Polypeptide with triple helix structure, recombinant XII type humanized collagen and application of recombinant XII type humanized collagen

Abstract

The invention relates to the technical field of synthetic biology, in particular to polypeptide with a triple helix structure, recombinant XII type humanized collagen and application of the recombinant XII type humanized collagen. The recombinant XII type humanized collagen is successfully expressed and prepared, has a triple-helix structure and good biological activity including promotion of cell proliferation activity, cell adhesion activity and inhibition of MMP-1, is used as a biological material derived from a human body, does not generate immunological rejection and anaphylactic reaction when applied to the human body, and has a good application prospect. The composition can be used for medical or non-medical application of a plurality of tissues and organs of a human body, filling, compatibilizing or repairing, and promotion of skin compactness or wrinkle resistance and other scenes.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, and in particular to polypeptides with triple helix structures, recombinant type XII humanized collagen, and their applications. Background Technology

[0002] Collagen is a type of protein widely distributed in human connective tissues and is the most abundant protein in the human body, accounting for 25% to 35% of total protein. At least 28 collagen subtypes have been discovered in the human body, each located in different tissues and organs.

[0003] However, natural collagen is present in low concentrations in organisms, its extraction process is complex, and animal-derived immune responses are a significant limitation in its applications. With the development of genetic engineering technology, recombinant collagen can be constructed by selecting short amino acid sequences from natural human collagen, aiming to reduce immunogenicity and improve bioactivity and stability. As the collagen industry grows, technologies for obtaining collagen, especially humanized collagen, through biosynthetic pathways are becoming increasingly mature. According to the latest nomenclature and classification, recombinant humanized collagen refers to the full-length or partial amino acid sequence functional region encoded by a specific type of human collagen gene prepared using DNA recombination technology, or a combination of functional regions containing human collagen functions.

[0004] Type XII collagen is a fibrous collagen mainly distributed in the dermis, tendons, ligaments, and other connective tissues, playing several important roles in the human body. First, Type XII collagen exists in the dermis of the skin, interacting with other types of collagen (such as Type I collagen) to form the skin's support network, maintaining its resilience and elasticity, allowing it to withstand stretching and pressure without easily becoming damaged or wrinkled. Second, it participates in tissue repair and regeneration. When the skin or other tissues are damaged, Type XII collagen guides cell migration, proliferation, and differentiation, promoting new tissue growth and repair. For example, during wound healing, it helps fibroblasts and other cells migrate to the wound site, synthesizing new extracellular matrix and accelerating wound healing. Furthermore, Type XII collagen stabilizes the musculoskeletal system. At the junctions of muscles and bones, such as tendons and ligaments, Type XII collagen helps maintain the structural integrity and mechanical properties of these tissues. It also works in conjunction with other collagens and proteins, enabling tendons and ligaments to withstand the forces generated by muscle contraction and skeletal movement, reducing the risk of injury. Furthermore, in articular cartilage, type XII collagen also participates in maintaining cartilage structure and function, playing a role in normal joint movement and cushioning. In addition, type XII collagen can interact with cell surface receptors, transmitting signals into the cell to regulate cell behavior, such as cell proliferation, differentiation, and apoptosis. Type XII collagen plays a regulatory role in cell growth and development, influencing the normal formation and maintenance of tissues.

[0005] In the existing technology, there is little research on recombinant type XII collagen. Furthermore, due to problems such as unmodified post-translational structure, failure to form a higher-order triple helix conformation, low biological activity, and poor stability, the application of recombinant collagen in cell adhesion, proliferation, and other fields is limited.

[0006] Therefore, designing a type XII collagen truncation sequence with good cell adhesion and cell proliferation activity and a typical triple helix structure will help overcome the shortcomings of existing technologies and meet the needs of related fields. Summary of the Invention

[0007] This invention provides peptides, recombinant type XII humanized collagen, and their applications.

[0008] This invention utilizes synthetic biology and structural biology techniques to develop recombinant type XII humanized collagen that can form a triple helix structure of collagen and perform the functions of human collagen.

[0009] Specifically, the present invention provides the following technical solutions.

[0010] In a first aspect, the present invention provides a polypeptide whose amino acid sequence comprises n repeating units; the repeating units comprise any one of the following (1)-(3) amino acid sequences or variant sequences thereof: (1) The amino acid sequence shown in any one of SEQ ID NO. 1-3; (2) An amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO. 1-3; (3) A variant sequence obtained by mutating one or more amino acid residues of the amino acid sequence shown in any one of SEQ ID NO. 1-3; Where n ≥ 1 and is an integer.

[0011] In some embodiments of the present invention, mutation is substitution, insertion, deletion, or addition.

[0012] In some embodiments of the present invention, the substitution is a conserved amino acid substitution.

[0013] In some embodiments of the present invention, the polypeptide is derived from human.

[0014] In some embodiments of the present invention, the polypeptide may form a triple helix structure.

[0015] In some embodiments of the present invention, the polypeptide has a flexible triple helix structure.

[0016] In some embodiments of the present invention, the polypeptide exists in the form of a trimer.

[0017] In some embodiments of the present invention, the triple helix structure of the polypeptide has one or more of the following activities or effects: cell adhesion activity, cell migration-promoting activity, and cell proliferation-promoting activity.

[0018] In some embodiments of the present invention, the variant sequences are as shown in any one of SEQ ID NO.10-11, 20-22.

[0019] Preferably, n is an integer between 1 and 20, more preferably an integer between 1 and 12; even more preferably 1, 5, 6, 7, 8 or 12.

[0020] In the polypeptide of the present invention, when n≥2, the repeating units can be directly linked or linked by a linker peptide composed of one or more amino acid residues.

[0021] Preferably, when n≥2, the repeating units are directly connected.

[0022] Preferably, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NO.4-6, 12-13, 16-17, 20-22.

[0023] In some embodiments of the present invention, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.4, 5, 6, 12, 13, 16, 17, 20, 21 or 22.

[0024] The aforementioned polypeptides can serve as monomers for recombinant type XII humanized collagen. This invention has revealed that these polypeptides not only form recombinant type XII humanized collagen with a triple helix structure, but the resulting recombinant type XII humanized collagen also exhibits excellent performance in cell adhesion, promoting cell proliferation, and improving the extracellular matrix.

[0025] Secondly, the present invention provides a fusion protein comprising the polypeptides described above.

[0026] Preferably, the fusion protein is obtained by fusing the polypeptide with other functional polypeptides. These other functional polypeptides include, but are not limited to, other collagen peptides; preferably, the other collagen peptides are derived from truncated sequences of human collagen.

[0027] Thirdly, the present invention provides recombinant type XII humanized collagen, wherein the recombinant type XII humanized collagen comprises the polypeptides described above.

[0028] The polypeptide described in the first aspect above can form a trimer, which in turn forms a recombinant type XII humanized collagen with a triple helix structure.

[0029] Preferably, the recombinant type XII humanized collagen has cell adhesion and / or cell proliferation promotion and / or extracellular matrix improvement activities.

[0030] Fourthly, the present invention provides a polynucleotide encoding the aforementioned polypeptide or the fusion protein or the recombinant type XII humanized collagen.

[0031] Preferably, the polynucleotide comprises the nucleotide sequence or degenerate sequence shown in any one of SEQ ID NO. 7-9, 14-15, 18-19, 23-25.

[0032] The degenerate sequence refers to a nucleotide sequence obtained based on the degeneracy of the codon that is different from the nucleotide sequences shown in SEQ ID NO.7-9, 14-15, 18-19, 23-25, but can still encode the polypeptide or the fusion protein described above.

[0033] The polynucleotides described above may also include sequences encoding purification tags and / or leader sequences.

[0034] Preferably, the purification tag is selected from His tag, GST tag, MBP tag, SUMO tag or NusA tag.

[0035] The leader sequence includes, but is not limited to, sequences such as signal peptides.

[0036] Fifthly, the present invention provides biological materials comprising the polynucleotides described above; said biological material is an expression cassette, a vector, or a host cell.

[0037] The expression cassette may be a recombinant DNA molecule obtained by operably linking a control element upstream or downstream of the polynucleotide.

[0038] The vectors include plasmid vectors, viral vectors, transposons, artificial chromosomes, etc. The vectors can be expression vectors or integration vectors.

[0039] Preferably, the vector is an expression vector. The vector preferably further comprises a control element operatively linked to the polynucleotide. The control element is preferably a promoter, terminator, and / or enhancer.

[0040] Preferably, the host cell is a bacterium, fungus, or animal cell; wherein the bacteria preferably include Escherichia coli; the fungus preferably includes yeast, more preferably Saccharomyces cerevisiae or Pichia pastoris.

[0041] In a sixth aspect, the present invention provides a method for producing the above-described polypeptide, the fusion protein, or the recombinant type XII humanized collagen, the method comprising: culturing host cells containing the polynucleotide, harvesting host cells and / or culture medium containing the polypeptide, the fusion protein, or the recombinant type XII humanized collagen, and isolating the polypeptide, the fusion protein, or the recombinant type XII humanized collagen therefrom.

[0042] In some embodiments of the present invention, the host cell is *Escherichia coli*. The host cell containing the polynucleotide is *E. coli* containing an expression vector carrying the polynucleotide. The expression vector may be a pET-28a series vector.

[0043] The above method may further include an enzyme cleavage tag step after isolating the polypeptide, the fusion protein, or the recombinant type XII humanized collagen.

[0044] In a seventh aspect, the present invention provides a composition comprising one or more selected from the polypeptide, the fusion protein, the recombinant type XII humanized collagen, the polynucleotide, and the biomaterial.

[0045] Preferably, the composition is selected from biological dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection filling / volume-enhancing materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, chronic wound repair materials, bone or cartilage regeneration materials, liver tissue materials, vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, cosmetics, pharmaceutical excipients, drugs, or food additives.

[0046] Preferably, the composition is a topical composition, an injectable composition, or an oral composition.

[0047] Preferably, the composition is a composition in the form of a solution, lyophilized powder, gel, sponge, or fiber.

[0048] Eighthly, the present invention provides the use of the above-described polypeptides, the fusion protein, the recombinant type XII humanized collagen, the polynucleotide, the biomaterial, or the composition in the preparation of bio-dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue filling / volume-enhancing materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, chronic wound repair materials, muscle or bone or cartilage or ligament regeneration materials, liver tissue materials, vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, cosmetics, pharmaceutical excipients, drugs, or food additives.

[0049] In a ninth aspect, the present invention provides the use of the polypeptide, the fusion protein, the recombinant type XII humanized collagen, the polynucleotide, the biomaterial, or the composition in the preparation of products for any one or more of the following: cell adhesion, promotion of cell proliferation, improvement of extracellular matrix, tissue repair, tissue filling or volume enhancement, inhibition of MMP-1, reduction of collagen degradation, promotion of collagen regeneration, skin tightening, skin anti-wrinkle, and skin anti-aging.

[0050] The tissue repair includes, but is not limited to, skin tissue repair, wound repair, connective tissue (such as tendons or ligaments) repair, bone or cartilage repair, etc.; and the tissue filling / volume enhancement includes, but is not limited to, tissues from various parts of the human body.

[0051] In a tenth aspect, the present invention provides the use of the above-described polypeptide, the fusion protein, or the recombinant type XII humanized collagen in any one or more of the following: cell adhesion, promotion of cell proliferation, improvement of extracellular matrix, tissue repair, tissue filling or volume enhancement, plastic surgery, inhibition of MMP-1, reduction of collagen degradation, promotion of collagen regeneration, skin tightening, skin anti-wrinkle, and skin anti-aging.

[0052] Preferably, the tissue repair, tissue filling or volume enhancement, or cosmetic surgery is performed by injecting the polypeptide, the fusion protein, or the recombinant type XII humanized collagen.

[0053] In one aspect, the present invention provides the use of the polypeptide, the fusion protein, the recombinant type XII humanized collagen, the polynucleotide, the biomaterial, or the composition in the preparation of a medicament for the prevention or treatment of diseases or conditions related to type XII collagen deficiency or for the supplementation of type XII collagen.

[0054] In a twelfth aspect, the present invention provides a method for promoting cell adhesion and / or proliferation and / or improving the extracellular matrix, the method comprising the step of contacting the polypeptide, the fusion protein, the recombinant type XII humanized collagen, or the composition with cells.

[0055] The cells are preferably animal cells; more preferably mammalian cells; and even more preferably human cells.

[0056] Preferably, the promotion of cell adhesion and / or proliferation and / or improvement of the extracellular matrix occurs in vivo / in vitro.

[0057] Optionally, the use of promoting cell adhesion and / or proliferation and / or improving the extracellular matrix is ​​for non-disease treatment purposes.

[0058] In a thirteenth aspect, the present invention provides a method for performing cosmetic surgery, tissue filling or augmentation, tissue repair, or ophthalmic treatment on subjects in need, the method comprising: administering the polypeptide, the fusion protein, or the recombinant type XII humanized collagen to the subject.

[0059] Preferably, the administration is oral or injectable.

[0060] Preferably, the subject is a human being.

[0061] In a fourteenth aspect, the present invention provides a product comprising the polypeptide, the fusion protein, the recombinant type XII humanized collagen, or the composition thereof.

[0062] Preferably, the product is a cosmetic.

[0063] The above products have any one or more of the following functions: cell adhesion, promoting cell proliferation, improving extracellular matrix, tissue repair, tissue filling or volume enhancement, inhibiting MMP-1, reducing collagen degradation, promoting collagen regeneration, skin tightening, skin anti-wrinkle, and skin anti-aging.

[0064] In a fifteenth aspect, the present invention provides a method for promoting skin firmness, anti-wrinkle effect, or anti-aging effect, the method comprising the step of administering the polypeptide, the fusion protein, the recombinant type XII humanized collagen, or the composition or the product to a subject.

[0065] The application refers to the application of an effective amount.

[0066] The beneficial effects of the present invention include at least the following: (1) The present invention provides the core functional region of recombinant type XII humanized collagen with a triple helix structure.

[0067] (2) This invention successfully expressed and prepared recombinant type XII humanized collagen for the first time. As a novel biomaterial derived from the human body, it can be used for medical or non-medical purposes and repair / filling / capacity enhancement of multiple tissues and organs of the human body.

[0068] (3) The amino acid composition of the recombinant type XII humanized collagen provided by the present invention is 100% identical to the corresponding part of the amino acid sequence of human natural collagen, and will not produce immune rejection and allergic reaction when applied to the human body.

[0069] (4) The present invention uses synthetic biology technology to produce high-yield recombinant type XII humanized collagen on a large scale.

[0070] (5) The recombinant type XII humanized collagen provided by the present invention has good biological activity, including cell proliferation activity and cell adhesion activity, and is suitable for a variety of medical or non-medical uses.

[0071] (6) The recombinant type XII humanized collagen provided by the present invention exhibits a unique and exquisite fibrous porous network structure under scanning electron microscopy. Overall, the fibers in the material are interwoven into a dense network, constructing an orderly and complex three-dimensional architecture, which directly improves the extracellular matrix and provides an ideal microenvironment for cell adhesion, growth and migration.

[0072] (7) The recombinant type XII humanized collagen provided by the present invention can effectively inhibit MMP-1, reduce the decomposition of collagen, retain the existing collagen fibers in the skin, slow down the loss of skin collagen, improve skin laxity, and has the effect of firming and anti-wrinkle. Attached Figure Description

[0073] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments will be briefly described below.

[0074] Figure 1A , Figure 1B , Figure 1C , Figure 2A , Figure 2B The figures are the electrophoretic detection results of the purified recombinant type XII humanized collagen XII-1, XII-2, XII-3, XII-11 and XII-12 in Example 1 of this invention; where M is the protein marker.

[0075] Figure 3A , Figure 3B , Figure 4A , Figure 4B , Figure 4C The figures 1 and 2 are the electrophoretic detection results of the purified recombinant type XII humanized collagen XII-1-5, XII-1-12, XII-13, XII-14, and XII-15 in Example 1 of this invention.

[0076] Figure 5A , Figure 5B , Figure 5C , Figure 5D , Figure 5E , Figure 5F , Figure 5G The results are shown in the circular dichroism ultraviolet scanning analysis of recombinant type XII humanized collagen XII-1, XII-2, XII-3, XII-11, XII-12, XII-1-5, and XII-1-12 in Example 2 of this invention.

[0077] Figure 6A and Figure 6B The results of cell adhesion activity detection in Example 3 of the present invention are shown, wherein 1-5, 1-12, 13, 14, and 15 represent recombinant collagen XII-1-5, XII-1-12, XII-13, XII-14, and XII-15, respectively.

[0078] Figure 7A and Figure 7B The results of cytotoxicity and proliferation activity detection in Example 4 of the present invention are shown, wherein 1-5, 1-12, 13, 14, and 15 represent recombinant collagen XII-1-5, XII-1-12, XII-13, XII-14, and XII-15, respectively.

[0079] Figure 8 The scanning electron microscopy results are for the recombinant type XII humanized collagen XII-1 (labeled as 1 in the figure), XII-11 (labeled as 11 in the figure), and XII-12 (labeled as 12 in the figure) in Example 5 of this invention.

[0080] Figure 9 The results of serum immunoglobulin and complement component determination of recombinant type XII humanized collagen in Example 6 of this invention are obtained by ELISA.

[0081] Figure 10The results show the detection of MMP-1 inhibition by recombinant type XII humanized collagen in Example 7 of this invention. Detailed Implementation

[0082] The following description provides further details to facilitate a better understanding of the invention.

[0083] Peptides and recombinant type XII humanized collagen This invention provides polypeptides and recombinant type XII humanized collagen comprising said polypeptides. The polypeptides of this invention may comprise one or more repeating units comprising the amino acid sequence shown in any one of SEQ ID NO. 1-3, 10-11, 20-22, or variant sequences having the same function obtained by substituting, deleting, or inserting one or more amino acids based on the amino acid sequence shown in any one of SEQ ID NO. 1-3, 10-11, 20-22.

[0084] For example, variants of the sequence shown in SEQ ID NO.1 may be SEQ ID NO.10-11, 20-22.

[0085] In some implementations, the number of repeating units can be an integer between 1 and 20.

[0086] In some implementations, the number of repeating units is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0087] In some implementations, the number of repeating units is 1, 5, 6, 7, 8, or 12.

[0088] In some implementations, the mutation can be a substitution, such as a conserved amino acid substitution. A conserved amino acid substitution refers to the replacement of one or more amino acids with amino acids that have similar or analogous properties.

[0089] In some implementations, the repeating units are directly connected.

[0090] In some implementations, one or more amino acid residues are spaced between each repeating unit.

[0091] In some embodiments, when the repeating unit is mutated (i.e., the aforementioned variant sequence), the resulting polypeptide or recombinant type XII humanized collagen retains comparable or superior cell adhesion or cell proliferation-promoting activity compared to the polypeptide or recombinant type XII humanized collagen obtained from the unmutated repeating unit.

[0092] In some embodiments, when there are spacer sequences between the repeating units, the obtained polypeptide or recombinant type XII humanized collagen has comparable or superior cell adhesion or cell proliferation-promoting activity compared to the polypeptide or recombinant type XII humanized collagen obtained by directly linking the repeating units.

[0093] In some embodiments, the recombinant type XII humanized collagen of the present invention is synthetic or recombinantly expressed.

[0094] Polynucleotides encoding peptides or recombinant type XII humanized collagen The present invention provides polynucleotides encoding the polypeptide or recombinant type XII humanized collagen for expression.

[0095] In some embodiments, the polynucleotide is codon-optimized for the host cell in which it is expressed.

[0096] In some implementations, the polynucleotide encoding recombinant type XII humanized collagen can be operatively linked to expression control elements, such as promoters, terminators, and / or enhancers, to form a recombinant nucleic acid molecule or an expression cassette.

[0097] In some embodiments, the polynucleotide encoding recombinant type XII humanized collagen may also include nucleotides encoding purification tags (e.g., His tag, GST tag, MBP tag, SUMO tag, or NusA tag) or nucleotides encoding leader sequences to facilitate peptide purification or secretion.

[0098] Compositions, kits and their applications The polypeptides or recombinant type XII humanized collagen of the present invention can be prepared into compositions or kits.

[0099] In some embodiments, the composition or kit may be a composition or kit for tissue filling and / or volume enhancement and / or repair.

[0100] In some embodiments, the composition or kit may also contain auxiliary substances.

[0101] In some embodiments, the compositions of the present invention may be tissue repair agents / tissue fillers / tissue volume enhancers (the tissues include, but are not limited to, muscle tissue, tendons and ligaments, cartilage / bone tissue, etc.), comprising the polypeptides or the recombinant type XII humanized collagen. The compositions of the present invention may be injectable / implantable and do not elicit an immune response in the human body.

[0102] Construction and production method of recombinant type XII humanized collagen The present invention also provides a method for constructing recombinant type XII humanized collagen, including functional region screening and construction of production strains, large-scale bio-fermentation, protein induction expression, purification and optional enzymatic digestion steps.

[0103] In some embodiments, the steps of functional region screening and production strain construction include: large-scale functional region screening to obtain the target gene functional region; inserting the obtained target gene functional region into the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid; and transforming the recombinant expression plasmid into Escherichia coli competent cells BL21(DE3) to screen for positive Escherichia coli genetically engineered bacteria.

[0104] The present invention also provides a method for producing the polypeptide or recombinant type XII humanized collagen, comprising: (1) culturing host cells containing polynucleotides encoding the polypeptide or recombinant type XII humanized collagen under suitable culture conditions; (2) harvesting the host cells and / or culture medium containing the polypeptide or protein; and (3) purifying the polypeptide or protein.

[0105] The host cells are cultured in a suitable nutrient medium for producing the peptide using methods known in the art. For example, cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermenter, in a suitable medium and under conditions that allow for peptide expression and / or isolation. Using procedures known in the art, the culture occurs in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the U.S. Center for Type Culture Collection). If the peptide is secreted into the nutrient medium, it can be recovered directly from the medium. If the peptide is not secreted, it can be recovered from cell lysates.

[0106] Peptides can be detected using methods known in the art that are specific to peptides. 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, enzymatic assays can be used to determine the activity of a peptide.

[0107] Peptides can be recovered using methods known in the art. For example, peptides can be recovered from nutrient media through conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, fermentation broth containing peptides can be recovered.

[0108] Peptides can be purified using a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, focusing chromatography, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric focusing electrophoresis), differential dissolution (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction, to obtain substantially pure peptides.

[0109] Step (1) may include one or more of the following steps: constructing an expression plasmid, for example, inserting the encoding nucleotide sequence into the pET-28a-Trx-His expression vector to obtain a recombinant expression plasmid. The successfully constructed expression plasmid can be transformed into Escherichia coli cells (e.g., Escherichia coli competent cells BL21(DE3)). The specific process may be as follows: (1) Take the plasmid to be transformed and add it into Escherichia coli competent cells BL21(DE3); (2) Place the mixture on ice (e.g., 10-60 min, e.g., 30 min), then heat shock in a water bath (e.g., at 40-50℃, e.g., 42℃, 45-90 s), remove it and place it on ice (e.g., 1-5 min, e.g., 2 min); (3) Add liquid LB medium and then culture (e.g., at 35-40℃, e.g., 37℃, 150-300 rpm, e.g., 220 rpm for 40-80 min, e.g., 60 min); (4) Spread the bacterial solution and select single colonies. For example, spread the bacterial suspension evenly on an LB agar plate containing ampicillin or kanamycin sulfate, and incubate the plate in a 37°C incubator for 15-17 hours until uniformly sized colonies grow.

[0110] Step (2) may include culturing single colonies in LB medium containing antibiotics (e.g., in a constant temperature shaker at 150-300 rpm, e.g., 220 rpm, 35-40°C, e.g., 37°C for 5-10 h, e.g., 7 h); then cooling to 10-30°C (e.g., 16°C), adding IPTG to induce expression for a period of time, and then collecting the cells (e.g., by centrifugation).

[0111] Step (3) may include resuspending the bacterial cells in a equilibration working solution, cooling the bacterial solution to ≤15°C, homogenizing (e.g., high-pressure homogenization, e.g., 1-5 times, e.g., 2 times) or sonicating to disrupt the cells, and separating the bacterial solution to obtain the supernatant. The equilibration working solution may contain 100-500 mM sodium chloride, 10-50 mM Tris, and 10-50 mM imidazole. For example, the concentration of sodium chloride can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 mM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM. The concentration of imidazole can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM. The pH can be 7, 7.5, 8, 8.5 or 9.

[0112] Step (3) may include purification and enzymatic digestion of the peptide. Purification may be crude purification, including Ni-agarose gel column purification of the supernatant to obtain an eluent containing the target protein. Crude purification may include washing the column material with water, for example, 2-10 column volumes (CV), such as 5 CV. The column material may be equilibrated with an equilibration buffer (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole), for example, 2-10 CV, such as 5 CV. The equilibration buffer may contain 100-500 mM sodium chloride, 10-50 mM Tris and 10-50 mM imidazole, pH 7-9. For example, the concentration of sodium chloride can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 mM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM. The concentration of imidazole can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM. The pH can be 7, 7.5, 8, 8.5 or 9.

[0113] 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 mM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM. The concentration of imidazole can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM. The pH can be 7, 7.5, 8, 8.5, or 9. Then, an eluent can be added and the flow-through collected. The eluent can contain 100-500 mM sodium chloride, 10-50 mM Tris, 100-500 mM imidazole, and pH 8.0. For example, the concentration of sodium chloride can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 mM. The concentration of Tris can be 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM. The concentration of imidazole can be 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, or 490 mM. The pH can be 7, 7.5, 8, 8.5, or 9.

[0114] Enzymatic digestion may include adding TEV enzyme (at a protein to TEV ratio of 10-100:1, e.g., 50:1, at 10-20°C, e.g., 16°C, for 2-8 hours, e.g., 4 hours). The digested protein solution is then dialyzed (e.g., placed in a dialysis bag and dialyzed at 1-6°C, e.g., 4°C, for 1-8 hours, e.g., 2 hours, then transferred to fresh dialysate and dialyzed overnight at 1-6°C, e.g., 4°C).

[0115] Purification may include fine purification (e.g., protein isoelectric point > 8.0). Preferably, fine purification includes gradient elution of the eluent containing the target protein or the enzymatically digested product (e.g., product after enzymatic digestion and dialyzing) using a strong anion exchange chromatography column (e.g., pH 7, 7.5, 8, 8.5, or 9). Gradient elution includes 0-15% solution B for 1-5 minutes followed by 1-5 (e.g., 3 column volumes), 15-30% solution B for 1-5 minutes followed by 1-5 (e.g., 3 column volumes), 30-50% solution B for 1-5 minutes followed by 1-5 (e.g., 3 column volumes), and 50-100% solution B for 1-5 minutes followed by 1-5 (e.g., 3 column volumes). Solution B may contain 10-50 mM Tris, 0.5-5 M sodium chloride, and pH 7-9. (For example, the concentration of Tris is 15, 20, 25, 30, 35, 40, or 45 mM; the concentration of sodium chloride is 1, 2, 3, or 4 M; the pH can be 7, 7.5, 8, 8.5, or 9). Purification may include equilibrating the column with solution A and loading the sample, followed by gradient elution. Solution A may contain 10-50 mM Tris, 10-50 mM sodium chloride, and pH 7-9 (for example, the concentration of Tris is 15, 20, 25, 30, 35, 40, or 45 mM; the concentration of sodium chloride is 15, 20, 25, 30, 35, 40, or 45 mM; the pH can be 7, 7.5, 8, 8.5, or 9).

[0116] Purification may include reverse-coated nickel column purification (e.g., protein isoelectric point < 8.0). Reverse-coated nickel column purification may include purification of the enzyme-digested product (e.g., the product after dialysis) onto a Ni-agarose gel column. The eluent may contain 10-50 mM (e.g., 15, 20, 25, 30, 35, 40, or 45 mM) Tris, 10-50 mM (e.g., 15, 20, 25, 30, 35, 40, or 45 mM) sodium chloride, 0.5-5 M (e.g., 1, 2, 3, or 4 M) imidazole, and pH 7-9 (e.g., 7, 7.5, 8, 8.5, or 9).

[0117] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Those skilled in the art should understand that the embodiments are merely exemplary and not restrictive.

[0118] Example 1: Construction and expression of recombinant type XII humanized collagen 1.1 Construction of recombinant type XII humanized collagen (1) Large-scale functional region screening of natural human type XII collagen (GenBank: XP_059970343.1) yielded the following different protein functional regions: C12h: GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER (SEQ ID NO. 1); C12f: GPSGKPGKPGDHGRPGPSGLKGEKGDRGDI (SEQ ID NO.2); C12d: GEQGRQGMKGDAGEPGLPGRTGTP (SEQ ID NO. 3).

[0119] To ensure the purification and stability of recombinant type XII humanized collagen, the amino acid fragments in these regions were optimized by repeated n times and directly linked to obtain recombinant collagen XII-1, recombinant collagen XII-2, and recombinant collagen XII-3, with the corresponding amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0120] The amino acid sequence of recombinant collagen XII-1 (8 repeats): GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGA RGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER (SEQ IDNO.4); The amino acid sequence of recombinant collagen XII-2 (6 repeats): GPSGKPGKPGDHGRPGPSGLKGEKGDRGDIGPSGKPGKPGDHGRPGPSGLKGEKGDRGDIGPSGKPGKPGDHGRPGPSGLKGEKGDRGDIGPSGKPGKPGDHGRPGPSGLKGEKGDRGDIGPSGKPGKPGDHGRPGPSGLKGEKGDRGDIGPSGKPGKPGDHGRPGPSGLKGEKGDRGDI (SEQ ID NO. 5); The amino acid sequence of recombinant collagen XII-3 (8 repeats): GEQGRQGMKGDAGEPGLPGRTGTPGEQGRQGMKGDAGEPGLPGRTGTPGEQGRQGMKGDAGEPGLPGRTGTPGEQGRQGMKGDAGEPGLPGRTGTPGEQGRQGMKGDAGEPGLPGRTGTPGEQGRQGMKGDAGEPGLPGRTGTPGEQGRQGMKGDAGEPGLPGRTGTPGEQGRQGMKGDAGEPGLPRTGTP (SEQ ID NO.6).

[0121] Codon optimization was performed using the E. coli expression system to obtain the encoding nucleic acid sequences of recombinant collagen XII-1, recombinant collagen XII-2, and recombinant collagen XII-3, as shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively.

[0122] Nucleic acid sequence of recombinant collagen XII-1: GGAGCTAGGGGGGAGCCGGGTCCCGGCGGTCGCCCAGGCTTTCCGGGCACCCCGGGCATGCAGGGTCCGCCGGGTGAACGCGGTTTGCCGGGTGAGAAGGGCGAACGTGGCGCTCGTGGTGAGCCGGGTCCGGGTGGTCGTCCGGGTTTTCCGGGCACCCCGGGCATGCAGGGTCCGCCGGGCGAAAGAGGCTTGCCGGGTGAAAAAGGTGAGCGCGGCGCGCGTGGGGAACCGGGCCCGGGAGGCCGTCCGGGCTTCCCGGGAACCCCGGGTATGCAAGGTCCGCCTGGCGAAAGAGGGCTGCCTGGCGAGAAAGGCGAACGCGGTGCGCGTGGTGAACCGGGCCCAGGTGGTCGTCCGGGTTTCCCGGGCACGCCGGGCATGCAGGGTCCGCCGGGCGAGCGCGGTCTGCCAGGCGAGAAAGGTGAACGTGGTGCGCGTGGTGAACCTGGTCCGGGTGGTCGTCCGGGCTTCCCGGGCACTCCGGGTATGCAGGGTCCGCCGGGCGAGCGCGGTCTGCCGGGCGAAAAGGGCGAGCGCGGTGCCCGTGGTGAACCGGGTCCGGGTGGTCGTCCGGGCTTTCCGGGTACGCCGGGTATGCAAGGCCCACCGGGCGAACGTGGCCTGCCGGGTGAGAAGGGTGAGCGTGGTGCTCGTGGTGAGCCTGGTCCGGGTGGCCGTCCGGGTTTCCCGGGCACCCCAGGCATGCAGGGCCCACCGGGTGAACGCGGTTTACCGGGCGAAAAGGGTGAGCGCGGCGCACGTGGCGAACCGGGCCCAGGTGGCCGTCCGGGCTTTCCGGGCACCCCGGGCATGCAAGGTCCACCGGGTGAGCGTGGTCTGCCGGGCGAGAAAGGTGAGCGC (SEQ ID NO.7); Nucleic acid sequence of recombinant collagen XII-2: GGGCCCTCAGGAAAGCCTGGCAAACCGGGCGACCACGGCCGTCCGGGTCCGTCGGGCTTGAAGGGCGAGAAGGGTGACCGCGGTGACATCGGCCCAAGCGGTAAACCGGGTAAACCGGGTGACCACGGTCGTCCCGGTCCGAGCGGTCTGAAAGGCGAGAAAGGTGATCGCGGTGACATCGGCCCATCTGGCAAGCCGGGTAAGCCGGGTGACCACGGTCGTCCGGGTCCGTCCGGTCTGAAGGGTGAAAAAGGTGACCGCGGTGATATCGGTCCGAGCGGTAAGCCGGGCAAACCGGGCGACCACGGTCGCCCAGGCCCATCTGGCCTGAAAGGTGAAAAAGGCGACCGTGGTGATATTGGTCCGAGCGGCAAGCCGGGCAAACCGGGGGATCACGGCCGTCCGGGTCCGAGCGGTCTCAAGGGCGAAAAGGGCGACAGAGGCGATATTGGCCCGTCCGGTAAGCCGGGTAAACCGGGTGATCACGGCCGTCCGGGTCCGAGCGGCCTGAAAGGCGAGAAGGGCGATCGTGGTGATATT(SEQ ID NO.8); Nucleic acid sequence of recombinant collagen XII-3: (SEQ ID NO.9).

[0123] (2) Regarding SEQ ID NO.1, in order to investigate the differences in amino acid sequences of different truncated amino acids in type XII collagen, the following truncated motifs were selected based on human type XII: GPPGPPGPPGSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER (C12g, SEQ ID NO. 10); GSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGTGSSGPRG (C12j, SEQ ID NO. 11); The above truncated motifs were optimized by repeating them n times and directly linking them to obtain recombinant collagen XII-11. The amino acid sequences corresponding to collagen XII-12 are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively.

[0124] The amino acid sequence of recombinant collagen XII-11 (5 repeats): GPPGPPGPPGSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GPPGPPGPPGSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GPPGPPGPPGSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GPPGPPGPPGSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GPPGPPGPPGSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER (SEQ ID NO.12); The amino acid sequence of recombinant collagen XII-12 (5 repeats): GSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGTGSSGPRG GSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGTGSSGPRG GSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGTGSSGPRG GSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGTGSSGPRG GSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGTGSSGPRG (SEQ ID NO.13).

[0125] Codon optimization was performed using the E. coli expression system to obtain the coding nucleic acid sequences for collagen XII-11 and collagen XII-12, as shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0126] Nucleic acid sequence of recombinant collagen XII-11: GGACCACCCGGGCCGCCCGGCCCGCCGGGCTCTGCCGGTGCTCGTGGCGAACCGGGCCCGGGCGGTCGTCCGGGTTTTCCGGGCACCCCTGGCATGCAAGGTCCGCCGGGCGAGCGTGGTCTGCCGGGCGAGAAGGGTGAACGTGGCCCACCGGGTCCGCCGGGCCCACCGGGCAGCGCGGGTGCACGTGGTGAGCCGGGTCCGGGTGGTCGTCCGGGTTTTCCGGGGACTCCGGGTATGCAGGGCCCGCCGGGTGAAAGAGGCTTACCGGGTGAAAAAGGTGAGCGCGGTCCGCCGGGCCCACCGGGTCCGCCAGGCAGCGCGGGTGCGCGTGGTGAACCGGGCCCGGGTGGCCGTCCGGGATTCCCGGGTACGCCGGGTATGCAGGGTCCGCCTGGTGAGCGCGGTCTGCCAGGCGAAAAGGGCGAGCGCGGTCCGCCCGGCCCACCGGGCCCGCCGGGCAGCGCGGGTGCGCGTGGTGAACCGGGTCCGGGCGGACGCCCTGGTTTCCCGGGAACCCCGGGCATGCAAGGTCCGCCGGGCGAGCGTGGTTTGCCGGGCGAGAAAGGCGAACGTGGTCCGCCGGGCCCACCGGGTCCGCCGGGCTCCGCTGGCGCACGTGGTGAGCCGGGCCCGGGCGGTCGCCCAGGCTTCCCGGGCACCCCGGGTATGCAGGGTCCGCCGGGTGAACGCGGTCTGCCTGGCGAGAAAGGTGAGCGC (SEQ ID NO.14); Nucleic acid sequence of recombinant collagen XII-12: (SEQ ID NO.15).

[0127] (3) Regarding the aforementioned repeating unit C12h: In order to explore the function of XII collagen amino acid sequences with different repeat numbers of the same repeat unit, GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER (SEQ ID NO.1) was designed with 5 and 12 repeats based on SEQ ID NO.1.

[0128] The amino acid sequence of recombinant collagen XII-1-5 (5 repeats): GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER (SEQ ID NO. 16).

[0129] The amino acid sequence of recombinant collagen XII-1-12 (12 repeats): GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER (SEQ IDNO.17).

[0130] Codon optimization was performed using the E. coli expression system to obtain the encoding nucleic acid sequences of recombinant collagen XII-1-5 and collagen XII-1-12, as shown in SEQ ID NO.18 and SEQ ID NO.19, respectively.

[0131] Nucleic acid sequence of recombinant collagen XII-1-5: ggagctaggggggagccgggtccgggtggtcgtccggggttcccgggcacgccgggcatgcagggtccaccgggcgagcgcggtttgccgggcgaaaagggtgaacgtggtgctcgtggtgagccgggcccaggcggtcgtccgggtttcccgggcaccccgggcatgcagggtccgcctggcgagcgcggtctgccgggtgagaagggcgaaagaggcgcacgtggtgaaccgggtccgggtggtcgtccgggctttccgggcaccccgggcatgcagggcccaccgggcgagcgcggcctgccgggggagaaaggtgaacgcggtgcgcgtggcgaaccgggtccgggcggacgcccaggcttcccgggtactccgggcatgcaaggtccgccaggcgaacgtggtttaccgggtgaaaaaggtgaacgtggtgcgcgtggcgagccgggtccgggcggtcgtccgggttttccgggcaccccgggcatgcaaggtccgcctggtgaacgtggtctgccgggcgagaagggtgagcgc (SEQ ID NO.18); Nucleic acid sequence of recombinant collagen XII-1-12: (4) Regarding the aforementioned repeating unit C12h: GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGER (SEQ ID NO.1), in order to investigate the differences in amino acid sequences with different truncations in human type XII collagen, the following truncation motifs were selected based on human type XII: The amino acid sequence of recombinant collagen XII-13: GDRGDIASQNMMRAVARQVCEQLISGQMNRFNQMLNQIPNDYQSSRNQPGPPGPPGPPGSAGARGEPGPGRPGFPGTPGMQGPPGERGLPGEKGER (SEQ ID NO. 20); The amino acid sequence of recombinant collagen XII-14: GARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGTGSSGPRGLPGPPGPQGESRTGPPGSTGSRGPPGPPGRPGNSGIRGPPGPPGYCDSSQC (SEQ ID NO. 21); The amino acid sequence of recombinant collagen XII-15: GDRGDIASQNMMRAVARQVCEQLISGQMNRFNQMLNQIPNDYQSSRNQPGPPGPPGPPGSAGARGEPGPGGRPGFPGTPGMQGPPGERGLPGEKGERGTGSSGPRGLPGPPGPQGESRTGPPGSTGSRGPPGPPGRPGNSGIRGPPGPPGYCDSSQC (SEQ ID NO. 22) Codon optimization was performed on the above sequences using the E. coli expression system to obtain the coding nucleic acid sequences for collagen XII-13, collagen XII-14, and collagen XII-15, as shown in SEQ ID NO.23, SEQ ID NO.24, and SEQ ID NO.25, respectively.

[0132] Nucleic acid sequence of recombinant collagen XII-13: ggagataggggggacatcgcctctcaaaacatgatgcgtgcagttgctcgtcaggtgtgcgaacagctgatcagcggtcagatgaaccgcttcaaccaaatgttgaatcaaattccgaatgattaccagtccagccgtaaccaaccgggccctccgggtccgccgggcccaccgggcagcgcgggtgcgcgtggtgagccgggcccaggcggtcgtccgggttttccgggaaccccgggcatgcagggtccgccgggcgagcgcggtctgccgggcgagaagggtgaacgc (SEQ ID NO.23); Nucleic acid sequence of recombinant collagen XII-14: ggagctaggggggagccgggcccgggcggtcgccctggcttcccgggtacgccgggcatgcagggtccgccgggcgagcgcggtttgccgggcgaaaagggtgaacgtggcaccggttccagcggtccgcgtggtctgccgggcccaccgggtccgcagggtgagagccgtaccggcccaccgggctccaccggttctcgtggtccgccgggtccgccgggccgtccgggaaacagcggcatccgcggtccgccgggtccgccaggctactgcgacagctcgcaatgt (SEQ ID NO.24); Nucleic acid sequence of recombinant collagen XII-15: ggggataggggagacatcgccagccaaaacatgatgcgtgcagttgcgcgtcaggtgtgcgaacagctgatcagcggtcagatgaatcgcttcaaccagatgctgaatcaaattccga atgattatcagagctcccgcaaccaaccgggtccgccaggcccaccgggcccgccgggcagcgcgggtgctcgtggtgagccgggccctggcggtcgtccgggttttccgggcaccccg ggcatgcaaggtccgccgggcgagcgcggtctgccgggcgaaaaaggtgaacgtggcaccggttcttctggtccgcgtggcttgccgggtccgccgggcccacagggtgagagcagaac gggtccgcctggctccaccggtagtcgcggtccgccgggcccaccgggccgtccgggaaacagcggtattcgtggtccgccgggtccgccgggctactgcgacagctcgcaatgt (seq ID NO.25).

[0133] 1.2 Expression of recombinant type XII humanized collagen The gene functional region synthesized in 1.1 above was inserted into the pET-28a-Trx-His expression vector to obtain the corresponding recombinant expression plasmid.

[0134] The successfully constructed expression plasmid was transformed into Escherichia coli competent cells BL21(DE3). The specific process was as follows: (1) The Escherichia coli competent cells BL21(DE3) were taken out of the ultra-low temperature freezer and placed on ice. When they were half-thawed, 2 μL of the plasmid to be transformed was added to the Escherichia coli competent cells BL21(DE3) and mixed slightly 2-3 times. (2) The mixture was placed on ice for 30 min, and then heat-shocked in a water bath at 42℃ for 45-90 s. After taking it out, it was placed on ice for 2 min. (3) It was transferred to a biosafety cabinet and 700 μL of liquid LB medium was added. Then it was cultured at 37℃ and 220 rpm for 60 min. (4) 200 μL of bacterial solution was evenly spread on LB plates containing kanamycin sulfate. (5) The plates were cultured in an incubator at 37℃ for 15-17 h until uniform colonies grew.

[0135] Pick 5-6 single colonies from the transformed LB agar plates and place them in a shake flask containing antibiotic stock solution (kanamycin sulfate 100 mg / L). Incubate at 220 rpm and 37°C in a constant temperature shaker for a certain period of time until the colonies appear as a mist. Then, cool the shake flasks to 16-30°C, add IPTG (0.5 mM) to induce expression for a period of time, aliquot the bacterial culture into centrifuge bottles, centrifuge at 6000 rpm and 4°C for 12 min, collect the bacterial cells, record the cell weight, and perform electrophoresis analysis.

[0136] The collected bacterial cells were resuspended in a balanced working solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole). The bacterial suspension was cooled to ≤15℃ and homogenized twice or sonicated to disrupt the cells. After the disruption, the bacterial suspension was collected. The disrupted bacterial suspension was aliquoted into centrifuge bottles and centrifuged at 17000 rpm and 4℃ for 30 min. The supernatant was collected.

[0137] The recombinant humanized type XII collagen was purified and enzymatically digested. The specific process is as follows: (1) Crude purification: a. Equilibrate the column: Equilibrate the column with equilibration buffer (200mM sodium chloride, 25mM Tris, 20mM imidazole) at a flow rate of 10mL / min. b. Load the sample: Add the supernatant after centrifugation to the column until the liquid is completely discharged at a flow rate of 5mL / min. c. Wash away contaminating proteins: Add 100 mL of washing buffer (200 mM sodium chloride, 25 mM Tris, 20mM imidazole) until the liquid is completely discharged at a flow rate of 10mL / min. d. Collect the target protein: Add 20 mL of elution buffer (200mM sodium chloride, 25mM Tris, 250mM imidazole) at a flow rate of 10mL / min, collect the flow-through and perform electrophoresis detection. e. Wash the column with 1M imidazole working solution at a flow rate of 10mL / min. (2) Enzyme digestion: Add TEV enzyme at a ratio of total protein to total TEV enzyme of 20:1, and digest at 16℃ for 2 h. Place the digested protein solution into a dialysis bag and dialyze at 4℃ for 2 h, then transfer to a new dialysis buffer (20 mM sodium chloride, 20 mM Tris) and dialyze overnight at 4℃. (3) Purification: a. Equilibrate the column: Equilibrate the column with solution A (20 mM Tris, 20 mM sodium chloride) at a flow rate of 10 mL / min. b. Load the sample: Load the sample at a flow rate of 5 mL / min and collect the flow-through sample, and perform electrophoresis detection. Store the protein at 4℃. c. Elute: Wash the column with solution B (1 M sodium chloride, 20 mM Tris) for 5 CVs. d. Wash the column.

[0138] The purified recombinant humanized type XII collagen was detected by SDS-PAGE. The electrophoretic results of recombinant collagen XII-1, XII-2, XII-3, XII-11, and XII-12 are shown below. Figure 1A , Figure 1B , Figure 1C , Figure 2A , Figure 2B The results showed that recombinant collagen XII-1, XII-2, XII-3, XII-11, and XII-12 were successfully expressed and prepared. The electrophoretic results of recombinant collagen XII-1-5, XII-1-12, XII-13, XII-14, and XII-15 are shown below. Figure 3A , Figure 3B , Figure 4A , Figure 4B , Figure 4C During expression, expression was initiated using the inducing agent IPTG. Host RNA polymerase bound to the promoter, transcribing the target gene into mRNA. Ribosomes then bound the mRNA, translating it into a polypeptide chain. Expression levels were verified by SDS-PAGE. After bacterial cell lysis, soluble collagen was released, and a band related to the target protein was detected in the bacterial supernatant. After loading and washing, the amount of contaminating proteins gradually decreased. The target protein was then eluted with elution buffer, and no band was observed in the loading and washing processes, indicating good binding of the target protein to the chromatography column. Treatment of the column with 1M imidazole removed some non-specifically bound contaminating proteins. After collagenase digestion, the tagged fusion protein was cleaved, and the target protein band appeared at a position consistent with the theoretical molecular weight. The digested protein was then transferred to solution A (replacement A) for further purification via ion exchange column chromatography (QFL). The QFL showed a high purity of the target protein, indicating good purification. Finally, the column material was washed with solution B to remove the cleaved tag and excess enzyme.

[0139] The theoretical molecular weight of recombinant collagen XII-1 after purification is 27.9 KD, and the apparent molecular weight after purification is consistent with the theoretical molecular weight. The theoretical molecular weight of recombinant collagen XII-2 after purification is 17.4 KD, and the apparent molecular weight after purification is consistent with the theoretical molecular weight. The theoretical molecular weight of recombinant collagen XII-3 after purification is 18.7 KD, and the apparent molecular weight after purification is consistent with the theoretical molecular weight. The theoretical molecular weight of recombinant collagen XII-11 is 22.27 KD, and the apparent molecular weight after purification is consistent with the theoretical molecular weight. Collagen XII-12 has a molecular weight of 22.28 KD, and the apparent molecular weight after purification is consistent with the theoretical molecular weight. The bands are clear and free of impurities. The theoretical molecular weights of XII-13, XII-14, and XII-15 are 10.06 KD, 9.07 KD, and 15.64 KD, respectively. After purification, the apparent molecular weights are consistent with the theoretical molecular weights, and the purified bands of XII-13 and XII-15 are clear and free of impurities.

[0140] Example 2: Circular dichroism UV-Vis analysis of recombinant type XII humanized collagen 1.1 Experimental Methods (1) Instrument parameter settings Bandwidth: 1.0nm; Step: 1.0nm; Measurement range: 190-260nm (far-UV region scanning) / 250-340nm (near-UV region scanning); Time per point: 0.5s; Repeats: 3 times; Cell Length: 10mm | 0.5mm; Temperature: Room temperature.

[0141] (2) Near and far ultraviolet scanning of standard samples The scanning wavelength was set to 180-340 nm for background testing and blank buffer testing. Then, the circular dichroism near and far UV absorption of 1 mg / mL CSA standard solution in the 180-340 nm range was collected.

[0142] (3) Sample processing Take protein samples and concentrate them to a protein concentration of 1 mg / mL using a 10 kDa ultrafiltration concentrator (Millibo).

[0143] (4) Far-ultraviolet scanning of samples Soak the cuvette in 2M HNO3 overnight, rinse it with deionized water and air dry it. First collect the background, then collect the blank buffer solution. Then add an appropriate amount of the test sample to the cuvette and perform a far-ultraviolet scan at 190-260 nm according to the above parameters and collect the data.

[0144] (5) Near-ultraviolet scanning of samples Soak the cuvette in 2M HNO3 overnight, rinse it with deionized water and air dry it. First collect the background, then collect the blank buffer solution. Then add an appropriate amount of the test sample to the cuvette and perform a near-ultraviolet scan at 250-340 nm according to the above parameters and collect the data.

[0145] (6) Scanning image processing All scanned spectra were processed using the software Pro-Data Viewer to extract baselines and smooth out the images.

[0146] (7) Experimental results and analysis The results show that ( Figure 5A , 5B (5C, 5D, 5E, 5F, 5G): The above three recombinant type XII humanized collagens XII-1, XII-2, XII-3, XII-11, and XII-12 all have positive peaks at 220-230nm, indicating that all five collagens have a triple helix structure.

[0147] Similarly, circular dichroism ultraviolet scanning analysis of recombinant collagen XII-1-5 and recombinant collagen XII-1-12 also revealed that they possess a triple helix structure.

[0148] Example 3: Detection of cell adhesion activity of recombinant type XII humanized collagen The specific implementation method is as follows: (1) The concentration of the protein samples to be tested was detected using ultraviolet absorption, including bovine type I collagen (China National Institutes for Food and Drug Control, No.: 380002) and recombinant type XII collagen provided in this invention. Specifically, the ultraviolet absorption of the samples at 215 nm and 225 nm was measured respectively, and the protein concentration was calculated using the empirical formula C (μg / mL) = 144 × (A215 - A225). Note that the detection must be performed when A215 < 1.5. The principle of this method is: to measure the characteristic absorption of peptide bonds under far-ultraviolet light, which is not affected by the content of chromophores, has few interfering substances, is simple to operate, and is suitable for detecting human collagen and its analogues that are not colorimetric by Coomassie Brilliant Blue. After the protein concentration was detected, the concentration of all the proteins to be tested was adjusted to 1 mg / mL with PBS.

[0149] (2) Sample preparation: The original sample solution was used directly for the experiment; the positive control bovine type I collagen (PC) was diluted to 1 mg / mL with D-PBS for later use; the negative control was D-PBS buffer (NC).

[0150] (3) Coating: Add 1 mg / mL collagen, positive control and negative control to the microplate, 100 μL per well, 5 replicates per group, and incubate overnight at 4°C.

[0151] (4) Blocking: Discard the supernatant, add 100 μL of 1% BSA (heat inactivation at 56℃ for 30 min), and incubate at 37℃ for 60 min. Discard the supernatant and wash 3 times with D-PBS solution.

[0152] (5) Cell seeding: Add 10g of the solution to each well. 5 3T3 / NIH cells in good condition, resuspended in D-PBS, were incubated at 37°C for 120 min. Each well was washed three times with D-PBS solution.

[0153] (6) Detection: The absorbance at OD450nm was measured using the CCK8 assay kit (manufacturer: Beyotime, product catalog number C0038). The cell adhesion degree was calculated according to the following formula. The cell adhesion rate reflects the cell adhesion ability of collagen. The higher the cell adhesion ability, the better the external environment can be provided to the cells in a short time, which helps the cells adhere.

[0154] Relative cell adhesion = {(OD1-OD0) / (OD2-OD0)}×100% Where: OD1: the average UV absorbance of each duplicate well of the collagen sample at 450nm; OD2: the average UV absorbance of each duplicate well of the control collagen sample at 450nm; OD0: the average UV absorbance of each duplicate well of the blank control group at 450nm.

[0155] (7) Statistical analysis: A two-tailed t-test was used to analyze the statistical differences between the target recombinant humanized collagen and the negative control. * P <0.05;**, P <0.01; ***, P <0.001, ****, P<0.0001. Results are as follows: Figure 6A and 6BAs shown, compared with the D-PBS group, the positive control had a significant promoting effect on cell adhesion. Recombinant humanized collagen XII-1, recombinant humanized collagen XII-2, recombinant humanized collagen XII-3, recombinant humanized collagen XII-11, and recombinant humanized collagen XII-12 also promoted cell adhesion, with cell adhesion activities of 442%, 424%, 426%, 403%, and 357% respectively compared to the negative control, significantly higher than the positive control. Figure 6A ).

[0156] Cell adhesion activity was also tested on recombinant collagen XII-1-5, recombinant collagen XII-1-12, and XII-13, XII-14, and XII-15. Compared with the negative control, the cell adhesion activities were 276%, 278%, 285%, 273%, and 255%, respectively, indicating that they possess biological activity for cell adhesion. Figure 6B ).

[0157] Example 4: Cell proliferation and toxicity assays of recombinant type XII humanized collagen CCK8 reagent contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt], which is reduced by cellular dehydrogenases to a highly water-soluble yellow formazan dye by the electron carrier 1-methoxy-5-methylphenazineonium sulfate (1-Methoxy PMS). The amount of formazan dye produced is directly proportional to the number of viable cells. Therefore, this property can be used to directly analyze HFB cell proliferation and toxicity.

[0158] (1) Experimental grouping: The culture medium containing 0.4% serum was the negative control (NC), 8% NaCl was the positive control (PC), the recombinant humanized collagen XII with a concentration of 1 mg / mL was the experimental group, and the complete culture medium without cells was the zeroing well.

[0159] (2) Sample preparation: Dilute the sample to the working concentration with 0.4% serum culture medium, filter it through a 0.22 µm microporous membrane for sterilization, and use it directly as a sample. Dissolve NaCl in 0.4% serum culture medium to a final concentration of 8%, and filter it for sterilization.

[0160] (3) Cell seeding: When the cell confluence reaches approximately 90%, digest and count the cells. Digest and count the cells at a rate of (5-10) × 10⁶ cells per well. 3 Cells were seeded in 96-well plates, and the edge wells were blocked with 100 µL of PBS. Cells were cultured for 24 h to allow adherence.

[0161] (4) Starvation treatment: After the cells adhered, the supernatant was aspirated, and 100 µL of culture medium containing 0.4% serum was added to each well and cultured for 24 h.

[0162] (5) Cell drug administration: Discard the supernatant and replace the corresponding group solutions in sequence, with 4 replicates per group, and culture for 24 h.

[0163] (6) Experimental detection: Discard the culture medium, add 100 µL (including 5 µL CCK8 solution) of basal culture medium, incubate in an incubator for 2-4 h, and detect using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. The cell viability calculation formula is as follows.

[0164] Cell viability = {(As-Ab) / (Ac-Ab)} × 100%; In the formula: As: Absorbance of experimental wells (including cells, culture medium, CCK8 solution and drug solution); Ac: Absorbance of control wells (containing cells, culture medium, and CCK8 solution, but excluding drugs); Ab: Absorbance of blank group (containing culture medium and CCK8 solution, but excluding cells and drugs). (7) Statistical analysis: The statistical difference between the sample and the negative control was analyzed using a two-tailed t-test. Among them, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0165] The results show that ( Figure 7A and Figure 7B Compared with the negative control group, the cell survival rate of the positive control group was significantly reduced, and the difference was statistically significant. The experiment demonstrated that the five recombinant humanized collagen proteins XII-1, XII-2, XII-3, XII-11, and XII-12 in the experimental group not only had no cytotoxicity but also exhibited significant cell proliferation-promoting activity. Cell proliferation activity assays of recombinant collagen proteins XII-1-5, XII-1-12, XII-13, XII-14, and XII-15 showed that they also had no cytotoxicity, and recombinant collagen proteins XII-1-5 and XII-1-12 had excellent cell proliferation-promoting effects.

[0166] Example 5: Scanning electron microscopy experiments of recombinant type XII humanized collagen For the preparation of lyophilized fiber samples of the above-mentioned recombinant type XII humanized collagen, please refer to the specific method described in Chinese patent application CN202210267088.8. A JEOL (JSM-7900F) scanning electron microscope was used, with a magnification of 1000× and a scale bar of 10µm. Three cross-sections (surface, inside, and side) of the lyophilized fiber samples of recombinant type XII humanized collagen were taken. Samples of suitable size and with smooth surfaces were selected, and the samples were fixed to the electron microscope stage with conductive tape. After gold sputtering, the samples were placed under the scanning electron microscope, and the magnification was adjusted for observation and photography to obtain the 3D microstructure of the samples. Electron microscope images show (…). Figure 8 The recombinant collagen materials XII-1, XII-11, and XII-12 exhibit a unique and sophisticated porous fibrous network structure. Overall, the fibers in the material intertwine to form a dense, intricate network, constructing an ordered and complex three-dimensional architecture. These fibers are relatively uniform in thickness and have smooth surfaces, demonstrating good uniformity and stability. Furthermore, the crisscrossing collagen fibers create numerous tiny pores at the microscale, and these orderly distributed pores constitute the porous characteristic. On the one hand, the abundant porosity provides an ideal microenvironment for cell adhesion, growth, and migration. Cells can easily embed themselves within these pores, obtaining sufficient space for metabolic activities. On the other hand, this structure significantly increases the specific surface area of ​​the material, helping to enhance its binding capacity with bioactive molecules, thereby better fulfilling its biological functions.

[0167] Furthermore, the fibrous morphology endows the material with certain mechanical properties, providing strength and toughness, making it less prone to breakage under certain external forces, and enabling it to maintain structural integrity in in vivo or in vitro environments. The porous fibrous network structure of recombinant collagen XII-1, XII-11, and XII-12 combines excellent biological and mechanical properties, showing great application potential in the biomedical field, such as tissue engineering scaffolds and wound dressings.

[0168] Verification showed that each group of recombinant type XII humanized collagen could exhibit a fibrous porous network structure to varying degrees.

[0169] Example 6: ELISA assay for serum immunoglobulins and complement components of recombinant type XII humanized collagen. Mice have a long history of being used to evaluate the immune response after in vivo application of medical device biomaterials. International standards, national standards, and related literature all recommend applying representative samples to mice to evaluate the effects of medical device biomaterials on the immune system. The test sample is injected subcutaneously into the back of mice, and peripheral blood serum is collected 30 days later. Changes in serum immunoglobulins and complement components (IgM, IgG, and C3a) are measured to evaluate the effect of the test sample on the humoral immune response.

[0170] Experimental group: Recombinant type XII humanized collagen was prepared at a concentration of 10 mg / mL using sterile saline; 0.08 mL of the sample solution was injected subcutaneously into the back of each mouse.

[0171] Positive control group (PC): Bovine serum albumin (BSA) and Freund's complete adjuvant were mixed in equal volumes in an emulsion, with a final BSA concentration of 1.67 mg / mL; 0.12 mL of BSA-CFA emulsion was subcutaneously injected into the back of each mouse.

[0172] Negative control group (NC): Each mouse was subcutaneously injected with 0.08 mL of 0.9% sodium chloride injection solution in its back. Thirty days after immunization, mice were sacrificed, and peripheral blood was collected from the orbital cavity into 1.5 mL centrifuge tubes. After standing at room temperature for 2 hours, the tubes were centrifuged at 1000g for 20 minutes, and the supernatant serum was collected and stored at -20℃ for later use. Each serum sample was diluted 10,000 times for IgM and C3a detection, and diluted 200,000 times for IgG detection. The diluted serum samples were tested according to the instructions of the commercially available ELISA kits for the target analytes (IgM, IgG, and C3a). Conventional statistical methods were used to analyze the data from the experimental and control groups. A 95% confidence level (P < 0.05) was used to determine whether there was a significant difference; * indicates P < 0.05; ** indicates P < 0.01.

[0173] The results are as follows Figure 9 As shown in this experiment, under the conditions of this study, after immunizing mice with different doses / concentrations of recombinant type XII humanized collagen for 30 days, the serum IgM, IgG, and complement fragment C3a levels in the positive control group were significantly higher than those in the negative control group; however, no significant changes in the serum IgG, IgM, and complement fragment C3a levels were observed in any of the experimental groups. This indicates that recombinant type XII humanized collagen does not cause an immune response in mice, has no adverse effects on the immune system, and is highly safe.

[0174] Example 7: Experimental Verification of Firming and Anti-aging Efficacy Matrix metalloproteinase-1 (MMP-1) is a protease that degrades type I and type III collagen in the dermis. Under normal circumstances, it can moderately clear away aged collagen, maintain collagen metabolic balance, and ensure normal skin renewal. However, when it is abnormally activated by factors such as ultraviolet radiation (the main trigger), aging, inflammation, or free radicals, the rate of collagen degradation will far exceed the rate of synthesis. Type I / III collagen is the "support network" for skin anti-wrinkle and firming; its significant loss and breakage will directly lead to a decrease in skin support and elasticity, thereby causing sagging and the transformation of dynamic wrinkles into static wrinkles. Therefore, the anti-wrinkle and firming function is achieved by inhibiting abnormal MMP-1 activity, preventing collagen degradation, and promoting collagen regeneration, thus maintaining a firm and wrinkle-free skin state and having an anti-aging effect.

[0175] The efficacy of recombinant type XII humanized collagen in inhibiting MMP-1 was verified, and the specific methods and results are described below.

[0176] Preparation of samples at various concentrations: Test sample (TA): Collagen XII was prepared with DMEM (Beijing Solarbio Science & Technology Co., Ltd.) containing 1% FBS to form three concentrations: 0.001%, 0.0005%, and 0.0001% (v / v).

[0177] Positive control (PC): Vc (L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, purchased from Sigma) was diluted to 200 μM using DMEM containing 1% FBS.

[0178] Negative control (NC): DMEM containing 1% FBS (Beijing Solarbio Science & Technology Co., Ltd.)

[0179] Experimental steps: 1. Normal human skin fibroblasts (purchased from the Kunming Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee) were cultured at 3.0~3.5×10⁻⁶ cells / mL. 4 Inoculate one well per cell into a 24-well plate and incubate for 18-24 hours.

[0180] 2. Wash the cell layer twice with PBS. Add maintenance medium (DMEM medium containing 1% FBS) containing different concentrations of the test sample and maintenance medium (DMEM medium containing 1% FBS) without the sample according to the group. Make 3 replicates for each group and retain for 24 hours.

[0181] 3. Remove the maintenance medium containing the test sample and the maintenance medium without the sample. Wash 2-3 times with PBS, then cover with a small amount of PBS (15 J / cm³). 2Human skin fibroblasts were irradiated with UVA. After irradiation, the cells were washed twice with PBS. The negative control group and the model control group were replaced with the same maintenance medium, while the other groups were added with maintenance medium containing the test sample or positive control and incubated for 48 hours.

[0182] 4. Collect the cell supernatant, store it at -80℃, and determine the MMP-1 content using an ELISA kit.

[0183] The experimental groups are shown in Table 1.

[0184] Table 1. Experimental Groups

[0185] MMP-1 content determination: Using the model control group (M) as 100%, the relative contents of MMP-1 in the negative control group (NT), positive control group (PC), and test sample groups of different concentrations (TA) were calculated. The results are as follows: Figure 10 As shown, compared with the model control group, *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0186] The results showed that the relative content of MMP-1 in the model control group (M) was significantly higher than that in the negative control group (NT); the relative content of MMP-1 in the positive control group (PC) was significantly lower than that in the model control group (M), indicating successful modeling; compared with the model control group (M), the relative content of MMP-1 in the test sample group (TA, collagen) decreased at all test concentrations, and the differences were significant. In particular, XII-1 and XII-3 were slightly better than PC group at a concentration of 0.001% (v / v), indicating that collagen has significant anti-wrinkle and firming effects.

[0187] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide contains n repeating units; The repeating unit comprises any one of the following amino acid sequences (1)-(3) or a variant thereof: (1) The amino acid sequence shown in any one of SEQ ID NO. 1-3; (2) An amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO. 1-3; (3) A variant sequence obtained by mutating one or more amino acid residues of the amino acid sequence shown in any one of SEQ ID NO. 1-3; Where n ≥ 1 and is an integer.

2. The polypeptide according to claim 1, characterized in that, The variant sequences are shown in any one of SEQ ID NO.10-11, 20-22.

3. The polypeptide according to claim 1 or 2, characterized in that, n is an integer between 1 and 20, preferably an integer between 1 and 12; more preferably 1, 5, 6, 7, 8 or 12; Preferably, when n≥2, the repeating units are directly connected; Preferably, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NO.4-6, 12-13, 16-17, 20-22.

4. A fusion protein, characterized in that, The fusion protein comprises the polypeptide according to any one of claims 1 to 3; preferably, it is obtained by fusing the polypeptide with other functional polypeptides.

5. Recombinant type XII humanized collagen, characterized in that, The recombinant type XII humanized collagen comprises the polypeptide according to any one of claims 1 to 3; Preferably, the recombinant type XII humanized collagen has cell adhesion and / or cell proliferation promotion and / or extracellular matrix improvement activities.

6. A polynucleotide, characterized in that, The polynucleotide encodes the polypeptide of any one of claims 1 to 3, the fusion protein of claim 4, or the recombinant type XII humanized collagen of claim 5; Preferably, the polynucleotide comprises the nucleotide sequence or degenerate sequence shown in any one of SEQ ID NO. 7-9, 14-15, 18-19, 23-25.

7. The polynucleotide according to claim 6, characterized in that, The polynucleotide further comprises a sequence encoding a purification tag and / or a leader sequence; preferably, the purification tag is selected from His tag, GST tag, MBP tag, SUMO tag or NusA tag.

8. A biomaterial, characterized in that, The biomaterial comprises the polynucleotide as described in claim 6 or 7; The biomaterial is an expression cassette, vector, or host cell; Preferably, the vector is an expression vector; the vector preferably further comprises a control element operatively linked to the polynucleotide; the control element is preferably a promoter, terminator, and / or enhancer. Preferably, the host cell is a bacterium, fungus, or animal cell; wherein the bacteria preferably include Escherichia coli; the fungus preferably includes yeast, more preferably Saccharomyces cerevisiae or Pichia pastoris.

9. A method for producing the polypeptide according to any one of claims 1 to 3, the fusion protein according to claim 4, or the recombinant type XII humanized collagen according to claim 5, characterized in that, The method includes: culturing host cells containing the polynucleotides of claim 6 or 7, harvesting host cells and / or culture medium containing the polypeptide or the fusion protein or the recombinant type XII humanized collagen, and isolating the polypeptide or the fusion protein or the recombinant type XII humanized collagen therefrom.

10. A composition, characterized in that, The composition comprises one or more selected from the polypeptides of any one of claims 1 to 3, the fusion protein of claim 4, the recombinant type XII humanized collagen of claim 5, the polynucleotide of claim 6 or 7, and the biomaterial of claim 8.

11. The composition according to claim 10, characterized in that, The composition is selected from biological dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue filling / volume-enhancing materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, chronic wound repair materials, bone or cartilage regeneration materials, liver tissue materials, vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, cosmetics, pharmaceutical excipients, drugs or food additives; Preferably, the composition is a topical composition, an injectable composition, or an oral composition; Preferably, the composition is a composition in the form of a solution, lyophilized powder, gel, sponge, or fiber.

12. The use of the polypeptide of any one of claims 1 to 3, the fusion protein of claim 4, the recombinant type XII humanized collagen of claim 5, the polynucleotide of claim 6 or 7, the biomaterial of claim 8, or the composition of claim 10 or 11 in the preparation of bio-dressings, human biomimetic materials, plastic and cosmetic materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue filling / volume-enhancing materials, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, chronic wound repair materials, muscle or bone or cartilage or ligament regeneration materials, liver tissue materials, vascular repair and regeneration materials, 3D printed artificial organ biomaterials, cosmetic raw materials, cosmetics, pharmaceutical excipients, drugs, or food additives.

13. The use of the polypeptide of any one of claims 1 to 3, the fusion protein of claim 4, the recombinant type XII humanized collagen of claim 5, the polynucleotide of claim 6 or 7, the biomaterial of claim 8, or the composition of claim 10 or 11 in the preparation of products for use in any one or more of the following: cell adhesion, promotion of cell proliferation, improvement of extracellular matrix, tissue repair, tissue filling or volume enhancement, inhibition of MMP-1, reduction of collagen degradation, promotion of collagen regeneration, skin tightening, skin anti-wrinkle, and skin anti-aging.

14. The use of the polypeptide of any one of claims 1 to 3, the fusion protein of claim 4, or the recombinant type XII humanized collagen of claim 5 in any one or more of the following: cell adhesion, promotion of cell proliferation, improvement of extracellular matrix, tissue repair, tissue filling or volume enhancement, plastic surgery, inhibition of MMP-1, reduction of collagen degradation, promotion of collagen regeneration, skin tightening, skin anti-wrinkle, and skin anti-aging.

15. The use of the polypeptide of any one of claims 1 to 3, the fusion protein of claim 4, the recombinant humanized type XII collagen of claim 5, the polynucleotide of claim 6 or 7, the biomaterial of claim 8, or the composition of claim 10 or 11 in the preparation of a medicament for the prevention and / or treatment of diseases or conditions related to type XII collagen deficiency or for the supplementation of type XII collagen.

16. A method for promoting cell adhesion and / or proliferation and / or improving the extracellular matrix, characterized in that, The method includes the step of contacting a cell with the polypeptide of any one of claims 1 to 3, the fusion protein of claim 4, the recombinant type XII humanized collagen of claim 5, or the composition of claim 10 or 11; the cell is preferably an animal cell; more preferably a mammalian cell; more preferably a human cell; preferably, the promotion of cell adhesion and / or proliferation and / or improvement of extracellular matrix is ​​performed in vivo / in vitro.

17. A product characterized in that, The product comprises the polypeptide of any one of claims 1 to 3, the fusion protein of claim 4, the recombinant type XII humanized collagen of claim 5, or the composition of claim 10 or 11.

18. The product according to claim 17, characterized in that, The product in question is a cosmetic product.

19. The product according to claim 17 or 18, characterized in that, The product has any one or more of the following functions: cell adhesion, promoting cell proliferation, improving extracellular matrix, tissue repair, tissue filling or volume enhancement, inhibiting MMP-1, reducing collagen degradation, promoting collagen regeneration, skin firming, anti-wrinkle skin, and anti-aging skin.

20. A method for promoting skin firmness, anti-wrinkle effects, or anti-aging effects, characterized in that, The method includes the step of administering to a subject the polypeptide of any one of claims 1 to 3, the fusion protein of claim 4, the recombinant type XII humanized collagen of claim 5, or the composition of claim 10 or 11, or the product of any one of claims 17 to 19.

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