Recombinant human XVII type collagen and application thereof in preparation of anti-wrinkle products

Abstract

The invention discloses recombinant human XVII type collagen and application thereof in preparation of anti-wrinkle products, and belongs to the technical field of bioengineering. The amino acid sequence of the recombinant human XVII type collagen is shown as SEQ ID NO.1, four fragments contained in the recombinant human XVII type collagen are derived from human, and the recombinant human XVII type collagen is low in immunogenicity risk and high in biocompatibility; only fragments with biological activity in the collagen are expressed, the molecular weight is smaller, and the expression efficiency is higher; meanwhile, the recombinant human XVII type collagen has the functions of promoting cell proliferation, resisting wrinkles and repairing. In addition, the fermentation and purification process of the recombinant human XVII type collagen is simple to operate, has conditions for expanding production, and can be used for large-scale industrial production. Therefore, the recombinant protein provided by the invention has relatively high popularization value and can be widely applied to the fields of medicines, biological materials, medical instruments or skin care products and the like.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to a recombinant human type XVII collagen and its application in the preparation of anti-wrinkle products. Background Technology

[0002] Collagen is a biological macromolecule and a major component of animal connective tissue. It is also the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of total protein, and even more than 80% in some organisms. There are many types of collagen. Currently, at least 30 genes encoding collagen chains have been discovered, which can form more than 16 types of collagen molecules. Based on their structure, they can be classified into fibrous collagen, basement membrane collagen, microfibrillary collagen, anchored collagen, hexagonal reticular collagen, non-fibrous collagen, transmembrane collagen, etc.

[0003] Type XVII collagen (also known as COL7) is a homogeneous trimer composed of three identical α1 (XVII) chains. It helps cells attach to the basement membrane and strengthens intercellular connections.

[0004] Skin aging is induced by either intrinsic aging (also known as time-related aging) or extrinsic aging caused by environmental factors such as air pollution and ultraviolet radiation. Adult stem cells (ASCs) are crucial for cell replacement in tissues. Their main function is to replace cells that may die due to disease or injury and to maintain intracellular homeostasis, but their ability declines with age. The self-renewal and differentiation capacity of stem cells is influenced by their environment (niche). Niche studies on resident stem cells have shown that transmembrane collagen COL17 mediates the interaction between stem cells and surrounding cells and the matrix, playing an important role in regulating skin homeostasis, aging, and wound repair.

[0005] The main methods for preparing type XVII collagen are: animal tissue extraction, cell culture, synthetic biology, and chemical synthesis. While animal tissue extraction can yield high levels of bioactive type XVII collagen, it is inherently scarce in tissues and, being a transmembrane protein, is difficult and costly to extract, hindering large-scale applications. Cell culture yields completely human proteins with low immunogenicity risk, but even in high-expression cell lines, expression levels remain relatively low. Synthetic biology, while less expensive and with shorter culture cycles, is largely unable to induce glycosylation or other modifications. Chemical synthesis, while producing extremely high purity and precise sequences, is limited to short peptides and lacks spatial structure.

[0006] Recombinant collagen is produced by introducing the natural or redesigned gene sequence of collagen into selected host cells, such as E. coli or yeast, followed by culturing, fermentation, separation, and purification processes to obtain a protein with certain characteristics and main functions of natural collagen. Recombinant collagen is preferred for industrial-scale production due to its simple molecular structure, clear definition, ease of control, high yield, lack of viral risks, low cytotoxicity, extremely low immunogenicity, and low likelihood of causing allergies, inflammation, fever, etc. Furthermore, it possesses good water solubility and emulsification properties. However, the full-length sequence of recombinant human XVII collagen is difficult to synthesize and effectively express, therefore, there is an urgent need to provide a recombinant human XVII collagen that can be effectively expressed and has anti-wrinkle effects.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to provide a recombinant human type XVII collagen and its application in the preparation of anti-wrinkle products. This recombinant human type XVII collagen can be effectively expressed and can provide a good anti-wrinkle effect.

[0009] This invention is implemented as follows: In a first aspect, the present invention provides a recombinant human type XVII collagen, the amino acid sequence of which is any one of the following (A1) to (A3): (A1) The amino acid sequence of recombinant human type XVII collagen is shown in SEQ ID NO.1; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the recombinant human type XVII collagen described in (A1); (A3) has an amino acid sequence that is at least 90% identical to the recombinant human type XVII collagen described in (A1) or the fusion protein described in (A2) and has anti-wrinkle effects.

[0010] Secondly, the present invention provides a biomaterial similar to the above-mentioned recombinant human type XVII collagen, which is any one of items (B1) to (B4): (B1) The nucleic acid molecule encoding the above-mentioned recombinant human type XVII collagen; (B2) An expression cassette containing the nucleic acid molecule described in (B1); (B3) A recombinant vector containing the nucleic acid molecule described in (B1) or the expression cassette described in (B2); (B4) A host cell containing the nucleic acid molecule described in (B1), the expression cassette described in (B2), or the recombinant vector described in (B3).

[0011] Thirdly, the present invention provides a method for preparing the above-mentioned recombinant human type XVII collagen, which includes: introducing a nucleic acid molecule encoding the above-mentioned recombinant human type XVII collagen into an expression vector, then introducing the obtained recombinant plasmid into a host cell, and then separating and purifying the recombinant human type XVII collagen after fermentation culture.

[0012] Fourthly, the present invention provides a composition comprising the above-described recombinant human type XVII collagen.

[0013] Fifthly, the present invention provides the application of the above-mentioned recombinant human type XVII collagen in the preparation of products that promote cell proliferation.

[0014] In a sixth aspect, the present invention provides the application of the above-mentioned recombinant human type XVII collagen in the preparation of anti-wrinkle products.

[0015] The present invention has the following beneficial effects: This invention splices and combines four fragments from the 15 collagen domains of human type XVII collagen to form a novel recombinant human type XVII collagen. Each fragment in this recombinant human type XVII collagen is derived from human tissue, resulting in low immunogenicity risk and high biocompatibility. Furthermore, it expresses only the biologically active fragments of collagen, has a smaller molecular weight, and higher expression efficiency. Simultaneously, this recombinant human type XVII collagen exhibits functions such as promoting cell proliferation, anti-wrinkle effects, and repair. In addition, the fermentation purification process of this invention for the recombinant human type XVII collagen is simple to operate and has the conditions for large-scale industrial production. Therefore, the recombinant protein provided by this invention has high promotional value and can be widely applied in pharmaceuticals, biomaterials, medical devices, and skincare products. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is an electrophoresis image of the target protein in Example 1; Figure 2 The effect of different experimental groups on the proliferation rate of HSF cells in Example 2; Figure 3 This illustrates the effect of different experimental groups in Example 2 on the secretion of hyaluronic acid by HSF cells. Figure 4 The effect of different experimental groups on MMP1 enzyme activity in HSF cells in Example 2; Figure 5 This describes the effect of different experimental groups on the activity of MMP2 enzyme in HSF cells in Example 2. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0020] As used in this invention, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of those embodiments.

[0021] When referring to a singular noun, the indefinite or definite article used, such as "a" or "a kind of," "the," includes the plural form of the noun.

[0022] As used in this invention, the term "nucleic acid molecule" is a collective term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), a biological macromolecular compound composed of many nucleotide monomers, and one of the most fundamental substances of life. A nucleotide sequence refers to the arrangement of bases in DNA or RNA. Nucleic acid molecules contain cDNA, and in some cases, nucleic acid molecules can be modified for use in the vectors of this invention, such as for codon optimization. In some cases, for the purpose of cloning into a vector, the sequence can be designed to contain terminal restriction sites. Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of encoding nucleic acids from one or more given cells or isolated from said one or more given cells.

[0023] As used herein, the term "expression cassette" refers to a nucleic acid construct comprising coding and regulatory sequences operable upon introduction into a host cell, resulting in transcription and / or translation of RNA or polypeptide, respectively. An expression cassette should be understood to include a promoter that allows transcription to begin, an open reading frame of the target gene, and a transcription terminator. Typically, the promoter sequence is positioned upstream of the target gene at a distance compatible with expression control.

[0024] As used herein, the term "expression vector" refers to a self-replicating DNA molecule used in recombinant DNA technology to transfer a DNA fragment (target gene) into a recipient cell. Besides the commonly used *E. coli* plasmid vector, there are many other artificially constructed plasmid vectors suitable for microorganisms, yeast, plants, etc. Vectors include, but are not limited to: single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. The most commonly used vector type is the "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, through standard molecular cloning techniques. Recombinant expression vectors may contain a form suitable for expressing nucleic acids in host cells, meaning that recombinant expression vectors include one or more regulatory elements that can be selected based on the host cell used for expression and which can be operatively linked to the nucleic acid sequence to be expressed.

[0025] As used in this invention, the term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc., of a nucleic acid construct or expression vector containing the polynucleotides of this invention. The host cell can be any cell useful in the production of the relevant protein. To produce the relevant protein, the nucleic acid encoding the protein can be isolated and inserted into one or more vectors for further cloning and / or expression in the host cell. Methods for introducing vectors into host cells are well known, such as using electroporation, and can also include transfection, microinjection, gene gun technology, liposome-mediated methods, etc.

[0026] The following is provided merely to aid in understanding the invention. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0027] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention. Any limited modifications made by any person within the scope of the claims of the present invention are still within the scope of the claims of the present invention.

[0028] Type XVII collagen is a transmembrane protein containing 1497 amino acids. Its most prominent structural feature is that the N-terminus is located inside the cell and consists of 486 amino acids, with four tandem repeat sequences of 24-26 amino acids in the middle. The C-terminus is a longer extracellular peptide chain that is connected to the anchor fibers of the lamina luminosilicate of the skin's basement membrane.

[0029] Human type XVII collagen contains 15 collagen domains (amino acid sequences as shown in SEQ ID NO: 3-17). These domains are located in the extracellular domain (ECD; aa 489-1497), which may be the structural sites where it interacts with extracellular matrix components to stabilize the connection between the epidermis and dermis. These collagen domains are separated from each other by short non-collagenous domains.

[0030] Based on this, the present invention, through analysis of the sequence and structure of human type XVII collagen and random combination of multiple fragments, screened a novel recombinant human type XVII collagen. The amino acid sequence of this recombinant protein originates from the GXY region of a triple helix structure and is composed of collagen domains from the three peptide chains that constitute collagen, without any non-collagen domains. Verification has shown that this recombinant human type XVII collagen can not only be effectively expressed but also exhibits good effects in promoting cell proliferation and anti-wrinkle properties.

[0031] The amino acid sequence of this recombinant human type XVII collagen is as follows: GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGP DGHQGPRGEQGLTGMPGIRGPPPGSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIGGAGSLGAGGAFGEAAGDRGPYGTDIGPGGGYGAAAEGGMYAGNGGLLGADFAGDLGPPGQPGPQGPPGISGPPGQKGEMGTPPGKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ (SEQ ID NO.1).

[0032] To facilitate subsequent separation and purification, the recombinant human type XVII collagen can also be tagged at the N-terminus and / or C-terminus to obtain a fusion protein. Protein tags can be broadly categorized into three types based on their function: detection tags, expression and purification tags, and tracer tags. Commonly used expression and purification tags include: His, GST, MBP, CBD, Strep-tag, Halo-Tag, SNAP-tag, SUMO, NusA, TrxA, DsbA, Flag, and c-Myc. This invention does not limit the type of tag; those skilled in the art can adjust it as needed.

[0033] In some embodiments, the amino acid sequence of the recombinant human type XVII collagen may also be: an amino acid sequence that has at least 90% identity with the recombinant human type XVII collagen or fusion protein and has the ability to promote cell adhesion and proliferation.

[0034] The percentage of sequence identity is the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with a reference polypeptide sequence, introducing vacancies where necessary to obtain the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0035] In some embodiments, having at least 90% sequence identity can specifically be a range of any one or any two of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0036] Based on the above-mentioned recombinant human type XVII collagen, the present invention may also provide a nucleic acid molecule that encodes the above-mentioned recombinant human type XVII collagen.

[0037] In specific implementation schemes, the aforementioned nucleic acid molecules can be optimized according to the codon preferences of the host cell before being used to artificially synthesize gene fragments. It should be understood that all nucleic acid molecules capable of being translated into the aforementioned amino acid sequences are within the scope of protection of this invention.

[0038] In some embodiments, the nucleotide sequence of the above-mentioned nucleic acid molecule is shown in SEQ ID NO.2.

[0039] Furthermore, the present invention can also provide an expression cassette or expression vector comprising the aforementioned nucleic acid molecule. The expression cassette further contains regulatory sequences, such as a promoter and a terminator; the expression vector comprises the aforementioned nucleic acid molecule or expression cassette.

[0040] In some embodiments, the expression vectors described above include, but are not limited to, pPICzαA.

[0041] The present invention may also provide a host cell comprising the above-mentioned nucleic acid molecule or the above-mentioned expression cassette or the above-mentioned expression vector, and the host cell is capable of expressing the above-mentioned recombinant human type XVII collagen.

[0042] In a specific implementation plan, the host cell can be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, and Bacillus licheniformis; eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells. Preferably, the host cell is Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, or Bacillus subtilis. More preferably, the host cell is Pichia pastoris X-33.

[0043] To obtain recombinant human type XVII collagen with this purity and stability, the present invention provides a corresponding preparation method, which includes: introducing the nucleic acid molecule of the above-mentioned recombinant human type XVII collagen into an expression vector, then introducing the obtained recombinant plasmid into a host cell, and then separating and purifying the above-mentioned recombinant human type XVII collagen after fermentation culture.

[0044] In the specific implementation plan, the preparation of recombinant human type XVII collagen using host cells refers to fermenting and culturing host cells, and the culture medium and culture conditions are well known to those skilled in the art.

[0045] In some embodiments, the culture medium described above may be YPD liquid culture medium.

[0046] The present invention does not impose any restrictions on the expression method, which can be selected and adjusted as needed. For example, the expression can be one or a combination of two of the following: compositional expression and induced expression. The inducer for induced expression can be IPTG, β-galactoside, methanol, ethanol, etc.

[0047] In some embodiments, recombinant human type XVII collagen is obtained by inducing expression in the host cells described above. Specifically, when the bacterial cell density reaches a certain value, methanol is added to induce expression.

[0048] As for the methods of separation and purification, they include one or a combination of several of the following: filtration, salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, membrane separation, etc.

[0049] In some embodiments, the separation and purification process includes: after fermentation, centrifuging to collect the supernatant, and then subjecting the supernatant to filtration, chromatography, and ultrafiltration. Preferably, the chromatography method is ion exchange chromatography; more preferably, the chromatography method is cation exchange chromatography.

[0050] The recombinant human type XVII collagen obtained through the specific fermentation and purification process described above not only has high yield and purity, but cell experiments have also demonstrated that this recombinant human type XVII collagen can promote cell proliferation and the secretion of hyaluronic acid by skin fibroblasts, and inhibit the activity of matrix metalloproteinase 1 (MMP1) and matrix metalloproteinase 2 (MMP2). Therefore, the recombinant human type XVII collagen of the present invention can be applied in the fields of cosmetics and pharmaceuticals.

[0051] In some embodiments, the recombinant human type XVII collagen described above can be used to prepare related products that promote cell proliferation.

[0052] In some embodiments, the recombinant human type XVII collagen described above can be used to prepare anti-wrinkle related products.

[0053] In some embodiments, the related products may be wound repair products, such as medical sprays, repair gels, and scar patches; they may also be wound dressings that promote healing, such as hydrogel dressings, sponge dressings, fibrin glue, and nanofiber scaffolds; they may also be scar management products, such as scar removal gels and scar softening injections; they may also be artificial dermis / skin substitutes, such as three-dimensional scaffolds constructed based on biomaterials such as collagen and chitosan; and they may also be anti-aging skincare products, such as serums, face creams, eye creams, and ampoule serums.

[0054] This specification is intended to enable those skilled in the art to practice the invention. Various modifications to the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0055] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0056] Example 1 This example describes the preparation of recombinant collagen, and the specific steps are as follows: (1) Preparation of shuttle plasmids In this embodiment, 15 domains of human type XVII collagen were randomly combined into various fragments, and codon optimization was performed on the yeast expression system to obtain the target gene sequence of recombinant human type XVII collagen. The obtained target gene sequence was synthesized by Qingke Biotechnology Co., Ltd. The synthesized gene was ligated into the pPicZαA plasmid, resulting in the following four amino acid sequences with relatively high expression yields. The corresponding plasmid is represented as pPicZαA-XVII-n (n represents one of a, b, c, and d): COL 17 a: It is formed by sequentially splicing the amino acid sequences described in SEQ ID NO: 17, 12, 7, 3; COL 17b: It is formed by sequentially splicing the amino acid sequences described in SEQ ID NO: 16, 6, 8, 5, 17; COL 17 c: It is formed by sequentially splicing the amino acid sequences described in SEQ ID NO: 17, 5, 4, 3; COL 17 d: It is formed by sequentially splicing the amino acid sequences described in SEQ ID NO: 10, 9, 17, 4.

[0057] (2) Preparation of yeast expression strains pPicZαA-XVII-n was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Transformants were screened using bleomycin resistance as a selection marker to obtain the yeast expression strain.

[0058] (3) Induced expression of target protein 1) Select a single colony of the constructed yeast expression strain and add it to 5 ml of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and incubate at 30℃ and 200 rpm for 48 h for activation; 2) Transfer 1% of the inoculum to a 500ml Erlenmeyer flask (containing 200ml of YPD culture medium), and incubate at 30℃ and 200rpm for 24h to serve as the seed for the next culture. 3) Prepare 3L of BSM medium and add it to a 5L fermenter. Sterilize at 121℃ for 20min. After cooling to 30℃, adjust the pH to 6.0. Add the seed prepared in (2) to the fermenter in the form of flame inoculation for fermentation culture. 4) When the OD600 reaches 64, start adding 50% glycerol. When the OD600 reaches about 100, stop adding glycerol and wait for the dissolved oxygen to rebound to 100% before starting to add methanol for induction. 5) During the induction process, the dissolved oxygen should be controlled at no less than 30% and the pH at around 6.0. Induction should be carried out for 40 hours. After fermentation, the culture medium should be centrifuged at 12,000 rpm for 2 minutes. The supernatant should be collected and the protein yield and purity should be detected by BCA method (Solarbio, Lot No. 242102001) and SDS-PAGE method.

[0059] Protein electrophoresis image as shown Figure 1 As shown in the figure. BCA results showed that the protein concentrations per liter of fermentation broth were COL 17a = 26.5 g / L, COL 17b = 22.8 g / L, COL 17c = 23.2 g / L, and COL 17d = 9.8 g / L.

[0060] (4) Purification of the target protein 1) The fermentation broth is centrifuged to remove the bacterial cells, resulting in a semi-clear liquid; 2) The fermentation supernatant is passed through hollow fiber to remove cell debris, and the fermentation broth is further clarified to remove pigments, resulting in a clear, pale yellow liquid. 3) After clarification, the fermentation broth is subjected to ion exchange chromatography. A cation exchange packing is used, and ion strength gradient elution is performed at pH 6.5 to collect the target protein with a purity ≥ 99%. The protein with a purity of ≥ 99% is obtained. Then, a 10 kDa ultrafiltration membrane is used to concentrate the protein to obtain the target protein solution. 4) The protein solution was freeze-dried to obtain the corresponding recombinant type XVII collagen lyophilized sponge. The freeze-dried protein sample was stored at -80℃ for later use.

[0061] Example 2 This example demonstrates the cellular anti-wrinkle effect of recombinant human type XVII collagen. (1) HSF cell proliferation rate 1) Select HSF cells in the logarithmic growth phase, digest with trypsin, centrifuge, discard the supernatant, and adjust the cell suspension concentration to 5 × 10⁻⁶. 5 Cells / mL, add 200 μL of cell suspension to each well of a 96-well plate, and incubate at 37°C and 5% CO2 for 24 h.

[0062] 2) Cells were treated for 24 h with DMEM medium (without antibiotics and fetal bovine serum) containing different concentrations (10 μg / mL, 50 μg / mL, and 100 μg / mL) of recombinant human type XVII collagen (COL 17n) and acetyl hexapeptide. The experimental groups were a blank control group (without samples) and an experimental group (treated with serum-free medium containing different concentrations of samples).

[0063] 3) Add 10 μL of CCK-8 solution and incubate in a 5% CO2, 37℃ incubator for 3 h. Measure the absorbance at 450 nm using a microplate reader to determine the cell proliferation rate. The formula for calculating the cell proliferation rate is as follows: Cell proliferation rate = ; In the formula: A1 represents the absorbance of the sample group; A2 represents the absorbance of the cell-free control group; A3 represents the absorbance of the blank group containing cells.

[0064] The effects of recombinant human type XVII collagen (COL 17n) and acetyl hexapeptide at concentrations of 10 μg / mL, 50 μg / mL, and 100 μg / mL on the proliferation rate of HSF cells were determined. The results are as follows: Figure 2 As shown.

[0065] Acetyl hexapeptide is an active polypeptide that can inhibit the release of neurotransmitters, block the transmission of electrical signals between synapses, relax skin muscles, and smooth dynamic wrinkles. It is often used as a reference in the daily chemical industry. Within the selected concentration range, acetyl hexapeptide did not have significant toxic effects on HSF cells (p>0.05).

[0066] COL 17a, COL 17b, COL 17c, and COL 17d all promoted the proliferation of HSF cells to varying degrees. The proliferation of HSF cells initially increased and then decreased with increasing sample concentration. COL 17a and COL 17d promoted the proliferation of HSF cells (P<0.05 compared to the blank control group), COL 17c promoted the proliferation of HSF cells significantly (P<0.01 compared to the blank control group), and COL 17b did not promote the proliferation of HSF cells (no significant difference compared to the blank control group). Furthermore, the proliferation effect of COL 17c on HSF cells was significantly better than that of COL 17a, COL 17b, and COL 17d.

[0067] (2) Measurement of hyaluronic acid secretion by HSF cells Procedure: Collect cells and detect intracellular ROS levels according to the assay method in the hyaluronic acid assay kit.

[0068] Hyaluronic acid is an important component of the extracellular matrix of dermal cells, playing a role in maintaining cell moisture, accelerating skin tissue repair, and improving wound healing and regeneration. However, with age, the content of hyaluronic acid in the extracellular matrix of skin cells gradually decreases, the skin's hydration capacity declines, and wrinkles appear.

[0069] like Figure 3 As shown, 10 μg / mL and 100 μg / mL of COL 17c can significantly increase the amount of hyaluronic acid secreted by HSF cells (p<0.05), and 50 μg / mL of COL 17c increases the amount of hyaluronic acid secreted by HSF cells from 0.45 ng / mL to 5.15 ng / mL, which has a highly significant effect and can increase the stability of the extracellular matrix of HSF cells, indicating that it has good potential to reduce wrinkle formation.

[0070] 3) MMP1 enzyme activity assay in HSF cells Procedure: Collect cells and detect changes in intracellular SOD enzyme activity according to the assay method in the MMP1 assay kit.

[0071] Human skin contains 23 different types of matrix metalloproteinases, and MMP1 plays an important role in the natural aging process of the skin. MMP1 can induce the degradation of collagen and inhibit the synthesis of collagen precursors.

[0072] like Figure 4 As shown, acetyl hexapeptide, COL 17a, COL 17b, COL 17c, and COL 17d all have a certain inhibitory effect on MMP1. Among them, 50 μg / mL of COL 17c can significantly inhibit the enzyme activity of MMP1 in HSF cells (p<0.01).

[0073] 4) MMP2 enzyme activity assay in HSF cells Procedure: Collect cells and detect changes in intracellular GSH-Px enzyme activity according to the assay method in the MMP2 assay kit.

[0074] HSF is a major source of MMPs, and MMP2 can degrade elastin and proteoglycans. For example... Figure 5 As shown, acetyl hexapeptide, COL 17a, COL 17b, COL 17c, and COL 17d can all inhibit the enzyme activity of MMP2 in HSF cells. Among them, COL 17c has a significant inhibitory effect, effectively inhibiting the degradation of collagen and elastin by inhibiting the activity of MMP2, thus delaying skin aging and reducing the formation of wrinkles.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant human type XVII collagen, characterized in that, The amino acid sequence of the recombinant human type XVII collagen is any one of the following (A1) to (A3): (A1) The amino acid sequence of the recombinant human type XVII collagen is shown in SEQ ID NO.1; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the recombinant human type XVII collagen described in (A1); (A3) has an amino acid sequence that is at least 90% identical to the recombinant human type XVII collagen described in (A1) or the fusion protein described in (A2) and has anti-wrinkle effects.

2. The biomaterial of recombinant human type XVII collagen as described in claim 1, characterized in that, It can be any one of items (B1) to (B4): (B1) A nucleic acid molecule encoding the recombinant human type XVII collagen as described in claim 1; (B2) An expression cassette containing the nucleic acid molecule described in (B1); (B3) A recombinant vector containing the nucleic acid molecule described in (B1) or the expression cassette described in (B2); (B4) A host cell containing the nucleic acid molecule described in (B1), the expression cassette described in (B2), or the recombinant vector described in (B3).

3. The biomaterial according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. The method for preparing recombinant human type XVII collagen as described in claim 1, characterized in that, include: The nucleic acid molecule encoding the recombinant human type XVII collagen as described in claim 1 is introduced into an expression vector, and then the obtained recombinant plasmid is introduced into a host cell. After fermentation culture, the recombinant human type XVII collagen is separated and purified to obtain the recombinant human type XVII collagen.

5. The preparation method according to claim 4, characterized in that, The expression vector includes pPICzαA.

6. The preparation method according to claim 4, characterized in that, The host cell includes Pichia pastoris.

7. A composition, characterized in that, Includes the recombinant human type XVII collagen as described in claim 1.

8. The use of the recombinant human type XVII collagen as described in claim 1 in the preparation of products that promote cell proliferation.

9. The use of the recombinant human type XVII collagen as described in claim 1 in the preparation of anti-wrinkle products.

10. The application according to claim 9, characterized in that, The product is used to promote the secretion of hyaluronic acid by cells and to inhibit the activity of matrix metalloproteinase 1 and matrix metalloproteinase 2.

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