collagen according to the first aspect of the present invention or the fusion protein according to the second aspect of the present invention.

[0018] In some embodiments, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:6 or SEQ ID NO:8 or a degenerate sequence thereof.

[0019] The fourth aspect of the present invention provides a vector comprising the polynucleotide according to the third aspect of the present invention.

[0020] In some embodiments, the vector is an expression vector; in some embodiments, the vector comprises a control element operably linked to the polynucleotide; in some embodiments, the control element is selected from a promoter, a terminator and / or an enhancer.

[0021] The fifth aspect of the present invention provides a host cell comprising the polynucleotide according to the third aspect of the present invention, or the vector according to the fourth aspect of the present invention.

[0022] In some embodiments, the host cell is a bacterium, a fungus or an animal cell; in some embodiments, the bacterium includes Escherichia coli; in some embodiments, the fungus includes yeast, such as Saccharomyces cerevisiae.

[0023] The sixth aspect of the present invention provides a trimeric collagen comprising the recombinant type II humanized collagen according to the first aspect of the present invention.

[0024] The seventh aspect of the present invention provides a method for producing the recombinant type II humanized collagen according to the first aspect of the present invention or the fusion protein according to the second aspect of the present invention, comprising the following steps:

[0025] (1) Culturing the host cell according to the fifth aspect of the present invention under suitable culture conditions;

[0026] (2) Harvesting the host cell and / or the culture medium containing the recombinant type II humanized collagen or the fusion protein; and optionally

[0027] (3) Purifying the recombinant type II humanized collagen or the fusion protein.

[0028] The eighth aspect of the present invention provides a composition comprising one or more of the recombinant type II humanized collagen described in the first aspect of the present invention, the fusion protein described in the second aspect, the polynucleotide described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, and the trimeric collagen described in the sixth aspect.

[0029] In some embodiments, the composition is one or more of a biological dressing, a human biomimetic material, a plastic or cosmetic material, an organoid culture material, a cardiovascular stent material, 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 and a blood vessel repair and regeneration material, a 3D printing artificial organ biological material, a cosmetic raw material, a pharmaceutical excipient, and a food additive; preferably, the composition is a surface composition, an injectable composition, or an oral composition; preferably, the composition is in the form of a solution, a lyophilized powder, a gel, a sponge, or a fiber.

[0030] The ninth aspect of the present invention provides the use of the recombinant type II humanized collagen described in the first aspect of the present invention, the fusion protein described in the second aspect, the polynucleotide described in the third aspect, the vector described in the fourth aspect, and the host cell described in the fifth aspect in the preparation of one or more of a biological dressing, a human biomimetic material, a plastic or cosmetic material, an organoid culture material, a cardiovascular stent material, 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 and a blood vessel repair and regeneration material, a 3D printing artificial organ biological material, a cosmetic raw material, a pharmaceutical excipient, and a food additive.

[0031] The tenth aspect of the present invention provides a method for promoting cell adhesion, the method comprising contacting cells with the recombinant type II humanized collagen described in the first aspect of the present invention, the fusion protein described in the second aspect, the polynucleotide described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, the trimeric collagen described in the sixth aspect, and / or the composition described in the seventh aspect of the present invention.

[0032] In some embodiments, the promoting of cell adhesion is carried out in vitro.

[0033] The eleventh aspect of the present invention provides the use of recombinant type II humanized collagen in the preparation of a product for promoting cartilage repair, wherein the recombinant type II humanized collagen comprises n repeating units, the repeating units are directly or connected through a linker, and the repeating unit comprises the amino acid sequence shown in any one of SEQ ID NO: 1-3 or a variant thereof, and the variant is (1) an amino acid sequence obtained by substituting, deleting or inserting one or more amino acids on the basis of any one of SEQ ID NO: 1-3; 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% identity with any one of SEQ ID NO: 1-3.

[0034] In some embodiments, n is an integer from 1 to 20; in some embodiments, n is 1, 4, 8 or 12.

[0035] In some embodiments, the linker comprises one amino acid or multiple amino acid residues; preferably, the linker comprises 2, 3, 4, 5, 6, 7 or 8 amino acid residues.

[0036] In some embodiments, the collagen comprises the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 5.

[0037] In some embodiments, the collagen comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 5.

[0038] In some embodiments, the collagen comprises an amino acid sequence obtained by substituting, deleting or inserting one or more amino acids on the basis of SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 5.

[0039] The advantages of the present invention include:

[0040] 1. Provide the core functional region of the novel recombinant type II humanized collagen, and successfully construct the recombinant type II humanized collagen with good biological activity based on the novel core functional region.

[0041] 2. The amino acid composition of the recombinant type II humanized collagen of the present invention is 100% identical to the corresponding part of the amino acid sequence of natural human type II collagen, and it will not cause immune rejection and allergic reactions when applied to the human body.

[0042] 3. The recombinant type II humanized collagen of the present invention has excellent cell adhesion activity and can be used for cartilage repair.

[0043] 4. The preparation method of the present invention is simple and can mass-produce recombinant type II humanized collagen with high yield. Description of the Drawings

[0044] Figure 1 Electrophoresis result diagram showing recombinant type II humanized collagen II-1.

[0045] Figure 2 Electrophoresis result diagram showing recombinant type II humanized collagen II-2.

[0046] Figure 3 Electrophoresis result diagram showing recombinant type II humanized collagen II-3.

[0047] Figure 4 CD diagram showing recombinant type II humanized collagen II-1.

[0048] Figure 5 CD diagram showing recombinant type II humanized collagen II-2.

[0049] Figure 6 CD diagram showing recombinant type II humanized collagen II-3.

[0050] Figure 7 Cell adhesion diagram showing three kinds of recombinant type II humanized collagen.

[0051] Figure 8 Diagram showing the promotion of the proliferation of human chondrocytes C28 / I2 by three kinds of recombinant type II humanized collagen.

[0052] Figure 9 Microscopic imaging of cell migration of three kinds of recombinant type II humanized collagen.

[0053] Figure 10 Cell migration rate of three kinds of recombinant type II humanized collagen. Detailed Embodiments

[0054] To make the objectives, technical solutions and advantages of the present invention more clear, the following will, in combination with the embodiments of the present invention, clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the scope of protection of the present invention.

[0055] Term Definitions

[0056] As used herein, "recombinant collagen" is a new type of biomaterial that is screened and prepared using cutting-edge structural biology, genetic engineering and other technologies, with the gene encoding the functional region of human-specific type collagen as a template, and is identical or similar to the amino acid sequence of human collagen.

[0057] As used herein, "recombinant humanized type II collagen" refers to a recombinant protein consisting of or substantially consisting of sequences derived from human type II collagen. Herein, recombinant humanized type II collagen can consist of or be substantially composed of fragments derived from human type II collagen or multiple repeats of the fragments.

[0058] As used herein, the term "expression" includes any step involved in the production of a polypeptide, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0059] As used herein, the term "vector" is a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the protein encoded by the inserted polynucleotide to be expressed, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic element carried by it can be expressed in the host cell. Vectors are well known to those skilled in the art and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as λ phage or M13 phage and animal viruses, etc. The vector can contain various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain an origin of replication. The vector can contain the nucleic acid of the present invention to facilitate its introduction into cells for expression. The vector can contain expression control elements operably linked to the nucleic acid, such as promoters, terminators and / or enhancers.

[0060] As used herein, the term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to a control sequence providing for its expression. Herein, the expression vector is an Escherichia coli expression vector.

[0061] As used herein, the term "recombinant expression vector" 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 otherwise found in nature, or synthetic, and that contains one or more control sequences.

[0062] As used herein, the term "control sequence" means a nucleic acid sequence that is necessary for the expression of a polynucleotide encoding the mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational termination signals. For the purpose of introducing specific restriction sites that facilitate the ligation of the control sequences to the coding region of the polynucleotide encoding the polypeptide, these control sequences may be provided with multiple linkers.

[0063] As used herein, the term "host cell" is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. The host cell may be a eukaryotic cell or a prokaryotic cell. For example, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. The prokaryotic cell may be an Escherichia coli cell. Detailed Description

[0065] Recombinant humanized type II collagen

[0066] The present invention provides recombinant humanized type II collagen. The recombinant humanized type II collagen of the present invention may comprise one or more repeating units, and the repeating unit comprises an amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or a variant sequence thereof.

[0067] In some embodiments, the variant sequence comprises an amino acid sequence obtained by mutation (substitution, addition, or deletion) of one or more amino acid residues based on any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3.

[0068] In some embodiments, the variant sequence comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3. In some embodiments, the number of repeat units can be 1-20.

[0069] In some embodiments, the number of repeat units is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0070] In some embodiments, the number of repeat units is 1, 4, 8 or 12.

[0071] In some embodiments, the mutation can be a substitution, such as a conservative amino acid substitution.

[0072] In some embodiments, each repeat unit is directly linked.

[0073] In some embodiments, there is a spacer of one or more (such as 2, 3, 4, 5, 6, 7, 8) amino acid residues between each repeat unit.

[0074] In some embodiments, in the case where there are mutations in the repeat units, the obtained recombinant collagen retains the ability of cell adhesion, cell proliferation promotion activity or cartilage repair equivalent to or superior to that of the collagen obtained from the non-mutated repeat units.

[0075] In some embodiments, the recombinant type II humanized collagen of the present invention is synthetic or recombinantly expressed.

[0076] Recombinant type II humanized collagen-encoding nucleic acid

[0077] The present invention provides a polynucleotide encoding recombinant type II humanized collagen for expression.

[0078] In some embodiments, the polynucleotide is codon-optimized for the host cell for expression.

[0079] In some embodiments, the polynucleotide encoding recombinant type II humanized collagen can be operably linked to expression control elements such as a promoter, a terminator and / or an enhancer to form a nucleic acid, or an expression cassette.

[0080] In some embodiments, the polynucleotide encoding recombinant humanized type II collagen may further comprise a nucleotide encoding a purification tag, such as a His tag, GST tag, MBP tag, SUMO tag or NusA tag, or a nucleotide encoding a leader sequence, for facilitating the purification or secretion of the polypeptide.

[0081] Production method

[0082] The present invention also provides a method for producing recombinant humanized type II collagen, which comprises: (1) culturing the host cells herein under suitable culture conditions; (2) harvesting the host cells and / or the culture medium containing recombinant humanized type II collagen or a fusion protein or its fusion protein; and (3) purifying the recombinant humanized type II collagen or the fusion protein. The method of the present invention may include the step of cleaving the tag with an enzyme.

[0083] Composition, kit and its application

[0084] The recombinant humanized type II collagen of the present invention can be prepared into a composition or a kit.

[0085] In some embodiments, the composition or the kit may be a composition or a kit for tissue filling and / or volume augmentation.

[0086] In some embodiments, the composition or the kit may further comprise an auxiliary substance.

[0087] In some embodiments, the composition of the present invention may be a cartilage repair agent, which comprises the polypeptide described herein. The composition of the present invention may be injectable. The composition of the present invention may be a human structural material, for example, it can be used for cartilage repair and does not cause an immune response in the human body.

[0088] Construction and production method of recombinant humanized type II collagen

[0089] The present invention also provides a method for constructing recombinant humanized type II collagen, which includes functional region screening and strain construction, large-scale bioreactor fermentation, protein induction expression, purification and optionally an enzyme cleavage step.

[0090] In some embodiments, the functional region screening and strain construction step includes: (1) large-scale functional region screening to obtain the functional region of the target gene; (2) inserting the obtained functional region of the target gene into an expression vector (such as PET-28a-Trx-His) to obtain a recombinant expression plasmid; (3) transferring the recombinant expression plasmid into Escherichia coli competent cells (such as BL21(DE3)), and screening to obtain a positive Escherichia coli genetic engineering strain.

[0091] In some embodiments, the large-scale bioreactor step includes: adding the screened positive recombinant Escherichia coli into a shake flask containing an antibiotic stock solution, and culturing it in a constant temperature shaker at 220 rpm and 37°C.

[0092] In some embodiments, the steps for induced expression of the protein include: (1) cooling the cultured shake flask to 16 - 30°C; (2) adding IPTG stock solution for induced expression; (3) transferring the induced bacterial solution into a centrifuge bottle, centrifuging at 6000 rpm and 4°C for 12 min, and collecting the bacterial cells.

[0093] In some embodiments, the steps for purification and optional enzymatic cleavage of humanized type II collagen include: (1) roughly purifying humanized type II collagen using a Ni affinity chromatography column; (2) adding TEV protease for enzymatic cleavage at a certain ratio; (3) highly purifying humanized type II collagen using an ion exchange column.

[0094] Examples

[0095] The following examples are provided to illustrate the present invention. Those skilled in the art should understand that the examples are merely illustrative and not restrictive. The present invention is solely defined by the scope of the appended claims.

[0096] Example 1: Construction and Expression of Recombinant Humanized Type II Collagen

[0097] 1.1 Construction of Recombinant Humanized Type II Collagen

[0098] Large-scale functional region screening of natural human type II collagen (GenBank: EAW57970.1) yielded the following different protein functional regions.

[0099] GEQGPKGEPGPAGPQGAPGPAGEE (SEQ ID NO.1)

[0100] GSPGPAGPTGKQGDRGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGLQGLPGPPGPSGDQGASGPAGPSGPR (SEQ ID NO.2)

[0101] GFQGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLP (SEQ ID NO.3)

[0102] To ensure the purification and stability of recombinant type II humanized collagen, the amino acid fragments in these regions were optimized by repeating them n times and directly linking them, resulting in recombinant collagen II-1, collagen II-2, and collagen II-3. The corresponding amino acid sequences are shown in SEQ ID NO:4, SEQ ID NO:2, and SEQ ID NO:6, respectively.

[0103] Amino acid sequence of collagen II-1 (12 repeats):

[0104] GEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEEGEQGPKGEPGPAGPQGAPGPAGEE(SEQ IDNO.4)

[0105] Amino acid sequence of collagen II-2 (1 repeat):

[0106] GSPGPAGPTGKQGDRGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGLQGLPGPPGPSGDQGASGPAGPSGPR(SEQ ID NO.2)

[0107] Amino acid sequence of collagen II-3 (4 repeats):

[0108] GFQGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLPGFQGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLPGFQGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLPGFQGLPGPPGPPGEGGKPGDQGVPGEAGAPGLVGPRGERGFPGERGSPGAQGLQGPRGLP(SEQ ID NO.5)

[0109] Codon optimization was performed according to the Escherichia coli expression system to obtain the coding nucleic acid sequences of collagen II-1, collagen II-2, and collagen II-3, as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

[0110] Collagen II-1 nucleic acid sequence:

[0111] GGAGAACAAGGGCCGAAAGGCGAACCGGGTCCGGCGGGTCCGCAGGGCGCACCGGGTCCGGCAGGCGAGGAAGGTGAGCAAGGTCCGAAAGGTGAGCCGGGTCCAGCTGGCCCTCAAGGCGCTCCGGGCCCGGCGGGCGAAGAAGGTGAGCAAGGCCCGAAGGGTGAGCCGGGTCCGGCCGGTCCGCAAGGCGCGCCAGGGCCGGCGGGTGAGGAAGGTGAGCAGGGTCCGAAAGGTGAGCCGGGTCCGGCGGGCCCGCAAGGTGCACCGGGCCCAGCTGGCGAAGAGGGCGAGCAGGGTCCGAAAGGCGAGCCGGGTCCGGCGGGCCCGCAGGGTGCGCCAGGCCCGGCGGGTGAAGAAGGCGAACAAGGCCCGAAAGGCGAACCGGGTCCGGCGGGTCCTCAAGGTGCCCCAGGCCCAGCGGGCGAAGAGGGCGAACAGGGTCCGAAGGGCGAGCCGGGGCCGGCGGGTCCGCAGGGCGCTCCGGGTCCGGCTGGCGAAGAAGGTGAACAGGGTCCGAAGGGCGAGCCGGGCCCGGCGGGTCCGCAAGGAGCACCGGGTCCGGCGGGCGAGGAAGGCGAACAGGGTCCCAAGGGTGAGCCGGGCCCGGCGGGTCCGCAAGGTGCCCCTGGCCCGGCGGGTGAAGAAGGTGAGCAGGGTCCCAAGGGCGAGCCGGGTCCGGCGGGTCCGCAGGGCGCACCGGGTCCGGCCGGCGAGGAGGGTGAACAAGGCCCAAAAGGCGAGCCGGGTCCGGCCGGCCCGCAGGGTGCTCCGGGTCCGGCTGGCGAAGAGGGCGAGCAGGGTCCGAAGGGTGAGCCGGGTCCGGCGGGCCCACAGGGCGCACCGGGTCCGGCCGGTGAGGAA(SEQ ID NO:6) Collagen II-2 nucleic acid sequence:

[0112] GGTAGCCCAGGTCCAGCAGGTCCGACAGGTAAACAAGGAGATCGTGGTGAAGCAGGAGCACAAGGACCAATGGGTCCAAGCGGTCCGGCAGGTGCAAGAGGTATTCAAGGGCCGCAAGGGCCACGCGGTGATAAAGGGGAAGCAGGTGAACCAGGTGAGAGAGGGTTAAAAGGACATCGTGGATTTACAGGACTGCAGGGTTTACCAGGTCCGCCGGGACCGAGCGGAGATCAAGGTGCAAGCGGTCCGGCGGGTCCGAGTGGTCCTCGT(SEQ ID NO:7)

[0113] Collagen II-3 nucleic acid sequence:

[0114] GGATTTCAGGGGTTGCCGGGCCCACCGGGTCCACCGGGCGAAGGTGGCAAGCCGGGCGACCAGGGCGTGCCGGGTGAGGCCGGTGCGCCAGGCCTGGTAGGTCCACGTGGTGAGCGCGGTTTCCCGGGTGAGCGCGGTTCTCCGGGTGCTCAAGGCCTGCAGGGTCCGCGTGGCCTGCCGGGCTTTCAGGGTTTGCCGGGCCCGCCGGGCCCGCCCGGCGAGGGTGGTAAGCCGGGCGACCAGGGCGTTCCGGGTGAAGCGGGTGCGCCAGGCCTGGTGGGCCCACGTGGTGAACGTGGTTTTCCGGGCGAACGCGGTAGCCCGGGCGCGCAGGGTCTGCAAGGACCGCGTGGCCTGCCGGGATTCCAGGGTCTCCCGGGTCCGCCTGGACCGCCGGGCGAAGGTGGCAAACCGGGTGATCAGGGTGTTCCGGGTGAAGCAGGCGCTCCGGGCCTGGTTGGTCCGCGTGGCGAACGTGGTTTTCCGGGCGAGCGCGGCTCCCCGGGTGCCCAAGGTTTACAAGGCCCGCGTGGTCTTCCGGGCTTCCAAGGCCTGCCGGGGCCGCCGGGCCCGCCTGGCGAGGGTGGTAAACCGGGCGATCAGGGTGTGCCGGGCGAGGCAGGTGCGCCGGGGTTGGTCGGTCCTAGAGGTGAACGTGGTTTCCCGGGTGAGCGCGGTAGCCCGGGTGCGCAAGGCCTGCAGGGCCCGCGTGGCCTGCCG(SEQ ID NO: 8)

[0115] 1.2 Expression of Recombinant Humanized Collagen Type II

[0116] The synthesized gene functional region was inserted into the pET-28a-Trx-His expression vector to obtain the corresponding recombinant expression plasmid.

[0117] The successfully constructed expression plasmid was transformed into Escherichia coli competent cell BL21(DE3). The specific process was as follows: (1) Take out Escherichia coli competent cell BL21(DE3) from the ultra-low temperature refrigerator and place it on ice. When it is half-thawed, add 2 μl of the plasmid to be transformed into Escherichia coli competent cell BL21(DE3), and mix gently 2-3 times. (2) Place the mixture on ice for 30 min, then heat shock it in a 42 °C water bath for 45-90 s, take it out and place it on ice for 2 min. (3) Transfer it to the biosafety cabinet, add 700 μl of liquid LB medium, and then culture it at 37 °C and 220 rpm for 60 min. (4) Take 200 μl of the bacterial solution and spread it evenly on the LB plate containing kanamycin sulfate. (5) Culture the plate in an incubator at 37 °C for 15-17 h until colonies of uniform size grow out.

[0118] Pick 5-6 single colonies from the transformed LB plate and transfer them to a shake flask containing an antibiotic stock solution (ampicillin 100 mg / L), culture them in a constant temperature shaker at 220 rpm and 37 °C for a certain period of time until it becomes foggy. Then cool the shake flask after culture to 16-30 °C, add IPTG (0.5 mM) to induce expression for a period of time, aliquot the bacterial solution into centrifuge bottles, centrifuge at 6000 rpm and 4 °C for 12 min, collect the bacterial cells, record the weight of the bacterial cells, and take samples for electrophoresis detection.

[0119] Resuspend the collected bacterial cells with the equilibration working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole), cool the bacterial solution to ≤15 °C, homogenize it twice or perform ultrasonic cell disruption, and collect the bacterial solution after completion. Aliquot the bacterial solution after cell disruption into centrifuge bottles, centrifuge at 17000 rpm and 4 °C for 30 min, and collect the supernatant.

[0120] Purify and digest recombinant humanized type II collagen. The specific process is as follows: (1) Crude purification: a. Equilibrate the column material: Use the equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) to equilibrate the column material at a flow rate of 10 mL / min. b. Load the sample: Add the centrifuged supernatant to the column material until the liquid has finished flowing, with a flow rate of 5 mL / min. c. Wash away impurities: Add 100 mL of the impurity washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) until the liquid has finished flowing, with a flow rate of 10 mL / min. d. Collect the target protein: Add 20 mL of the elution solution (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole) at a flow rate of 10 mL / min, and collect the flow-through solution. Detect the protein concentration using ultraviolet-visible spectrophotometry, and calculate the protein concentration according to the following formula (C (mg / ml) = A280 × dilution factor × extinction coefficient), and perform electrophoresis detection). e. Wash the column material with 1 M imidazole working solution at a flow rate of 10 mL / min. (2) Digestion: Add TEV enzyme according to the ratio of the total protein amount to the total amount of TEV enzyme of 20:1, and digest at 16 °C for 2 h. Put the digested protein solution into a dialysis bag, dialyze at 4 °C for 2 h, and then transfer it to a new dialysis solution (20 mM sodium chloride, 20 mM Tris) for overnight dialysis at 4 °C. (3) Fine purification: a. Equilibrate the column material: Use solution A (20 mM Tris, 20 mM sodium chloride) to equilibrate the column material at a flow rate of 10 ml / min. b. Load the sample: The flow rate is 5 ml / min, load the sample and collect the flow-through sample, and perform electrophoresis detection. Store the protein in an environment at 4 °C. c. Elute: Wash the column material with solution B (1 M sodium chloride, 20 mM Tris) for 5 column volumes. d. Wash the column material.

[0121] The purified recombinant protein was detected by SDS-PAGE, and the electrophoresis detection results are shown in Figures 1-3 . As Figures 1-3 can be seen, Collagen II-1, Collagen II-2, and Collagen II-3 were all successfully expressed and prepared; at the same time, control collagen B1 (also from type II collagen) was prepared, and the amino acid and nucleic acid sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 10 respectively:

[0122] GEPGREGSPGADGPPGRDGAAGVKGDRGETGAVGAPGAPGPPGSPGPAGPTGKQGDRGEAGAQGPMGPSGPAGARGIQGPQGPRGDKGEAGEPGERGLKGHRGFTGLQGLPGPPGPSGDQGASGPAGPSGPRGPPGPVGPSGKDGANGIPGPIGPPGPRGRSGETGPA (SEQ ID NO: 9)

[0123] GGTGAACCAGGTCGTGAAGGTAGCCCAGGTGCAGATGGACCACCAGGTCGTGATGGTGCAGCAGGAGTGAAAGGAGATCGTGGTGAAACCGGTGCAGTAGGTGCACCTGGTGCGCCAGGTCCGCCAGGTAGCCCTGGTCCAGCCGGTCCTACCGGAAAACAAGGGGATAGAGGAGAAGCAGGAGCACAGGGTCCGATGGGTCCGTCAGGTCCGGCGGGTGCACGTGGTATTCAGGGTCCGCAGGGTCCGCGTGGTGATAAAGGTGAAGCAGGTGAACCGGGGGAAAGAGGATTAAAAGGGCATCGTGGTTTTACGGGTCTGCAGGGTCTGCCTGGTCCGCCTGGTCCGAGCGGTGATCAGGGTGCAAGCGGTCCGGCAGGTCCGAGCGGACCTCGTGGACCTCCGGGTCCTGTGGGTCCTAGTGGTAAGGATGGGGCAAATGGTATTCCTGGTCCTATTGGTCCGCCGGGTCCGCGTGGGAGATCAGGTGAAACCGGACCGGCA(SEQ ID NO:10)

[0124] After purification, the protein yield of Collagen II-1 was 0.59%. The amount of the target protein was small after fine purification. The theoretical molecular weight after fine purification was 25.9 KD, and the apparent molecular weight after purification was consistent with the theoretical molecular weight ( Figure 1 ); the protein yield of Collagen II-2 was 0.55%. The theoretical molecular weight after fine purification was 8.3 KD, and the apparent molecular weight after purification was consistent with the theoretical molecular weight ( Figure 2 ); Collagen II-3 was completely digested. The purity was high and the expression level was high after fine purification. The purification yield was 0.65%. The theoretical molecular weight after fine purification was 22.4 KD, and the apparent molecular weight after purification was consistent with the theoretical molecular weight ( Figure 3 ). Collagen II-1, Collagen II-2, and Collagen II-3 were selected as the target proteins for subsequent detection.

[0125] Example 2: Circular Dichroism Ultraviolet Scanning Analysis of Recombinant Humanized Type II Collagen

[0126] Experimental Method

[0127] (1) Instrument Parameter Setting

[0128]

[0129] (2) Far and near ultraviolet scanning of the standard product

[0130] Set the scanning wavelength to 180 - 340 nm for background testing and blank buffer testing, and then collect the circular dichroism far and near ultraviolet absorption of the 1 mg / mL CSA standard product solution in the range of 180 - 340 nm.

[0131] Sample treatment

[0132] Take the protein sample and concentrate the protein to a concentration of 1 mg / ml using a 10KD ultrafiltration concentrator tube (Millipore).

[0133] (4) Far ultraviolet scanning of the sample

[0134] Soak the cuvette in 2M HNO3 overnight, rinse it thoroughly with deionized water and dry it. First, collect the background, then the blank buffer, and then add an appropriate amount of the test sample to the cuvette and perform far ultraviolet scanning at 190 - 260 nm according to the above parameters and collect the data.

[0135] (5) Near ultraviolet scanning of the sample

[0136] Soak the cuvette in 2M HNO3 overnight, rinse it thoroughly with deionized water and dry it. First, collect the background, then the blank buffer, and then add an appropriate amount of the test sample to the cuvette and perform near ultraviolet scanning at 250 - 340 nm according to the above parameters and collect the data.

[0137] (6) Processing of the scanned spectra

[0138] Perform subtract baseline and smoothing processing on all the scanned spectra using the software Pro-Data Viewer.

[0139] The results of circular dichroism ultraviolet scanning analysis of recombinant collagen II-1, II-2, and II-3 are respectively as Figure 4 , Figure 5 and Figure 6 shown. The expression of recombinant humanized type II collagen has positive peaks at 220 - 230 nm, indicating that collagen II-1, II-2, and II-3 all have a triple helix structure.

[0140] Example 3: Biological activity of recombinant humanized type II collagen

[0141] The method for detecting the activity of collagen can refer to the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004). The specific implementation method is as follows:

[0142] (1) Use the ultraviolet absorption method to detect the concentration of the protein sample to be tested, including bovine type I collagen (National Institutes for Food and Drug Control, No.: 380002), collagen II-1 provided by the present invention, collagen II-2, collagen II-3 and control collagen B1. Measure the ultraviolet absorption of the sample at 215 nm and 225 nm respectively, and calculate the protein concentration using the empirical formula C (μg / mL) = 144×(A215 - A225), where C in the formula represents the protein concentration in the sample to be tested, A215 represents the absorbance of the sample to be tested at a wavelength of 215 nm, and A225 represents the absorbance of the sample to be tested at a wavelength of 225 nm. Note that the detection needs to be carried out when A215 < 1.5. The principle of this method is: measure the characteristic absorption of peptide bonds under far ultraviolet light, which is not affected by the content of chromophores, has few interfering substances, and is easy to operate. It is suitable for detecting human collagen and its analogs that do not show color with Coomassie Brilliant Blue. (The reference is Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45.). After detecting the protein concentration, adjust the concentration of all proteins to be tested to 0.5 mg / mL with PBS.

[0143] (2) Add 100 μL of various protein solutions and blank PBS solution control (NC) to a 96-well plate, and let it stand at room temperature for 60 min.

[0144] (3) Add 10 5 well-grown 3T3 / NIH cells to each well, and incubate at 37 °C for 60 min.

[0145] (4) Wash each well 4 times with PBS.

[0146] (5) The absorbance at OD450nm was detected using a CCK8 detection kit (Beyotime, product catalog number C0038). According to the value of the blank control, the cell adhesion rate could be calculated. The calculation formula was as follows: Cell adhesion rate = {(test well - blank well) / (positive well - blank well)} × 100%. The cell adhesion rate could reflect the activity of collagen. The higher the protein activity, the better the external environment it could provide for cells in a short time to help cells adhere.

[0147] The results were as Figure 7 shown. It could be seen from the comparison that compared with the positive control (PC) bovine type I collagen (B ColI group, 0.5mg / ml) and control collagen B1, the humanized collagens II-1, II-2, and II-3 of the present invention had more excellent bioadhesive activity.

[0148] Example 4: Cartilage repair effect of recombinant humanized type II collagen

[0149] 4.1. Cell proliferation experiment

[0150] The method for detecting the activity of collagen to promote cell increase can refer to the pharmaceutical industry standard YYT1849-2022 recombinant collagen of the People's Republic of China. The specific implementation method was as follows:

[0151] (1) The principle of this method was that the CCK8 reagent contained WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazolium monosodium salt], which was reduced by dehydrogenases in cells to a highly water-soluble yellow formazan product (Formazan dye) under the action of the electron carrier 1-methoxy-5-methylphenazinium sulfate dimethyl ester (1-Methoxy PMS). The amount of formazan produced was proportional to the number of live cells. Therefore, this property could be used for direct cell proliferation analysis.

[0152] (2) Experimental grouping: Complete medium DMEM was used as the negative control, control collagen Native Human Collagen II protein (Abcam, #ab134522) was used as the positive control, collagen II-1, II-2, II-3 solutions were used as test articles, and complete culture medium without cells was used as the zero-adjustment well.

[0153] (3) Specimen preparation: Collagen was dissolved in complete medium to the highest concentration for use, filtered and sterilized with a 0.22μm microporous filter membrane, and various concentrations were prepared.

[0154] (4) Cell seeding: When the confluence of human chondrocytes C28 / I2 cells in a T75 culture flask reaches about 90%, they are digested and counted with trypsin. 1×10 5 cells are seeded into each well of a 96-well plate. The edge wells are filled with 100 μL of PBS for sealing, and the cells are cultured for 24 h to adhere to the wall.

[0155] (5) Cell drug administration: After the cells adhere to the wall, the supernatant is aspirated and discarded, and the corresponding group solutions are replaced in turn. There are 4 replicate wells in each group, and the cells are cultured for 48 h.

[0156] (6) Experimental detection: The culture medium is discarded, 100 μL (including 5 μL of CCK8 solution) of basal medium is added, and the cells are incubated in an incubator for 1 - 2 h. Detection is carried out using an enzyme-linked immunosorbent detector at a wavelength of 450 nm. The calculation formula for cell viability is as follows.

[0157] Cell viability = {(As - Ab) / (Ac - Ab)} × 100%.

[0158] Where As is the absorbance of the recombinant collagen test well, Ab is the absorbance of the zero-adjustment well, and Ac is the absorbance of the negative control well.

[0159] Collagen II-1, Collagen II-2, Collagen II-3, control collagen, and bovine serum albumin BSA (Beyotime product number: ST023, irrelevant protein control) were detected using the above method. Figure 8 It was shown that compared with type II collagen in the control group, Collagen II-1, Collagen II-2, and Collagen II-3 showed significant proliferative effects on human chondrocytes C28 / I2.

[0160] 4.2 Cell migration experiment

[0161] (1) Human chondrocytes C28 / I2 were cultured in a 48-well plate. After the cells grew confluently, a uniform scratch wound was created on the monolayer cells using a 10 μL pipette tip. The cells were washed 1 - 2 times with sterile PBS to ensure that there was no cell debris or residual suspended cells, which would affect subsequent experiments.

[0162] (2) 1 mg / mL of Collagen II-1, Collagen II-2, Collagen II-3, and control collagen (Abcam, #ab134522) were added. The cells in the non-drug-added medium group were used as the blank control. The closure of the scratch was observed at 0, 6, and 12 hours.

[0163] (3) Calculate the scratch area. The percentage of scratch closure rate was obtained through the following formula:

[0164] Scratch closure rate (%) = (A0 - At) / A0 × 100%, where A0 is the scratch area at 0 h and At is the scratch area at the specified time.

[0165] The migration ability of chondrocytes was verified by cell migration experiments. The chondrocytes of collagen II-1 and collagen II-2 had a strong ability to promote proliferation and both reached closure after 12 h, while the proliferation activity of chondrocytes of collagen II-3 was slightly inferior. By Figures 9-10 It can be seen that under the action of the three types of collagen II, the scratch closure rate increased with the treatment duration. When the treatment time reached 12 h, the three types of type II collagen had a significantly higher scratch closure rate than the control collagen. In summary, collagen II-1, collagen II-2, and collagen II-3 of the present invention all have good repair and healing abilities; within a 12-hour period, they showed a significant promoting effect on the migration of chondrocytes, and this difference was very obvious compared with the control collagen. The three types of collagen II of the present invention have very significant potential application value and prospects in promoting chondrocyte regeneration, improving cell migration ability, and treating diseases such as osteoarthritis.

Claims

1. Recombinant type II humanized collagen, characterized in that, Comprising n repeating units, said repeating units being linked directly or via a linker, said repeating units comprising the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or a variant thereof, said variant being (1) an amino acid sequence obtained by substitution, deletion or insertion of one or more amino acids on the basis of SEQ ID NO:1 or SEQ ID NO:3; 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% identity with SEQ ID NO:1 or SEQ ID NO:3; preferably, said n is an integer from 1 to 20; preferably, said n is 1, 4, 8 or 12; Preferably, said collagen comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5 or an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:4 or SEQ ID NO:5 or an amino acid sequence obtained by substitution, deletion or insertion of one or more amino acids on the basis of SEQ ID NO:4 or SEQ ID NO:

5.

2. A fusion protein, which comprises the recombinant type II humanized collagen according to claim 1 and a purification tag and / or a leader, preferably, said purification tag is selected from His tag, GST tag, MBP tag, SUMO tag or NusA tag.

3. A polynucleotide, which encodes the collagen according to claim 1 or the fusion protein according to claim 2; preferably, said polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:6 or SEQ ID NO:8 or a degenerate sequence thereof.

4. A vector, which comprises the polynucleotide according to claim 3; preferably, said vector is an expression vector; preferably, said vector comprises control elements operably linked to the polynucleotide; preferably, said control elements are selected from promoters, terminators and / or enhancers.

5. A host cell, which comprises the polynucleotide according to claim 3, or the vector according to claim 4; preferably, said host cell is a bacterium, a fungus or an animal cell; preferably, said bacterium includes Escherichia coli; preferably, said fungus includes yeast, such as Saccharomyces cerevisiae.

6. A trimeric collagen, which comprises the recombinant type II humanized collagen of claim 1.

7. A method for producing the recombinant type II humanized collagen according to claim 1 or the fusion protein according to claim 2, characterized in that, Comprising the following steps: (1) Culturing the host cell according to claim 5 under suitable culture conditions; (2) Harvesting the host cell and / or the culture medium containing the recombinant type II humanized collagen or the fusion protein; and optionally (3) Purifying the recombinant type II humanized collagen or the fusion protein.

8. A composition, characterized in that, Comprising one or more of the recombinant type II humanized collagen according to claim 1, the fusion protein according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, the host cell according to claim 5, and the trimeric collagen according to claim 6.

9. The composition according to claim 8, wherein the composition is one or more of a biological dressing, a human biomimetic material, a plastic or cosmetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological and obstetric biomaterial, a nerve repair and regeneration material, a liver tissue material and a blood vessel repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient, and a food additive; preferably, the composition is a surface composition, an injectable composition, or an oral composition; preferably, the composition is in the form of a solution, a freeze-dried powder, a gel, a sponge, or a fiber.

10. Use of the recombinant type II humanized collagen according to claim 1, the fusion protein according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, the host cell according to claim 5, and / or the trimeric collagen according to claim 6 in the preparation of one or more of a biological dressing, a human biomimetic material, a plastic or cosmetic material, an organoid culture material, a cardiovascular stent material, a coating material, a tissue injection filling material, an ophthalmic material, a gynecological and obstetric biomaterial, a nerve repair and regeneration material, a liver tissue material and a blood vessel repair and regeneration material, a 3D printed artificial organ biomaterial, a cosmetic raw material, a pharmaceutical excipient, and a food additive.

11. A method for promoting cell adhesion, characterized in that, The method comprises contacting the cell with the polypeptide according to claim 1, the fusion protein according to claim 2, the host cell according to claim 5, the trimeric collagen according to claim 6, and / or the composition according to claim 8 or 9; preferably, the promoting of cell adhesion is carried out in vitro.

12. Use of recombinant type II humanized collagen in the preparation of a product for promoting cartilage repair, wherein, The recombinant type II humanized collagen comprises n repeating units, which are directly or connected through a linker, and the repeating unit comprises the amino acid sequence shown in any one of SEQ ID NO: 1-3 or a variant thereof, and the variant is (1) an amino acid sequence obtained by substituting, deleting, or inserting one or more amino acids on the basis of any one of SEQ ID NO: 1-3; 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% identity with any one of SEQ ID NO: 1-3; Preferably, the n is an integer from 1 to 20; Preferably, the n is 1, 4, 8, or 12; Preferably, the collagen comprises the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 5, or an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 5, or an amino acid sequence obtained by substitution, deletion or insertion of one or more amino acids based on SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 5.

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